U.S. patent application number 10/809860 was filed with the patent office on 2004-08-05 for dna molecule encoding a variant alpha-2b-adrenoaceptor protein and uses thereof.
This patent application is currently assigned to Oy Juvantia Pharma Ltd.. Invention is credited to Alhopuro, Pia, Heinonen, Paula, Karvonen, Matti, Kauhanen, Jussi, Koulu, Markku, Lakka, Timo A., Nyyssonen, Kristiina, Pesonen, Ullamari, Salonen, Jukka T., Salonen, Riitta, Scheinin, Mika, Snapir, Amir, Tuomainen, Tomi-Pekka, Valkonen, Veli-Pekka.
Application Number | 20040152637 10/809860 |
Document ID | / |
Family ID | 23677216 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152637 |
Kind Code |
A1 |
Snapir, Amir ; et
al. |
August 5, 2004 |
DNA molecule encoding a variant alpha-2B-adrenoaceptor protein and
uses thereof
Abstract
This invention relates to a DNA sequence comprising a nucleotide
sequence encoding a variant .alpha..sub.2B-adrenoceptor protein and
to the variant .alpha..sub.2B-adrenoceptor protein as well as a
method for screening a subject to determine if the subject is a
carrier of a variant gene that encodes the variant
.alpha..sub.2B-adrenoceptor protein. This invention also relates to
a method for treating a mammal suffering from vascular contraction
of coronary arteries, the method comprising the step of
administering a selective .alpha..sub.2B-adrenoceptor antagonist to
the mammal. This invention further relates to transgenic animals
comprising a human DNA molecule encoding human
.alpha..sub.2B-adrenoceptor protein or the variant
.alpha..sub.2B-adrenoceptor protein.
Inventors: |
Snapir, Amir; (Turku,
FI) ; Heinonen, Paula; (Turku, FI) ; Alhopuro,
Pia; (Turku, FI) ; Karvonen, Matti; (Turku,
FI) ; Koulu, Markku; (Turku, FI) ; Pesonen,
Ullamari; (Turku, FI) ; Scheinin, Mika;
(Naantali, FI) ; Salonen, Jukka T.; (Jannevirta,
FI) ; Tuomainen, Tomi-Pekka; (Kuopio, FI) ;
Lakka, Timo A.; (Kuopio, FI) ; Nyyssonen,
Kristiina; (Kuopio, FI) ; Salonen, Riitta;
(Jannevirta, FI) ; Kauhanen, Jussi; (Kuopio,
FI) ; Valkonen, Veli-Pekka; (Kuopio, FI) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Oy Juvantia Pharma Ltd.
Turku
FI
|
Family ID: |
23677216 |
Appl. No.: |
10/809860 |
Filed: |
March 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10809860 |
Mar 26, 2004 |
|
|
|
09422985 |
Oct 22, 1999 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/320.1; 435/325; 435/69.1; 514/16.4; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 9/10 20180101; A61K
38/00 20130101; A01K 2217/05 20130101; C07K 14/70571 20130101 |
Class at
Publication: |
514/012 ;
530/350; 435/006; 435/069.1; 435/320.1; 435/325; 536/023.5 |
International
Class: |
A61K 038/17; C12Q
001/68; C07H 021/04 |
Claims
1. A nucleic acid encoding a variant C2B-adrenoceptor protein, the
variant protein comprises a deletion of at least 1 glutamate from a
glutamic acid repeat element of 12 glutamates, amino acids 298-309,
in an acidic stretch of 18 amino acids 294-311, located in a
3.sup.rd intracellular loop of the receptor protein.
2. The nucleic acid of claim 1, wherein the variant protein
comprises a deletion of 3 glutamates, amino acids 307-309, from
said glutamic acid repeat element of 12 glutamates, amino acids
298-309, in said acidic stretch of 18 amino acids 294-311, located
in the 3.sup.rd intracellular loop of the receptor polypeptide.
3. The nucleic acid of claim 2 comprising a genomic nucleotide
sequence as set forth in SEQ ID NO:1.
4. The nucleic acid of claim 1, wherein said nucleic acid is
cDNA.
5. An RNA sequence fully complementary to the DNA sequence of claim
1.
6. A variant .alpha..sub.2B-adrenoceptor protein comprising a
deletion of at least 1 glutamate from said glutamic acid repeat
element of 12 glutamates, amino acids 298-309, in said acidic
stretch of 18 amino acids 294-311, located in the 3.sup.rd
intracellular loop of the receptor polypeptide.
7. The variant .alpha..sub.2B-adrenoceptor protein of claim 6,
wherein the protine comprises a deletion of 3 glutamates, amino
acids 307-309, from said glutamic acid repeat element of 12
glutamates, amino acids 298-309, in said acidic stretch of 18 amino
acids 294-311, located in the 3.sup.rd intracellular loop of the
receptor polypeptide.
8. The variant .alpha..sub.2B-adrenoceptor protein of claim 7
comprising an amino acid sequence set forth in SEQ ID NO: 2.
9. An assay for determining the presence or absence of the nucleic
acid of claim 1.
10. The assay of claim 9, wherein the assay is a DNA-assay.
11. A method for determining the presence or absence in a
biological sample of the nucleic acid of claim 1 comprising
contacting a single-stranded form of said nucleic acid if present
in the sample with a capturing nucleic acid probe and a detector
nucleic acid probe to form a complex and detecting the presence or
absence of the complex.
12. The method of claim 11, wherein the capturing nucleic acid
probe is attached or capable of attaching to a solid phase, and
comprises a cDNA encoding the variant .alpha..sub.2B-adrenoceptor
protein, wherein a detected signal from the solid phase is an
indication of the presence in the sample of said nucleic acid.
13. The method according to claim 11, wherein the capturing nucleic
acid probe is attached or capable of attaching to a solid phase,
and comprises a cDNA encoding a non-variant
.alpha..sub.2B-adrenoceptor protein, wherein a detected signal from
the solid phase is an indication of the absence in the sample of
said nucleic acid.
14. A method for screening a subject to determine if the subject is
at risk for developing a disease involving vascular contraction of
coronary arteries or is in need of .alpha..sub.2B-selective or
.alpha..sub.2B-nonselective .alpha..sub.2-adrenoceptor antagonist
therapy, said method comprises obtaining a biological sample of the
subject and determining whether the subject (i) has the
insertion/insertion (I/I) or deletion/insertion (D/I) genotypes of
the human .alpha..sub.2B-adrenoceptor protein or (ii) has the D/D
genotype of the human .alpha..sub.2B-adrenoceptor protein, wherein
if the subject has the D/D genotype, the subject is at risk for
developing a disease involving vascular contraction of coronary
arteries or is in need of .alpha..sub.2B-selective
.alpha..sub.2-adrenoceptor antagonist therapy.
15. The method of claim 14, wherein the assay is a DNA-assay.
16. A capturing probe which comprises a single strand of the cDNA
of claim 4.
17. A capturing probe which comprises a single strand of a cDNA
encoding a non-variant .alpha..sub.2B-adrenoceptor protein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a division of U.S. patent
application Ser. No. 09/422,985 filed on 22 Oct. 1999. This
application is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a DNA molecule encoding a variant
human .alpha..sub.2B-adrenoceptor, said variant
.alpha..sub.2B-adrenoceptor protein and a method to assess the risk
of individuals to suffer from vascular contraction of coronary
arteries in mammals as well as a method for the treatment of
vascular contraction of coronary arteries. This invention also
relates to transgenic animals comprising a human DNA molecule
encoding human .alpha..sub.2B-adrenoceptor or said variant
.alpha..sub.2B-adrenoceptor.
BACKGROUND OF THE INVENTION
[0003] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated by reference.
