JP2002527079A - Genes for assessing cardiovascular status and compositions for their use - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for assessing cardiovascular status in an individual. The method comprises determining the sequence of one or more polymorphic positions in a human gene encoding ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor. Including. The present invention also provides an isolated nucleic acid encoding a polymorphism in a gene encoding ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor, Also provided are nucleic acid probes that hybridize to the location, kits for predicting cardiovascular status, and nucleic acid and peptide targets for use in identifying cardiovascular drug candidates.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to polymorphisms and polymorphism patterns useful for assessing cardiovascular status in humans. More specifically, the present invention provides ACE, AT1, AGT, renin to predict the outcome of treatment or the likelihood of developing cardiovascular disease, and to assist in diagnosing and prescribing an effective treatment regimen. Aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor. .

BACKGROUND OF THE INVENTION The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular physiology in mammals. Specifically, RAAS is a salt-
Controls water homeostasis and normal maintenance of blood vessels. Stimulation or inhibition of the system raises or lowers blood pressure, respectively, and confusion in the system
For example, it may be involved in the pathogenesis of hypertension, stroke and myocardial infarction. The RAAS system may also have other functions, such as, for example, controlling cell growth. The renin-angiotensin system includes renin, angiotensin converting enzyme (ACE), angiotensinogen (AGT), type 1 angiotensin II receptor (AT1), type 2 angiotensin II receptor (AT2) and aldosterone synthase.

International Patent Application No. PCT / IB98 / 00475 (filed on April 1, 1998)
Disclose for the first time the association of polymorphic patterns in the ACE, AGT and AT1 genes with cardiovascular status, especially with the ability to predict the outcome of a particular treatment regimen.

[0004] Other genes that play a role in the control of cardiovascular physiology are R
Expresses polypeptides involved in pathways other than AAS, but prior to the present invention, no known association between polymorphism patterns in these genes and cardiovascular status was observed and would not be reported Can be Among these other regulators of cardiovascular physiology are endothelin, endothelin receptors and β
-There are adrenergic receptors 1 and 2.

[0005] AGT, a component of the RAAS pathway , is a specific substrate for the aspartyl protease, renin. The human AGT gene contains 5 exons and 4 introns, spanning 13 Kb (Gailard et al., DNA, 1989, 8: 87-).
99; Fukamizu et. al. Biol. Chem. ,
1990, 265: 7576-7582). The first exon (37 bp)
Encodes the 5 'untranslated region of the mRNA. The second exon encodes the signal peptide and the first 252 amino acids of the mature protein. Exons 3 and 4 are shorter and encode 90 and 48 amino acids, respectively. Exon 5
It contains a short coding sequence (62 amino acids) and a 3 'untranslated region.

[0006] Plasma AGT is synthesized primarily in the liver, the expression of which is positively regulated by estrogens, glucocorticoids, thyroid hormones and angiotensin II (Ang II) (Clauser et al., Am. H
hypertension, 1989, 2: 403-410). Cleavage of the amino terminal segment of AGT by renin releases the decapeptide prohormone, angiotensin-I. This is further attributed to the angiotensin converting enzyme (AC
It is processed by the dipeptidyl carboxypeptidase designated E) to become the active octapeptide angiotensin II. AG by Renin
Cleavage of T is the rate-limiting step in the activation of the renin-angiotensin system.

[0007] Several epidemiological observations indicate that AGT may play a role in blood pressure control. A very important correlation between plasma AGT concentration and blood pressure has been observed in epidemiological studies (Walker et al., J. Hypert.
enhancement, 1979, 1: 287-291). Interestingly, many allelic dimorphisms have been identified in the AGT gene. The frequency of at least two of them (174M and 235T) is partially characterized and has been shown to be significantly elevated in hypertensive patients in certain populations (
Jeunemaitre et al. , Cell, 1992, 71: 169.
-180). In addition, certain polymorphisms, 235T, have been suggested to be directly involved in coronary atherosclerosis (Ishigami et al.,
Circulation, 1995, 91: 951-4). In addition, AGT
The presence of A or G at position 1218 within the regulatory region has been linked to differences in the transcriptional ability of this gene in vitro (Inoue et al., J. Am.
Clin. Invest. , 1997, 99: 1786). However, the above are studies involving only one or at most two polymorphisms. Further, the only disclosed use is susceptibility to disease.

[0008] The human ACE gene is also a candidate marker for hypertension and myocardial infarction. AC
E inhibitors constitute an important and effective therapeutic method in the control of human hypertension (
Sassaho et al. , Am. J. Med. , 1987, 83
: 227-235). In plasma and on the surface of endothelial cells, ACE converts inactive angiotensin I molecules (AngI) to active angiotensin II (AngII).
(Bottari et al., Front. Neuroen
docrinology, 1993, 14: 123-171). Other ACE
The substrate is bradykinin, a potent vasodilator and inhibitor of smooth muscle cell proliferation, which is inactivated by ACE (Ehlers et a
l. , Biochemistry, 1989, 28: 5311-5318;
Erdos, E .; G. FIG. , Hypertension, 1990, 16:
363-370; Johnston, C.I. I. Drugs (Supplement 1),
1990, 39: 21-31).

[0009] ACE levels are very stable in individuals, but vary greatly between individuals. Greater risk of myocardial infarction has been identified in a group of patients with the ACE polymorphism, designated ACE-DD (Cambian et al.,
Nature, 1992, 359: 641-644), and a 12-fold greater risk of myocardial infarction is due to the ACE polymorphism ACE-DD and the AGT polymorphism described above (235
T) in a subgroup of patients having a combination with one of (T) (K
amitani et al. , Hypertension, 1994,
24: 381). Recently, six ACE polymorphisms have been identified and characterized (Vil
lard et al. , Am. J. Human Genet. , 19
96, 58: 1268-1278).

The vasoconstrictive, cell growth-promoting and salt-converting effects of angiotensin II are mediated through binding to and activation of the angiotensin receptor, and at least two forms of that receptor have been cloned ( AT1 and AT
2). Type 1 AngII, seven transmembrane domain proteins to which G proteins are bound
The receptor (AT1) is widely distributed in the body and almost all known Ang
Mediates the II effect (Fyhrquist et al., J. Hum.
Hypertension, 1995, 5: 519-524).

[0011] Several polymorphisms have been identified in the AT1 receptor gene. Early studies suggest that at least one of them is more frequent in hypertensive patients (AT ¹¹⁶⁶ C) (Bonnardeaux et al.,
Hypertension, 1994, 24: 63-69). This polymorphism
It has been shown that, together with the ACE-DD polymorphism, strongly correlates with the risk of myocardial infarction (Tiret et al., Lancet, 1994, 344:
910-913).

Endothelin (ET) regulation of cardiovascular physiology Endothelin is a potent vasoconstrictor peptide characterized by a long lasting effect. It was first discovered as a vasoconstrictor in conditioned medium (Hickey et al., Am. J.
. Physiol. , 1985, 248: C550), then purified.
(Yanagisawa et al., Nature, 1
988, 332: 411). ET is produced as preproendothelin,
It is cleaved after removal of the signal sequence by endopeptidase and subsequently by endothelin converting enzyme (Xu et al., Cell).
Shimada et al., 1994, 78: 473; , J. et al. Bi
ol. Chem. , 1994, 269: 18274). Analysis of the human ET gene revealed the presence of two additional ET-like peptides, called ET-2 and ET-3, that are expressed in various tissues (Inoue et a
l. , Proc. Natl. Acad. Sci. USA, 1989
, 86: 2863). Therefore, the first endothelin was named ET-1.

One of the important discoveries following the characterization of ETs is that two ET receptors, ET _A And ET_B(Arai et al., Nature, 1).
990, 348: 730; Sakumi et al. , Nature,
1990, 348: 782). Both are 7-helical G protein-coupled receptors
Belongs to the family. 68% amino acid between the two receptor subtypes
There is acid identity. ET_AIs a single copy gene located on human chromosome 4.
Exist (Hosoda et al., J. Biol. Chem.,
1992, 267: 18797; Cyr et al. , Biochem.
 Biophys. Res. Commun. , 1991, 181: 18.
4). ET_BSplicing variants have been found (Shyamala e
t al. , Cell. Mol. Biol. Res. , 1994,
40: 285-96) But ET_BIs a single gene located on human chromosome 13
Exists as a pea gene (Arai et al., J. Biol. C
hem. , 1993, 268: 3463-70). ET_AThe cDNA sequence of
, GenBank under the accession number S57498.

In the early stages of ET studies, a great number of pharmacological studies suggested that responses to ETs could be divided into two groups according to the pharmacological efficacy of the three peptides. In fact, these two receptors, ET _A and ET _B , differ in their ligand binding affinity and distribution in tissues and cells. ET _A has a high affinity for ET-1 and ET-2, but a low affinity for ET-3. ET _B has equally strong affinity for all three endogenous ETs. ET _A is present on the smooth muscle, which medium vasoconstriction. In contrast, ET _B is present on the endothelium, mediates the release of relaxing factor, such as nitric oxide and prostacyclin. However, some reports, ET _B on certain vascular smooth muscle, showed that also mediates vasoconstriction.

[0015] Cloning of the ET receptor gene was performed using BE-18257B, and BE-
Such as two derivatives of 18257B, BQ-123 and FR139317
Accelerated development of ET receptor antagonists (Masaki, Cardiova)
scular Res. , 1998, 39: 530). ET _A And ET_BMany selective and non-selective antagonists have emerged.

Although the pathophysiological role of ET is still unknown, ET antagonists are
Heart failure, pulmonary hypertension, cerebral vasospasm after subarachnoid hemorrhage, acute renal failure and essential high
It has shown important and beneficial effects in pathological conditions including blood pressure. ET or ET reception
Putter knockout mice have also been implicated in the physiological and pathophysiological significance of ET.
Provided important information (Masaki, supra). Specifically, ET-3 or ET _B The mouse in which the receptor gene was knocked out was Hirschsprung.
Disease, ET_BA phenotype resembling the human hereditary syndrome with a missense mutation in the gene
(Piffeffenberger et al., Cell, 1).
994, 79: 1257).

Despite advances in understanding the role of the endothelin pathway in treating cardiovascular disease, questions remain. Not all experimental models of hypertension respond to endothelin antagonists. And it remains unclear whether endothelin antagonists improve the structure and action of inotropic drugs beyond the benefits of lowering blood pressure (M
oreau, Cardiovascular Res. , 1998, 39
: 534). Accordingly, there is a need in the art for a reliable and effective means for predicting whether an endothelin antagonist is effective in treating hypertension in an individual.

Β-Adrenergic receptors (β-adrenoceptors ) Adrenoceptors are divided into three main groups, α ₁ , α ₂ and β,
In each of these, additional subtypes can be pharmacologically distinguished (
Lullmann et al. , In Color Atlas of P
pharmacology, New York, 1993). Adrenergic receptors are all bound to G proteins. Adrenergic receptors are involved in the control of the cardiovascular system and in the control of metabolic activity, such as insulin secretion and glucose release. Adrenergic receptors also
Mediates the contraction and relaxation of smooth muscle cells in the respiratory tract, gastrointestinal tract and genitourinary tract (Berne and Levy, Principles of Physiolo)
gy (2nd edition), Mosby-Year Books, Inc. , 199
6, 691-696).

Adrenoceptors are targets for epinephrine and norepinephrine, which are representative of a family of monoamine neurotransmitters. Epinephrine is
Has equally high affinity to all the α- and β- receptors, whereas, norepinephrine, beta ₂ - due to its low affinity for the receptor, different from epinephrine (The Biochemical Basis of Neuroph
armacology (7th edition), New York, 1996, 226
-292 pages). Adrenoceptors themselves are in three different classes of G proteins: G _s (β-adrenoceptors), which mediate the activation of adenylate cyclase, and G _i (which mediate the inhibition of adenylate cyclase). alpha ₂ - adrenoceptor acids), and _{G q} to mediate activation of phospholipase C (α ₁ -. a adrenoceptors acids), preferentially interact (Hieble et al, J.
of Med. Chem. , 1995, 38: 3415-3444).

[0020] Pharmacological interest adrenoceptor class is mainly example, beta antagonists for treating hypertension, through the development of alpha ₁ antagonists and alpha ₂ agonists, for the treatment of cardiovascular diseases is there. However, they also can be considered important for the treatment of asthma (beta ₂ agonists).

Β ₂ -adrenergic receptors are expressed on many cell types, such as bronchial smooth muscle. In bronchial smooth muscle, its activation leads to relaxation and bronchodilation. These receptors are also expressed on epithelial cells, vascular endothelium, alveolar walls, immune cells, and presynaptic terminal nerves (Liggett,
Am. J. Respier. Crit. Care. Med. , 1
997, 156: S156-S162). Cardiac cells primarily express β ₁ -adrenoceptors, but also slightly express β ₂ -adrenoceptors (
Collins et al. , Biochimica et Biophys
ica Acta, 1993, 1172: 171-174). β ₁ -adrenoceptors are also expressed in brain and pineal gland.

