EP1121462A2 - Genes for assessing cardiovascular status and compositions for use thereof - Google Patents

Genes for assessing cardiovascular status and compositions for use thereof

Info

Publication number
EP1121462A2
EP1121462A2 EP99947799A EP99947799A EP1121462A2 EP 1121462 A2 EP1121462 A2 EP 1121462A2 EP 99947799 A EP99947799 A EP 99947799A EP 99947799 A EP99947799 A EP 99947799A EP 1121462 A2 EP1121462 A2 EP 1121462A2
Authority
EP
European Patent Office
Prior art keywords
polymoφhic
positions
numbered
receptor
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99947799A
Other languages
German (de)
French (fr)
Inventor
Leif Torbjorn Norberg
Maria Kristina Andersson
Per Harry Rutger Lindstrom
Lena Jonsson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MEADOWLAND BUSINESS PARTNERS AB
Original Assignee
Eurona Medical AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eurona Medical AB filed Critical Eurona Medical AB
Publication of EP1121462A2 publication Critical patent/EP1121462A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0079—Steroid 11 beta monooxygenase (P-450 protein)(1.14.15.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C12N9/6486—Renin (3.4.23.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23015—Renin (3.4.23.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to genetic polymorphisms and polymorphism patterns useful for assessing cardiovascular status in humans. More particularly, the invention relates to identifying and using polymo ⁇ hism patterns comprising a polymo ⁇ hism in the a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor to predict a treatment outcome or likelihood of developing cardiovascular disease, and to assist in diagnosis and in prescription of an effective therapeutic regimen.
  • RAAS renin-angiotensin-aldosterone system
  • RAAS plays an important role in cardiovascular physiology in mammals. Specifically, RAAS regulates salt-water homeostasis and the maintenance of vascular tone. Stimulation or inhibition of this system raises or lowers blood pressure, respectively, and disturbances in this system may be involved in the etiology of, for example, hypertension, stroke, and myocardial infarction.
  • the RAAS system may also have other functions such as, e.g., control of cell growth.
  • the renin-angiotensin system includes renin, angiotensin converting enzyme (ACE), angiotensinogen (AGT), type 1 angiotensin II receptor (ATI), type 2 angiotensin II receptor (AT2) and aldosterone synthase.
  • ACE angiotensin converting enzyme
  • AGT angiotensinogen
  • ATI type 1 angiotensin II receptor
  • AT2 type 2 angiotensin II receptor
  • aldosterone synthase aldosterone synthase
  • genes express polypeptides involved in pathways other than RAAS that also play a role in the regulation of cardiovascular physiology, although it is believed that prior to the present invention, no known association of polymo ⁇ hism patterns in these genes with cardiovascular status has been observed or reported.
  • these other regulators of cardiovascular physiology are endothelin, endothelin receptor, and ⁇ -adrenergic receptors 1 and 2.
  • AGT is the specific substrate of renin, an aspartyl protease.
  • the human AGT gene contains five exons and four introns which span 13Kb (Gaillard et al, DNA, 1989, 8:87-99; Fukamizu et al. J. Biol. Chem., 1990, 265:7576-7582).
  • the first exon (37 bp) codes for the 5' untranslated region of the mRNA.
  • the second exon codes for the signal peptide and the first 252 amino acids of the mature protein.
  • Exons 3 and 4 are shorter and code for 90 and 48 amino acids, respectively.
  • Exon 5 contains a short coding sequence (62 amino acids) and the 3'-untranslated region.
  • Plasma AGT is synthesized primarily in the liver and its expression is positively regulated by estrogens, glucocorticoids, thyroid hormones, and angiotensin II (Ang II) (Clauser et al, Am. J. Hypertension, 1989, 2:403-410). Cleavage of the amino- terminal segment of AGT by renin releases a decapeptide prohormone, angiotensin-I, which is further processed to the active octapeptide angiotensin II by the dipeptidyl carboxypeptidase designated angiotensin-converting enzyme (ACE). Cleavage of AGT by renin is the rate-limiting step in the activation of the renin-angiotensin system.
  • ACE angiotensin-converting enzyme
  • the human ACE gene is also a candidate as a marker for hypertension and myocardial infarction.
  • ACE inhibitors constitute an important and effective therapeutic approach in the control of human hypertension (Sassaho et al, Am. J. Med., 1987, 83:227- 235).
  • ACE converts the inactive angiotensin I molecule (Ang I) into active angiotensin II (Ang II) (Bottari et al, Front. Neuroendocrinology, 1993, 14: 123-171).
  • ACE substrate is bradykinin, a potent vasodilator and inhibitor of smooth muscle cell proliferation, which is inactivated by ACE (Ehlers et al, Biochemistry, 1989, 28:531 1-5318; Erdos, E.G., Hypertension, 1990, 16:363-370; Johnston, C.I. Drugs (suppl. 1), 1990, 39:21-31).
  • vasoconstrictive, cell growth-promoting and salt conserving actions of angiotensin II are mediated through binding to and activation of angiotensin receptors, of which at least two types have been cloned (ATI and AT2).
  • angiotensin receptors of which at least two types have been cloned (ATI and AT2).
  • ATI type 1 Ang II receptor
  • AT2 AT2
  • ATI Ang II receptor
  • Endothelin is a potent vasoconstrictive peptide characterized by long lasting action. It was first discovered as a vasoconstricting factor in conditioned medium (Hickey et al, Am. J. Physiol., 1985, 248:C550), and subsequently purified and characterized (Yanagisawa et al, Nature, 1988, 332:411). ET is produced as preproendothelin, which is cleaved after removal of the signal sequence by an endopeptidase, followed by cleavage with endothelin converting enzyme (Xu et al, Cell, 1994, 78:473: Shimada et al, J. Biol. Chem., 1994, 269: 18274).
  • ET A and ET B Two ET receptors, ET A and ET B (Arai et al, Nature, 1990, 348:730; Sakumi et al, Nature, 1990, 348:782). Both belong to the family of heptahelical G-protein coupled receptors. There is 68% amino acid identity between the two receptor subtypes.
  • ET A exists as a single copy gene located on human chromosome 4 (Hosoda et al, J. Biol. Chem., 1992, 267: 18797; Cyr et a/., Biochem. Biophys. Res. Commun., 1991, 181: 184).
  • ET B exists as a single copy gene located on human chromosome 13 (Arai et al, J. Biol. Chem., 1993, 268:3463-70), although a splice variant of ET B has been found (Shyamala et al, Cell. Mol. Biol. Res., 1994, 40:285-96).
  • the cDNA sequence of ET A has been deposited with GenBank with accession number S57498.
  • ET ⁇ and ET B are distinct in their ligand binding affinity and distribution in tissues and cells.
  • ET A has a high affinity to ET-1 and ET-2, but a low affinity to ET-3.
  • ET B has equally potent affinities to all three endogenous ETs.
  • ET A exists on smooth muscle and mediates vasoconstriction.
  • ET B exists on endothelium and mediates the release of relaxing factors such as nitric oxide and prostacycline.
  • ET B exists on some vascular smooth muscle also mediated vasoconstriction.
  • ET-receptor antagonists such as BE-18257B, and BQ-123 and FR139317, two derivatives of BE-18257B (See Masaki, Cardiovascular Res., 1998, 39:530). Many selective and non-selective antagonists for ET A and ET B have emerged.
  • ET antagonists demonstrated significant beneficial effects in pathological conditions, including congestive heart failure, pulmonary hypertension, cerebrovascular spasm after subarachnoid hemorrhage, acute renal failure, and essential hypertension.
  • ET or ET receptor knockout mice have also provided important information regarding the physiological and pathophysiological significance of ET (Masaki, supra).
  • mice with a knockout of ET-3 or the ET B receptor genes exhibit phenotypic changes that resemble Hirschsprung's disease, a human hereditary syndrome associated with a missense mutation of the ET B gene (Pfiffenberger, et al, Cell, 1994, 79: 1257).
  • ⁇ -Adrenergic Receptors ⁇ -Adrenergic Receptors
  • the adrenoceptors fall into three major groups, ,, ⁇ 2 , ⁇ , within each of which further subtypes can be distinguished pharmacologically (L ⁇ llmann, et al. in Color Atlas of Pharmacology, New York, 1993).
  • Adrenergic receptors are all G-protein linked. They are involved in regulation of the cardiovascular system, and in the control of metabolic activity, e.g., insulin secretion and glucose release.
  • Adrenoceptors are targets for epinephrine and norepinephrine, which are representatives of the family of monoamine neurotransmitters. Epinephrine has equally high affinity for all ⁇ - and ⁇ -receptors while norepinephrine differs from epinephrine by its low affinity for ⁇ 2 -receptors (The Biochemical Basis of Neuropharmacology, (7 th Ed.) New York, 1996, pp. 226-292).
  • the adrenoceptors themselves interact preferentially with three different classes of G-proteins: G s ( ⁇ -adrenoceptors) mediating activation of adenylate cyclase, G, (cc 2 -adrenoceptors) mediating inhibition of adenylate cyclase, and G q ( ⁇ , -adrenoceptors) mediating activation of phospholipase C (Hieble et al, ]. of Med. Chem., 1995, 38:3415-3444).
  • the pharmacological interest of adrenoceptors is mainly for the treatment of cardiovascular diseases, e.g.. through the development of ⁇ -antagonists, ,-antagonists and ⁇ 2 -agonists to treat hypertension, but they are also considered important for the treatment of asthma ( ⁇ 2 -agonists).
  • the ⁇ 2 -adrenergic receptor is expressed on a number of cell types, e.g., bronchial smooth muscle, where its activation results in relaxation and bronchial dilatation. These receptors are also being expressed on epithelial cells, vascular endothelium, alveolar walls, immune cells, and presynaptic nerve terminals (Liggett, Am. J. Respir. Crit. Care. Med., 1997, 156:S156-S162). Cardiac cells express mainly ⁇ ,-, but also a small fraction of ⁇ 2 -adrenoceptors (Collins et al, Biochimica et Biophysica Acta, 1993, 1172:171-174). ⁇ , -adrenoceptors are also expressed in brain and pineal gland.