[0004] The .alpha..sub.2-adrenoceptors (.alpha..sub.2-ARs) mediate
many of the physiological effects of the catecholamines
norepinephrine and epinephrine. Three genetic subtypes of
.alpha..sub.2-adrenoceptors are known in humans and other mammals,
denoted as .alpha..sub.2A-, .alpha..sub.2B- and
.alpha..sub.2C-adrenoceptors. The human genes encoding the
receptors are located on chromosomes 10, 2 and 4, respectively. No
splice variants are known to exist of these receptors, as the genes
are intronless. The tissue distributions and physiological and
pharmacological functions of the receptor subtypes have been
reviewed e.g. by MacDonald et al. (1997) and Docherty (1998). Based
on recent studies with gene-targeted and transgenic mice,
.alpha..sub.2A-adrenocept- ors mediate most of the pharmacological
actions ascribed to currently available .alpha..sub.2-adrenoceptor
agonists, including inhibition of neurotransmitter release, central
hypotensive and bradycardic effects, sedation and anesthesia, and
analgesia. The same studies indicate that
.alpha..sub.2B-adrenoceptors mediate peripheral vasoconstriction in
response to agonist activation (Link et al. 1996, Macmillan et al.
1996). Other physiological or pharmacological effects have not been
associated with certainty with this receptor subtype. The
.alpha..sub.2C-adrenocepto- r subtype appears to be involved in
regulation of complex behaviors. It is not known that this subtype
would have important functions in peripheral tissues outside the
central nervous system or in cardiovascular regulation.
[0005] Coronary heart disease (CHD), like many other common
disorders, arises from complex interactions between genetic and
environmental factors. It is reasonable to assume that functionally
important genetic variation in mechanisms important for the
regulation of vascular functions, including the coronary
vasculature, will be found to be associated with the pathogenesis
and therapy of CHD. A variant form of the human .alpha..sub.2B-AR
gene was recently identified (Heinonen et al., 1999). The variant
allele encodes a receptor protein with a deletion of three
glutamate residues in an acidic stretch of 18 amino acids (of which
15 are glutamates) located in the third intracellular loop of the
receptor polypeptide. This acidic stretch is a unique feature in
the primary structure of .alpha..sub.2B-AR in comparison to
.alpha..sub.2A-AR and .alpha..sub.2C-AR, suggesting that the motif
has a distinct role in the function of .alpha..sub.2B-AR. Amino
acid sequence alignment of .alpha..sub.2B-AR polypeptides of
different mammals reveals that the acidic stretch is highly
conserved among the .alpha..sub.2B-ARs of mammals and that the
acidic stretch is long in humans in comparison to other species.
This suggests that the motif is important for the functionality of
the receptor, and that the short form (D for "deletion") probably
represents the ancestral form and the long form (I for "insertion")
could well represent a more recent allelic variant in humans.
Jewell-Motz and Liggett (1995) studied the in vitro functions of
this stretch using site-directed mutagenesis to delete as well as
to substitute 16 amino acids of the stretch. Their results suggest
that this acidic motif is necessary for full agonist-promoted
receptor phosphorylation and desensitization.
[0006] Based on the vasoconstrictive property of .alpha..sub.2B-AR
in mice and the involvement of this acidic region in the
desensitization mechanism of the receptor, we hypothesized that the
deletion variant confers reduced receptor desensitization and
therefore augmented vasoconstriction that could be associated with
cardiovascular pathologies. To test this hypothesis, we carried out
a 4-year prospective study in 912 middle-aged Finnish men.
OBJECT AND SUMMARY OF THE INVENTION
[0007] One object of this invention is to provide a DNA sequence of
a variant human .alpha..sub.2B-adrenoceptor gene and the
corresponding variant .alpha..sub.2B-adrenoceptor protein.
[0008] Another object of the invention is to provide a method for
screening a subject to assess if an individual is at risk to suffer
from vascular contraction of coronary arteries.
[0009] A third object of the invention is to provide a method for
the treatment of vascular contraction of coronary arteries of
mammals.
[0010] A fourth object of the invention is to provide a transgenic
animal with a gene encoding a human .alpha..sub.2B-adrenoceptor or
said variant thereof.
[0011] Thus, according to one aspect the invention concerns a DNA
sequence comprising a nucleotide sequence encoding a variant
.alpha..sub.2B-adrenoceptor protein with a deletion of at least 1
glutamate from a glutamic acid repeat element of 12 glutamates,
amino acids 298-309, in an acidic stretch of 18 amino acids
294-311, located in the 3.sup.rd intracellular loop of the receptor
polypeptide.
[0012] The invention further concerns a variant
.alpha..sub.2B-adrenocepto- r protein with a deletion of at least 1
glutamate from a glutamic acid repeat element of 12 glutamates,
amino acids 298-309, in an acidic stretch of 18 amino acids
294-311, located in the 3.sup.rd intracellular loop of the receptor
polypeptide.
[0013] According to another aspect the invention concerns a method
for screening a subject to determine if said subject is a carrier
of a said variant gene with both alleles encoding a said variant
.alpha..sub.2B-adrenoceptor, i.e. to determine if said subject's
genotype of the human .alpha..sub.2B-adrenoceptor is of the
deletion/deletion (D/D) type, comprising the steps of
[0014] a) providing a biological sample of the subject to be
screened,
[0015] b) providing an assay for detecting in the biological sample
the presence of
[0016] i) the insertion/insertion (I/I) or deletion/insertion (D/I)
genotypes of the human .alpha..sub.2B-adrenoceptor, or
[0017] ii) the D/D genotype of the human
.alpha..sub.2B-adrenoceptor, and
[0018] c) assessing at least one of the two following
[0019] i) an individual's risk to develop a disease involving
vascular contraction of coronary arteries, or
[0020] ii) an individual's need for .alpha..sub.2B-selective or
.alpha..sub.2B-nonselective .alpha..sub.2-adrenoceptor antagonist
therapy,
[0021] based on whether said subject is of said D/D genotype or
not.
[0022] According to a third aspect the present invention concerns a
method for treating a mammal suffering from vascular contraction of
coronary arteries, said method comprising the step of administering
a selective .alpha..sub.2B-adrenoceptor antagonist to said
mammal.
[0023] According to a fourth aspect the present invention concerns
a transgenic animal which carries a human DNA sequence comprising a
nucleotide sequence encoding a human .alpha..sub.2B-adrenoceptor
protein or a variant thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention relates to a DNA molecule encoding a
variant human .alpha..sub.2BB-adrenoceptor, said variant
.alpha..sub.2B-adrenocep- tor protein and a method to assess the
risk of individuals to suffer from vascular contraction of coronary
arteries in mammals as well as a method for the treatment of
vascular contraction of coronary arteries. The present invention
also relates to transgenic animals comprising a human DNA molecule
encoding a human .alpha..sub.2B-adrenoceptor or said variant
.alpha..sub.2B-adrenoceptor protein.
[0025] The word treating shall also be understood to include
preventing.
[0026] The concept "a deletion of at least 1 glutamate from a
glutamic acid repeat element of 12 glutamates" refers to any
deletion of 1 to 12 glutamates irrespective of the specific
location in, or how many glutamates from said repeat element of 12
glutamates, amino acids 298-309 (SEQ ID NO: 4), in an acidic
stretch of 18 amino acids 294-311 located in the 3.sup.rd
intracellular loop of the receptor polypeptide are deleted.