Function of β-Adrenoceptors β ₁ -and β ₂ -adrenergic receptors are bound to the G _s -protein complex, which activates denylate cyclase. Agonist binding to β ₁ -receptors on cardiomyocytes mediates enhanced contractility and cardiac output (Lullmann, supra). The binding of agonists to β ₂ -receptors in the peripheral vascular arteries causes vasodilation by increasing the amount of cAMP and thereby inhibiting the activation of myosin kinase, which is required for smooth muscle cell contraction. Mediate (ibid.). Activation of cAMP in cardiac cells by agonists that bind to both β ₁ -and β ₂ -adrenoceptors activates cAMP-dependent protein kinase (PKA) (Castellano and Bohm, Hyper).
tension, 1997, 29: 715-722). β-Adrenoceptors also regulate the control of melatonin production in the pineal gland by cAMP activation of one of the enzymes involved in melatonin synthesis (5-HT-N-acyltransferase).

Β ₂ -receptors mediate enhanced conversion of glycogen to glucose (glycolysis) in both liver and skeletal muscle (H. Lulmann et al., Ibid.)
Stimulates the influx of potassium into muscle cells to prevent hyperkalemia (Ber
ne and Levy, ibid.).

Β-receptors are regulated at the protein level by desensitization. The initial desensitization process is effected by phosphorylation of serine and threonine residues in the cytoplasmic tail or third intracellular loop by several protein kinases, including βARK and PKA (Hieble et al., Supra). βARK specifically phosphorylates serine and threonine residues at the C-terminus of receptors blocked by agonists. Phosphorylation is cytostriated (cytost)
olic) induces binding of the protein to β-arrestin and results in _dissociation from _Gsα . PKA is activated by cAMP, and by relatively slow process, beta ₂ - phosphorylates adrenoceptors. Phosphorylated receptors, lose the ability to activate _{G s} (Castellano
And Bohm, ibid.). Long interactions between agonists and adrenoceptors generally lead to receptor desensitization.

The β- adrenoceptor genetic makeup human beta ₁ - adrenoceptor gene, alpha _{2A -} adrenoceptor gene identical to the chromosome of, are located on the longer arm of ten chromosomes. The coding sequence for this gene has been deposited with GenBank under accession number X69168. The regulatory region has also been deposited with GenBank under accession number J03019. This is 1431
Encodes a gene product without base pair introns (Hall, Thor
ax, 1996, 51: 351-353). Both the promoter and coding region of the gene are rich in G and C residues, with G and C residues comprising 70% of the bases.
Make up the super. The promoter does not contain a paired consensus TATA box and CAAT box element, but instead has an inverted CAAT box.
Including clusters having box and SP ₁ or AP-2 binding motif. Receptors of this type that are reminiscent of "housekeeping genes" have also been described for other G protein-coupled receptors (Collins et al., Supra).

The human β ₂ -adrenoceptor gene is located on the long arm of chromosome 5, the same chromosome as the α _1B -adrenoceptor gene. The coding sequence is GenB
deposit with the accession number M15169, J02728 or M16106. The regulatory region sequence has also been deposited with GenBank under the accession number Y00106. It encodes a 1239 base pair intronless gene product (Hall, supra). The promoter region is located between 200 and 30 of the translation initiation codon.
0 base 5 'side, which can form a strong secondary structure due to high GC content. Approximately 30 and 8 respectively from the mRNA start region
There are two TATA boxes (separated by approximately 10 base pairs) and a CAAT box located 0 base pairs upstream.

Gene Regulation of β-Adrenoceptor There are several regulatory regions identified in the promoter region of the β ₁ -adrenoceptor gene. It consists of cAMP response element (CRE), consensus thyroid response element (TRE) and glucocorticoid response element (G
RE). This is consistent with evidence that both thyroid hormones and corticosteroids affect adrenergic susceptibility in both heart and adipose tissue. CRE regions, beta ₂ - as shown for adrenoceptors genes, may have a self-regulating function (Colling et al
. , Ibid.).

[0028] β ₂ - of adrenoceptor 5 'flanking region, there are some of the regulatory domain.
Among these are the cAMP responsive elements (CRE), which are recognized and stimulated by a phosphoprotein called CRE binding protein (CREB). CREB is partly under the control of the PKA-dependent phosphorylation process. This is, in the early stage after exposure to β agonists, β ₂ - adrenoceptor m
Seen as an increase in the amount of RNA. However, the amount of mRNA decreases after prolonged exposure to agonists, which is probably mediated by a shortened mRNA half-life (Castellano and Bohm, supra). Stimulation with glucocorticoids in various tissues has also been shown to upregulate transcription of the β ₂ -adrenoceptor gene (Collins et al., Ibid.). In the 3 'adjacent area,
There are sequences homologous to the glucocorticoid response element. These are the β ₂ − observed in cells transfected after treatment with hydrocortisone.
May be associated with increased expression of adrenoceptors (Emorine and Marullo, Proc. Natl. Acad. Sci. USA
, 1987, 84: 6995-6999).

Protein Structure of β-Adrenoceptors The model proposed for β-adrenoceptors, like most of the G protein-coupled receptors, has seven α-helical transmembrane structures (where 7 One α
The helix is radially arranged around a central "hole" to which the receptor's ligand binds). β-adrenoceptors have an extracellular glycosylated N-terminus and an intracellular C-terminus. The β ₁ -receptor consists of 477 amino acids and β _2-
The receptor consists of 413 amino acids.

The amino acid identity of human β ₁ -and β ₂ -adrenoceptors as a whole
Only 54%. However, the pharmacological differences between β ₂ -receptors and β ₁ -receptors are due to slight changes in the orientation of the main binding site, resulting in slightly different binding sites rather than specific amino acid substitutions. (Hieble et al., J. Med. Chem., 19)
95, 38: 3415-3444).

[0031] Site-directed mutagenesis involves Asp-113, an aspartic acid residue located in a third spanning helix, and two serine residues, Ser-204 and Ser-207. Was required for complete agonist binding to β ₂ -adrenoceptors. β ₁ -adrenoceptors are
beta ₂ - address exists at a position corresponding to the position shown to be important for the binding of agonist to Bruno Scepter, it contains the same amino acid residue. Another aspartic acid residue, Asp-79, located in the second α-helix of both β-receptors, is highly conserved in G protein-coupled receptors (Hieble et al.).
al. , Ibid.). Ser-319 has a potential role in binding agonists to β ₂ -adrenoceptors.

The beta ₂ - mutation of Tyr-350 is located in the cytoplasmic tail of the receptor, which interferes with the binding of _{G s} of the receptor (. Hieble et al, supra). Further, palmitoylation of Cys-341 in the C-terminus, beta ₂ - adrenoceptors is it possible to form a fourth cytoplasmic loop, this loop,
Enhance the capacity of agonist-coupled receptors with respect to mediating adenylyl cyclase stimulation (Strosberg, Preotein Science, 19
93, 2: 1198-1209).

Β-Adrenoceptors as Pharmaceutical Targets In 80-90% of hypertensive patients, the cause of the disease cannot be confirmed. Such patients have so-called essential hypertension. Essential hypertension affects 5-10% of the general population, and essential hypertension is responsible for the most common disease in developed countries (J. Axford, Medicine, Blackwell).
l Science Ltd. , 1996, 10.1199-10.130
). β-blockers are widely used in the treatment of hypertension. β-blockers are particularly useful for treating hypertension in young people with tachycardia and high cardiac output. β
-Blockers or β-adrenergic blockers were first introduced in 1964 as a treatment for essential hypertension, and β-blockers are inexpensive (this is a (Improves compliance) and is still recommended as a first-line drug. β-blockers act on cardiac smooth muscle cells by binding to β ₁ -receptors. This leads to reduced cardiac output. Most β-blockers are not specific β ₁ -receptor antagonists, but also bind to β ₂ -receptors. Although inhibition of β ₂ -receptors leads to vasoconstriction,
beta ₂ - binding to the receptor class, the desired effect gives the opposite effect. This has the side effect of cooling the limbs because most of the β ₂ -receptors are in the peripheral vascular arteries.

Β-blockers are also known to cause bronchospasm, as well as some central nervous system side effects (nightmares, somnolence). β-blockers reduce insulin secretion, and therefore, for the treatment of hypertension with diabetes mellitus, β-blockers
Blockers are not appropriate. Β-blockers can also cause heart failure and peripheral arterial occlusive disease (Velaseco and Rodrigues, J.
own of Human Hypertension 10, Sup
pl. 1, S77-S80, 1996).

Β-adrenoceptor agonists such as dopamine and dobutamine are used to stimulate myocardial β ₁ -adrenoceptors in the treatment of the acute phase of congestive heart failure. Beta-adrenoceptor agonists act by increasing contractility and cardiac output.

[0036] Many different β ₂ -agonists are used in the treatment of asthma. beta ₂ - agonists, beta ₂ of the bronchial smooth muscle - the adrenergic receptor, exert their primary action of, resulting in relaxation and bronchodilation. β ₂ -agonists also protect against the attack of bronchoconstrictor factors (Hall, supra).

Thus, in the art, for an improved understanding of the effects of β-blockers in different patients, and which patients have a better response to treatment with β-blockers. There is a clear need to predict what to show.

The Need for Efficient Cardiovascular Status Assessment The high morbidity and mortality associated with heart disease can lead to the most effective treatment outcome in the art for patients suffering from cardiovascular disease. 2 illustrates the need for methods and compositions that allow for the determination and / or prediction of a likely treatment regimen. This includes, for example, identifying individuals who respond more or less to a particular treatment regimen, including certain drugs conventionally used in the treatment of cardiovascular disease.

In addition, heterogeneity in response to cardiovascular treatment underscores the need for other approaches for rational drug development. In particular, a population that has been identified as unresponsive to a particular treatment regimen may be qualified for the development and testing of alternative regimens. Thus, effective treatment regimens can be developed for a larger proportion of the affected population.

In summary, there is a need to reduce or eliminate trial and error in selecting a treatment regimen for a particular individual. Whether a given individual is responsive, for example, to a certain type of drug or just a certain drug, or whether he / she is susceptible to adverse reactions or side effects Would be desirable instead.

Also, in the art, to facilitate early intervention and disease prevention,
There is a need for methods and compositions that allow the identification of individuals who are predisposed to cardiovascular diseases such as myocardial infarction, hypertension, atherosclerosis and stroke.

The present invention provides polymorphisms and polymorphism patterns that are characteristic of cardiovascular status, and to prescribe or develop more effective treatments or to assist in diagnosis By using these polymorphisms and polymorphism patterns,
It focuses on these and other needs in the art.

Citation of any reference in this application shall not be construed as a confession that the reference is prior art to the present invention. Each cited reference is hereby incorporated by reference in its entirety.

[0044] In summary one aspect of the invention, the present invention provides certain processing regimen (particular pharmaceutical, certain types of medicament, or, not the case or a pharmacological other treatment regimens Is effective in improving cardiovascular conditions in certain human individuals,
Or reagents and methods are provided for predicting whether it is ineffective for that purpose, or whether there are any adverse reactions or undesirable side effects associated with its use.

A particular advantage of the present invention is that one or more polymorphic markers provide a basis for predicting the outcome of a treatment regimen. By comparing a patient's polymorphism pattern in need of treatment for cardiovascular abnormalities, such as hypertension, to a pre-established reference pattern to correlate with responsiveness to a treatment regimen,
A physician can predict whether a treatment regimen, such as administration of an ACE inhibitor, will be effective or not before letting the patient take the treatment regimen. For example, a comparison of a test polymorphism pattern from an individual with a reference polymorphism pattern in a population of individuals that exhibits different responses to a particular therapeutic intervention indicates the individual's responsiveness to such intervention. Can be used to predict the type or degree of Thus, the present invention represents an important discovery in the treatment of cardiovascular pathologies in reducing or eliminating trial and error in selecting a patient's treatment for a particular individual cardiovascular.

An additional benefit of the present invention derives from the ability to remove patients from clinical trials that are predicted to be unresponsive or at risk of adverse response to a particular treatment regimen. In addition, adverse results in early trials can be evaluated to identify polymorphic patterns, and the adverse results can be correlated with a sub-population of the test population, and such Subgroups can be excluded. Thus, the present invention can ensure that beneficial medicaments can be approved for use in the appropriate population, and reduce the number of patients needed, and therefore the duration and cost of clinical trials. Can be reduced. The present invention may also lead to the identification of other subpopulations that may be targets for the development of other therapeutic regimens.

All of the foregoing applications within the scope of the present invention can be considered to be an assessment of an individual's cardiovascular status, as the term is broadly defined below.

The above method of the present invention comprises ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-
A test polymorphism pattern established by at least one polymorphism position in the gene encoding the adrenoceptor is compared to a polymorphism pattern (a reference pattern) in a population that exhibits a predetermined response to the regimen. It is done by doing. If the test pattern matches the reference pattern, the individual has the same cardiovascular status as the cardiovascular status associated with the reference pattern.
There is a statistically significant probability.

The polymorphism pattern preferably consists of at least two (more preferably at least three) polymorphic positions, at least one of which comprises ACE, AT1, AGT,
A gene encoding a polypeptide from the group consisting of renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor, the second polymorphism being ACE, AT1, AGT, renin, Aldosterone synthase, a gene encoding a polypeptide selected from the group consisting of type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor.