  • the ⁇ ,- and ⁇ 2 -adrenergic receptors are coupled to a G s -protein complex, which activates adenylate cyclase.
  • ⁇ 2 -receptor located in peripheral vascular arteries, mediates vasodilation by increasing the amount of cAMP, and thereby inhibiting activation of myosin kinase, which is necessary for smooth muscle cell constriction (ibid.)
  • Activation of cAMP in cardiac cells by agonist binding to both ⁇ ,- and ⁇ - 2 -adrenoceptors activates the cAMP-dependent protein kinase (PKA) (Castellano and B ⁇ hm, Hypertension, 1997,
  • PKA cAMP-dependent protein kinase
  • ⁇ -adrenoceptors also regulate the control of melatonin production in the pineal gland, by the cAMP activation of one of the enzymes (5-HT-N-acetyl transferase) involved in the synthesis of melatonin (Collins et al, supra).
  • the ⁇ 2 -receptors mediate increased conversion of glycogen to glucose (glycogenolysis) in both the liver and skeletal muscle (H. Lullmann, et al, supra), and stimulate influx of potassium into muscle cells to prevent hyperkalemia (Berne and Levy, supra).
  • the ⁇ -receptors are regulated on the protein level by desensitization.
  • the initial desensitization process results from the phosphorylation of serine and threonine residues in the cytoplasmic tail or third intracellular loop by several protein kinases, including ⁇ ARK and PKA (Hieble et al, supra).
  • ⁇ ARK phosphorylates specific serine or threonine residues in the C-terminal of receptors that are occupied by an agonist.
  • the phosphorylation triggers binding of the cytostolic protein ⁇ -arrestin and results in the uncoupling from G s ⁇ .
  • PKA is activated by cAMP and phosphorylates the ⁇ 2 -adrenoceptor by a relatively slow process.
  • the phosphorylated receptor loses the ability to activate G s (Castellano and B ⁇ hm, supra). Prolonged interaction of agonists with adrenoceptors generally results in receptor desensitization.
  • ⁇ -Adrenoceptor Gene Structure The human ⁇ ,-adrenoceptor gene is located on the long arm of chromosome 10, the same chromosome as for the ⁇ 2A -adrenoceptor gene.
  • the coding sequence of this gene is deposited with GenBank, accession number X69168.
  • the regulatory region is also deposited with GenBank, accession number J03019. It codes for an intronless gene product of 1431 base pairs (Hall, Thorax, 1996, 51 :351-353). Both the promoter and the coding region of the gene are rich in G and C residues, which make up greater than 70%> of the bases.
  • the promoter does not contain any paired consensus TATA box and CAAT box elements but instead clusters with an inverted CAAT box and SP, or AP-2 binding motifs.
  • This type of receptor reminiscent of "housekeeping genes", has been described for other G-protein coupled receptors as well (Collins et al, supra).
  • the human ⁇ 2 -adrenoceptor gene is located on the long arm of chromosome 5, the same chromosome as the I B -adrenoceptor gene.
  • the coding sequence has been deposited with GenBank, with accession numbers Ml 5169, J02728, or Ml 6106.
  • the regulatory region sequence is also deposited with GenBank, accession number Y00106. It codes for an intronless gene product of 1239 base pairs (Hall, supra).
  • the promoter region is 200-300 bases 5' of the translation initiation codon, and it can form strong secondary structures due to high G-C content.
  • ⁇ -Adrenoceptor Gene Regulation There are some regulatory regions identified in the promoter region of the ⁇ ,-adrenoceptor gene: a cAMP response element (CRE), a consensus thyroid response element (TRE), and a glucocorticoid response element (GRE). This is consistent with the evidence that both thyroid hormone and corticosteroids affect adrenergic sensitivity in both heart and adipose tissue.
  • CRE region might have a self-regulatory function, as has been shown for the ⁇ 2 -adrenoceptor gene (Collins et al., supra).
  • CRE CRE binding protein
  • ⁇ 2 -adrenoceptor gene is upregulated by stimulation with glucocorticoids in a variety of tissues (Collins et al., supra). In the 3' flanking region there are sequences homologous to glucocorticoid response elements. These might be responsible for the increased expression of ⁇ 2 adrenoceptor observed in transfected cells after treatment with hydrocortisone (Emorine and Marullo, Proc. Natl. Acad. Sci. USA, 1987, 84:6995-6999).
  • the proposed model for ⁇ -adrenoceptors is like most of the G-protein binding receptors, a seven -helical transmembrane structure, where the seven ⁇ -helices are radially arranged around a central "pore", in which the receptor ligands bind.
  • the ⁇ -adrenoceptors have an extracellular glycocylated N-terminus, and an intracellular C-terminus.
  • the ⁇ ,-receptor consists of 477 amino acids; the ⁇ 2 -receptor consists of 413 amino acids.
  • Site-directed mutagenesis has demonstrated that an aspartic acid residue, Asp-113, located in the third transmembrane-spanning helix, and two serine residues, Ser-204 and Ser-207, are required for full agonist binding to the ⁇ 2 -adrenoceptor.
  • the ⁇ ,-adrenoceptor contains identical amino acid residues located in corresponding positions to those shown to be important for agonist binding to the ⁇ 2 -adrenoceptor.
  • Another aspartic acid residue, Asp-79 located in the second -helix of both ⁇ -receptors is highly conserved in G-protein coupled receptors (Hieble et al, supra). Ser-319 has a potential role in agonist binding to the ⁇ ⁇ -adrenoceptor.
  • Betablockers have been widely used in the treatment of hypertension. They are particularly useful for the treatment of juvenile hypertension with tachycardia and high cardiac output. Betablockers or beta-adrenergic blockers were first introduced as a treatment for essential hypertension in 1964, and are still recommended as first choice because the cost for betablockers is low, which improves patient compliance. They act by binding to ⁇ , -receptors on the cardiac smooth muscle cells, which leads to decreased cardiac output.
  • betablockers are not specific ⁇ ,-receptor antagonists but binds to ⁇ 2 -receptors as well.
  • the binding to ⁇ 2 -receptors gives the opposite of the desired effect though inhibition of ⁇ 2 -receptors leads to vasoconstriction. This gives a side effect with cold hands and feet because most of the ⁇ 2 -receptors are located in the peripheral vascular arteries.
  • ⁇ -adrenoceptor agonists such as dopamine and dobutamine are used to stimulate myocardial ⁇ , -adrenoceptors in the acute management of congestive heart failure. They act by increasing contractility and cardiac output.
  • ⁇ 2 -agonists are used in the treatment of asthma. They exert their primary effect on the ⁇ 2 -adrenergic receptor of bronchial smooth muscle, resulting in relaxation and bronchial dilatation. They also protect against bronchoconstrictor challenge (Hall, supra).
  • cardiovascular disease demonstrate a need in the art for methods and compositions that allow the determination and/or prediction of the therapeutic regimen that will result in the most effective treatment outcome in a patient suffering from cardiovascular disease. This includes identification of individuals who are more or less responsive to particular therapeutic regimens, including, e.g., particular drugs that are conventionally used to treat cardiovascular disease.
  • heterogeneity in responses to cardiovascular therapies emphasizes a need for another approach to rational drug development.
  • populations that are identified as non-responsive to a particular therapeutic regimen can be identified for development and testing of alternative regimens.
  • effective treatment regimens could be developed for a larger percentage of the affected population.
  • the present invention provides reagents and methods for predicting whether a particular therapeutic regime (such as a specific drug, a class of drugs or any other therapeutic regime, pharmacological or not) would be effective in improving a cardiovascular condition in a human individual, or would be ineffective for that pu ⁇ ose, or its use would be associated with adverse reactions or undesirable side-effects.
  • a particular therapeutic regime such as a specific drug, a class of drugs or any other therapeutic regime, pharmacological or not
  • a particular advantage of the invention is that one or more polymo ⁇ hic markers provide a basis for predicting the outcome of a treatment regimen.
  • a physician can predict whether a treatment plan, such as administration of an ACE inhibitor, is likely or not to be effective before subjecting the subject to the treatment plan.
  • a comparison of the test polymo ⁇ hic pattern from an individual with reference polymo ⁇ hic patterns of individuals exhibiting differing responses to a particular therapeutic intervention can be used to predict the type or degree of responsivity of the individual to such intervention.
  • the present invention thus represents a significant breakthrough in treating cardiovascular pathologies in that it reduces or eliminates trial and error in selecting a treatment for a particular individual cardiovascular patient.
  • An additional advantage of the invention derives from the ability to eliminate subjects from clinical trials who are predictably non-responsive, or at risk for an adverse response, to a particular treatment regimen. Furthermore, adverse results in an early trial can be evaluated to identify polymorphic patterns, so that the adverse results can be correlated with a sub-population of the test population permitting exclusion of such sub-population from the treatment group.
  • the invention may thus ensure that a beneficial drug can be approved for use in the appropriate population, and decrease the number of required patients and therefore the duration and cost of clinical trials. It may also lead to identification of another subgroup which can be the target for development of another therapeutic regimen.
  • the foregoing methods of the invention are carried out by comparing a test polymo ⁇ hic pattern established by at least one polymo ⁇ hic position within a gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor with a polymo ⁇ hic pattern of a population of individuals exhibiting a predetermined responsivity to the regimen (reference pattern). If the test pattern matches the reference pattern, there is a statistically significant probability that the individual has the same cardiovascular status as that correlated with the reference pattern.
  • the polymo ⁇ hic pattern preferably consists of at least two (and more preferably at least three) polymo ⁇ hic positions, at least one of which is in the gene encoding a polypeptide from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor, and a second polymo ⁇ hism in a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor.
  • the invention provides methods for assessing whether a particular individual has a genetic predisposition to a cardiovascular pathology.