[0027] The concept "deletion/deletion (D/D) genotype of the human
.alpha..sub.2B-adrenoceptor", in short "D/D genotype", refers to a
genotype of an individual having both .alpha..sub.2B-adrenoceptor
alleles code for a variant .alpha..sub.2B-adrenoceptor with a
deletion of at least 1 glutamate from a glutamic acid repeat
element of 12 glutamates, amino acids 298-309, in an acidic stretch
of 18 amino acids 294-311 (SEQ ID NO: 4), located in the 3.sup.rd
intracellular loop of the receptor polypeptide. Correspondingly
"deletion/insertion (D/I) genotype" refers to a genotype having one
of the gene alleles code for an .alpha..sub.2B-adrenoceptor with a
said deletion and the other without a said deletion, i.e. with a
respective insertion, and thus the "insertion/insertion (I/I)
genotype" refers to a genotype having both alleles code for an
.alpha..sub.2B-adrenoceptor without said deletion or deletions.
[0028] We recently identified a common variant form (SEQ ID NO: 1)
of the human .alpha..sub.2B-AR gene (SEQ ID NO: 3). This variant
gene encodes a receptor protein (SEQ ID NO: 2) with a deletion of 3
glutamates, amino acids 307-309, from a glutamic acid (Glu) repeat
element of 12 glutamates, amino acids 298-309, in an acidic stretch
of 18 amino acids 294-311 (SEQ ID NO: 4), located in the 3.sup.rd
intracellular loop of the receptor polypeptide. This variant gene
(SEQ ID NO: 1) was associated with decreased basal metabolic rate
(BMR) in a group of obese Finnish subjects (Heinonen et al. 1999).
Of the 166 obese subjects, 47 (28%) were homozygous for the long 12
glutamate repeat element (Glu.sup.12/Glu.sup.12), whereas 90 (54%)
were heterozygous (Glu.sup.12/Glu.sup.9) and 29 (17%) were
homozygous for the short form (Glu.sup.9/Glu.sup.9).
[0029] The results to be presented below show that in a
population-based cohort of 912 Finnish middle-aged men subjects
homozygous for the short form (Glu.sup.9/Glu.sup.9) described
above, thus representing a deletion/deletion (D/D) genotype of the
.alpha..sub.2B-adrenoceptor, have a significantly elevated risk for
acute coronary events in a four-year follow-up study. The risk for
an acute coronary event, defined as definite or possible acute
myocardial infarction (AMI) or prolonged (>20 min) chest pain
requiring hospitalization, was increased 2.5 fold in subjects who
had this D/D genotype. This increase in the risk for acute coronary
events is as great as so far observed for any other genetic risk
factor for acute coronary events or acute myocardial infarction in
a prospective population study. Also the frequency of a study
subject having a history of coronary heart disease (CHD) as well as
CHD in an exercise test was associated with this D/D genotype.
[0030] Based on these results and previous publications referred to
above it can be postulated that this D/D genotype is related to an
impaired capacity to downregulate .alpha..sub.2B-adrenoceptor
function during sustained receptor activation. Since altered
.alpha..sub.2B-adrenoceptor function seems to be of relevance in
the pathogenesis of a significant fraction of all cases of acute
coronary events in subjects with this D/D genotype (homozygous
Glu.sup.9/Glu.sup.9) we believe it could also be of relevance in
subjects with the insertion/deletion (IID) (heterozygous
Glu.sup.12/Glu.sup.9) and insertion/insertion (I/I) (homozygous
Glu.sup.12/Glu.sup.12) genotypes when other risk factors for AMI
are present. Further, since this specific deletion of 3 glutamates,
amino acids 307-309, from said glutamic acid repeat element of 12
glutamates, amino acids 298-309, in said acidic stretch of 18 amino
acids 294-311, located in the .sub.3rd intracellular loop of the
receptor polypeptide seems to be of relevance in cases of AMI we
believe that also other deletions, i.e. deletions of at least 1
glutamate, from said glutamic acid repeat element of 12 glutamates,
amino acids 298-309, could be of relevance in the pathogenesis of
AMI, because the 3.sup.rd intracellular loop of the receptor
polypeptide it is located in seems to have an essential role in the
downregulation of the .alpha..sub.2B-adrenoceptor.
[0031] Thus based on the results to be presented below and the
publications referred to above an .alpha..sub.2B-adrenoceptor
antagonist would be useful for treating a mammal suffering from
vascular contraction of coronary arteries.
[0032] Furthermore, an .alpha..sub.2B-adrenoceptor antagonist
selective for the .alpha..sub.2B-adrenoceptor subtype would be
therapeutically beneficial for the treatment of a disease involving
said vascular contraction of coronary arteries. Such a disease
could be clinically expressed as chronic angina pectoris,
specifically e.g. AMI, unstable angina pectoris or Prinzmetal's
variant form of angina pectoris. If .alpha..sub.2B-adrenoceptor
dependent vasoconstriction is a causative factor in some cases of
AMI, then antagonism of these receptors should restore coronary
circulation and reduce the ischemic myocardial damage. An
.alpha..sub.2B-adrenoceptor antagonist will relieve the
vasoconstrictive component in the sustained ischemic episode of
unstable angina pectoris, thus alleviating the symptoms and
preventing AMI. Vasoconstriction is a key factor in the
pathogenesis of Prinzmetal's angina, and an
.alpha..sub.2B-adrenoceptor antagonist may resolve and prevent
attacks. An .alpha..sub.2B-adrenoceptor antagonist will help to
alleviate the vasoconstrictive component in all types of CHD,
providing both symptomatic relief and protection from AMI.
[0033] .alpha..sub.2B-adrenoceptors mediate vascular contraction of
coronary arteries, and genetic polymorphism present in the
.alpha..sub.2B-adrenoceptor gene renders some subjects more
susceptible to .alpha..sub.2B-adrenoceptor mediated
vasoconstriction of coronary arteries and associated clinical
disorders. These subjects will especially benefit from treatment
with an .alpha..sub.2B-adrenoceptor antagonist, and will be at
increased risk for adverse effects if subtype-nonselective
.alpha..sub.2-agonists are administered to them. Therefore, a gene
test recognizing subjects with a deletion variant of the
.alpha..sub.2B-adrenoceptor gene will be useful in diagnostics and
patient selection for specific therapeutic procedures. A gene test
recognizing the D/D genotype of the .alpha..sub.2B-adrenoceptor is
useful in assessing an individual's risk to develop AMI and other
clinical disorders involving vascular contraction of coronary
arteries related to the D/D genotype. A gene test recognizing the
D/D genotype of the .alpha..sub.2B-adrenoceptor is useful in
selecting drug therapy for patients with diseases involving
vascular contraction of coronary arteries associated with the D/D
genotype; subjects with the D/D genotype will especially benefit
from therapy with .alpha..sub.2-adrenoceptor antagonists
(.alpha..sub.2B-selective or nonselective). A gene test recognizing
the D/D genotype of the .alpha..sub.2B-adrenoceptor is useful in
selecting drug therapy for patients who might be at increased risk
for adverse effects of .alpha..sub.2-adrenergic agonists; either,
it will be possible to avoid the use of .alpha..sub.2-agonists in
such patients, or it will be possible to include a specific
.alpha..sub.2B-antagonist in their therapeutic regimen.
[0034] The DNA sequence can be used for screening a subject to
determine if said subject is a carrier of a variant gene. The
determination can be carried out either as a DNA analysis according
to well known methods, which include direct DNA sequencing of the
normal and variant gene, allele specific amplification using the
polymerase chain reaction (PCR) enabling detection of either normal
or variant sequence, or by indirect detection of the normal or
variant gene by various molecular biology methods including e.g.