In addition, the present invention provides methods for assessing whether a particular individual has a genetic predisposition for cardiovascular disease. This aspect of the invention relates to ACE, AT1, AGT
, Renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor, at least one, preferably at least two, most preferably at least three polymorphic positions, and ACE , AT1, AGT, renin, aldosterone synthase, test polymorphism established by one or more, preferably two or more other polymorphic positions in type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor Comparing the pattern with a polymorphic pattern of a population that is predisposed to cardiovascular syndrome. The conclusion drawn depends on whether the individual's polymorphism pattern matches the reference pattern.

The present invention also provides ACE, AT1, AGT, renin, aldosterone synthase,
An isolated nucleic acid having a sequence corresponding to part or all of a gene encoding angiotensin II receptor type 2, an endothelin receptor and a β-adrenoceptor, comprising ACE, AT1, AGT, renin, aldosterone synthase, 2
Also provided are nucleic acids comprising a polymorphism in the type angiotensin II receptor, endothelin receptor and β-adrenoceptor genes. In a preferred embodiment, the polymorphism is the same gene or a protein selected from the group consisting of human ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor. Combination of the encoding gene with one or more other polymorphisms in the sequence may be predictive of a particular type or level of responsiveness to a given treatment or may be associated with a cardiovascular disease.
Evidence of one or more clinical syndromes, or both.

The isolated polymorphisms (described using the numbering shown in Table 1 below) in accordance with the present invention include, but are not limited to, the following: Exon (exon 10) contains one or more polymorphic positions (3
(A translocation of cytosine to thymine which results in a premature stop codon at position 87).

Nucleic acid encoding the promoter of aldosterone synthase at position -344.

In GenBank, accession number X69168 is assigned,
2238, 2440, 2493, 2502, 2577, 2585, 2693, 2
Nucleic acids encoding β-adrenergic receptor-1 regulatory regions at positions 724 and 2775. In a preferred embodiment, the base at a specific position is 2238
G, 2238A, 2577C, 2257T, 2557A and 2557G.

In GenBank, accession number J03019 is assigned,
Β- at positions 231, 758, 1037, 1251, 1403 and 1528
A nucleic acid encoding an adrenergic receptor-1 coding region. In a preferred embodiment, the base at a specific position is 231A, 231G, 1251C, 1
251G, 1403A, 1403G, 1528C and 1528A.

In GenBank, accession numbers M15169 or J02728 or M16
932, 934, 987, 1006, 112, numbered 106
Nucleic acid encoding a β-adrenergic receptor-2 regulatory region at positions 0, 1221, 1541 and 1568. In a preferred embodiment, the base at a specific position is 934A, 934G, 987C, 987G, 1006A, 1006G,
1120C, 1120G, 1221C, 1221T, 1541C, 1541T,
1568C and 1568T.

In GenBank, accession number Y00106 is assigned.
839, 872, 1045, 1284, 1316, 1846, 1891, 203
Β-adrenergic receptor-2 at positions 2,2068 and 2070
A nucleic acid encoding a coding region. In a preferred embodiment, the base at a specific position is 839A, 839G, 872C, 872G, 1045A, 1045G, 1
284C, 1284T, 1316A, 1316C, 1846C, 1846G, 2
032A, 2032G, 2068 No insertion, 2068G, 2068C, 2070
No insertion and 2070C.

In GenBank, accession number S57498 is attached.
Nucleic acids encoding endothelin-A receptors at positions 969, 1005, 1146 and 2485. In a preferred embodiment, the base at a specific position is 969
C, 969T, 1005A, 1005G, 1146A, 1146G, 2485T
And 2485C.

To establish a polymorphism pattern, encode a polypeptide selected from the group consisting of ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor Nucleic acids containing polymorphisms present in other genes, which can be used in combination with polymorphisms derived from the gene, are disclosed in International Patent Application No. PCT / IB98 / 00475, which includes: : (I) positions in the regulatory region numbered 5106; 375, 582, 7
Nucleic acids encoding ACEs having one or more polymorphic positions at positions 1451; positions in the coding regions numbered 31, 1060, 2741, 3132, 3387, 3503, and 3906. All positions are GenBank
As numbered in kRegistration X62855. In a preferred embodiment, the sequence at the polymorphic position in the ACE regulatory region is one or more of 5106C and 5106T, and the sequence at the polymorphic position in the coding region is 375A,
375C, 582C, 582T, 731A, 731G, 1060G, 1060A
, 2741G, 2741T, 3132C, 3132T, 3387T, 3387C
, 3503G, 3503C, 3906G and 3906A. The invention also includes a nucleic acid encoding a deletion of nucleotides 1451 to 1783, numbered entry X62855 in GenBank.

(Ii) 395, 412, 432, 449, 692, 839, 1007, 10
Positions in the regulatory regions numbered 72 and 1204; 273, 912, 9
Positions in the coding regions numbered 97, 1116 and 1174;
And AGT encoding nucleic acid having one or more polymorphic positions at positions 49 (as numbered in GenBank Accession M24688). In a preferred embodiment, the sequence at the polymorphic position in the AGT regulatory region is 395T, 395A
, 412C, 412T, 432G, 432A, 449T, 449C, 692C,
692T, 839G, 839A, 1007G, 1007A, 1072G, 107
2A, 1204C and 1204A, wherein the sequence of the polymorphic position in the coding region is 273C, 273T, 912C, 912T, 997G,
The sequence at position 49 in one or more of 997C, 1116G, 1116A, 1174C and 1174A and in GenBank entry M24688 is A or G.

(Iii) one or more positions in the regulatory regions numbered 1427, 1756, 1853, 2046, 2354, 2355 and 2415; and in the coding region numbered 449; A nucleic acid encoding AT1 having a polymorphic position. In a preferred embodiment, the sequence at the polymorphic position in the AT1 regulatory region is:
1427A, 1427T, 1756T, 1756A, 1853T, 1853G,
2046T, 2046C, 2354A, 2354C, 2355G, 2355C,
2415A and 2415G, and the sequence of the polymorphic position in the coding region is 449G, 449C, 678T, 678C, 1167A, 11
67G, 1271A and 1271C.

The present invention also encompasses libraries of isolated nucleic acid sequences, such as arrays on solid surfaces. Here, each sequence in the library is ACE, AT1
, AGT, renin, aldosterone synthase, a type 2 angiotensin II receptor, an endothelin receptor, or a polymorphic position in a gene encoding a β-adrenoceptor, including polymorphic positions and sequences disclosed herein ( But not limited thereto), and other polymorphic positions in other genes. Also, nucleic acid probes that specifically hybridize to the identified polymorphic positions, peptides and polypeptides containing the polymorphic positions, and polymorphic-specific antibodies that can be used to identify particular polymorphic variants, i.e. Also provided are sequence-specific antibodies that specifically bind to polymorphic variants of the aforementioned genes.

In another aspect, the present invention provides a kit for determining a polymorphism pattern in a gene of an individual. The kit includes means for detecting a polymorphic sequence, including, but not limited to, an oligonucleotide probe that hybridizes at or adjacent to the polymorphic position and a polymorphic-specific antibody.

In yet another aspect, the invention provides nucleic acid and polypeptide targets for use in a screening method for identifying a cardiovascular drug candidate. The nucleic acid target can be, for example, DNA or RNA, the length of which is preferably at least about 10, most preferably at least about 15 residues,
It contains one or more polymorphic positions in a gene encoding a polypeptide from the group consisting of ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor. Peptide targets are at least about 5 amino acids in length and may be as large or larger than the full length polypeptide.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates, in part, to the discovery that polymorphisms in certain genes in the RAAS, endothelin and β-adrenoceptor pathways define polymorphic patterns that correlate with cardiovascular status. Based on Most importantly, the test individual correlates with the reference polymorphism pattern by comparing the test individual's polymorphism pattern with a reference polymorphism pattern, which is a polymorphism pattern from a population of individuals with a known cardiovascular status. You can predict if you have an increased likelihood of sharing the same cardiovascular status as you do. In particular, certain patterns correlate with responsiveness to ACE inhibitors, unresponsiveness to ACE inhibitors, and predisposition to cardiovascular disease or dysfunction, including myocardial infarction and stroke.

The present invention provides a powerful predictive tool for clinical testing and treatment of cardiovascular disease. For clinical trials, the present invention provides smaller, more efficient clinical trials by identifying individuals likely to be poorly responsive to treatment regimens and reducing the amount of uninterpretable data. enable. By assessing the polymorphism pattern of a test individual, a physician can prescribe a prophylactic or therapeutic regimen tailored to the individual's cardiac status. Adverse responses to a particular therapy can be avoided by eliminating individuals at risk for that therapy due to their cardiovascular status. Appropriate changes in lifestyle, including diet, environmental stress and exercise levels, can be prescribed for individuals whose test polymorphism pattern matches a reference pattern that correlates with an increased predisposition to cardiovascular disease.

Definitions As used herein, "cardiovascular status" refers to one that includes the genotype.
Or the physiological status of the cardiovascular system of an individual as reflected in two or more status markers or indicators. The cardiovascular status can be, without limitation, the presence or absence of a pathology or disease of one or more components of the individual's cardiovascular system and the individual's predisposition to produce such a condition, as well as the individual's responsiveness, That is, the ability or inability of an individual to respond (positively or negatively) to a particular prophylactic or therapeutic regimen or treatment of a cardiovascular condition, such as a drug or a class of drugs. Will be recognized. Negative responses include one or more adverse reactions and side effects. Status markers include, without limitation, clinical measurements, such as, for example, blood pressure, electrocardiographic profile, differential blood flow analysis, and the presence of increased levels of cellular proteins associated with cardiovascular events. Examples of such proteins (also called diagnostic markers) that are important in cardiac events include myosin light chain, myosin heavy chain, myoglobin, troponin I, troponin T, CK-MB, etc. (U.S. Pat. No. 5,604,105). No. 5,744,35
No. 8). A status marker according to the present invention is evaluated using conventional methods well known in the art. Further included in the assessment of cardiovascular status would be, for example, use in determining the onset of a final cardiovascular condition in a predisposed individual or in determining an individual's response to a particular therapeutic regimen. Qualitative or quantitative changes in the status marker over time.

Examples of cardiovascular conditions included in the above definition of cardiovascular status include, for example, hypertension, acute myocardial infarction, asymptomatic myocardial infarction, unstable angina, stroke and atherosclerosis. Such a diagnosis or predisposition for one or more cardiovascular syndromes. It will be appreciated that the diagnosis of cardiovascular syndrome by healthcare professionals is not only a clinical measurement but also a medical judgment.

As used herein, “responsiveness” refers to the type and extent of an individual's response to a particular therapeutic regimen, ie, the effect of treatment on the individual. Responsiveness is divided into three main categories: therapeutic effect; no effect; and adverse effects. Naturally, there may be different degrees of therapeutic effect, for example, between complete and partial disappearance of symptoms. Further, adverse effects or side effects may be observed even when the treatment is beneficial, ie, therapeutically effective. Indeed, the invention may allow to identify individuals with complex responsive properties or patterns.

“Predisposition to develop cardiovascular syndrome” refers to the increased likelihood of developing the cardiovascular syndrome as defined above, as compared to the general population. Predisposition does not mean certainty, and the development of the syndrome can be prevented or prevented by prevention, for example by employing a modified diet, exercise program or treatment with gene therapy or medicine. Inherently, one advantage of the present invention is that it allows the identification of individuals who are predisposed to develop cardiovascular syndrome, based on the genotype of the individual, and for which preventive intervention may be of particular importance. .

As used herein, “polymorphism” refers to a mutation in the nucleotide sequence of a gene in an individual. Genes that have different nucleotide sequences as a result of a polymorphism are "aryls (alleles)". "Polymorphic positions" are predetermined nucleotide positions within a sequence. In some cases, the genetic polymorphism is reflected in a mutation in the amino acid sequence, such that the polymorphic position refers to the position of the polymorphism in the amino acid sequence at a predetermined position in the sequence of the polypeptide. Can bring. An individual "homozygous" for a particular polymorphism is one in which both copies of the gene contain the same sequence at the polymorphic location. An individual "heterozygous" for a particular polymorphism is one in which two copies of the gene contain sequences that differ at the polymorphic location.

As used herein, a “polymorphism pattern” refers, without limitation, to one or more than one or more that can be included in the sequence of a single gene or multiple genes, including single nucleotide polymorphisms. Means a set of polymorphisms. In the simplest case, the polymorphic pattern may consist of a single nucleotide polymorphism at the position of only one of the two alleles of the individual. However, both copies of the gene must be seen. A polymorphism pattern suitable for assessing a particular aspect of cardiovascular status (eg, predisposition to hypertension) assesses another aspect of cardiovascular status (eg, responsiveness to ACE inhibitors for control of hypertension) The same polymorphism number as the polymorphism pattern (
Of course, the same does not have to be included. As used herein, a "test polymorphism pattern" is a polymorphism pattern determined for a human subject with an undefined cardiovascular status. As used herein, a “reference polymorphism pattern” is determined from a statistically significant correlation of patterns in a population of individuals having a predetermined cardiovascular status.