  • This aspect of the invention comprises comparing a test polymo ⁇ hic pattern established by at least one and preferably at least two and most preferably at least three polymo ⁇ hic positions within a gene encoding ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor in conjunction with one or more, and preferably two or more, other polymo ⁇ hic positions in ACE, ATI , AGT, renin, WO 00/22166 _ ⁇ 3 _ PCT/IB99/01678
  • aldosterone synthase type-2 angiotensin II receptor, endothelin receptor, or ⁇ - adrenoceptor
  • a polymo ⁇ hic pattern of individuals exhibiting a predisposition to a cardiovascular syndrome The conclusion drawn depends on whether the individual's polyymo ⁇ hism pattern matches the reference pattern.
  • the invention also provides an isolated nucleic acid having a sequence corresponding to part or all of the gene encoding ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor, the nucleic acid comprising a polymo ⁇ hism in the ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor gene.
  • the polymo ⁇ hism in combination with one or more other polymo ⁇ hisms in the sequence of the same gene or a gene encoding a protein selected from the group consisting of human ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor, is predictive of a particular type or level of responsivity to a given treatment, or indicates a predisposition to one or more clinical syndromes associated with cardiovascular disease, or both.
  • the isolated polymo ⁇ hisms according to the invention include without limitation:
  • Nucleic acids encoding aldosterone synthase promoter at position -344 Nucleic acids encoding ⁇ -adrenergic receptor- 1 regulator region, at positions 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724, and 2757, as numbered in GenBank accession number X69168. In preferred embodiments, the bases at specific positions are 2238 G, 2238 A, 2577 C, 2257 T, 2757 A, and 2757 G.
  • Nucleic acids encoding ⁇ -adrenergic receptor- 1 coding region at positions 231, 758, 1037, 1251, 1403, and 1528, as numbered in GenBank accession number J03019.
  • the bases at specific positions are 231 A, 231 G, 1251 C, 1251 G, 1403 A, 1403 G, 1528 C, and 1528 A.
  • the bases at specific positions are 934 A. 934 G, 987 C, 987 G, 1006 A, 1006 G, 1 120 C, 1 120 G, 1221 C, 1221 T, 1541 C, 1541 T, 1568 C, and 1568 T..
  • Nucleic acids encoding ⁇ -adrenergic receptor-2 coding region at positions 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070, as numbered in GenBank accession number Y00106.
  • the bases at specific positions are 839 A, 839 G, 872 C, 872 G. 1045A, 1045 G, 1284 C, 1284 T, 1316 A, 1316 C, 1846 C, 1846 G, 2032 A, 2032 G, 2068 no insert, 2068 G, 2068 C, 2070 no insert, and 2070 C.
  • the bases at specific positions are 969 C, 969 T, 1005 A, 1005 G, 1146 A, 1146 G, 2485 T, and 2485 C.
  • Nucleic acids comprising polymo ⁇ hisms present in other genes which can be used in combination with a polymo ⁇ hism from a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor to establish a polymorhism pattern, have been disclosed in International Patent Application No. PCT/IB98/00475, and include:
  • sequences at the polymo ⁇ hic positions in the ACE regulatory region are one or more of 5106C and 5106T; and the sequences at the polymo ⁇ hic positions in the coding region are one or more of 375A, 375C, 582C, 582T, 731A, 731G, 1060G, 1060A, 2741G, 2741T, 3132C, 3132T, 3387T, 3387C. 3503G, 3503C, 3906G, and 3906A.
  • the invention also encompasses a nucleic acid encoding a deletion of nucleotides 1451-1783 as numbered in GenBank entry X62855.
  • sequences at the polymo ⁇ hic positions in the AGT regulatory region are one or more of 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 692C, 692T, 839G, 839A, 1007G, 1007A, 1072G, 1072A, 1204C, and 1204A;
  • sequences at the polymo ⁇ hic position in the coding region are one or more of 273C, 273T, 912C, 912T, 997G, 997C, 1116G, 1 116A, 1 174C and 1174A; and the sequence at position 49 in GenBank entry M24688 is either A or G.
  • sequences at the polymo ⁇ hic positions in the ATI regulatory region are one or more of 1427A, 1427T, 1756T, 1756A, 1853T, 1853G, 2046T, 2046C, 2354A, 2354C, 2355G, 2355C, 2415A and 2415G; and the sequences at the polymo ⁇ hic positions in the coding region are one or more of 449G, 449C, 678T, 678C, 1167A, 1167G, 1271 A, and 1271C.
  • the invention also encompasses libraries of isolated nucleic acid sequences, such as arrays on a solid surface, wherein each sequence in the library comprises a polymo ⁇ hic position in the gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ - adrenoceptor and other polymo ⁇ hic positions in the other genes, including without limitation the polymo ⁇ hic positions and sequences disclosed herein.
  • nucleic acid probes that hybridize specifically to the identified polymo ⁇ hic positions; peptides and polypeptides comprising polymo ⁇ hic positions; and polymo ⁇ hism-specific antibodies, i.e., sequence-specific antibodies that bind differentially to polymo ⁇ hic variants of the foregoing genes, that can be used to identify particular polymo ⁇ hic variants.
  • kits for the determination of polymo ⁇ hic patterns in an individual's genes comprise a means for detecting polymo ⁇ hic sequences, including without limitation oUgonucleotide probes that hybridize at or adjacent to the polymo ⁇ hic positions and polymo ⁇ hism-specific antibodies.
  • nucleic acid targets for use in screening methods to identify candidate cardiovascular drugs.
  • Nucleic acid targets may be, e.g., DNA or RNA and are preferably at least about 10, and most preferably at least about 15, residues in length and comprise one or more polymo ⁇ hic positions in a gene encoding a polypeptide from the group consisting of ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ - adrenoceptor.
  • Peptide targets are at least about 5 amino acids in length and may be as large or larger than the full-length polypeptides.
  • the invention in based, in part, on the discovery that polymo ⁇ hisms in certain genes in the RAAS, endothelin, and ⁇ -adrenoceptors pathways define polymo ⁇ hism patterns that correlate with cardiovascular status. Most significantly, by comparing a test individual's polymo ⁇ hism pattern with a reference polymo ⁇ hism pattern, which is a polymo ⁇ hism pattern from a population of individuals with known cardiovascular status, one is able to predict whether the test individual has an increased likelihood to share the same cardiovascular status as that correlated with the reference polymo ⁇ hism pattern. In particular, particular patterns correlate with responsiveness to ACE inhibitors, non-responsiveness to ACE inhibitors, and predisposition to cardiovascular diseases or dysfunctions, including myocardial infarction and stroke.
  • the invention provides a powerful predictive tool for clinical testing and treatment of cardiovascular disease.
  • the present invention permits smaller, more efficient clinical trials by identifying individuals who are likely to respond poorly to a treatment regimen and reducing the amount of uninte ⁇ retable data.
  • a physician can prescribe a prophylactic or therapeutic regimen customized to that individual's cardiac status.
  • Adverse responses to particular therapies can be avoided by excluding those individuals whose cardiovascular status puts them at risk for that therapy.
  • Appropriate changes in lifestyle, including diet, environmental stress, and exercise levels can be prescribed for individuals whose test polymo ⁇ hic pattern matches a reference pattern that correlates with increased predisposition to cardiovascular disease.
  • Cardiovascular status refers to the physiological status of an individual's cardiovascular system, as reflected in one or more status markers or indicators including genotype. Cardiovascular status shall be deemed to include without limitation not only the absence or presence of a pathology or disease in one or more components of the individual's cardiovascular system and the individual's predisposition to developing such a condition, but also the individual's responsivity, i.e., the ability or inability of the individual to respond (positively or negatively) to a particular prophylactic or therapeutic regimen or treatment for a cardiovascular condition, such as a drug or a class of drugs. A negative response includes one or more adverse reactions and side effects.
  • Status markers include without limitation clinical measurements such as, e.g., blood pressure, electrocardiographic profile, differentiated blood flow analysis, and the presence of increased levels of cellular proteins associated with a cardiovascular event.
  • diagnostic markers which are important in cardiac events include myosin light chain, myosin heavy chain, myoglobin, troponin I, troponin T, CK-MB, etc. (see U.S. Patents No. 5,604,105 and No. 5,744,358).
  • Status markers according to the invention are assessed using conventional methods well known in the art. Also included in the evaluation of cardiovascular status are quantitative or qualitative changes in status markers with time, such as would be used, e.g., in the determination of an individual's response to a particular therapeutic regimen or of a predisposed individual's eventual development of a cardiovascular condition.
  • cardiovascular syndromes that are included in the foregoing definition of cardiovascular status include diagnosis of, or predisposition to, one or more cardiovascular syndromes, such as, e.g., hypertension, acute myocardial infarction, silent myocardial infarction, unstable angina, stroke, and atherosclerosis. It will be understood that a diagnosis of a cardiovascular syndrome made by a medical practitioner encompasses not only clinical measurements but also medical judgment.
  • Responsivity refers to the type and degree of response an individual exhibits to a particular therapeutic regimen, i.e., the effect of a treatment on an individual. Responsivity breaks down into three major categories: therapeutic effect; no effect; and adverse effect. Naturally, there can be differing degrees of a therapeutic effect, e.g., between full elimination and partial elimination of symptomology. In addition, adverse effects, or side effects, may be observed even though the treatment is beneficial, i.e., therapeutically effective. Indeed, the present invention may permit identification of individuals with complex responsivity traits or patterns.
  • a "predisposition to develop a cardiovascular syndrome” refers to an increased likelihood, relative to the general population, to develop a cardiovascular syndrome, as defined above.
  • a predisposition does not signify certainty, and development of the syndrome may be forestalled or prevented by prophylaxis, e.g., adopting a modified diet, exercise program, or treatment with gene therapy or pharmaceuticals.
  • prophylaxis e.g., adopting a modified diet, exercise program, or treatment with gene therapy or pharmaceuticals.