PCR-single stranded conformation polymorphism (SSCP) method or
denaturing gradient gel electrophoresis (DGGE). Determination of
the normal or variant gene can also be done by using a restriction
fragment length (polymorphism (RFLP) method, which is particularly
suitable for genotyping large numbers of samples. Similarly, a test
based on gene chip technology can be easily developed in analogy
with many currently existing such tests for single-nucleotide
polymorphisms.
[0035] The determination can also be carried out at the level of
RNA by analyzing RNA expressed at tissue level using various
methods. Allele specific probes can be designed for hybridization.
Hybridization can be done e.g. using Northern blot, RNase
protection assay or in situ hybridization methods. RNA derived from
the normal or variant gene can also be analyzed by converting
tissue RNA first to cDNA and thereafter amplifying cDNA by an
allele specific PCR method.
[0036] As examples of useful .alpha..sub.2B-adrenoceptor
antagonists can be mentioned imiloxan
[2-(1-ethyl-2-imidazoyl)methyl-1,4-benzodioxan, ARC-239
[2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3--
(2H,4H)-isoquinolindione], prazosin
[1-(4-amino-6,7-dimethoxy-2-quinazo-li-
nyl)-4-(2-furanylcarbonyl)piperazine] and chlorpromazine
[2-chloro-N,N-dimethyl-10H-pheno-thiazine-10-propanamine].
[0037] The required dosage of the compounds will vary with the
particular condition being treated, the severity of the condition,
the duration of the treatment, the administration route and the
specific compound being employed. A typical therapeutically
effective daily dose administered, e.g. orally or by infusion, can
vary from e.g. 0.1 .mu.g to 10 mg per kilogram body weight of an
adult person.
[0038] Influence of the variant gene sequence can be investigated
in transgenic animals. A transgenic animal can be generated e.g.
using targeted homologous recombination methodology. This will
provide an ideal preclinical model to investigate and screen new
drug molecules, which are designed to modify the influence of the
variant gene.
[0039] The invention will be described in more detail in the
experimental section.
[0040] Experimental Section
[0041] Determination of Genomic Alleles Encoding the
.alpha..sub.2B-Adrenoceptor
[0042] PCR-SSCA Analysis
[0043] The polymerase chain reaction-single stranded conformational
analysis (PCR-SSCA) used to identify the genomic alleles encoding
the .alpha..sub.2B-adrenoceptor was carried out as follows: The
genomic DNA encoding the .alpha..sub.2B-adrenergic receptor was
amplified in two parts specific for the intronless
.alpha..sub.2B-adrenoceptor gene sequence (Lomasney et al. 1990).
The PCR primer pairs for PCR amplification were as follows: Pair 1:
5'-GGGGCGACGCTCTTGTCTA-3' (SEQ ID NO: 5) and
5'-GGTCTCCCCCTCCTCCTTC-3' (SEQ ID NO: 6) (product size 878 bp), and
pair 2: 5'-GCAGCAACCGCAGAGGTC-3' (SEQ ID NO: 7) and
5'-GGGCAAGAAGCAGGG-TGAC-3' (SEQ ID NO: 8) (product size 814 bp).
The primers were delivered by KeboLab (Helsinki, Finland). PCR
amplification was conducted in a 5 .mu.l volume containing 100 ng
genomic DNA (isolated from whole blood), 2.5 mmol/l of each primer,
1.0 mmol/l deoxy-NTPs, 30 nmol/l .sup.33P-dCTP and 0.25 U AmpliTaq
DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn.). PCR conditions
were optimized using the PCR Optimizer.TM. kit (Invitrogen, San
Diego, Calif.). Samples were amplified with a GeneAmp PCR System
9600 (Perkin Elmer Cetus). PCR products were digested with
restriction enzymes for SSCA analysis. The product of primer pair 1
was digested with Dde I and Dra III (Promega Corp., Madison, Wis.).
The product of primer pair 2 was digested with Alu I and Hinc II
(Promega Corp.). The digested samples were mixed with SSCA buffer
containing 95% formamide, 10 mmol/l NaOH, 0.05% xylene cyanol and
0.05% bromophenol blue (total volume 25 .mu.l). Before loading, the
samples were denatured for 5 min at 95.degree. C. and kept 5 min on
ice. Three microliters of each sample were loaded on MDE.TM.
high-resolution gel (FMC, BioProducts, Rockland, Mass.). The gel
electrophoresis was performed twice, at two different running
conditions: 6% MDE gel at +4.degree. C. and 3% MDE gel at room
temperature, both at 4 W constant power for 16 h. The gels were
dried and autoradiography was performed by apposing to Kodak BioMax
MR film for 24 h at room temperature.
[0044] Sequencing and Genotyping
[0045] DNA samples migrating at different rates in SSCA were
sequenced with the Thermo Sequenas.TM. Cycle Sequencing Kit
(Amersham Life Science, Cleveland, Ohio). For genotyping the
identified 3-glutamic acid deletion, DNA was extracted from
peripheral blood using standard methods. The .alpha..sub.2B-AR I/D
genotype was determined by separating PCR-amplified DNA fragments
with electrophoresis. Based on the nature of the I/D variant,
identification of the long and short alleles was achieved by their
different electrophoretic migration rates due to their 9 bp size
difference.
[0046] The region of interest was amplified using a sense primer
5'-AGGGTGTTTGTGGG-GCATCT-3' (SEQ ID NO: 9) and an anti-sense primer
5'-CAAGCTGAGGCCGGAGACACT-3' (SEQ ID NO: 10) (Oligold, Eurogentec,
Belgium), yielding a product size of 112 bp for the long allele (I)
and 103 bp for the short allele (D). PCR amplification was
conducted in a 10 .mu.L volume containing .about.100 ng genomic
DNA, 1.times. buffer G (Invitrogen, San Diego, Calif., USA), 0.8 mM
dNTPs, 0.3 .mu.M of each primer and 0.25 units of AmpliTaq DNA
polymerase (Perkin Elmer Cetus, Norwalk, Conn., USA). Samples were
amplified with a GeneAmp PCR System 9600 (Perkin Elmer Cetus).
After initial denaturation at 94.degree. C. for 2 minutes, the
samples were amplified over 35 cycles. PCR amplification conditions
were 96.degree. C. (40 s), 69.degree. C. (30 s) and 72.degree. C.
(30 s) followed by final extension at 72.degree. C. for 6 minutes.
The PCR products representing the long and short alleles were
identified by two alternative methods.
[0047] 1)The amplified samples were mixed with 4 .mu.l of stop
solution (Thermo Sequenase.TM. Cycle Sequencing kit), heated to
95.degree. C. for 2 min, and loaded hot onto sequencing gels (Long
Ranger.TM., FMC). The gels were dried and autoradiography was
performed as previously described.
[0048] Separation of the amplified PCR products was performed with
electrophoresis on a high-resolution 4% Metaphor agarose gel (FMC
Bioproducts, Rockland, Me.) and the bands were visualized by
ethidium bromide staining. In both methods, the long (Glu.sup.12)
and short (Glu.sup.9) alleles were identified based on their
different electrophoretic migration rates.
[0049] Follow-Up Study
[0050] The above referred four-year follow-up study of 912 Finnish
middle-aged men subjects including 192 subjects with a specific
deletion/deletion (D/D) genotype of the .alpha..sub.2B-adrenoceptor
is described in more detail in the following:
[0051] Knowing the vasoconstrictive property of .alpha..sub.2B-AR
in mice and the possible involvement of the investigated acidic
region in the desensitization mechanism of the receptor we
hypothesized that the observed insertion/deletion allelic variation
could be associated with cardiovascular pathologies such as AMI. To
test this hypothesis, we carried out a four-year follow-up study in
912 middle-aged Finnish men with no prior history of AMI. The study
was carried out as part of the Kuopio Ischemic Heart Disease Risk
Factor Study (KIHD), which is an ongoing population-based study
designed to investigate risk factors for cardiovascular diseases
and related outcomes in men from eastern Finland (Salonen 1988).