As used herein, “nucleic acid” or “polynucleotide” refers to a purine and pyrimidine containing polymer of any length of polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribonucleotides. Nucleic acids include, without limitation, single- and double-stranded molecules, ie, DNA-DNA, DN.
Includes A-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by attaching bases to an amino acid backbone. This also includes nucleic acids containing modified bases and unnatural phosphoester analog linkages, such as phosphorothioates and thioesters. The term nucleic acid molecule, especially DNA
Or, an RNA molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, the term especially refers to linear or circular DNA molecules (
Double-stranded DNA found on, for example, restriction fragments), plasmids, and chromosomes
including. In discussing the structure of a particular double-stranded DNA molecule, the sequence herein refers only to the sequence in the 5 'to 3' direction along the non-transcribed strand of DNA (ie, the strand having a sequence homologous to the mRNA). And may be described according to normal conventions. A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

As used herein, the term “oligonucleotide” refers to a genomic DNA molecule, a cDNA molecule or an mRNA molecule encoding a gene, cDNA, mRNA,
Generally at least 10, capable of hybridizing to RNA, or other nucleic acid of interest.
A nucleic acid of preferably at least 15, and more preferably at least 20 nucleotides. Oligonucleotides can be labeled using nucleotides covalently linked to a label such as, for example, ³² P-nucleotides or biotin. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, the oligonucleotide, one or both of which may be labeled, is used as a PCR primer to clone the full length or fragment of the gene of interest or to detect the presence of a nucleic acid encoding the gene of interest. be able to. In a further aspect, the oligonucleotides of the invention are capable of forming a triple helix with the desired double-stranded sequence of a DNA molecule. In yet another embodiment, a library of oligonucleotides located on a solid support, such as a silicon wafer or chip, can be used to detect various polymorphisms of interest. Generally, oligonucleotides are prepared by synthesis, preferably on a nucleic acid synthesizer. Thus, oligonucleotides can be prepared using non-natural phosphoester analog linkages, such as thioester linkages.

As used herein, “isolated” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that has been removed from its original environment (eg, the natural environment, if natural). An isolated nucleic acid or polypeptide contains less than about 50%, preferably less than about 75%, and most preferably less than about 90% of the cellular components with which it was originally associated.

A nucleic acid or polypeptide sequence “derived from” a specified sequence refers to a sequence that corresponds to a region of the specified sequence. For a nucleic acid sequence, this includes sequences identical or complementary to the sequence.

“Probe” refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target nucleic acid due to complementarity between the sequence in the target nucleic acid and at least one sequence in the probe. Generally, probes are labeled and can be detected after hybridization.

A nucleic acid molecule is capable of annealing to another nucleic acid molecule under appropriate conditions of temperature and solution ionic strength, such as cDNA, genomic DNA or RNA, if the single-stranded form of the nucleic acid molecule is capable of annealing. Is "hybridizable" to nucleic acid molecules (Sa
mbrook et al. , 1989, Molecular Cloni
ng: A Laboratory Manual, Second Edit
ion, Cold Spring Harbor Laboratory P
res: see Cold Spring Harbor, New York). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, it can be used low stringency hybridization conditions, corresponding to the _{T m} of 55 ° C. (e.g. 5 × SSC, 0.1% SDS, 0.2
5% milk and no formamide; or 30% formamide, 5 × SSC,
0.5% SDS). Hybridization conditions of moderate stringency is equivalent to a higher T _m which, for example 40% formamide 5 × or 6 × SC
It contains C. Hybridization conditions of high stringency is equivalent to the highest T _m, e.g., 50% formamide, 5 × or 6 × SCC. Hybridization requires that the two nucleic acids contain complementary sequences,
Mismatches between bases are possible depending on the stringency of the hybridization. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, ie, variables well known in the art. 2
The greater the degree of homology or similarity between two nucleotide sequences, the greater the _Tm value for hybrids of nucleic acids having those sequences. Nucleic acid hybridization relative stability (corresponding to higher _{T m),} RNA: RNA,
DNA: RNA, DNA: DNA decrease in that order. For hybrids greater than 100 nucleotides in length, formulas for calculating _Tm have been derived (see Sambrook et al., Supra, 9.50-9.51). For the hybridization of shorter nucleic acids, i.e., oligonucleotides, the location of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8). I want to.) The minimum length of a hybridizable nucleic acid is at least about 10 nucleotides, preferably at least about 15 nucleotides, and most preferably, the length is at least about 2 nucleotides.
0 nucleotides.

In one specific embodiment, “standard hybridization conditions” include 5
It refers to 5 ° C. in T _m, using the above conditions. In some preferred embodiments, T _m is 6
0 ° C., and in a more preferred embodiment, the T _m is 65 ° C. In certain specific embodiments, "high stringency" refers to 42 string in 0.2 x SSC at 68 ° C.
At 50 ° C. in 50% formamide, 4 × SSC, or conditions that give equivalent levels of hybridization as observed under either of these two conditions.
Refers to hybridization and / or washing conditions.

As used herein, a “gene” for a particular protein is a contiguous nucleic acid sequence corresponding to a sequence present in the genome, comprising (i) an exon (ie, a sequence encoding a polypeptide sequence; Or "coding (or transcribed) region" comprising "protein coding" or "transcribed sequence"), introns, sequences of junctions between exons and introns, and 5 '
And 3 ′ untranslated regions (uTRs); and (ii) regulatory sequences flanking the coding region at the 5 ′ and 3 ′ ends. For example, "ACE gene" as used herein includes the regulatory and coding regions of a human gene encoding angiotensin converting enzyme. Similarly, the "AGT gene" includes the regulatory and coding regions of the human gene encoding angiotensinogen,
The "T1 gene" comprises the regulatory and coding regions of a human gene encoding the type 1 angiotensin II receptor. Typically, the regulatory sequences of the present invention are located 5 '(ie, upstream) of the coding region segment. The reference sequences obtained from GenBank used in the practice of the present invention are shown in Table 1.

[0081]

[Table 1]

We have surprisingly and unexpectedly evaluated cardiovascular status, alone or in combination, depending on which component of cardiovascular status is being evaluated. Of polymorphisms in the human gene encoding ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor, which can be used for E. coli. According to the present invention, the polymorphic pattern of the gene, alone or in an individual, is determined by renin, ACE, AGT, AT1, AT2, aldosterone synthase, endothelin receptor, and β-adrenergic receptors 1 and 2.
In combination with other genes that encode can predict the individual's responsiveness to a particular therapeutic intervention and serve as an indicator of a predisposition to various forms of cardiovascular disease. The present invention provides a method for assessing cardiovascular status by detecting a polymorphic pattern in an individual. The present invention also includes isolated nucleic acids derived from genes containing these polymorphisms, including probes that specifically hybridize to the polymorphic positions and primers that amplify the region of the gene where the polymorphism is located; Isolated polypeptides and peptides comprising a type residue; and ACE, AT1, AGT, renin, aldosterone synthase comprising one or more polymorphic amino acids;
Antibodies that specifically recognize the type 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor polypeptide are also provided.

Method for Evaluating Cardiovascular Status The present invention provides a diagnostic method for evaluating cardiovascular status in a human individual. The method of the present invention relates to polymorphic positions or patterns in the gene of an individual encoding ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor ("
The test polymorphism pattern ") is compared to a human polymorphism pattern exhibiting a predetermined cardiovascular status (" reference polymorphism pattern "). If the cardiovascular status is a prediction of responsiveness to therapy, a single polymorphic location can provide a pattern for comparison. However, it is preferred to use more than one polymorphic position for the pattern to improve the accuracy of the prediction. If the cardiovascular status is predisposed to cardiovascular syndrome, a pattern is created using at least two, and preferably at least three, polymorphic locations. Furthermore, angiotensin converting enzyme (ACE), angiotensinogen (AGT), type 1 angiotensin II receptor (AT1), type 2 angiotensin II receptor, renin, aldosterone synthase, endothelin, receptor or β-adrenergic receptor 1 and 2 Other polymorphisms in the gene encoding can be used to establish a polymorphism pattern for that individual.

For any meaningful prediction, the individual's polymorphism pattern is the same as the individual's polymorphism pattern indicating a particular status marker, cardiovascular syndrome, and / or a particular response pattern to a particular therapeutic intervention. It is.

In one embodiment, the method comprises comparing the test polymorphism pattern of the individual with a reference polymorphism pattern of the individual that has been shown to respond positively or negatively to a particular treatment regimen. Including. As used herein, treatment regimen refers to treatment aimed at eliminating or ameliorating events and symptoms associated with cardiovascular disease. Such treatments include, without limitation, changes in diet, lifestyle, and exercise regimens; invasive or non-invasive surgical techniques, such as resection, angioplasty, and coronary bypass surgery; And ACE inhibitors, angiotensin II receptor antagonists, diuretics, α-adrenoceptor antagonists, cardiotonic glycosides, phosphodiesterase inhibitors, β-adrenoceptor antagonists, calcium channel blockers, HMG-C
one or more of a pharmaceutical intervention such as administration of an oA reductase inhibitor, an imidazoline receptor blocker, an endothelin receptor blocker, and an organic nitrite. Also included are interventions with medicaments whose activity is not yet known to correlate with specific polymorphic patterns associated with cardiovascular disease. The present inventors have discovered that certain polymorphic patterns correlate with an individual's responsiveness to ACE inhibitors (see, eg, Example 3 below). For example, it is contemplated to screen patients who are candidates for a particular therapeutic regimen for polymorphic patterns that correlate with responsiveness to that particular regimen.

In another embodiment, the method includes determining the polymorphic pattern of the individual, for example, a cardiovascular disease such as hypertension, abnormal electrocardiographic profile, myocardial infarction, unstable angina, stroke, or atherosclerosis. Comparing to the polymorphism pattern of an individual who showed or exhibited one or more markers of the individual (see eg, Example 2 below), and the responsiveness of the individual to the therapies detailed above, syndromes And making similar conclusions about the likelihood of developing.

Identification of Polymorphism Patterns In practicing the method of the present invention, the polymorphism pattern of an individual can be determined, for example, by obtaining DNA from the individual and identifying a predetermined polymorphic location within a gene or two or more genes. It can be established by determining the sequence.

[0088] DNA can be obtained from any cell source. Non-limiting examples of cell sources available in clinical practice include, without limitation, blood cells, cheek cells,
Examples include cervical and vaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Cells may be obtained from body fluids including, without limitation, blood, saliva, sweat, urine, cerebrospinal fluid, stool, and tissue exudate at sites of infection or inflammation. DNA is extracted from cell sources or body fluids using any of a number of methods standard in the art. It will be appreciated that the particular method used to extract the DNA will depend on the nature of the source.

The sequencing of the extracted DNA at the polymorphic positions can be accomplished by any means known in the art, including direct sequencing, hybridization with allele specific oligonucleotides, allele specific. PCR, ligase-PCR, HO
Including but not limited to T-cleavage, denaturing gradient gel electrophoresis (DGGE), and single-stranded conformational polymorphism (SSCP). Direct sequencing can be performed in any manner, including, without limitation, chemical sequencing using the Maxam-Gilbert method; enzymatic sequencing using the Sanger method; mass spectrometry sequencing; and using chip-based techniques. Including sequencing. See, for example, Little et al., Genet.
Anal. 1996, 6: 151. Preferably, D from the subject
NA is first analyzed using the polymerase chain reaction (PCR) using specific amplification primers.
).

[0090] In an alternative embodiment, a biopsy tissue is obtained from the subject. ACE, AT1, AGT
An antibody capable of distinguishing different polymorphic forms of renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor is then applied to a sample of tissue and identified by the antibody. Determine the presence or absence of the polymorphic form. Antibodies may be polyclonal or monoclonal, but are preferably monoclonal. Measurement of the binding of the specific antibody to the cells can be achieved by any known method, such as, for example, quantitative flow cytometry, or enzyme-linked or fluorescent-linked immunoassay. The presence or absence of a particular polymorphism or polymorphic pattern and its allelic distribution (ie, homozygous versus heterozygous) establishes values obtained from the patient from a population of patients with a known polymorphic pattern. Is determined by comparison with the given criteria.

In another alternative embodiment, standard methods well known to those skilled in the art, such as guanidine thiocyanate-phenol-chloroform extraction (Chomocy)
Znski et al., Anal. Biochem. 1987, 162: 156) to isolate RNA from biopsy tissue. The isolated RNA is then subjected to coupled reverse transcription and polymerase chain reaction amplification (RT-PCR) using specific oligonucleotide primers specific for the selected polymorphism. Conditions for annealing the primers are chosen to ensure specific reverse transcription and amplification. Thus, the appearance of an amplification product is diagnostic for the presence of a particular polymorphism. In another embodiment, the RNA is reverse transcribed and amplified, and then
The amplified sequence is identified, for example, by direct sequencing. In yet another embodiment, cDNA from RNA can be cloned and sequenced to identify polymorphisms.