  • an advantage of the present invention is that it permits identification of individuals who are, based on their genotype, predisposed to develop a cardiovascular syndrome, and for whom prophylactic intervention can be especially important.
  • a "polymo ⁇ hism” as used herein denotes a variation in the nucleotide sequence of a gene in an individual. Genes that have different nucleotide sequences as a result of a polymo ⁇ hism are "alleles".
  • a "polymo ⁇ hic position” is a predetermined nucleotide position within the sequence.
  • genetic polymo ⁇ hisms are reflected by an amino acid sequence variation, and thus a polymo ⁇ hic position can result in location of a polymo ⁇ hism in the amino acid sequence at a predetermined position in the sequence of a polypeptide.
  • An individual "homozygous” for a particular polymo ⁇ hism is one in which both copies of the gene contain the same sequence at the polymo ⁇ hic position.
  • An individual “heterozygous” for a particular polymo ⁇ hism is one in which the two copies of the gene contain different sequences at the polymo ⁇ hic position.
  • polymo ⁇ hism pattern denotes a set of one or more polymo ⁇ hisms, including without limitation single nucleotide polymo ⁇ hisms, which may be contained in the sequence of a single gene or a plurality of genes.
  • a polymo ⁇ hism pattern can consist of a single nucleotide polymb ⁇ hism in only one position of one of two alleles of an individual . However, one has to look at both copies of a gene.
  • a polymo ⁇ hism pattern that is appropriate for assessing a particular aspect of cardiovascular status need not contain the same number (nor identity, of course) of polymo ⁇ hisms as a polymo ⁇ hism pattern that would be appropriate for assessing another aspect of cardiovascular status (e.g., responsivity to ACE inhibitors for control of hypertension).
  • a "test polymo ⁇ hism pattern” as used herein is a polymo ⁇ hism pattern determined for a human subject of undefined cardiovascular status.
  • a “reference polymo ⁇ hism pattern” as used herein is determined from a statistically significant correlation of patterns in a population of individuals with predetermined cardiovascular status.
  • Nucleic acid or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.
  • Nucleic acids include without limitation single- and double-stranded molecules, i.e., DNA-DNA, DNA- RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases and non-naturally occurring phosphoester analog bonds, such as phosphorothioates and thioesters.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • oligonucleotide refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, cDNA, mRNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
  • a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
  • oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a gene of interest, or to detect the presence of nucleic acids encoding the gene of interest.
  • an oligonucleotide of the invention can form a triple helix with a double stranded sequence of interest in a DNA molecule.
  • a library of oligonucleotides arranged on a solid support can be used to detect various polymo ⁇ hisms of interest.
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds.
  • An "isolated" nucleic acid or polypeptide as used herein refers to a nucleic acid or polypeptide that is removed from its original environment (for example, its natural environment if it is naturally occurring). 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 that is "derived from" a designated sequence refers to a sequence that corresponds to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are identical to or complementary to the sequence.
  • a “probe” refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target nucleic acid due to complementarity of at least one sequence in the probe with a sequence in the target nucleic acid. Generally, a probe is labeled so it can be detected after hybridization.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55 °C, can be used, e.g., 5x SSC, 0.1%> SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5%) SDS).
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40%) formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50%> formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T m have been derived (see Sambrook et al, supra, 9.50-9.51).
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides: preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
  • standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60°C; in a more preferred embodiment, the T m is 65 °C.
  • high stringency refers to hybridization and/or washing conditions at 68°C in 0.2XSSC, at 42°C in 50% formamide, 4XSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
  • a “gene” for a particular protein as used herein refers to a contiguous nucleic acid sequence corresponding to a sequence present in a genome which comprises (i) a "coding (or transcribed) region," which comprises exons (i.e., sequences encoding a polypeptide sequence, or “protein-coding” or “transcribed sequences"), introns, sequences at the junction between exons and introns, and 5' and 3' untranslated regions (uTRs); and (ii) regulatory sequences, which flank the coding region at both 5' and 3' termini.
  • the "ACE gene” as used herein encompasses the regulatory and coding regions of the human gene encoding angiotensin converting enzyme.
  • the "AGT gene” encompasses regulatory and coding regions of the human gene encoding angiotensinogen and the "ATI gene” encompasses regulatory and coding regions of the human gene encoding type I angiotensin II receptor.
  • regulatory sequences according to the invention are located 5' (i.e., upstream) of the coding region segment.
  • GenBank GenBank
  • the present inventors have su ⁇ risingly and unexpectedly discovered the existence of genetic polymo ⁇ hisms within the human gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ - adrenoceptor which, singly or in combination, can be used to assess cardiovascular status, depending on which component of cardiovascular status is under evaluation.
  • the polymo ⁇ hic pattern of the gene can predict the responsivity of the individual to particular therapeutic interventions and serve as an indicator of predisposition to various forms of cardiovascular disease.
  • the invention provides methods for assessing cardiovascular status by detecting polymo ⁇ hic patterns in an individual.
  • the present invention also provides isolated nucleic acids derived from the gene which comprise these polymo ⁇ hisms, including probes which hybridize specifically to polymo ⁇ hic positions and primers that amplify the region of the gene in which the polymo ⁇ hism is located; isolated polypeptides and peptides comprising polymo ⁇ hic residues; and antibodies which specifically recognize ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor polypeptides containing one or more polymo ⁇ hic amino acids.
  • the present invention provides diagnostic methods for assessing cardiovascular status in a human individual.
  • the methods are carried out by comparing a polymo ⁇ hic position or pattern ("test polymo ⁇ hic pattern") within the individual's gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor with the polymo ⁇ hic patterns of humans exhibiting a predetermined cardiovascular status ("reference polymo ⁇ hic pattern"). If the cardiovascular status is the prediction of responsivity to a therapy, a single polymo ⁇ hic position can provide a pattern for comparison.
  • polymo ⁇ hic position for the pattern to improve the accuracy of the prediction.
  • at least two, and preferably at least three, polymo ⁇ hic positions are used to make the pattern.
  • other polymo ⁇ hisms in genes encoding angiotensin converting enzyme (ACE), angiotensinogen (AGT), type 1 angiotensin II receptor (ATI), type 2 angiotensin II receptor, renin, aldosterone synthase, endothelin, receptor or ⁇ -adrenergic receptors 1 and 2 can be used to establish a polymo ⁇ hic pattern for the individual.
  • the polymo ⁇ hic pattern of the individual is identical to the polymo ⁇ hic pattern of individuals who exhibit particular status markers, cardiovascular syndromes, and/or particular patterns of response to therapeutic interventions.
  • the method involves comparing an individual's test polymo ⁇ hic pattern with reference polymo ⁇ hic patterns of individuals who have been shown to respond positively or negatively to a particular therapeutic regimen.
  • Therapeutic regimen refers to treatments aimed at the elimination or amelioration of symptoms and events associated cardiovascular disease.
  • Such treatments include without limitation one or more of alteration in diet, lifestyle, and exercise regimen; invasive and noninvasive surgical techniques such as atherectomy, angioplasty, and coronary bypass surgery; and pharmaceutical interventions, such as administration of ACE inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antagonists, cardiac glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor antagonists, calcium channel blockers, HMG-CoA reductase inhibitors, imidazoline receptor blockers, endothelin receptor blockers, and organic nitrites. Interventions with pharmaceutical agents not yet known whose activity correlates with particular polymo ⁇ hic patterns associated with cardiovascular disease are also encompassed.
  • the present inventors have discovered that particular polymo ⁇ hic patterns correlate with an individual's responsivity to ACE inhibitors (see, e.g., Example 3 below). It is contemplated, for example, that patients who are candidates for a particular therapeutic regimen will be screened for polymo ⁇ hic patterns that correlate with responsivity to that particular regimen.
  • the method involves comparing an individual's polymo ⁇ hic pattern with polymo ⁇ hic patterns of individuals who exhibit or have exhibited one or more markers of cardiovascular disease, such as, e.g., high blood pressure, abnormal electrocardiographic profile, myocardial infarction, unstable angina, stroke, or atherosclerosis (see. e.g., Example 2 below) and drawing analogous conclusions as to the individual's responsivity to therapy, predisposition to developing a syndrome, etc., as detailed above.
  • markers of cardiovascular disease such as, e.g., high blood pressure, abnormal electrocardiographic profile, myocardial infarction, unstable angina, stroke,
  • an individual's polymo ⁇ hic pattern can be established e.g. by obtaining DNA from the individual and determining the sequence at a predetermined polymo ⁇ hic position or positions in a gene, or more than one gene.
  • the DNA may be obtained from any cell source.
  • Non-limiting examples of cell sources available in clinical practice include without limitation blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Cells may also be obtained from body fluids, including without limitation blood, saliva, sweat, urine, cerebrospinal fluid, feces, and tissue exudates at the site of infection or inflammation.
  • DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source.
  • Determination of the sequence of the extracted DNA at polymo ⁇ hic positions is achieved by any means known in the art, including but not limited to direct sequencing, hybridization with allele-specific oligonucleotides, allele-specific PCR, ligase-PCR, HOT cleavage, denaturing gradient gel electrophoresis (DGGE), and single- stranded conformational polymo ⁇ hism (SSCP).
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam- Gilbert method; by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; and sequencing using a chip-based technology. See, e.g., Little et al, Genet. Anal., 1996, 6: 151.
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • biopsy tissue is obtained from a subject.
  • Antibodies that are capable of distinguishing between different polymo ⁇ hic forms of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor are then applied to samples of the tissue to determine the presence or absence of a polymo ⁇ hic form specified by the antibody.
  • the antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, or enzyme-linked or fluorescence-linked immunoassay.
  • the presence or absence of a particular polymo ⁇ hism or polymo ⁇ hic pattern, and its allelic distribution is determined by comparing the values obtained from a patient with norms established from populations of patients having known polymo ⁇ hic patterns.
  • RNA is isolated from biopsy tissue using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al, Anal. Biochem., 1987, 162:156).