This area is known for its homogenous population (Sajantila et al.
1996) and high coronary morbidity and mortality rates (Keys
1980).
[0052] Of the 912 subjects, 192 (21%) had the D/D genotype, 256
(28%) had the I/I genotype and 464 (51%) were heterozygous i.e.
I/D. This genotype distribution is in Hardy-Weinberg equilibrium
(p=0.46).
[0053] Of the 37 cases that had an acute coronary event during the
follow-up, 18 were classified as definite AMI, 12 as possible AMI
and seven as prolonged chest pain. Among the subjects with the D/D
genotype, 15 (8%) had an acute coronary event during the follow-up
time. The corresponding incidences for the I/I and the heterozygous
genotypes i.e. I/D were 10 (4%) and 12 (3%). The observed
cumulative incidence of acute coronary events differed
significantly among the different genotypes (p=0.008). No
significant difference in the cumulative incidence of acute
coronary events was found between the I/D and the H genotypes
(p=0.4) (table 1). There was a significant difference (log-rank
p=0.0045) between the D/D subgroup and the other two genotypes
combined in the cumulative event-free time in the Kaplan-Meier
survival function, demonstrating that there is a consistently
increased incidence of acute coronary events in the D/D
subgroup.
[0054] The D/D genotype was associated with a 2.5 fold increased
risk for an acute coronary event (95% CI=1.3-4.8, p=0.006) in
comparison to the other two genotypes combined. The relative risk
remained above 2 after adjustment for major CHD risk factors (table
2).
[0055] The D/D subgroup was not significantly different from the
I/D+I/I subgroup in terms of many known major risk factors for CHD.
From 87 variables in the study database only 5 were significantly
different between the D/D and the I/D+I/I genotype subgroups: 1.
there were more acute coronary events in the D/D subgroup (8% vs.
3%, p=0.006), 2. history of CHD was more prevalent in the D/D
subgroup (37% vs. 29%, p=0.043), 3. the prevalence of CHD in
exercise test was higher in the D/D subgroup (30% vs. 22%,
p=0.036), 4. mean hemoglobin level was higher in the D/D subgroup
(149.0 g/l vs. 146.8 g/l, p=0.005) and 5. mean dietary cholesterol
intake (4-days) was lower in the D/D subgroup (411.6 mg vs. 440.1
mg, p=0.033) (table 3). The first four observed differences support
our hypothesis that the D/D genotype confers reduced receptor
desensitization and therefore augmented vasoconstriction. This
augmented vasoconstriction is the reason for the increased
incidence of acute coronary events, the higher prevalence of CHD in
exercise and history of CHD. We hypothesize that the increased
level of hemoglobin is due to relative anoxia of tissues because of
this augmented vasoconstriction.
[0056] To examine the possibility that the D/D genotype is a
genetic marker for acute coronary events rather than a causative
factor, we have searched the literature for known genetic risk
factors for acute coronary events and AMI and their chromosomal
localization. All but one (Apo-B) are on different chromosomes than
the .alpha..sub.2B-AR gene (chromosome 2) and the gene for Apo-B is
neither in the physical nor the genetic vicinity of the
.alpha..sub.2B-AR gene. Cox regression analysis revealed that the
increased RR for acute coronary events in the D/D subgroup is not
affected by the serum Apo-B concentration.
[0057] Taken together, the known biological properties of the
.alpha..sub.2B-AR, the homogeneity of the Finnish population with
its relatively high incidence of CHD, the study design, the
relatively large representative study population and the clustering
of the findings around one trait suggest that the D/D receptor
allele is a causal genetic risk factor for acute coronary
events.
1TABLE 1 The cumulative incidence of acute coronary events among
men with different genotypes of the .alpha..sub.2B-AR (p values are
stated below) Genotype Events (% of men at risk) Men at risk (% of
all) D/D observed 15 (8) 192 (21) expected 7.8 I/D observed 12 (3)
464 (51) expected 18.8 I/I observed 10 (4) 256 (28) expected 10.4
I/D + I/I observed 22 (3) 720 (79) expected 29.2 Total observed 37
(4) 912 (100) P values for the above table: D/D vs. I/D vs. I/I p =
0.008 D/D vs. I/D p = 0.002 D/D vs. I/I p = 0.038 I/D vs. I/I p =
0.389 D/D vs. I/D + I/I p = 0.005
[0058]
2TABLE 2 Relative risk (RR) and its 95% confidence interval (CI)
for an acute coronary event-a comparison of each of the genotypes
with the other two combined. Results of a Cox regression model for
37 acute coronary events in a population sample of 912 subjects RR
(95% CI) Adjusted RR (95% CI) Genotype Events/men at risk P P D/D
15/192 2.5 (1.3-4.8) 2.3 (1.2-4.5) 0.006 0.014 I/D 12/464 0.44
(0.2-0.9) 0.5 (0.2-1.0) 0.020 0.052 I/I 10/256 1.03 (0.5-2.1) 0.96
(0.5-2.0) 0.940 0.901 Adjustment was done for age, CHD in the
family, high cholesterol in the family, hypertension and
smoking
[0059]
3TABLE 3 List of all significant differences (p < 0.05) between
the D/D and the I/D + I/I genotype subgroups among 87 variables in
the study database Variable D/D I/D + I/I P Acute coronary events
[event/n (%)] 15/192 (8) 22/720 (3) 0.006 Ischemic findings in
exercise 57/192 (30) 160/720 (22) 0.036 test [case/n (%)] History
of CHD [case/n (%)] 71/192 (37) 209/720 (29) 0.043 Mean blood
haemoglobin [g/L] 149.0 146.8 0.005 Mean 4 day dietary cholesterol
411.6 440.1 0.033 intake [mg] % = Percent of men at risk
[0060] It will be appreciated that the methods of the present
invention can be incorporated in the form of a variety of
embodiments, only a few of which are disclosed herein. It will be
apparent for the specialist in the field that other embodiments
exist and do not depart from the spirit of the invention. Thus, the
described embodiments are illustrative and should not be construed
as restrictive.
[0061] References
[0062] Docherty J R: Subtypes of functional .alpha..sub.1- and
.alpha..sub.2-receptors. Eur J Pharmacol 1998;361:1-15
[0063] Heinonen P, Koulu M, Pesonen U, Karvonen M, Rissanen A,
Laakso M, Valve R, Uusitupa M, Scheinin M: Identification of a
three amino acid deletion in the alpha-2B-adrenergic receptor which
is associated with reduced basal metabolic rate in obese subjects.
J Clin Endocrinol Metab 1999;84:2429-2433
[0064] Jewell-Motz E, Liggett S B: An acidic motif within the third
intracellular loop of the alpha2C2 adrenergic receptor is required
for agonist-promoted phosphorylation and desensitization.
Biochemistry 1995;34:11946-11953
[0065] Keys A: Seven Countries: A Multivariate Analysis of Death
and Coronary Heart Disease. Cambridge, Mass, Harvard University
Press, 1980
[0066] Link R E, Desai K, Hein L, Stevens M E, Chruscinski A,
Bernstein D, Barsh G S, Kobilka B K: Cardiovascular regulation in
mice lacking alpha2-adrenergic receptor subtypes b and c. Science
1996;273:803-805
[0067] Lomasney J W, Lorenz W, Allen L F, King K, Regan J W,
Yang-Feng T L, Caron M C, Lefkowitz R J: Expansion of the alpha-2
adrenergic receptor family: cloning and characterization of a human
alpha-2 adrenergic receptor subtype, the gene for which is located
on chromosome 2. Proc Natl Acad Sci USA. 1990;87:5094-5098.