Establishing a Reference Polymorphism Pattern In the practice of the present invention, the distribution of the polymorphism pattern in a number of individuals exhibiting a particular cardiovascular status is determined by any of the methods described above,
Compared to the distribution of polymorphic patterns in patients with quantitative or qualitatively different cardiovascular status, matched for ethnic origin and / or any other statistically or medically relevant parameters . The correlation is nominal (nominal)
) Achieved using any method known in the art, including logistic regression, or standard least squares regression analysis. In this way, it is possible to establish a statistically significant correlation between a particular polymorphism pattern and a particular cardiovascular status. Further, it is possible to establish a statistically significant correlation between a particular polymorphism pattern and a change in cardiovascular status, for example, resulting from a particular treatment regimen. Therefore, it is possible to correlate the polymorphism pattern with the responsiveness to a specific process.

A statistically significant correlation preferably has a "p" value of 0.05 or less. Any standard statistical method can be used to calculate these values, such as the Normal Student's T test or Fisher's Exact test.

The identity and number of polymorphisms to be included in the reference pattern depend not only on the incidence of the polymorphism and its predicted value for a particular application, but also on the value of that application and the requirements on the accuracy of the prediction. I do. The larger the predicted value of a polymorphism, the smaller the need to include two or more polymorphisms in the reference pattern. However, if a polymorphism is very rare, its absence in an individual's pattern may not provide any indication as to whether the individual has a particular status. Under these circumstances, it may be advisable to select two or more polymorphisms with a higher incidence instead.
The presence of both (or all three) can be sufficiently predictive for that particular purpose, even if none of them have high predictive values on their own.

For example, if the use of a reference pattern is to predict the response to a drug, and if only a 30% response to the drug is observed among the affected population, then the need for the reference pattern is It only allows the selection of a population that improves the response rate by 10% to provide a significant improvement in level. On the other hand,
If the use for the reference pattern is to select a subject for a particular clinical study, the pattern should be as selective as possible, and thus, together, Multiple polymorphisms that provide high predictive accuracy should be included.

In establishing a reference polymorphism pattern, it is desirable to use a defined population. For example, tissue libraries that are collected and maintained by state or national ministries of health are of value since the genotypes determined from these samples can be matched to the individual's medical history, especially cardiovascular status. Certain sources can be provided. Such tissue libraries can be found, for example, in Sweden, Iceland, Norway, and Finland. As will be readily appreciated by those skilled in the art, a particular polymorphism may be associated with a closely related population. However, other polymorphisms in the same gene may correlate with the cardiovascular status of other genetically related populations. Thus, in addition to the specific polymorphisms provided in this application, the present invention provides for establishing reference and test polymorphism patterns therein to assess the cardiovascular status of individuals within a population. Identify a gene that can use any polymorphism.

In certain embodiments, DNA samples can be obtained from a well-defined population, such as 277 Caucasian men born in Uppsala, Sweden between 1920 and 1924. In specific embodiments, such individuals are identified based on their medical history (ie, they are (i) healthy and have no signs of cardiovascular disease (100), or (ii) have acute myocardial infarction ( 68), asymptomatic myocardial infarction (
34), seizures (18), seizures and acute myocardial infarction (19), or hypertension at the age of 50 (39)). DN
A sample is obtained from each individual.

In one specific embodiment, the DNA is obtained by (i) amplifying each short fragment of the gene using the polymerase chain reaction (PCR) and (ii) sequencing the amplified fragment. Sequence analysis can be performed. Sequences obtained from each individual can then be compared to a first known sequence, such as those shown in Table 1, to identify polymorphic locations.

Comparison of Test Pattern with Reference Pattern As described above, a test pattern from an individual can be compared to a reference pattern established for a predetermined cardiovascular status. The identity between the test pattern and the reference pattern means that there is a certain probability that the tested individual has the same cardiovascular status as represented by the reference pattern. As described above, this probability depends on the incidence of the polymorphism and the statistical significance of its correlation with cardiovascular status.

Polymorphism Positions The polymorphism positions in the genes encoding ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor included in the present invention are determined within the population. Can be identified by determining the DNA sequence of all or a part of the gene in a plurality of individuals. DNA sequencing can be accomplished using any conventional method, including, for example, chemical or enzymatic sequencing.

The polymorphic positions of the genes for use in the present invention include, without limitation, those listed below (their numbering corresponds to the GenBank sequences listed in Table 1).

(I) ACE: Positions in regulatory regions (referred to as ACRs) numbered 5106, 5349 and 5496; 375, 582, 731, 1060, 12
15, 2193, 2328, 2741, 3132, 3387, 3503 and 39
Positions in the coding region numbered 06 (called ACE);
And 1451 numbered Registration X62855 in GenBank
position.

(Ii) AGT: 395, 412, 432, 449, 692, 839, 100
Regulatory regions numbered 7,1072, 1204 and 1218 (AGR
273, 620, 803, 912, 997, 1116 and 1174 in the coding region (called AGT); and 4 in GenBank, numbered M24688.
9 positions.

(Iii) AT1: 1427, 1756, 1853, 2046, 2354, 2
Positions in the regulatory regions numbered 355 and 2415 (called ATR); and positions in the coding regions numbered 449, 678, 1167 and 1271 (called AT1).

(Iv) renin: a mutant renin gene in increasing familial prorenin,
A point mutation in the last exon (exon 10) of the gene has been identified (Villard et al., J. Biol. Chem. 1994, 269: 3).
0307-12). The translocation of cytosine to thymine creates a premature stop codon at position 387 that results in a truncated form of renin with 20 amino acids deleted from the carboxy terminus.

(V) Aldosterone synthase: the position in the promoter region of aldosterone synthase, assuming that the start codon starts with 1, at position -344 is the same as in June 1998.
International Meeting in Amsterdam in April
on Hypertension reported by Cambien et al.

Β-adrenergic receptor-1, 2238, 2440, 2493, 2
Positions in the regulatory regions (designated BP1) numbered 502, 2577, 2585, 2693, 2724 and 2557; and 231, 758,
Positions in the coding regions numbered 1037, 1251, 1403 and 1528 (designated BR1).

Β-adrenergic receptor-2, 932, 934, 987, 1006
Positions in regulatory regions (designated B2P), numbered 1120, 1221, 1541 and 1568; and 839, 872, 1045, 128
Positions in the coding regions numbered 4,1316, 1846, 1891, 2032, 2068 and 2070 (named B2R).

[0109] 969,1005,1146 and numbered 2485 is (are named ET _A) endothelin receptor type A coding region attached.

In a preferred embodiment, the base at each of the above polymorphic positions is any of the following: (i) ACE regulatory region: 5106C, 5106T, 5349A, 5349T,
5496T and 5496C.

(Ii) ACE coding region: 375A, 375C, 582C, 582T
, 731A, 731G, 1060G, 1060A, 1215C, 1215T, 2
193G, 2193A, 2328A, 2328G, 2741G, 2741T, 3
132C, 3132T, 3387T, 3387C, 3503G, 3503C, 3
906G, and 3906A; and deletions of nucleotides 1451-1782, numbered accession X62855 in GenBank.

(Iii) AGT regulatory region: 395T, 395A, 412C, 412T, 43
2G, 432A, 449T, 449C, 692C, 692T, 839G, 839
A, 1007G, 1007A, 1072G, 1072A, 1204C, 1204
A, 1218A, 1218G.

(Iv) AGT coding region: 273C, 273T, 620C, 620T
, 803T, 803C, 912C, 912T, 997G, 997C, 1116G
, 1116A, 1174C and 1174A; and GenBank registration M2468
A or G at position 49 in 8.

(V) AT1 regulatory region: 1427A, 1427T, 1756T, 1756A
, 1853T, 1853G, 2046T, 2046C, 2354A, 2354C
, 2355G, 2355C, 2415A and 2415G; and (vi) AT1 coding region: 449G, 449C, 678T, 678C.
, 1167A, 1167G, 1271A and 1271C.

(Vii) β-adrenergic receptor-1 regulatory region: 2238G, 2
238A, 2577C, 2257T, 2557A, and 2557G.

(Viii) β-adrenergic receptor-1 coding region: 231
A, 231G, 758C, 758T, 1251C, 1251G, 1403A, 1
403G, 1528C, and 1528A.

(Ix) β-adrenergic receptor-2 regulatory region: 934A, 934
G, 987C, 987G, 1006A, 1006G, 1120C, 1120G,
1221C, 1221T, 1541C, 1541T, 1568C, and 1568
T.

(X) β-adrenergic receptor-2 coding region: 839A
, 839G, 872C, 872G, 1045A, 1045G, 1284C, 12
84T, 1316A, 1316C, 1846C, 1846G, 2032A, 20
32G, no 2068 insertion, 2068G, 2068C, 2070 no insertion, and 2070C.

(Xi) Endothelin receptor type A: 969C, 969T, 1005A, 1
005G, 1146A, 1146G, 2485T, and 2485C.

An individual may be homozygous or heterozygous for a particular polymorphic location (see, eg, Table 6 below).

[0121] Non-limiting examples of polymorphism patterns comprising one or more polymorphisms in the ACE, AGT, and / or AT1 genes according to the present invention include the following: Correlated with an increased incidence of clinical signs of the disease: ACR 5349A / T, AGR 1218A; ACR 5496C, AGR
1204A / C; ACR 5496C / T, AGR 1218A, AGT 6
20C / T; ACE 2193A, AGR 1204C, ACE 2328G;
ACE 2193A, AGR 1204A / C; ACE 3387T, AGR
1218A; ACE 3387T, AGT 620C / T; AGR 1204A
/ C, AT1 678C / T; AGR 1204A / C, AT1 1271A /
C; ACE 1215C, AGR 1204A / C; AGR 1204A / C,
AT1 1167A, ACE 3906A / G; AGR 1204A, AGT
620C, AT1 1271A, AT1 1167A, AGR 395A / T;
AGR 1204A / C, AGT 620C / T, AT1 1271A / C, A
T1 1167A, AGR 395T; AGR 1204A / C, AGT 62
0C / T, AT1 1271A / C, AT1 1167A / G, AGR 395
T; AGR 1204A, AT1 678C, AT1 1167A, AGR 3
95A / T; AGR 1204A / C, AT1 678C / T, AT1 116
7A, AGR 395T; AGT 620C / T, AT1 1271A / C, A
T1 1167A, AGR 395T; AGT 620C / T, AT1 127
1A / C, AT1 1167A / G, AGR 395T; AGT 620C,
AT1 1271A, AT1 1167A, AGR 395A / T; AGT 6
20C, AT1 678A, AT1 1167A, AGR 395A / T; AG
T620C / T, AT1 678C / T; AT1 1167A, AGR 39
5T; ACE 2193A, AGR 1218A, AGT 803A; ACE
2193A, AGT 620C / T; ACE 2328G, AGT 620C /
T; ACE 3387T, AGR 1204A / C; ACE 2193A, AC
E 2328G, AGR 1204C; ACE 2193A / G, AGR 10
72G / G, AT1 1167A / A. A further polymorphism pattern is described in Example 4 below.
And 5 in the table.

Polymorphism Patterns Correlating with ACE Inhibitor Responsiveness The following table lists a set of polymorphic patterns that have been found to correlate with responsiveness to ACE inhibitor treatment.

[0123]

[Table 2]

Polymorphism Patterns Correlating with ACE Inhibitor Unresponsiveness The following table lists the set of polymorphism patterns that have been found to correlate with unresponsiveness to ACE inhibitor treatment.

[0125]

[Table 3]

Polymorphism Patterns Correlating to Predisposition to MI The following table lists the set of polymorphism patterns that have been found to correlate with predisposition to myocardial infarction.

[0127]

[Table 4]

Polymorphism Patterns Correlating to Predisposition to Seizure The following table lists the set of polymorphism patterns that have been found to correlate with predisposition to seizure.

[0129]

[Table 5]

Polymorphism Patterns in β-Adrenergic Receptor Gene The following table lists the set of polymorphism patterns found in β-adrenergic receptor genes.

[0131]

[Table 6]

A number of different polymorphisms have been identified in type 2 β-adrenoceptors. All of these differed from the wild-type sequence by a single base change. Four of the polymorphisms alter the amino acid sequence of the receptor protein (Hall, Th
orax, 1996, 51: 351-353). Amino acid sequence alterations are described in more detail below.

Arg16 → Gly: The Gly16 mutant undergoes higher agonist-promoted down-regulation as compared to the wild-type but retains binding to adenylyl cyclase and agonist binding (Liggett, Am .J.Respir.Cr
it. Care Med. 1997, 156: S156-S162).

Gln27 → Glu: The Glu27 mutant shows little agonist-promoted down-regulation and maintains binding to adenylyl cyclase and agonist binding (Id.).

Val34 → Met: Met34 is extremely rare. No change in receptor function has been found (Id.).

Thr164 → Ile: Not common (about 5%). The Ile164 mutant exhibits weaker binding to adenylyl cyclase and reduced affinity for agonists having a hydroxyl group on the β-carbon (such as epinephrine, norepinephrine and isoproterenol) compared to wild type (Id.)

A polymorphism in nucleic acid 523 (CGG → AGG) may be linked to one of the other functional polymorphisms (Id.).

Although there is no difference in the frequency of these polymorphisms between the normal group and the group with asthma, these polymorphisms are associated with differences in response to treatment with agonists in asthma. For example, the Gly16 mutant undergoes a higher agonist-promoted down-regulation compared to the wild type (Id.).