  • the isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a selected polymo ⁇ hism.
  • RT-PCR polymerase chain reaction
  • Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic of the presence of a particular polymo ⁇ hism.
  • RNA is reverse-transcribed and amplified, after which the amplified sequences are identified by, e.g., direct sequencing.
  • cDNA obtained from the RNA can be cloned and sequenced to identify a polymo ⁇ hism.
  • the distribution of polymo ⁇ hic patterns in a large number of individuals exhibiting particular cardiovascular status is determined by any of the methods described above, and compared with the distribution of polymo ⁇ hic patterns in patients that have been matched for age, ethnic origin, and/or any other statistically or medically relevant parameters, who exhibit quantitatively or qualitatively different cardiovascular status. Correlations are achieved using any method known in the art, including nominal logistic regression or standard least squares regression analysis. In this manner, it is possible to establish statistically significant correlations between particular polymo ⁇ hic patterns and particular cardiovascular statuses. It is further possible to establish statistically significant correlations between particular polymo ⁇ hic patterns and changes in cardiovascular status such as, would result, e.g., from particular treatment regimens. Thus, it is possible to correlate polymo ⁇ hic patterns with responsivity to particular treatments.
  • a statistically significant correlation preferably has a "p" value of less than or equal to 0.05. Any standard statistical method can be used to calculate these values, such as the normal Student's T Test, or Fischer's Exact Test.
  • the identity and number of polymo ⁇ hisms to be included in a reference pattern depends not only on the prevalence of a polymo ⁇ hism and its predictive value for the particular use, but also on the value of the use and its requirement for accuracy of prediction.
  • the greater the predictive value of a polymo ⁇ hism the lower the need for inclusion of more than one polymo ⁇ hism in the reference pattern.
  • a polymo ⁇ hism is very rare, then its absence from an individual's pattern might provide no indication as to whether the individual has a particular status. Under these circumstances, it might be advisable to select instead two or more polymo ⁇ hisms which are more prevalent. Even if none of them has a high predictive value on its own, the presence of both (or all three) of them might be sufficiently predictive for the particular pu ⁇ ose.
  • the reference pattern need only permit selection of a population that improves the response rate by 10% to provide a significant improvement in the state of the art.
  • the use for the reference pattern is selection of subjects for a particular clinical study, the pattern should be as selective as possible and should therefore include a plurality of polymo ⁇ hisms that together provide a high predictive accuracy for the intended response. In establishing reference polymo ⁇ hism patterns, it is desirable to use a defined population.
  • tissue libraries collected and maintained by state or national departments of health can provide a valuable resource, since genotypes determined from these samples can be matched with medical history, and particularly cardiovascular status, of the individual.
  • tissue libraries are found, for example, in Sweden, Iceland, Norway, and Finland.
  • specific polymo ⁇ hisms may be associated with a closely linked population.
  • other polymo ⁇ hisms in the same gene may correlate with cardiovascular status of other genetically related populations.
  • the invention identifies genes in which any polymo ⁇ hisms can be used to establish reference and test polymo ⁇ hism patterns for evaluating cardiovascular status of individuals in the population.
  • DNA samples can be obtained form a well defined population, such as 277 Caucasian males born in Uppsala, Sweden between 1920 and 1924.
  • individuals are selected for the test population based on their medical history, i.e., they were either (i) healthy, with no signs of cardiovascular disease (100); or (ii) had suffered one of acute myocardial infarction (68), silent myocardial infarction (34), stroke (18), stroke and acute myocardial infarction (19), or high blood pressure at age 50 (39).
  • DNA samples are obtained from each individual.
  • DNA sequence analysis can be carried out by: (i) amplifying short fragments of each of the genes using polymerase chain reaction (PCR) and (ii) sequencing the amplified fragments.
  • PCR polymerase chain reaction
  • sequences obtained from each individual can then be compared with the first known sequences, e.g., as set forth in Table 1, to identify polymo ⁇ hic positions.
  • test pattern from an individual can be compared to a reference pattern established for a predetermined cardiovascular status.
  • Identity between the test pattern and the reference pattern means that the tested individual has a probability of having the same cardiovascular status as that represented by the reference pattern. As discussed above, this probability depends on the prevalence of the polymo ⁇ hism and the statistical significance of its correlation with a cardiovascular status.
  • Polymo ⁇ hic positions in the genes encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ - adrenoceptor which are encompassed by the invention can be identified by determining the DNA sequence of all or part of the gene in a multiplicity of individuals in a population. DNA sequence determination may be achieved using any conventional method, including, e.g., chemical or enzymatic sequencing.
  • the polymo ⁇ hic positions of genes for use in the invention include without limitation those listed below, whose numbering corresponds to the GenBank sequences listed in Table 1.
  • ACE positions in the regulatory region (designated ACR) numbered 5106, 5349, and 5496; positions in the coding region (designated ACE) numbered 375, 582, 731 , 1060. 1215, 2193, 2328, 2741, 3132, 3387, 3503, and 3906; and position 1451 as numbered in GenBank entry X62855.
  • AGT positions in the regulatory region (designated AGR) numbered 395, 412, 432, 449. 692, 839, 1007, 1072, 1204, and 1218; positions in the coding region (designated AGT) numbered 273, 620, 803, 912, 997, 1116, and 1174; and position 49 as numbered in GenBank entry M24688.
  • ATI positions in the regulatory region (designated ATR) numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; and positions in the coding region (designated ATI) numbered 449, 678, 1167, and 1271.
  • Renin A mutant renin gene in familial elevation of prorenin, a point mutation in the last exon of the gene (exon 10), has been identified (Villard et al, J. Biol. Chem., 1994, 269:30307-12). A cytosine to thymine transition creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus.
  • Aldosterone synthase A position in the promoter region of aldosterone synthase, position -344 (with the initiation codon starting at 1) has been reported by Cambien et al. at the International Meeting on Hypertension held in Amsterdam in June 1998.
  • ⁇ -adrenergic receptor- 1 positions in the regulatory region (designated BP1) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724, and 2757; and positions in the coding region (designated BR1) numbered 231, 758, 1037, 1251, 1403, and 1528.
  • ⁇ -adrenergic receptor-2 positions in the regulatory region (designated B2P) numbered 932, 934, 987, 1006. 1120, 1221, 1541, and 1568; and positions in the coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070.
  • Endothelin receptor type A coding region (designated ET A ) numbered 969, 1005, 1 146, and 2485.
  • the base at each of the above polymo ⁇ hic positions is one of:
  • ACE Regulatory Region 5106C, 5106T, 5349A, 5349T, 5496T, and 5496C;
  • ACE Coding Region 375A, 375C, 582C, 582T, 731A, 731G,
  • AGT Regulatory Region 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 692C, 692T, 839G, 839A, 1007G, 1007A, 1072G, 1072A, 1204C, 1204A, 1218A, 1218G;
  • AGT Coding Region 273C, 273T, 620C, 620T, 803T, 803C, 912C, 912T, 997G, 997C, 11 16G, 1116A, 1174C, and 1174 A; and A or G at position 49 in GenBank entry M24688;
  • _ ATI Regulatory Region 1427A, 1427T, 1756T, 1756A, 1853T,
  • ATI Coding Region 449G, 449C, 678T, 678C, 1167A, 1167G, 1271A, and 1271C.
  • ⁇ -adrenergic receptor- 1 regulatory region 2238 G, 2238 A, 2577 C, 2257 T, 2757 A, and 2757 G.
  • ⁇ -adrenergic receptor-1 coding region 231 A, 231 G, 758 C, 758 T, 1251 C, 1251 G, 1403 A, 1403 G, 1528 C, and 1528 A.
  • (ix) ⁇ -adrenergic receptor-2 regulatory region 934 A, 934 G, 987 C, 987 G, 1006 A, 1006 G, 1120 C, 1120 G, 1221 C, 1221 T, 1541 C, 1541 T, 1568 C, and 1568 T.
  • (x) ⁇ -adrenergic receptor-2 coding region 839 A, 839 G, 872 C, 872 G, 1045A, 1045 G, 1284 C, 1284 T, 1316 A, 1316 C, 1846 C, 1846 G, 2032 A, 2032 G, 2068 no insert, 2068 G, 2068 C, 2070 no insert, and 2070 C.
  • Endothelin receptor type A 969 C, 969 T, 1005 A, 1005 G, 1 146 A, 1 146 G, 2485 T, and 2485 C.
  • An individual may be homozygous or heterozygous for a particular polymo ⁇ hic position (see, e.g., Table 6 below).
  • polymo ⁇ hic patterns comprising one or more polymo ⁇ hism in ACE, AGT, and/or ATI genes according to the invention include the following, which were correlated with an increased incidence of clinical signs of cardiovascular disease: ACR 5349 A/T, AGR 1218 A; ACR 5496 C, AGR 1204 A/C; ACR 5496
  • Beta adrenergic receptors 1 and 2 Positions carrying genetic variation in the Beta adrenergic receptors 1 and 2
  • B1P Beta adrenergic receptor 1, regulatory promoter region.
  • B1R Beta adrenergic receptor 1, coding region.
  • B2P Beta adrenergic receptor 2, regulatory promoter region.
  • B2R Beta adrenergic receptor 2, coding region.
  • polymo ⁇ hisms A number of different polymo ⁇ hisms have been identified in the type 2 ⁇ - adrenoceptor. All of these differed from the wild type sequence by a single base change. Four of the polymo ⁇ hisms alter the amino acid sequence of the receptor protein (Hall, Thorax, 1996, 51:351-353). The amino acid sequence modifications are described in greater detail below:
  • Argl6-*Gly The Glyl ⁇ variant undergoes an enhanced agonist-promoted down regulation as compared to wild type but the coupling to adenylyl cyclase and agonist binding are maintained (Liggett, Am. J. Respir. Crit. Care Med., 1997, 156:S 156-S162).
  • Gln27 ⁇ Glu The Glu27 variant displays very little agonist-promoted downregulation and the coupling to adenylyl cyclase and agonist binding are maintained (id.).