[0068] MacDonald E, Kobilka B K, Scheinin M: Gene targeting--homing
in on .alpha..sub.2-adrenoceptor subtype function. Trends Pharmacol
Sci 1997; 18:211-219
[0069] MacMillan L B, Hein L, Smith M S, Piascik M T, Limbird L E:
Central hypotensive effects of the alpha2a-adrenergic receptor
subtype. Science 1996;273:801-803
[0070] Sajantila A, Salem A H, Savolainen P, Bauer K, Gierig C,
Paabo S: Paternal and maternal DNA lineages reveal a bottleneck in
the founding of the Finnish population. Proc.Natl.Acad.Sci. U.S.A.
1996;93:12035-12039
[0071] Salonen J T: Is there a continuing need for longitudinal
epidemiologic research? The Kuopio Ischaemic Heart Disease Risk
Factor Study. Ann. Clin Res 1988;20:46-50
Sequence CWU 1
1
10 1 1344 DNA Homo sapiens CDS (1)..(1341) Coding sequence for
variant human alpha-2B-adrenoceptor protein 1 atg gac cac cag gac
ccc tac tcc gtg cag gcc aca gcg gcc ata gcg 48 Met Asp His Gln Asp
Pro Tyr Ser Val Gln Ala Thr Ala Ala Ile Ala 1 5 10 15 gcg gcc atc
acc ttc ctc att ctc ttt acc atc ttc ggc aac gct ctg 96 Ala Ala Ile
Thr Phe Leu Ile Leu Phe Thr Ile Phe Gly Asn Ala Leu 20 25 30 gtc
atc ctg gct gtg ttg acc agc cgc tcg ctg cgc gcc cct cag aac 144 Val
Ile Leu Ala Val Leu Thr Ser Arg Ser Leu Arg Ala Pro Gln Asn 35 40
45 ctg ttc ctg gtg tcg ctg gcc gcc gcc gac atc ctg gtg gcc acg ctc
192 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp Ile Leu Val Ala Thr Leu
50 55 60 atc atc cct ttc tcg ctg gcc aac gag ctg ctg ggc tac tgg
tac ttc 240 Ile Ile Pro Phe Ser Leu Ala Asn Glu Leu Leu Gly Tyr Trp
Tyr Phe 65 70 75 80 cgg cgc acg tgg tgc gag gtg tac ctg gcg ctc gac
gtg ctc ttc tgc 288 Arg Arg Thr Trp Cys Glu Val Tyr Leu Ala Leu Asp
Val Leu Phe Cys 85 90 95 acc tcg tcc atc gtg cac ctg tgc gcc atc
agc ctg gac cgc tac tgg 336 Thr Ser Ser Ile Val His Leu Cys Ala Ile
Ser Leu Asp Arg Tyr Trp 100 105 110 gcc gtg agc cgc gcg ctg gag tac
aac tcc aag cgc acc ccg cgc cgc 384 Ala Val Ser Arg Ala Leu Glu Tyr
Asn Ser Lys Arg Thr Pro Arg Arg 115 120 125 atc aag tgc atc atc ctc
act gtg tgg ctc atc gcc gcc gtc atc tcg 432 Ile Lys Cys Ile Ile Leu
Thr Val Trp Leu Ile Ala Ala Val Ile Ser 130 135 140 ctg ccg ccc ctc
atc tac aag ggc gac cag ggc ccc cag ccg cgc ggg 480 Leu Pro Pro Leu
Ile Tyr Lys Gly Asp Gln Gly Pro Gln Pro Arg Gly 145 150 155 160 cgc
ccc cag tgc aag ctc aac cag gag gcc tgg tac atc ctg gcc tcc 528 Arg
Pro Gln Cys Lys Leu Asn Gln Glu Ala Trp Tyr Ile Leu Ala Ser 165 170
175 agc atc gga tct ttc ttt gct cct tgc ctc atc atg atc ctt gtc tac
576 Ser Ile Gly Ser Phe Phe Ala Pro Cys Leu Ile Met Ile Leu Val Tyr
180 185 190 ctg cgc atc tac ctg atc gcc aaa cgc agc aac cgc aga ggt
ccc agg 624 Leu Arg Ile Tyr Leu Ile Ala Lys Arg Ser Asn Arg Arg Gly
Pro Arg 195 200 205 gcc aag ggg ggg cct ggg cag ggt gag tcc aag cag
ccc cga ccc gac 672 Ala Lys Gly Gly Pro Gly Gln Gly Glu Ser Lys Gln
Pro Arg Pro Asp 210 215 220 cat ggt ggg gct ttg gcc tca gcc aaa ctg
cca gcc ctg gcc tct gtg 720 His Gly Gly Ala Leu Ala Ser Ala Lys Leu
Pro Ala Leu Ala Ser Val 225 230 235 240 gct tct gcc aga gag gtc aac
gga cac tcg aag tcc act ggg gag aag 768 Ala Ser Ala Arg Glu Val Asn
Gly His Ser Lys Ser Thr Gly Glu Lys 245 250 255 gag gag ggg gag acc
cct gaa gat act ggg acc cgg gcc ttg cca ccc 816 Glu Glu Gly Glu Thr
Pro Glu Asp Thr Gly Thr Arg Ala Leu Pro Pro 260 265 270 agt tgg gct
gcc ctt ccc aac tca ggc cag ggc cag aag gag ggt gtt 864 Ser Trp Ala
Ala Leu Pro Asn Ser Gly Gln Gly Gln Lys Glu Gly Val 275 280 285 tgt
ggg gca tct cca gag gat gaa gct gaa gag gag gaa gag gag gag 912 Cys
Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu Glu Glu Glu Glu Glu 290 295
300 gag gag tgt gaa ccc cag gca gtg cca gtg tct ccg gcc tca gct tgc
960 Glu Glu Cys Glu Pro Gln Ala Val Pro Val Ser Pro Ala Ser Ala Cys
305 310 315 320 agc ccc ccg ctg cag cag cca cag ggc tcc cgg gtg ctg
gcc acc cta 1008 Ser Pro Pro Leu Gln Gln Pro Gln Gly Ser Arg Val
Leu Ala Thr Leu 325 330 335 cgt ggc cag gtg ctc ctg ggc agg ggc gtg
ggt gct ata ggt ggg cag 1056 Arg Gly Gln Val Leu Leu Gly Arg Gly
Val Gly Ala Ile Gly Gly Gln 340 345 350 tgg tgg cgt cga cgg gcg cag
ctg acc cgg gag aag cgc ttc acc ttc 1104 Trp Trp Arg Arg Arg Ala
Gln Leu Thr Arg Glu Lys Arg Phe Thr Phe 355 360 365 gtg ctg gct gtg
gtc att ggc gtt ttt gtg ctc tgc tgg ttc ccc ttc 1152 Val Leu Ala
Val Val Ile Gly Val Phe Val Leu Cys Trp Phe Pro Phe 370 375 380 ttc
ttc agc tac agc ctg ggc gcc atc tgc ccg aag cac tgc aag gtg 1200
Phe Phe Ser Tyr Ser Leu Gly Ala Ile Cys Pro Lys His Cys Lys Val 385
390 395 400 ccc cat ggc ctc ttc cag ttc ttc ttc tgg atc ggc tac tgc
aac agc 1248 Pro His Gly Leu Phe Gln Phe Phe Phe Trp Ile Gly Tyr
Cys Asn Ser 405 410 415 tca ctg aac cct gtt atc tac acc atc ttc aac
cag gac ttc cgc cgt 1296 Ser Leu Asn Pro Val Ile Tyr Thr Ile Phe