Polymorphism Patterns in the Endothelin Receptor Type A Gene The following table lists the set of polymorphism patterns found in the coding region of the endothelin receptor A type gene.

[0140]

[Table 7]

Isolated Polymorphic Nucleic Acids, Vectors, Probes and Primers and Vectors for Expression of Array Polymorphic Variants The present invention relates to ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, An isolated nucleic acid comprising the polymorphic positions described above for a human gene encoding an endothelin receptor and a β-adrenoceptor;
Vectors comprising the nucleic acids; and transformed host cells comprising the vectors. The present invention also provides probes useful for detecting these polymorphisms.

Nucleic acids encoding genes containing polymorphisms useful for determining an individual's cardiovascular status can be obtained by direct screening for nucleic acids having the polymorphism, or by screening polypeptides expressed by the nucleic acid. Regardless, it is particularly valuable for screening.

In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. Such techniques are well known and are described, for example, in Sambrook.
"Molecular Cloning: A Laboratory"
Manual, 2nd edition, 1989 (Cold Spring Harbor)
Laboratory Press, Cold Spring Harbor
, New York); "DNA Cloning: A Practica.
l Approach, "Volumes 1 and 2, 1985 (DN Glove)
r)) "Oligoncleotide Synthesis", 1984
Year (ML Gait Edition); “Nucleic Acid Hybridiz
nation ", 1985 (Hames and Higgins); Ausu
Bel et al., "Current Protocols in Molecular."
Biology ", 1997 (John Wiley and Sons)
And "Methods in Enzymology" Vols. 154 and 15;
Five volumes, each of Wu and Grossman and Wu, are fully described.

The insertion of a nucleic acid (typically a DNA) comprising a sequence of the present invention into a vector is performed by DN
This is easily achieved if both ends of A and the vector contain compatible restriction sites.
If this is not done, the ends of the DNA and / or vector may be modified by digesting single stranded DNA overhangs generated by restriction endonucleases to create blunt ends, or to achieve the same result. It may be necessary to modify the single-stranded ends by filling in the single-stranded ends with a unique DNA polymerase.

Alternatively, any desired site may be created, for example, by linking a nucleotide sequence (linker) to the termini. Such a linker may include a specific oligonucleotide sequence that defines the desired restriction site. Restriction sites can also be created by using the polymerase chain reaction (PCR). See, for example, Saiki et al., Science, 1988, 239: 48. Truncated vectors and DNA fragments may be modified, if necessary, by forming a homopolymer tail.

[0146] Nucleic acids may be isolated directly from cells or may be chemically synthesized using known methods. Alternatively, the nucleic acid of the invention can be produced using the polymerase chain reaction (PCR) method, using either chemically synthesized strands or genomic material as a template. Primers used for PCR can be synthesized using the sequence information provided herein and, if desired, to facilitate incorporation into a given vector for recombinant expression. Can be further designed to introduce appropriate new restriction sites.

The nucleic acids of the invention may be flanked by native gene sequences and may contain heterologous sequences including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5 ′ and 3 ′ non-coding regions, and the like. It may be linked to an array.

The present invention also provides nucleic acid vectors containing the disclosed genes or derivatives or fragments thereof. A number of vectors have been described for replication and / or expression in various eukaryotic and prokaryotic hosts, including plasmids and fungal vectors, and can be used for gene therapy and simple cloning or protein expression. Non-limiting examples of suitable vectors include, without limitation, the pUC plasmid, the pET plasmid (Novagen, Inc., Madison).
, WI) or pRSET or pREP (Invitrogen, San
(Diego, CA), and there are many suitable host cells using the methods disclosed or cited herein or other methods known to those of skill in the relevant art. The choice of a particular vector / host is not critical to the practice of the present invention.

Suitable host cells include electroporation, CaCl ₂ -mediated DNA uptake, calcium phosphate precipitation, fungal or viral infection, lipofection, microinjection, microprojectile, or other established methods. The cells can be transformed, transfected, and infected as appropriate by any suitable method, including: Suitable host cells include bacteria, archaebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Numerous transcription initiation and termination control regions have been isolated and have been shown to be effective in transcription and translation of heterologous proteins in various hosts. Examples of these regions, methods of isolation, modes of operation and the like are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced peptides and polypeptides from each of ACE, AGT, or AT1.

[0150] Nucleic acids encoding gene sequences from ACE, AGT or AT1 can also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell, thereby causing homologous recombination at that site of an endogenous gene, or a sequence having substantial identity to the gene. Other recombination-based methods, such as non-homologous recombination, or deletion of endogenous genes by homologous recombination can also be used.

Oligonucleotides The nucleic acids of the invention find use as probes for the detection of polymorphisms, as primers for the expression of polymorphisms, or in molecular library arrays for high-throughput screening. ing.

The probes according to the invention can have, without limitation, about 10 to 100 bp, preferably 15
~ 75 bp, and most preferably 17-25 bp in length, to one or more of the polymorphic sequences derived from the genes or sequences immediately adjacent to the polymorphic positions disclosed herein. , Hybridize at high stringency. Further, in some embodiments, the full-length gene sequence may be used as a probe. In one series of embodiments, the probes span the polymorphic positions in the genes disclosed above. In another series of embodiments, the probes correspond to sequences immediately adjacent to the polymorphic location.

[0153] Oligonucleotide nucleic acids can also be modified by a number of means known in the art. Non-limiting examples of such modifications include methylation, “caps”, replacement of one or more natural nucleotides with an analog, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters) Kind,
Intermediate nucleotide modifications using phosphoramidates, carbamates, etc., and using charged linkages (eg, phosphorothioates, phosphorodithioates, etc.). Nucleic acids include, for example, proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (eg, acridine, psoralen, etc.), chelating agents (eg, metals, radiometals, iron Oxidizing metals, etc.) and alkylating agents,
One or more additional covalent linking moieties may be included. PNAs are also included. Nucleic acids can be derivatized by formation of a methyl or ethyl phosphotriester or alkyl phosphoramidate linkage. In addition, the nucleic acid sequences of the present invention can be modified, directly or indirectly, with a label capable of providing a detectable signal. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.

PCR amplification of a gene segment containing a polymorphism provides a powerful tool for detecting the polymorphism. The oligonucleotide of the present invention can also be used as a PCR primer for amplifying a gene segment containing the target polymorphism. The amplified segments can be evaluated for the presence or absence of the polymorphism by restriction endonuclease activity, SSCP, or by direct sequencing. In another embodiment, the primer is specific for a polymorphic sequence on the gene. If a polymorphism is present, the primers can hybridize and DNA is generated by PCR. However, if the polymorphism is not present, the primer will not hybridize and no DNA will be produced. Thus, the presence or absence of a polymorphism can be directly assessed using PCR.

Oligonucleotides (including oligonucleotides with modifications as described above)
The molecular library array of ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor gene, preferably in combination with other genes, Or another powerful tool for rapidly assessing whether two or more polymorphisms are present. Molecular library arrays are disclosed in U.S. Patent Nos. 5,677,195, 5,599,695, 5,545,531, and 5,510,270.

Polymorphic Polypeptides and Polymorph-Specific Antibodies The present invention relates to ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-containing polymorphic positions disclosed above. -Isolated peptides and polypeptides encoded by all or part of a gene encoding a polypeptide selected from the group consisting of adrenoceptors. In one preferred embodiment, peptides and polypeptides are useful screening targets for identifying cardiovascular drugs. In another preferred embodiment, the peptides and polypeptides react specifically with the polypeptide comprising the polymorphic position to distinguish an antibody that distinguishes it from other polypeptides having a different amino acid sequence at that position into a suitable host. It can be induced in animals.

The polypeptides of the present invention are preferably at least 5 residues or more in length, preferably at least 15 residues. The method for obtaining these polypeptides is described below. Many conventional techniques in protein biochemistry and immunology are used. Such techniques are well known and are described in "Immunochemical Methods".
in Cell and Molecular Biology ", 1987 (edited by Mayer and Waller; Academic Press, L.
ondon); Scopes, "Protein Purification"
: Principles and Practice, "Second Edition, 1987 (
Springer-Verlag, N.W. Y. ) And "Handbook of
Experimental Immunology, Vol. I-IV, 1986 (edited by Weir and Blackwell).

[0158] Nucleic acids comprising a protein coding sequence are useful for recombinant expression of polypeptides derived from ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor in intact cells. Alternatively, it can be used to direct in a cell-free translation system.
The known genetic code can be adapted for more efficient expression in a given host organism, if desired, and used to synthesize an oligonucleotide encoding the desired amino acid sequence. Polypeptides may be isolated from human cells, or may contain heterologous organisms or cells (including bacteria, fungi, insects, plants and mammalian cells, where the appropriate protein coding sequence has been introduced and expressed). But not limited thereto). Further, the polypeptide may be part of a recombinant fusion protein.

Peptides and polypeptides may be chemically synthesized by commercially available automated procedures, including, but not limited to, exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation or classical solution synthesis. Can be synthesized. The polypeptide is preferably Merrifield, J. et al. Am. Chem. Soc. , 1963
85: 2149, prepared by solid phase peptide synthesis.

Methods for polypeptide purification are well known in the art and include disc gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography, and chromatography. Including but not limited to flow distribution. For some purposes, it is preferred that the polypeptide be produced in a recombinant system, in which case the protein may comprise additional sequences, such as, but not limited to, polyhistidine sequences, which facilitate purification. Including tags. The polypeptide can then be purified from the crude lysate of the host cells by chromatography on a suitable solid phase matrix. Alternatively, an antibody produced against ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor, or a peptide derived therefrom is used as a purification reagent. Can be used. Other purification methods are possible.

The present invention also includes derivatives and homologs of those polypeptides. For some purposes, the nucleic acid sequence encoding the peptide may be altered by substitution, addition or deletion providing a functionally equivalent molecule, ie, a function-conservative variant.
For example, one or more amino acid residues in the sequence may be replaced with, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (asparagine and glutamine); polar neutral amino acids; Can be replaced by another amino acid of similar properties.

The isolated polypeptide may be modified, for example, by phosphorylation, sulfation, acylation, or other protein modifications. The polypeptides may be directly or indirectly modified with a label that provides a detectable signal, including but not limited to radioisotopes and fluorescent compounds.

The present invention also includes antibodies that specifically recognize a polymorphic position of the invention and distinguish peptides or polypeptides containing a particular polymorphism from those that contain different sequences at that position. Such polymorphic site-specific antibodies of the present invention include polyclonal and monoclonal antibodies. Antibodies may be induced in an animal host by immunizing with an immunogenic component derived from ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor. Or by in vitro immunization of immune cells.
The immunogenic components used to elicit the antibodies may be isolated from human cells or may be produced in recombinant systems. Antibodies can also be produced in recombinant systems, programmed using the appropriate antibody-encoding DNA. Alternatively, antibodies may be constructed by biochemical rearrangement of purified heavy and light chains. Antibodies include hybrid antibodies (ie, comprising two sets of heavy / light chain combinations, each of which recognizes a different antigen), chimeric antibodies (ie,
Heavy or light chains, or both, are fusion proteins) and monovalent (Univalent) antibodies (ie, consisting of a heavy / light chain complex attached to the constant region of a second heavy chain). . Also included are Fab fragments, including Fab ′ fragments and F (ab) ₂ fragments of antibodies. Methods for the production of all of the above types of antibodies and derivatives are well known in the art and are detailed below. For example, techniques for producing and processing polyclonal antisera are described in Maye
r and Walker, "Immunochemical Methods in
Cell and Molecular Biology ", 1987 (A
Cademic Press, London). The general methodology for making monoclonal antibodies by hybridomas is well known. Immortalized antibody-producing cell lines can be created by cell fusion, or by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. Can be created. For example, Schreier et al., "Hybridoma Technologies".
", 1980; U.S. Patent Nos. 4,341,761; 4,399,121;
No. 4,427,783; No. 4,444,887; No. 4,466,917;
Nos. 4,472,500; 4,491,632; and 4,493,890.
Please refer to the issue. Panels of monoclonal antibodies produced against epitopes derived from ACE, AGT or AT1 can be screened for various properties, ie, isotype, epitope affinity, and the like.

The antibodies of the present invention can be purified by standard methods, including preparative disk gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography. , And countercurrent distribution. An antibody purification method is described in, for example, “The Art of Antibody.
Purification ", 1989 (Amicon Division,
W. R. Grace & Co. ). A general protein purification method is described in “Protein Purification: Principles”.
and Practice, "R. K. Scopes, 1987 (Sp
Ringer-Verlag, New York, NY).

[0165] Methods for determining the immunogenic potential of the disclosed sequences and the characteristics of the resulting sequence-specific antibodies and immune cells are well known in the art. For example, an antibody elicited in response to a peptide containing a particular polymorphic sequence will have its ability to specifically recognize that polymorphic sequence, i.e., will bind differently to a peptide or polypeptide containing that polymorphic sequence. , Whereby it can be tested for its ability to distinguish it from similar peptides or polypeptides containing different sequences at the same position.