  • Val34-Met Met34 is very rare. No altering of receptor function has been found (id.). Thrl64 ⁇ Ile: Uncommon (about 5%). The He 164 variant shows depressed coupling to adenylyl cyclase and decreased affinities for agonists with hydroxyl groups on their ⁇ -carbons, such as epinephrine, norepinephrine, and isoproterenol compared to wild type (id.).
  • the polymo ⁇ hism at nucleic acid 523 (CGG ⁇ AGG) might be linked with one of the other functional polymo ⁇ hisms (id.).
  • the present invention provides isolated nucleic acids comprising the polymo ⁇ hic positions described above for the human genes encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ - adrenoceptor; vectors comprising the nucleic acids; and transformed host cells comprising the vectors.
  • the invention also provides probes which are useful for detecting these polymo ⁇ hisms.
  • nucleic acids encoding a gene comprising a polymo ⁇ hism that is useful for determining cardiovascular status of an individual is particularly valuable for screening, whether by direct screening of the nucleic acid with the polymo ⁇ hism, or by screening the polypeptide expressed by that nucleic acid.
  • many conventional techniques in molecular biology, microbiology, and recombinant DNA are used. Such techniques are well known and are explained fully in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, 1989 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D.N.
  • any site desired may be produced, e.g., by ligating nucleotide sequences (linkers) onto the termini.
  • linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated by the use of the polymerase chain reaction (PCR). See, e.g., Saiki et al., Science, 1988, 239:48.
  • PCR polymerase chain reaction
  • the nucleic acids may be isolated directly from cells or may be chemically synthesized using known methods.
  • the polymerase chain reaction (PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates.
  • Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate inco ⁇ oration into a given vector for recombinant expression.
  • the nucleic acids of the present invention may be flanked by native gene sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like.
  • the invention also provides nucleic acid vectors comprising the disclosed genes or derivatives or fragments thereof.
  • a large number of vectors, including plasmid and fungal vectors have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple cloning or protein expression.
  • Non-limiting examples of suitable vectors include without limitation pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP (Invitrogen, San Diego, CA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • the particular choice of vector/host is not critical to the practice of the invention.
  • Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl 2 mediated DNA uptake, calcium phosphate precipitation, fungal or viral infection, lipofection, microinjection, microprojectile, or other established methods.
  • Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells.
  • a large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art.
  • host cells can be used as a source of recombinantly produced ACE-, AGT-, or ATI -derived peptides and polypeptides.
  • Nucleic acids encoding ACE-, AGT-, or ATI-derived gene sequences may also be introduced into cells by recombination events.
  • such a sequence can be introduced into a cell and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene.
  • Other recombination-based methods such as nonhomologous recombinations or deletion of endogenous genes by homologous recombination may also be used.
  • the nucleic acids of the present invention find use as probes for the detection of genetic polymo ⁇ hisms, as primers for the expression of polymo ⁇ hisms, or in molecular library arrays for high throughput screening.
  • Probes in accordance with the present invention comprise without limitation isolated nucleic acids of about 10 - 100 bp, preferably 15-75 bp and most preferably 17-25 bp in length, which hybridize at high stringency to one or more of the gene-derived polymo ⁇ hic sequences disclosed herein or to a sequence immediately adjacent to a polymo ⁇ hic position.
  • a full-length gene sequence may be used as a probe.
  • the probes span the polymo ⁇ hic positions in the genes disclosed above.
  • the probes correspond to sequences immediately adjacent to the polymo ⁇ hic positions.
  • oligonucleotide nucleic acids may also be modified by many means known in the art.
  • modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. PNAs are also included.
  • the nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • PCR amplification of gene segments that contain a polymo ⁇ hism provides a powerful tool for detecting the polymo ⁇ hism.
  • the oligonucleotides of the invention can also be used as PCR primers to amplify gene segments containing a polymo ⁇ hism of interest.
  • the amplified segment can be evaluated for the presence or absence of a polymo ⁇ hism by restriction endonuclease activity, SSCP, or by direct sequencing.
  • the primer is specific for a polymo ⁇ hic sequence on the gene. If the polymo ⁇ hism is present, the primer can hybridize and DNA will be produced by PCR. However, if the polymo ⁇ hism is absent, the primer will not hybridize, and no DNA will be produced. Thus, PCR can be used to directly evaluate whether a polymo ⁇ hism is present or absent.
  • Molecular library arrays of oligonucleotides are another powerful tool for rapidly assessing whether one or more polymo ⁇ hisms are present in a ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor gene, preferably in combination with other genes.
  • Molecular library arrays are disclosed in US Patents No. 5,677,195, No. 5,599,695, No. 5,545,531, and No. 5,510,270.
  • the present invention encompasses isolated peptides and polypeptides encoded by all or a portion of a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor, comprising polymo ⁇ hic positions disclosed above.
  • a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ⁇ -adrenoceptor, comprising polymo ⁇ hic positions disclosed above.
  • the peptides and polypeptides are useful screening targets to identify cardiovascular drugs.
  • the peptides and polypeptides are capable of eliciting antibodies in a suitable host animal that react specifically with a polypeptide comprising the polymo ⁇ hic position and distinguish it from other polypeptides having a different amino acid sequence at that position.
  • Polypeptides according to the invention are preferably at least five or more residues in length, preferably at least fifteen residues. Methods for obtaining these polypeptides are described below. Many conventional techniques in protein biochemistry and immunology are used.
  • Nucleic acids comprising protein-coding sequences can be used to direct the recombinant expression of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor-derived polypeptides in intact cells or in cell-free translation systems.
  • the known genetic code tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences.
  • polypeptides may be isolated from human cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an appropriate protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins. Peptides and polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis as described by Merrifield, J. Am. Chem. Soc, 1963, 85:2149.
  • polypeptide purification is well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution.
  • the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies produced against ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.
  • nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e., function-conservative variants.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids.
  • the isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to. radioisotopes and fluorescent compounds.
  • the present invention also encompasses antibodies that specifically recognize the polymo ⁇ hic positions of the invention and distinguish a peptide or polypeptide containing a particular polymo ⁇ hism from one that contains a different sequence at that position.
  • Such polymo ⁇ hic position-specific antibodies according to the present invention include polyclonal and monoclonal antibodies.
  • the antibodies may be elicited in an animal host by immunization with ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor-derived immunogenic components or may be formed by in vitro immunization of immune cells.
  • the immunogenic components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems.
  • the antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA.
  • the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains.
  • the antibodies include hybrid antibodies (i.e., containing two sets of heavy chain/light chain combinations, each of which recognizes a different antigen), chimeric antibodies (i.e., in which either the heavy chains, light chains, or both, are fusion proteins), and univalent antibodies (i.e., comprised of a heavy chain/light chain complex bound to the constant region of a second heavy chain).
  • Fab fragments including Fab' and F(ab) 2 fragments of antibodies.
  • Epstein-Barr virus See, e.g., Schreier et al, Hybridoma Techniques, 1980; U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491 ,632; and 4,493,890.
  • Panels of monoclonal antibodies produced against ACE, AGT, or ATI-derived epitopes can be screened for various properties; i.e. for isotype, epitope affinity, etc.
  • the antibodies of this invention can be purified by standard methods, including but not limited to preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. Purification methods for antibodies are disclosed, e.g., in The Art of Antibody Purification, 1989 (Amicon Division, W.R. Grace & Co.) General protein purification methods are described in Protein Purification: Principles and
  • antibodies elicited in response to a peptide comprising a particular polymo ⁇ hic sequence can be tested for their ability to specifically recognize that polymo ⁇ hic sequence, i.e., to bind differentially to a peptide or polypeptide comprising the polymo ⁇ hic sequence and thus distinguish it from a similar peptide or polypeptide containing a different sequence at the same position.
  • the kits comprise a means for determining the sequence at the polymo ⁇ hic positions, and may optionally include data for analysis of polymo ⁇ hic patterns.
  • the means for sequence determination may comprise suitable nucleic acid-based and immunological reagents (see below).
  • kits also comprise suitable buffers, control reagents where appropriate, and directions for determining the sequence at a polymo ⁇ hic position.
  • the kits may also comprise data for correlation of particular polymo ⁇ hic patterns with desirable treatment regimens or other indicators.
  • the invention provides nucleic acid-based methods for detecting polymo ⁇ hic patterns in a biological sample.
  • the sequence at particular polymo ⁇ hic positions in the genes is determined using any suitable means known in the art, including without limitation hybridization with polymo ⁇ hism-specific probes and direct sequencing.
  • diagnostic kits suitable for nucleic acid-based diagnostic applications.
  • diagnostic kits include the following components:
  • Probe DNA The probe DNA may be pre-labelled; alternatively, the probe DNA may be unlabelled and the ingredients for labelling may be included in the kit in separate containers; and (ii) Hybridization reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • diagnostic kits include: (i) Sequence determination primers: Sequencing primers may be pre-labelled or may contain an affinity purification or attachment moiety; and
  • the kit may also contain other suitably packaged reagents and materials needed for the particular sequencing protocol.
  • the kit comprises a panel of sequencing primers, whose sequences correspond to sequences adjacent to the polymo ⁇ hic positions.
  • the invention also provides antibody-based methods for detecting polymo ⁇ hic patterns in a biological sample.
  • the methods comprise the steps of: (i) contacting a sample with one or more antibody preparations, wherein each of the antibody preparations is specific for a particular polymo ⁇ hic form of the gene under conditions in which a stable antigen-antibody complex can form between the antibody and antigenic components in the sample; and (ii) detecting any antigen-antibody complex formed in step (i) using any suitable means known in the art, wherein the detection of a complex indicates the presence of the particular polymo ⁇ hic form in the sample.
  • immunoassays use either a labelled antibody or a labelled antigenic component (e.g., that competes with the antigen in the sample for binding to the antibody).
  • Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules.
  • Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labelled immunoassays, such as ELISA assays.
  • kits suitable for antibody-based diagnostic applications typically include one or more of the following components: (i) Polymorphism-specific antibodies: The antibodies may be pre-labelled; alternatively, the antibody may be unlabelled and the ingredients for labelling may be included in the kit in separate containers, or a secondary, labelled antibody is provided; and (ii) Reaction components: The kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.
  • kits referred to above may include instructions for conducting the test. Furthermore, in preferred embodiments, the diagnostic kits are adaptable to high- throughput and/or automated operation.
  • nucleotide sequences derived from the gene encoding a polymo ⁇ hic form of ACE, ATI, AGT, renin, aldosterone synthase, type- 2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor, and peptide sequences derived from that polymo ⁇ hic for are useful targets to identify cardiovascular drugs, i.e., compounds that are effective in treating one or more clinical symptoms of cardiovascular disease.
  • Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor and (ii) isolated peptides and polypeptides derived from ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor polypeptides, each of which comprises one or more polymo ⁇ hic positions.
  • an isolated nucleic acid comprising one or more polymo ⁇ hic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner.
  • the methods comprise: (i) providing a first nucleic acid containing a particular sequence at a polymo ⁇ hic position and a second nucleic acid whose sequence is identical to that of the first nucleic acid except for a different sequence at the same polymo ⁇ hic position;
  • Selective binding refers to any measurable difference in any parameter of binding, such as, e.g., binding affinity, binding capacity, etc.
  • an isolated peptide or polypeptide comprising one or more polymo ⁇ hic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner.
  • the screening methods involve:
  • high-throughput screening protocols are used to survey a large number of test compounds for their ability to bind the genes or peptides disclosed above in a sequence-specific manner.
  • Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • Intact cells or whole animals expressing polymo ⁇ hic variants of a gene encoding ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor can be used in screening methods to identify candidate cardiovascular drugs.
  • a permanent cell line is established from an individual exhibiting a particular polymo ⁇ hic pattern.
  • cells including without limitation mammalian, insect, yeast, or bacterial cells
  • Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to particular polymo ⁇ hic variants of the gene; (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of the gene; and (iii) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions the gene.
  • transgenic animals are created in which (i) a human ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ⁇ -adrenoceptor gene having different sequences at particular polymo ⁇ hic positions are stably inserted into the genome of the transgenic animal; and/or (ii) the endogenous genes are inactivated and replaced with human genes having different sequences at particular polymo ⁇ hic positions.
  • Such animals can be treated with candidate compounds and monitored for one or more clinical markers of cardiovascular status.
  • populations that are not amenable to an established treatment for a cardiovascular disease or disorder can be selected for testing of alternative treatments.
  • treatments that are not as effective in the general population, but that are highly effective in the selected population, may be identified that otherwise would be overlooked. This is an especially powerful advantage of the present invention, since it eliminates some of the randomness associated with clinical trials.
  • Example 1 Methods for Identification of Polymorphic Positions in Human Genes Encoding ACE. AGT. and ATI
  • DNA samples were obtained from 277 individuals. The individuals were Caucasian males bom in Uppsala, Sweden between 1920 and 1924. Individuals were selected for the test population based on their medical history, i.e., they were either (i) healthy, with no signs of cardiovascular disease (100); or (ii) had suffered one of acute myocardial infarction (68), silent myocardial infarction (34), stroke (18), stroke and acute myocardial infarction (19), or high blood pressure at age 50 (39). DNA samples were obtained from each individual.
  • DNA sequence analysis was carried out by: (i) amplifying short fragments of each of the ACE, AGT, and ATI genes using polymerase chain reaction (PCR) and (ii) sequencing the amplified fragments. The sequences obtained from each individual were then compared with known ACE, AGT, and ATI genomic sequences (see Table 1).
  • the designation "i-4: 1-200” indicates that the primer sequence is located within the sequence extending 200 bp upstream of, and including, the nucleotide immediately upstream of the first coding nucleotide of exon 4.
  • the designation "i+4: 1-200” indicates that the primer sequence is located within the sequence extending from the nucleotide that is located immediately downstream of the last coding nucleotide of exon 4 downstream for 200 bp.
  • the specific location of the primer sequence is indicated in Table 2 in the column marked "Nucleotides”.
  • the amplified fragments are described in Table 5 below with respect to the primers and PCR reaction conditions used for amplification.
  • CGACGTTGTAAAACGACGGCCAGT-3' SEQ ID NO: 122
  • the primer was fluorescently labeled with a Cy-5-molecule on the 5 '-nucleotide.
  • the positions carrying a genetic variation were identified by determination of the nucleotide sequence by the use of the ALFexpressTM system commercially available from Pharmacia Biotech.
  • the detection of the fragment ACEDI was performed by analyzing the sizes of the amplified fragments by gel electrophoresis, where the presence of a shorter PCR product (192 base pairs) indicated the D-allele and a longer PCR product (479 base pairs) indicated the I-allele. The presence of both bands indicated a heterozygote for the two alleles.
  • the detection of the allele-specific reaction of position ATI- 1271 was performed by separately running two parallel PCR reactions on the same sample and comparing the sizes of the amplified fragments.
  • a PCR product of 501 base pairs should always be present as a control in both parallel runs, whereas the presence of a PCR product of 378 base pairs in the reaction designated ATI -spec. 1 indicated the presence of an A in this position.
  • the presence of a PCR product of 378 base pairs in the reaction designated AT1- spec. 2 indicated a C in this position. If the shorter PCR product was present in both reactions, the individual is a heterozygote for A and C.
  • results The analysis described above resulted in the identification of polymorphic positions within the regulatory and coding/intron segments of the human genes encoding ACE, AGT, and ATI .
  • the polymo ⁇ hic positions, the variant nucleotides found at each of the positions, and the PCR fragment in which the polymo ⁇ hism was identified are shown in Table 6 below. Also shown are the frequencies of each genotype in a population of 90 individuals, expressed as the percent of the study population having that genotype. Polymo ⁇ hisms that resulted in alternate amino acids in ACE, AGT, or ATI are also indicated.
  • Table 7 shows the polymo ⁇ hic positions, the sequence at these positions, and the genotype frequencies for each position in a population of 277 as described in Example 1 above.
  • polymo ⁇ hic positions identified as in Example 1 were correlated with the following markers of cardiovascular status present in the study population: healthy (100 individuals evaluated); myocardial infarction (MI) (120 individuals); stroke (37 individuals); and high blood pressure (BP) (39 individuals).
  • MI myocardial infarction
  • BP high blood pressure
  • Two groups of hypertensive patients were studied, 41 in the first group and 20 in the second group.
  • the groups were analyzed independently and in combination.
  • the patients in this population were each treated with one of the following five ACE inhibitors: Captopril, Trandolapril, Lisinopril, Fosinopril, or Enalapril.
  • Captopril Captopril
  • Trandolapril Lisinopril
  • Fosinopril or Enalapril.
  • the effect of the drugs on mean arterial blood pressure was quantified.
  • Mean arterial blood pressure was defined as 2/3 of the diastolic blood pressure + 1/3 of systolic blood pressure.
  • the individuals were also categorized as "high responders,” i.e., those exhibiting a decrease of more than 16 mm Hg during treatment with an ACE inhibitor drug, and "low responders,” i.e., those not exhibiting a decrease of more than 16 mm Hg.
  • Example 4 Correlation Between a Specific Polymorphism Pattern and Treatment Response or Predisposition to a Cardiovascular Syndrome
  • Polymo ⁇ hic patterns i.e., combinations of sequences at particular polymo ⁇ hic positions, that show a statistically significant correlation with myocardial infarction and stroke are shown below:
  • Example 5 Correlation Between a Specific Polymorphism Pattern and High Blood Pressure Treatment with a ⁇ -Blocker
  • BlP putative promoter region of the gene encoding the beta adrenergic receptor 1 BlR
  • ADBR2 the protein coding region of the gene encoding the beta adrenergic receptor 1 B2P
  • ADBR2 putative promoter region of the gene encoding the beta adrenergic receptor 2 B2R
  • ADBR2 the protein coding region of the gene encoding the beta adrenergic receptor 2
  • Beta-blockers These positions, one or a combination of several, can be used to predict a good response to anti-hypertensive treatment with Beta-blockers.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Neurology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides methods for assessing cardiovascular status in an individual, which comprise determining the sequence at one or more polymorphic positions within the human genes encoding ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor. The invention also provides isolated nucleic acids encoding polymorphisms in genes encoding ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor and β-adrenoceptor, nucleic acid probes that hybridized to polymorphic positions, kits for the prediction of cardiovascular status, and nucleic acid and peptide targets for use in identifying candidate cardiovascular drugs.

Description

GENES FOR ASSESSING CARDIOVASCULAR STATUS AND COMPOSITIONS FOR USE THEREOF
FIELD OF THE INVENTION The present invention relates to genetic polymorphisms and polymorphism patterns useful for assessing cardiovascular status in humans. More particularly, the invention relates to identifying and using polymoφhism patterns comprising a polymoφhism in the a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor to predict a treatment outcome or likelihood of developing cardiovascular disease, and to assist in diagnosis and in prescription of an effective therapeutic regimen.
BACKGROUND OF THE INVENTION The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular physiology in mammals. Specifically, RAAS regulates salt-water homeostasis and the maintenance of vascular tone. Stimulation or inhibition of this system raises or lowers blood pressure, respectively, and disturbances in this system may be involved in the etiology of, for example, hypertension, stroke, and myocardial infarction. The RAAS system may also have other functions such as, e.g., control of cell growth. The renin-angiotensin system includes renin, angiotensin converting enzyme (ACE), angiotensinogen (AGT), type 1 angiotensin II receptor (ATI), type 2 angiotensin II receptor (AT2) and aldosterone synthase.