Asn Gln Asp Phe Arg Arg 420 425 430 gcc ttc cgg agg atc ctg tgc cgc
ccg tgg acc cag acg gcc tgg tga 1344 Ala Phe Arg Arg Ile Leu Cys
Arg Pro Trp Thr Gln Thr Ala Trp 435 440 445 2 447 PRT Homo sapiens
2 Met Asp His Gln Asp Pro Tyr Ser Val Gln Ala Thr Ala Ala Ile Ala 1
5 10 15 Ala Ala Ile Thr Phe Leu Ile Leu Phe Thr Ile Phe Gly Asn Ala
Leu 20 25 30 Val Ile Leu Ala Val Leu Thr Ser Arg Ser Leu Arg Ala
Pro Gln Asn 35 40 45 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp Ile
Leu Val Ala Thr Leu 50 55 60 Ile Ile Pro Phe Ser Leu Ala Asn Glu
Leu Leu Gly Tyr Trp Tyr Phe 65 70 75 80 Arg Arg Thr Trp Cys Glu Val
Tyr Leu Ala Leu Asp Val Leu Phe Cys 85 90 95 Thr Ser Ser Ile Val
His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Trp 100 105 110 Ala Val Ser
Arg Ala Leu Glu Tyr Asn Ser Lys Arg Thr Pro Arg Arg 115 120 125 Ile
Lys Cys Ile Ile Leu Thr Val Trp Leu Ile Ala Ala Val Ile Ser 130 135
140 Leu Pro Pro Leu Ile Tyr Lys Gly Asp Gln Gly Pro Gln Pro Arg Gly
145 150 155 160 Arg Pro Gln Cys Lys Leu Asn Gln Glu Ala Trp Tyr Ile
Leu Ala Ser 165 170 175 Ser Ile Gly Ser Phe Phe Ala Pro Cys Leu Ile
Met Ile Leu Val Tyr 180 185 190 Leu Arg Ile Tyr Leu Ile Ala Lys Arg
Ser Asn Arg Arg Gly Pro Arg 195 200 205 Ala Lys Gly Gly Pro Gly Gln
Gly Glu Ser Lys Gln Pro Arg Pro Asp 210 215 220 His Gly Gly Ala Leu
Ala Ser Ala Lys Leu Pro Ala Leu Ala Ser Val 225 230 235 240 Ala Ser
Ala Arg Glu Val Asn Gly His Ser Lys Ser Thr Gly Glu Lys 245 250 255
Glu Glu Gly Glu Thr Pro Glu Asp Thr Gly Thr Arg Ala Leu Pro Pro 260
265 270 Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln Gly Gln Lys Glu Gly
Val 275 280 285 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu Glu Glu
Glu Glu Glu 290 295 300 Glu Glu Cys Glu Pro Gln Ala Val Pro Val Ser
Pro Ala Ser Ala Cys 305 310 315 320 Ser Pro Pro Leu Gln Gln Pro Gln
Gly Ser Arg Val Leu Ala Thr Leu 325 330 335 Arg Gly Gln Val Leu Leu
Gly Arg Gly Val Gly Ala Ile Gly Gly Gln 340 345 350 Trp Trp Arg Arg
Arg Ala Gln Leu Thr Arg Glu Lys Arg Phe Thr Phe 355 360 365 Val Leu
Ala Val Val Ile Gly Val Phe Val Leu Cys Trp Phe Pro Phe 370 375 380
Phe Phe Ser Tyr Ser Leu Gly Ala Ile Cys Pro Lys His Cys Lys Val 385
390 395 400 Pro His Gly Leu Phe Gln Phe Phe Phe Trp Ile Gly Tyr Cys
Asn Ser 405 410 415 Ser Leu Asn Pro Val Ile Tyr Thr Ile Phe Asn Gln
Asp Phe Arg Arg 420 425 430 Ala Phe Arg Arg Ile Leu Cys Arg Pro Trp
Thr Gln Thr Ala Trp 435 440 445 3 1353 DNA Homo sapiens CDS
(1)..(1350) Coding sequence for human alpha-2B-adrenoceptor protein
3 atg gac cac cag gac ccc tac tcc gtg cag gcc aca gcg gcc ata gcg
48 Met Asp His Gln Asp Pro Tyr Ser Val Gln Ala Thr Ala Ala Ile Ala
1 5 10 15 gcg gcc atc acc ttc ctc att ctc ttt acc atc ttc ggc aac
gct ctg 96 Ala Ala Ile Thr Phe Leu Ile Leu Phe Thr Ile Phe Gly Asn
Ala Leu 20 25 30 gtc atc ctg gct gtg ttg acc agc cgc tcg ctg cgc
gcc cct cag aac 144 Val Ile Leu Ala Val Leu Thr Ser Arg Ser Leu Arg
Ala Pro Gln Asn 35 40 45 ctg ttc ctg gtg tcg ctg gcc gcc gcc gac
atc ctg gtg gcc acg ctc 192 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp
Ile Leu Val Ala Thr Leu 50 55 60 atc atc cct ttc tcg ctg gcc aac
gag ctg ctg ggc tac tgg tac ttc 240 Ile Ile Pro Phe Ser Leu Ala Asn
Glu Leu Leu Gly Tyr Trp Tyr Phe 65 70 75 80 cgg cgc acg tgg tgc gag
gtg tac ctg gcg ctc gac gtg ctc ttc tgc 288 Arg Arg Thr Trp Cys Glu
Val Tyr Leu Ala Leu Asp Val Leu Phe Cys 85 90 95 acc tcg tcc atc
gtg cac ctg tgc gcc atc agc ctg gac cgc tac tgg 336 Thr Ser Ser Ile
Val His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Trp 100 105 110 gcc gtg
agc cgc gcg ctg gag tac aac tcc aag cgc acc ccg cgc cgc 384 Ala Val
Ser Arg Ala Leu Glu Tyr Asn Ser Lys Arg Thr Pro Arg Arg 115 120 125
atc aag tgc atc atc ctc act gtg tgg ctc atc gcc gcc gtc atc tcg 432
Ile Lys Cys Ile Ile Leu Thr Val Trp Leu Ile Ala Ala Val Ile Ser 130
135 140 ctg ccg ccc ctc atc tac aag ggc gac cag ggc ccc cag ccg cgc
ggg 480 Leu Pro Pro Leu Ile Tyr Lys Gly Asp Gln Gly Pro Gln Pro Arg
Gly 145 150 155 160 cgc ccc cag tgc aag ctc aac cag gag gcc tgg tac
atc ctg gcc tcc 528 Arg Pro Gln Cys Lys Leu Asn Gln Glu Ala Trp Tyr
Ile Leu Ala Ser 165 170 175 agc atc gga tct ttc ttt gct cct tgc ctc
atc atg atc ctt gtc tac 576 Ser Ile Gly Ser Phe Phe Ala Pro Cys Leu
Ile Met Ile Leu Val Tyr 180 185 190 ctg cgc atc tac ctg atc gcc aaa
cgc agc aac cgc aga ggt ccc agg 624 Leu Arg Ile Tyr Leu Ile Ala Lys
Arg Ser Asn Arg Arg Gly Pro Arg 195 200 205 gcc aag ggg ggg cct ggg
cag ggt gag tcc aag cag ccc cga ccc gac 672 Ala Lys Gly Gly Pro Gly
Gln Gly Glu Ser Lys Gln Pro Arg Pro Asp 210 215 220 cat ggt ggg gct
ttg gcc tca gcc aaa ctg cca gcc ctg gcc tct gtg 720 His Gly Gly Ala
Leu Ala Ser Ala Lys Leu Pro Ala Leu Ala Ser Val 225 230 235 240 gct
tct gcc aga gag gtc aac gga cac tcg aag tcc act ggg gag aag 768 Ala