Diagnostic Methods and Kits The present invention relates to the determination of an individual's ACE in combination with the determination of the sequence of a polymorphic position in another gene.
, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II
A kit is provided for determining the sequence of a polymorphic position in a receptor, endothelin receptor or β-adrenoceptor gene. The kit includes a means for determining the sequence of the polymorphic position, and may optionally include data for analysis of the polymorphic pattern. Means for sequencing can include suitable nucleic acid-based reagents and immunological reagents (see below). Preferably, the kit comprises
Also includes suitable buffers, control reagents where appropriate, and instructions for determining the sequence of the polymorphic position. The kit can also include data on the correlation between a particular polymorphism pattern and a desired treatment regimen or other indicator.

Diagnostic Methods and Kits Based on Nucleic Acids The present invention provides nucleic acid-based methods for the detection of polymorphic patterns in biological samples. The sequence of a particular polymorphic position in a gene can be determined using any suitable means known in the art, including but not limited to hybridization with a polymorphism-specific probe and direct sequencing. You.

The present invention also provides suitable kits for nucleic acid-based diagnostic applications. In one embodiment, the diagnostic kit comprises the following components: (i) Probe DNA: The probe DNA can be pre-labeled. Alternatively, the probe DNA may be unlabeled, and the components for labeling may be included in the kit in a separate container; and (ii) a hybridization reagent: the kit may be adapted to a particular hybridization protocol. It may also contain other suitably packaged reagents and materials as required, including solid phase matrices and, where applicable, standards.

In another embodiment, the diagnostic kit comprises: (i) sequencing primers: the sequencing primers may be pre-labeled and may include affinity purification or attachment moieties. And (ii) sequencing reagents: the kit may also include other suitably packaged reagents and materials required for the particular sequencing protocol. In one preferred embodiment, the kit comprises a panel of sequencing primers whose sequences correspond to sequences flanking the polymorphic position.

Antibody-Based Diagnostic Methods and Kits The present invention also provides antibody-based methods for detecting polymorphic patterns in biological samples. The method comprises the steps of (i) contacting a sample with one or more antibody preparations, wherein each of the antibody preparations comprises a stable antigen-antibody complex between the antibody and an antigenic component in the sample. Specific to a particular polymorphic form of the gene under conditions that can form between it) and step (ii) formed in step (i) using any suitable means known in the art. Detecting the antigen-antibody complex (where detection of the complex indicates that the particular polymorphic form is present in the sample).

[0171] Typically, immunoassays involve labeled antibodies or labeled antigenic components (
For example, a component that competes with the antigen for binding to the antibody in the sample is used. Suitable labels include, but are not limited to, enzyme-based, fluorescent, chemiluminescent, radioactive or dye molecules. For example, assays utilizing biotin and avidin and enzyme-labeled immunoassays such as ELISA assays,
Assays that amplify the signal from the probe are also known.

The present invention also provides kits suitable for use in antibody-based diagnostics. Diagnostic kits typically include one or more of the following components: (i) Polymorph-specific antibodies: The antibodies may be pre-labeled. Alternatively, the antibody may be unlabeled, the component for labeling may be included in a separate container in the kit, or a second labeled antibody is provided; and (ii) Reaction Components: The kit may also include other suitable packaged reagents and materials required for the particular immunoassay protocol, including solid matrix (if applicable) and standards.

The kits referred to above may include instructions for performing the test. Further, in a preferred embodiment, the diagnostic kit is adaptable for high-throughput and / or automated operation.

Pharmaceutical Targets and Screening Methods According to the present invention, they are derived from genes encoding polymorphic forms of ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor. A nucleotide sequence, and
Peptide sequences derived from the polymorphic form are cardiovascular drugs, ie, one of cardiovascular diseases.
It is a useful target for identifying compounds that are effective in treating one or more clinical conditions. The pharmaceutical target is ACE, AT1, AGT, renin, aldosterone synthase, an isolated nucleic acid derived from a gene encoding type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor; and (ii) ACE, AT1
Isolated peptides and polypeptides derived from, but not limited to, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor polypeptide;
Each of these contains one or more polymorphic positions.

In Vitro Screening Methods In one series of embodiments, an isolated nucleic acid containing one or more polymorphic positions is tested in vitro for its ability to bind a test compound in a sequence-specific manner. The method comprises the steps of (i) a first nucleic acid containing a specific sequence at a polymorphic position, and a second nucleic acid, wherein the sequence is different from the first at a different position at the same polymorphic position. Providing a nucleic acid that is the same as the nucleic acid; (ii) contacting the nucleic acid (s) with a plurality of test compounds under conditions suitable for binding; and (iii) a first nucleic acid sequence or a second nucleic acid. Identifying compounds that selectively bind to the sequence.

[0176] As used herein, selective binding refers to any measurable difference in any binding parameter, such as, for example, binding affinity, binding capacity, and the like.

[0177] In another series of embodiments, an isolated peptide or polypeptide comprising one or more polymorphic positions is tested in vitro for its ability to bind a test compound in a sequence-specific manner. The screening method comprises: (i) a first peptide or polypeptide containing a specific sequence at a polymorphic position, and a second peptide or polypeptide, the sequences of which are different at the same polymorphic position. Providing a peptide or polypeptide that is the same as the first peptide or polypeptide except that (ii) contacting the polypeptide (s) with a plurality of test compounds under conditions suitable for binding; And (iii) identifying a compound that selectively binds to one of the nucleic acid sequences.

In a preferred embodiment, a high-throughput screening protocol is used to examine a large number of test compounds for their ability to bind to the gene or peptide in a sequence-specific manner.

Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random or directed synthesis of compounds based on sugars, peptides and nucleic acids. The synthetic compound library is available from Mayb
ride Chemical Co. (Trevillet, Cornwal
1, UK), Comgenex (Princeton, NJ), Brandon
Associates (Merrimack, NH) and Microsoft
Commercially available from ce (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI).
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available, for example, from Pan Laboratories (Bothell, WA) or M
It is available from ycoSearch (NC) or can be easily made. In addition, natural and synthetic libraries and compounds are readily modified through conventional chemical, physical and biochemical means.

In Vivo Screening Method Intact cells expressing polymorphic variants of the gene encoding ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor Whole animals can be used in screening methods to identify candidates for cardiovascular drugs.

[0181] In one series of embodiments, permanent cell lines are established from individuals expressing a particular polymorphic pattern. Alternatively, cells (including but not limited to mammalian, insect, yeast or bacterial cells) are programmed to express a gene comprising one or more polymorphic sequences by introduction of appropriate DNA. Identification of candidate compounds
(i) an assay that measures the selective binding of a test compound to a particular polymorphic variant of the gene; (ii) a test compound that alters (ie, inhibits or enhances) a measurable activity or function of the gene. And (iii) an assay that measures the ability of a compound to alter (ie, inhibit or enhance) the transcriptional activity of a sequence derived from the promoter (ie, regulatory) region of a gene. Can be achieved using a suitable assay, including but not limited to

In another series of embodiments, transgenic animals are generated wherein (i) human ACE, AT1, AGT, having different sequences at specific polymorphic positions,
The renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor or β-adrenoceptor gene is stably inserted into the genome of the transgenic animal; and / or (ii) the endogenous gene is inactivated. And is replaced by a human gene having a different sequence at a particular polymorphic position. See, for example, Coffman, Semin. Nephrol. , 1997, 17
: 404; Esther et al. , Lab. Invest. , 1
996, 74: 953; Murakami et al. , Blood P
ress. Suppl. , 1996, 2:36. Such animals can be treated with the candidate compound and monitored for one or more clinical markers of cardiovascular status.

In addition, populations that are not amenable to treatment that have been established due to cardiovascular disease or failure,
Can be selected for testing alternative treatments. In addition, treatments that are not effective in the general population but are very effective in the selected population can be identified, as would otherwise have been missed. This is a particularly powerful advantage of the present invention as it eliminates some of the randomness associated with clinical trials.

The following are intended to be non-limiting examples of the present invention.

[0185]

【Example】

Example 1: Polymorphisms in human genes encoding ACE, AGT and AT1
The following studies were performed to identify polymorphic residues in the genes encoding human ACE, AGT and AT1.

DNA samples were obtained from 277 individuals. The individuals were Caucasian men born between 1920 and 1924 in Uppsala, Sweden. Individuals were selected for the test population based on their medical history. That is,
Are they (i) healthy without signs of cardiovascular disease (100);
Or (ii) acute myocardial infarction (68), asymptomatic myocardial infarction (34), stroke (18),
Either had seizures and acute myocardial infarction (19), or one of hypertension at the age of 50 (39). DNA samples were obtained from each individual.

DNA sequence analysis was performed by (i) amplifying short fragments of each of the ACE, AGT and AT1 genes using the polymerase chain reaction (PCR) and (ii) sequencing the amplified fragments. Was. Sequences obtained from each individual were then compared to known ACE, AGT and AT1 gene sequences (see Table 1).
.

(I) Amplification: In the PCR reaction, the primers shown in Table 2 below were used.

[0189]

[Table 8]

[0190]

[Table 9]

[0191]

[Table 10]

[0192]

[Table 11]

[0193]

[Table 12]

[0194]

[Table 13]

[0195]

[Table 14]

As indicated, primers were modified in one of the following ways: (i)
A biotin molecule was conjugated to the 5 'end of the indicated sequence (B); (ii) a sequence of nucleotides from M13, 5'-CAGGAAACAGCTATGACT-3'
(SEQ ID NO: 120) was added to the 5 ′ end of the indicated sequence (MT); or (iii) the sequence 5′-AGTCACGACGTTGTTAAAACGACGGCCA
GT-3 '(SEQ ID NO: 121) was added to the 5' end of the indicated sequence (T = tail). As shown in Table 1, according to the GenBank sequence listed in Table 1,
The nucleotides were numbered. If the sequence involved was not publicly available, numbering was performed as in the following example. The designation "i-4: 1-200" means that the primer sequence is located within a sequence that includes the nucleotide immediately upstream of the first coding nucleotide of exon 4 and extends 200 base pairs upstream of this nucleotide. To indicate that Similarly, the designation "i + 4: 1-200" means that the primer sequence is located within a sequence extending 200 base pairs downstream from the nucleotide immediately downstream of the last coding nucleotide of exon 4. To indicate that In each case, the specific position of the primer sequence is shown in Table 2 in the column labeled "nucleotide".

In the following, the reaction components used for the PCR are described in Table 3,
The reaction conditions for CR are described in Table 4.

[0198]

[Table 15]

[0199]

[Table 16]

Some differences are shown in the “change” column in Table 5 below.

The amplified fragments are listed in Table 5 below, in relation to the primers used for amplification and the PCR reaction conditions.

[0202]

[Table 17]

[0203]

[Table 18]

[0204]

[Table 19]

[0205]

[Table 20]

All of the PCR products (ACED1, AT1-spec.1 and AT1-spec)
c. 2 fragments) were subjected to solid phase sequencing according to a protocol commercially available from Pharmacia Biotech. The sequencing reaction is performed using a sequencing primer having a sequence complementary to the "tail" sequence already described in Table 2. The nucleotide sequence of the sequencing primer was 5'-CGACGTTGTAAAACGACGGCCAGT-
3 '(SEQ ID NO: 122) and the primer was fluorescently labeled with a Cy-5 molecule at the 5'-nucleotide. The location with the genetic mutation was found in ALFexpress ™ commercially available from Pharmacia Biotech.
The system was used to identify by nucleotide sequence determination.

The detection of the ACED1 fragment was performed by analyzing the size of the amplified fragment by gel electrophoresis. Here, the presence of the shorter PCR product (192 base pairs) indicates the D-allele and the longer PCR product (4
79 base pairs) indicated the I-allele. The presence of both bands indicated a heterozygote of the two alleles. Detection of the allele-specific reaction at position AT1-1271 was performed by performing two separate parallel PCR reactions on the same sample and comparing the sizes of the amplified fragments. A 501 base pair PCR product should always be present as a control in both parallel reactions, while A
T1-spec. The presence of a 378 bp PCR product in the reaction designated 1 indicated the presence of A at this position. AT1-spec. The presence of a 378 bp PCR product in the reaction designated 2 indicated the presence of a C at this position. If a shorter PCR product was present in both reactions, the individual is heterozygous for A and C.

Results The above analysis resulted in the identification of polymorphic positions within the regulatory and coding / intron segments of the human genes encoding ACE, AGT and AT1. The polymorphic positions, the mutant nucleotides found at each of those positions, and the PCR fragment where the polymorphism was identified are shown in Table 6 below. Also shown is the frequency of each genotype in the population of 90 individuals, expressed as a percentage of the study population having that genotype. Polymorphisms that resulted in alternative amino acids in ACE, AGT or AT1 have also been shown. As used herein below, the designations “AGR”, “ACR” and “ATR” refer to human AG
T, ACE and the regulatory region of the AT1 gene, and refer to "AGT", "ACE"
The designations "" and "AT1" refer to the coding regions of the AGT, ACE and AT1 genes.

[0209]

[Table 21]

[0210]

[Table 22]

[0211]

[Table 23]

[0212]

[Table 24]

A subset of these polymorphic positions was further analyzed in additional 187 individuals. Table 7, as described in Example 1 above, shows the polymorphic positions, the sequences of these positions, and the genotype frequencies for each position in the 277 population.