Ser Ala Arg Glu Val Asn Gly His Ser Lys Ser Thr Gly Glu Lys 245 250
255 gag gag ggg gag acc cct gaa gat act ggg acc cgg gcc ttg cca ccc
816 Glu Glu Gly Glu Thr Pro Glu Asp Thr Gly Thr Arg Ala Leu Pro Pro
260 265 270 agt tgg gct gcc ctt ccc aac tca ggc cag ggc cag aag gag
ggt gtt 864 Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln Gly Gln Lys Glu
Gly Val 275 280 285 tgt ggg gca tct cca gag gat gaa gct gaa gag gag
gaa gag gag gag 912 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu Glu
Glu Glu Glu Glu 290 295 300 gag gag gag gaa gag tgt gaa ccc cag gca
gtg cca gtg tct ccg gcc 960 Glu Glu Glu Glu Glu Cys Glu Pro Gln Ala
Val Pro Val Ser Pro Ala 305 310 315 320 tca gct tgc agc ccc ccg ctg
cag cag cca cag ggc tcc cgg gtg ctg 1008 Ser Ala Cys Ser Pro Pro
Leu Gln Gln Pro Gln Gly Ser Arg Val Leu 325 330 335 gcc acc cta cgt
ggc cag gtg ctc ctg ggc agg ggc gtg ggt gct ata 1056 Ala Thr Leu
Arg Gly Gln Val Leu Leu Gly Arg Gly Val Gly Ala Ile 340 345 350 ggt
ggg cag tgg tgg cgt cga cgg gcg cag ctg acc cgg gag aag cgc 1104
Gly Gly Gln Trp Trp Arg Arg Arg Ala Gln Leu Thr Arg Glu Lys Arg 355
360 365 ttc acc ttc gtg ctg gct gtg gtc att ggc gtt ttt gtg ctc tgc
tgg 1152 Phe Thr Phe Val Leu Ala Val Val Ile Gly Val Phe Val Leu
Cys Trp 370 375 380 ttc ccc ttc ttc ttc agc tac agc ctg ggc gcc atc
tgc ccg aag cac 1200 Phe Pro Phe Phe Phe Ser Tyr Ser Leu Gly Ala
Ile Cys Pro Lys His 385 390 395 400 tgc aag gtg ccc cat ggc ctc ttc
cag ttc ttc ttc tgg atc ggc tac 1248 Cys Lys Val Pro His Gly Leu
Phe Gln Phe Phe Phe Trp Ile Gly Tyr 405 410 415 tgc aac agc tca ctg
aac cct gtt atc tac acc atc ttc aac cag gac 1296 Cys Asn Ser Ser
Leu Asn Pro Val Ile Tyr Thr Ile Phe Asn Gln Asp 420 425 430 ttc cgc
cgt gcc ttc cgg agg atc ctg tgc cgc ccg tgg acc cag acg 1344 Phe
Arg Arg Ala Phe Arg Arg Ile Leu Cys Arg Pro Trp Thr Gln Thr 435 440
445 gcc tgg tga 1353 Ala Trp 450 4 450 PRT Homo sapiens 4 Met Asp
His Gln Asp Pro Tyr Ser Val Gln Ala Thr Ala Ala Ile Ala 1 5 10 15
Ala Ala Ile Thr Phe Leu Ile Leu Phe Thr Ile Phe Gly Asn Ala Leu 20
25 30 Val Ile Leu Ala Val Leu Thr Ser Arg Ser Leu Arg Ala Pro Gln
Asn 35 40 45 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp Ile Leu Val
Ala Thr Leu 50 55 60 Ile Ile Pro Phe Ser Leu Ala Asn Glu Leu Leu
Gly Tyr Trp Tyr Phe 65 70 75 80 Arg Arg Thr Trp Cys Glu Val Tyr Leu
Ala Leu Asp Val Leu Phe Cys 85 90 95 Thr Ser Ser Ile Val His Leu
Cys Ala Ile Ser Leu Asp Arg Tyr Trp 100 105 110 Ala Val Ser Arg Ala
Leu Glu Tyr Asn Ser Lys Arg Thr Pro Arg Arg 115 120 125 Ile Lys Cys
Ile Ile Leu Thr Val Trp Leu Ile Ala Ala Val Ile Ser 130 135 140 Leu
Pro Pro Leu Ile Tyr Lys Gly Asp Gln Gly Pro Gln Pro Arg Gly 145 150
155 160 Arg Pro Gln Cys Lys Leu Asn Gln Glu Ala Trp Tyr Ile Leu Ala
Ser 165 170 175 Ser Ile Gly Ser Phe Phe Ala Pro Cys Leu Ile Met Ile
Leu Val Tyr 180 185 190 Leu Arg Ile Tyr Leu Ile Ala Lys Arg Ser Asn
Arg Arg Gly Pro Arg 195 200 205 Ala Lys Gly Gly Pro Gly Gln Gly Glu
Ser Lys Gln Pro Arg Pro Asp 210 215 220 His Gly Gly Ala Leu Ala Ser
Ala Lys Leu Pro Ala Leu Ala Ser Val 225 230 235 240 Ala Ser Ala Arg
Glu Val Asn Gly His Ser Lys Ser Thr Gly Glu Lys 245 250 255 Glu Glu
Gly Glu Thr Pro Glu Asp Thr Gly Thr Arg Ala Leu Pro Pro 260 265 270
Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln Gly Gln Lys Glu Gly Val 275
280 285 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu Glu Glu Glu Glu
Glu 290 295 300 Glu Glu Glu Glu Glu Cys Glu Pro Gln Ala Val Pro Val
Ser Pro Ala 305 310 315 320 Ser Ala Cys Ser Pro Pro Leu Gln Gln Pro
Gln Gly Ser Arg Val Leu 325 330 335 Ala Thr Leu Arg Gly Gln Val Leu
Leu Gly Arg Gly Val Gly Ala Ile 340 345 350 Gly Gly Gln Trp Trp Arg
Arg Arg Ala Gln Leu Thr Arg Glu Lys Arg 355 360 365 Phe Thr Phe Val
Leu Ala Val Val Ile Gly Val Phe Val Leu Cys Trp 370 375 380 Phe Pro
Phe Phe Phe Ser Tyr Ser Leu Gly Ala Ile Cys Pro Lys His 385 390 395
400 Cys Lys Val Pro His Gly Leu Phe Gln Phe Phe Phe Trp Ile Gly Tyr
405 410 415 Cys Asn Ser Ser Leu Asn Pro Val Ile Tyr Thr Ile Phe Asn
Gln Asp 420 425 430 Phe Arg Arg Ala Phe Arg Arg Ile Leu Cys Arg Pro
Trp Thr Gln Thr 435 440 445 Ala Trp 450 5 19 DNA Artificial
Sequence Description of Artificial Sequence PCR primer pair 5
ggggcgacgc tcttgtcta 19 6 19 DNA Artificial Sequence Description of
Artificial Sequence PCR primer pair 6 ggtctccccc tcctccttc 19 7 18
DNA Artificial Sequence Description of Artificial Sequence PCR
primer pair 7 gcagcaaccg cagaggtc
18 8 19 DNA Artificial Sequence Description of Artificial Sequence
PCR primer pair 8 gggcaagaag cagggtgac 19 9 20 DNA Artificial
Sequence Description of Artificial Sequence PCR primer pair 9
agggtgtttg tggggcatct 20 10 21 DNA Artificial Sequence Description
of Artificial Sequence PCR primer pair 10 caagctgagg ccggagacac t
21
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