[0214]

[Table 25]

Example 2: Correlation of Polymorphism Pattern with Cardiovascular Disease Polymorphic locations identified as in Example 1 were associated with the following markers of cardiovascular status present in the study population: Myocardial infarction (MI) (120 individuals); stroke (37 individuals); and hypertension (BP)
) (39 individuals). Polymorphism patterns that show a statistically significant correlation with one or more of these markers, ie, combinations of sequences at specific polymorphic positions, are shown below.

[0216]

[Table 26]

[0219]

[Table 27]

[0218]

[Table 28]

[0219]

[Table 29]

[0220]

[Table 30]

[0221]

[Table 31]

[0222]

[Table 32]

[0223]

[Table 33]

[0224]

[Table 34]

Example 3: Correlation between specific polymorphism patterns and treatment response The following study predicts the efficacy of treatment of cardiovascular disease, human ACE, AGT
And / or to define polymorphism patterns in the AT1 gene.

Two groups of hypertensive patients were studied, the first group having 41 individuals and the second group having 20 individuals. The groups were analyzed independently and in combination.

Patients in this population were each treated with the following five ACE inhibitors: Captopr
il, Trandolapril, Lisinopril, Fosinopri
1, treated with one of Enalapril. The effect of the drug on the mean arterial blood pressure was quantified. Mean arterial blood pressure was defined as 2/3 of diastolic blood pressure + 1/3 of systolic blood pressure. Individuals are also “high responders”, ie, individuals who show a reduction of more than 16 mmHg during treatment with an ACE inhibitor, and “low responders”, ie, 16 mmHg.
Individuals that did not show a decrease of more than g were classified.

One particular polymorphism pattern, ACE, that was present in 51% of the first study population
2193 A / G, AGR 1072 G / G, AT1 1167 A / A were distinguished between high responders and low responders. In a second group of 20 patients, the pattern was less pronounced (25%). However, the correlation with reduced blood pressure was still statistically significant. Individuals with this polymorphic pattern (hereinafter designated "1") experience greater reductions in blood pressure than individuals lacking this polymorphic pattern (hereinafter designated "0"). did.

[0229]

[Table 35]

Further, the distribution of high responders and low responders (as defined above) was as follows.

[0231]

[Table 36]

Taken together, the results from the two groups show that the presence of this polymorphic pattern correlates with an increased reduction of 6.4-7.3 mmHg compared to individuals without this polymorphic pattern Is shown.

[0233] The incidence of this polymorphic pattern was 41% in this hypertensive population. This means that by testing this polymorphic pattern in hypertensive patients and then prescribing an ACE inhibitor only to hypertensive patients with this polymorphic pattern,
It suggests that it can be increased from 43% (in the general hypertensive population) to 76% in the hypertensive population selected according to the method of the present invention.

Even if one polymorphism is missing from the pattern, a high degree of analysis of the variables and hence the value of the prediction will be lost.

Example 4 Specific Polymorphism Patterns and Processing Responses or Dispositions for Cardiovascular Syndrome
In accordance with the methods disclosed in the above examples, additional correlations between polymorphism patterns and responsiveness to ACE inhibitors, unresponsiveness to ACE inhibitors, predisposition to myocardial infarction, or predisposition to stroke were derived. Was. These correlations are listed in the following table.

[0236]

[Table 37]

[0237]

[Table 38]

[0238]

[Table 39]

The polymorphism patterns that show a statistically significant correlation with myocardial infarction and stroke are shown below, that is, combinations of sequences at specific polymorphic locations.

[0240]

[Table 40]

[0241]

[Table 41]

The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

In addition, all base sizes or amino acid sizes, and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate and are provided for explanation. It is understood that there is.

Various patents, patent applications, method studies and publications are cited herein, the disclosures of which are incorporated by reference in their entirety.

Example 5: Between certain polymorphism patterns and hypertension treatment with β-blockers
Using the techniques described in Correlation Examples 1 and 2, we determined that ADBP1 and ADBP1
Polymorphisms in the BP2 gene (regulatory / promoter region and coding region) were investigated. The source material was as described above.

Initially, we identified a set of key polymorphisms. Some of these are known and others are new. They are summarized in Table 14.

[0247]

[Table 42]

Abbreviations used in gene naming: Putative promoter region of gene encoding B1P (ADBR1) -β-adrenergic receptor 1 Protein coding of gene encoding B1R (ADBR1) -β-adrenergic receptor 1 Region Putative promoter region of gene encoding B2P (ADBR2) -β-adrenergic receptor 2 Protein coding region of gene encoding B2R (ADBR2) -β-adrenergic receptor 2 One or more of these positions Can be used to predict a good response to anti-hypertensive treatment with β-blockers.

Indeed, we have identified various polymorphism / polymorphism patterns that can be used to predict high response to anti-hypertensive treatment with β-blockers. These are shown in Table 15.

[0250]

[Table 43]

Not only positions in the β-adrenergic receptor but also positions in the renin-angiotensin-aldosterone system can be used to predict response to anti-hypertensive treatment by β-blockers (see Table 16). . The numbering is given according to the GenBank numbering as defined in Table 1 above.

[0252]

[Table 44]

The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further understood that all sizes, and all molecular weights or mass values, are approximate and are provided for description.

The patents, patent applications, methods and publications cited throughout this application are hereby incorporated by reference in their entirety.

[Sequence list]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Lindstrom, Pair Harry Ratga, Sweden, S-756-33-Apsala, Svankarasbergen 26Ai (72) Inventor Johnson, Lina Sweden, S-756-42-Apsala, Gustav Kuslbergsberg 20 F term (reference) 4B024 AA01 AA11 BA14 BA17 BA63 CA09 HA14 HA19 4B063 QA01 QA08 QA19 QQ12 QQ36 QQ43 QR32 QR55 QR62 QS16 QS25 QS33 QS34

Claims (16)

[Claims] 1. A method for assessing cardiovascular status in an individual to be tested, comprising: (a) renin, aldosterone synthase, type 2 angiotensin II in the individual.
Testing a polymorphic pattern comprising at least one polymorphic position in a gene encoding a polypeptide selected from the group consisting of a receptor, an endothelin receptor and a β-adrenoceptor; Comparing to a reference polymorphism pattern from the indicated population of individuals, and determining whether the individual has that cardiovascular condition based on whether the test pattern matches the reference pattern. A method that includes 2. The method of claim 1, wherein the polymorphic pattern of (a) comprises a polymorphic position in a second gene encoding a polypeptide selected from the group consisting of ACE, AT1, and AGT. 3. The method of claim 1, wherein the predetermined cardiovascular status is predisposed to cardiovascular syndrome. 4. The method of claim 3, wherein the cardiovascular syndrome is selected from the group consisting of myocardial infarction, unstable angina, hypertension, atherosclerosis and stroke. 5. The method according to claim 3, wherein the reference pattern comprises at least two polymorphisms. 6. The method of claim 5, wherein the reference pattern includes at least three polymorphisms. 7. The method of claim 1 or 2, wherein the predetermined cardiovascular status is a response to a cardiovascular treatment regimen. 8. A treatment regimen comprising an ACE inhibitor, an angiotensin II receptor antagonist, a diuretic, an α-adreno receptor antagonist, cardiac glycoside, a phosphodiesterase inhibitor, a β-adreno receptor antagonist, a calcium channel Blockers, HMG-CoA reductase inhibitors, imidizolin receptor blockers,
10. The method of claim 7, comprising administering a cardiovascular drug selected from the group consisting of an endothelin receptor blocker and an organic nitrite. 9. A transposition of cytosine to thymine in exon 10 wherein the polymorphic position of the gene results in a premature stop codon at position 387 resulting in a truncated form of renin with a deletion of 20 amino acids from the carboxy terminus. A position in the promoter region of the aldosterone synthase, numbered -344 (assuming the start codon starts at 1); 2
238, 2440, 2493, 2502, 2577, 2585, 2693, 27
Β-adrenergic receptors numbered 24 and 2775
Position in the regulatory region of one gene (designated B1P); 231, 758, 1
Positions in the β-adrenergic receptor-1 coding region (designated B1R), numbered 037, 1251, 1403 and 1528; 932, 934, 987, 1006, 1120, 1221, 1541 and 1
Positions in the β-adrenergic receptor-2 gene regulatory region (designated B2P), numbered 568; 839, 872, 1045, 12
Positions in the β-adrenergic receptor-2 gene coding region (designated B2R), numbered 84, 1316, 1846, 1891, 2032, 2068 and 2070; 969, 1005, 1146 and 24
Endothelin receptor type A coding region numbered 85 (E
T _A and named by being) located in; and is selected from the group consisting of any combination thereof, The method of claim 1, wherein. 10. The polymorphic position of the second gene is 5106, 5349 and 54.
Positions in the ACE regulatory region numbered 96; 375, 582, 731
, 1060, 1215, 2193, 2328, 2741, 3132, 3387,
Positions in the ACE coding regions numbered 3503 and 3906; 1451 positions in the ACE gene numbered accession X62855 in GenBank; 395, 412, 432, 449, 692, 839, 10
Positions in the AGT regulatory regions numbered 07, 1072, 1204 and 1218; positions in the AGT coding regions numbered 273, 620, 803, 912, 997, 1116 and 1174; registration M24688 in GenBank Position 49 in the AGT gene, numbered 142;
Positions in the AT1 regulatory region, numbered 7, 1756, 1853, 2046, 2354, 2355 and 2415; and 449, 678, 1167 and 1
The method of claim 2, wherein the method is selected from the group consisting of positions in the AT1 coding region numbered 271. 11. An isolated nucleic acid encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor in an individual,
An isolated nucleic acid wherein the nucleic acid comprises a polymorphic position, wherein the polymorphic position is a position in a polymorphic pattern from a population of individuals exhibiting a predetermined cardiovascular status. 12. A probe that hybridizes with high stringency to the polymorphic position defined in claim 11. 13. A nucleic acid library containing a nucleic acid, wherein (a) encodes a polypeptide selected from the group consisting of renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adrenoceptor. A polymorphic position of the gene, wherein the polymorphic position is a position in a polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status, and (b) an individual's cardiovascular A nucleic acid comprising another polymorphic position of a human gene that is an indicator of status, wherein the human gene is ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin II receptor, endothelin receptor and β-adreno. A nucleic acid library comprising a nucleic acid selected from the group consisting of sceptors. 14. The library according to claim 13, wherein the nucleic acid is an oligonucleotide. 15. A kit for assessing cardiovascular status, comprising: (a) a sequencing primer, and (b) a sequencing reagent, wherein the primer comprises: (i) renin, aldosterone synthase, type 2 Polymorphic positions in a gene encoding a polypeptide selected from the group consisting of angiotensin II receptor, endothelin receptor and β-adrenoceptor; and (ii) ACE, AT1, AGT, renin, aldosterone synthase, type 2 angiotensin Selected from the group consisting of primers that hybridize at or immediately adjacent to the second polymorphic position in the gene encoding a polypeptide selected from the group consisting of II receptor, endothelin receptor and β-adrenoceptor Done, kit. 16. The kit of claim 15, wherein the second polymorphic position is 5
Positions in the ACE regulatory region numbered 106, 5349 and 5496; 375, 582, 731, 1060, 1215, 2193, 2328, 274
ACEs numbered 1, 3, 132, 3387, 3503 and 3906
Position in the coding region; position 1451 in the ACE gene numbered accession X62855 in GenBank; 395, 412, 432, 44
Positions in the AGT regulatory region numbered 9, 692, 839, 1007, 1072, 1204 and 1218; 273, 620, 803, 912, 997
Positions in the AGT coding region numbered 1116 and 1174; 49 positions in the AGT gene numbered M24688 in GenBank; 1427, 1756, 1853, 2046, 2354, 23
Positions in the AT1 regulatory region, numbered 55 and 2415;
Positions in the AT1 coding region numbered 78, 1167 and 1271; in exon 10 resulting in a premature stop codon at position 387 resulting in a truncated form of renin with a deletion of 20 amino acids from the carboxy terminus. Positions in the renin coding region, including the transfer of cytosine to thymine;
Positions in the promoter region of aldosterone synthase numbered -344 (assuming the start codon starts at 1); 2238, 2440, 2493, 2
Positions in the regulatory region of the β-adrenergic receptor-1 gene (designated B1P), numbered 502, 2577, 2585, 2693, 2724 and 2557; 231, 758, 1037, 1251, 1403 And positions in the β-adrenergic receptor-1 gene coding region (designated B1R), numbered 932, 934, 98
Positions in the β-adrenergic receptor-2 regulatory region (designated B2P), numbered 7, 1006, 1120, 1221, 1541 and 1568; 839, 872, 1045, 1284, 1316, 1846 , 18
Positions in the β-adrenergic receptor-2 gene coding region (designated B2R), numbered 91, 2032, 2068 and 2070; endothelin numbered 969, 1005, 1146 and 2485 receptor type a coding region (which is named ET _a) position in; and is selected from the group consisting of any combination thereof, kits.