International Patent Application No. PCT/IB98/00475, filed April 1, 1998, discloses for the first time an association of polymoφhism patterns in ACE, AGT, and ATI genes with cardiovascular status, particularly with the ability to predict the therapeutic outcome of a particular treatment regimen.
Other genes express polypeptides involved in pathways other than RAAS that also play a role in the regulation of cardiovascular physiology, although it is believed that prior to the present invention, no known association of polymoφhism patterns in these genes with cardiovascular status has been observed or reported. Among these other regulators of cardiovascular physiology are endothelin, endothelin receptor, and β-adrenergic receptors 1 and 2.
RAAS Pathway Components AGT is the specific substrate of renin, an aspartyl protease. The human AGT gene contains five exons and four introns which span 13Kb (Gaillard et al, DNA, 1989, 8:87-99; Fukamizu et al. J. Biol. Chem., 1990, 265:7576-7582). The first exon (37 bp) codes for the 5' untranslated region of the mRNA. The second exon codes for the signal peptide and the first 252 amino acids of the mature protein. Exons 3 and 4 are shorter and code for 90 and 48 amino acids, respectively. Exon 5 contains a short coding sequence (62 amino acids) and the 3'-untranslated region.
Plasma AGT is synthesized primarily in the liver and its expression is positively regulated by estrogens, glucocorticoids, thyroid hormones, and angiotensin II (Ang II) (Clauser et al, Am. J. Hypertension, 1989, 2:403-410). Cleavage of the amino- terminal segment of AGT by renin releases a decapeptide prohormone, angiotensin-I, which is further processed to the active octapeptide angiotensin II by the dipeptidyl carboxypeptidase designated angiotensin-converting enzyme (ACE). Cleavage of AGT by renin is the rate-limiting step in the activation of the renin-angiotensin system.
Several epidemiological observations indicate a possible role of AGT in blood pressure regulation. A highly significant correlation between plasma AGT concentration and blood pressure has been observed in epidemiological studies (Walker et al, J. Hypertension, 1979, 1 :287-291 ). Interestingly, a number of allelic dimoφhisms have been identified in the AGT gene. The frequency of at least two of them (174M and 235T) have been partially characterized and in certain populations shown to be significantly elevated in hypertensive subjects (Jeunemaitre et al, Cell, 1992, 71 : 169- 180). In addition, a specific polymoφhism, 235T, has been suggested to be directly involved in coronary atherosclerosis (Ishigami et al, Circulation, 1995, 91 :951-4). Futhermore, the presence of A or G at position 1218 in the AGT regulatory region has been correlated with differences in in vitro transcriptional capacity for this gene (Inoue et. al., J. Gin. Invest., 1997, 99: 1786). However, the foregoing are studies involving only one or at most two polymorphisms. Furthermore, the sole disclosed use is susceptibility to disease.
The human ACE gene is also a candidate as a marker for hypertension and myocardial infarction. ACE inhibitors constitute an important and effective therapeutic approach 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 the inactive angiotensin I molecule (Ang I) into active angiotensin II (Ang II) (Bottari et al, Front. Neuroendocrinology, 1993, 14: 123-171). Another ACE substrate is bradykinin, a potent vasodilator and inhibitor of smooth muscle cell proliferation, which is inactivated by ACE (Ehlers et al, Biochemistry, 1989, 28:531 1-5318; Erdos, E.G., Hypertension, 1990, 16:363-370; Johnston, C.I. Drugs (suppl. 1), 1990, 39:21-31).
Levels of ACE are very stable within individuals, but differ greatly between individuals. A greater risk of myocardial infarction has been identified in a group of subjects with an ACE polymoφhism designated ACE-DD (Cambien et al, Nature, 1992, 359:641-644), and a 12-fold greater risk of myocardial infarction has been identified in a subgroup of patients having a combination of the ACE polymoφhism ACE-DD and one of the AGT polymoφhisms (235T) described above (Kamitani et al, Hypertension, 1994, 24:381). Recently, six ACE polymoφhisms were identified and characterized (Villard et al, Am. J. Human Genet., 1996, 58: 1268-1278).
The vasoconstrictive, cell growth-promoting and salt conserving actions of angiotensin II are mediated through binding to and activation of angiotensin receptors, of which at least two types have been cloned (ATI and AT2). The type 1 Ang II receptor (ATI), a G-protein-coupled seven transmembrane domain protein, is widely distributed in the body and mediates almost all known Ang II effects (Fyhrquist et al, J. Hum.
Hypertension, 1995, 5:519-524).
Several polymoφhisms have been identified in the ATI receptor gene. Initial studies suggest that at least one of them is more frequent in hypertensive subjects (AT"66C)(Bonnardeaux et al, Hypertension, 1994, 24:63-69). This polymoφhism, combined with the ACE-DD polymoφhism, has been shown to correlate strongly with the risk of myocardial infarction (Tiret et al, Lancet, 1994, 344:910-913).
Endothelin (ET) Regulation of Cardiovascular Physiology
Endothelin is a potent vasoconstrictive peptide characterized by long lasting action. It was first discovered as a vasoconstricting factor in conditioned medium (Hickey et al, Am. J. Physiol., 1985, 248:C550), and subsequently purified and characterized (Yanagisawa et al, Nature, 1988, 332:411). ET is produced as preproendothelin, which is cleaved after removal of the signal sequence by an endopeptidase, followed by cleavage with endothelin converting enzyme (Xu et al, Cell, 1994, 78:473: Shimada et al, J. Biol. Chem., 1994, 269: 18274). Analysis of the human Eu gene has revealed the existence of two additional ΕT-like peptides expressed in various tissues, termed ΕT-2 and ET-3 (Inoue et al, Proc. Natl. Acad. Sci. USA, 1989, 86:2863). The first endothelin was accordingly termed ET-1.
One of the important discoveries following characterization of ETs was the discovery of two ET receptors, ETA and ETB (Arai et al, Nature, 1990, 348:730; Sakumi et al, Nature, 1990, 348:782). Both belong to the family of heptahelical G-protein coupled receptors. There is 68% amino acid identity between the two receptor subtypes. ETA exists as a single copy gene located on human chromosome 4 (Hosoda et al, J. Biol. Chem., 1992, 267: 18797; Cyr et a/., Biochem. Biophys. Res. Commun., 1991, 181: 184). ETB exists as a single copy gene located on human chromosome 13 (Arai et al, J. Biol. Chem., 1993, 268:3463-70), although a splice variant of ETB has been found (Shyamala et al, Cell. Mol. Biol. Res., 1994, 40:285-96). The cDNA sequence of ETA has been deposited with GenBank with accession number S57498.
In the early stage of ET research, a great number of pharmacological studies suggested that the responses to ETs could be divided into two groups according to the pharmacological potency of the three peptides. Indeed, these two receptors, ETΛ and ETB, are distinct in their ligand binding affinity and distribution in tissues and cells. ETA has a high affinity to ET-1 and ET-2, but a low affinity to ET-3. ETB has equally potent affinities to all three endogenous ETs. ETA exists on smooth muscle and mediates vasoconstriction. In contrast, ETB exists on endothelium and mediates the release of relaxing factors such as nitric oxide and prostacycline. However, several reports demonstrated that ETB on some vascular smooth muscle also mediated vasoconstriction.
Cloning of the ET receptor gene facilitated the development of ET-receptor antagonists, such as BE-18257B, and BQ-123 and FR139317, two derivatives of BE-18257B (See Masaki, Cardiovascular Res., 1998, 39:530). Many selective and non-selective antagonists for ETA and ETB have emerged.
Although the pathophysiological role of ET is still unclear, ET antagonists demonstrated significant beneficial effects in pathological conditions, including congestive heart failure, pulmonary hypertension, cerebrovascular spasm after subarachnoid hemorrhage, acute renal failure, and essential hypertension. ET or ET receptor knockout mice have also provided important information regarding the physiological and pathophysiological significance of ET (Masaki, supra). In particular, mice with a knockout of ET-3 or the ETB receptor genes exhibit phenotypic changes that resemble Hirschsprung's disease, a human hereditary syndrome associated with a missense mutation of the ETB gene (Pfiffenberger, et al, Cell, 1994, 79: 1257).
Despite advances in understanding the role of the endothelin pathway in the treatment of cardiovascular diseases, questions remain. Not all experimental models of hypertension respond to endothelin antagonists, and it remains a uncertain whether endothelin antagonists improve cardiac structure and function beyond the benefits of blood pressure reduction (Moreau, Cardiovascular Res., 1998, 39:534). Thus, there is a need in the art for a reliable and effective means for predicting whether endothelin antagonists will be effective for treating hypertension in a given individual.
β-Adrenergic Receptors (β-Adrenoceptors) The adrenoceptors fall into three major groups, ,, α2, β, within each of which further subtypes can be distinguished pharmacologically (Lϋllmann, et al. in Color Atlas of Pharmacology, New York, 1993). Adrenergic receptors are all G-protein linked. They are involved in regulation of the cardiovascular system, and in the control of metabolic activity, e.g., insulin secretion and glucose release. They also mediate constriction or relaxation of smooth muscle cells in the respiratory, gastrointestinal, and genitourinary tracts (Berne and Levy, Principles of Physiology (2nd Ed.), Mosby-Year Books, Inc., 1996, pp. 691-696). Adrenoceptors are targets for epinephrine and norepinephrine, which are representatives of the family of monoamine neurotransmitters. Epinephrine has equally high affinity for all α- and β-receptors while norepinephrine differs from epinephrine by its low affinity for β2-receptors (The Biochemical Basis of Neuropharmacology, (7th Ed.) New York, 1996, pp. 226-292). The adrenoceptors themselves interact preferentially with three different classes of G-proteins: Gs (β-adrenoceptors) mediating activation of adenylate cyclase, G, (cc2-adrenoceptors) mediating inhibition of adenylate cyclase, and Gq (α, -adrenoceptors) mediating activation of phospholipase C (Hieble et al, ]. of Med. Chem., 1995, 38:3415-3444). The pharmacological interest of adrenoceptors is mainly for the treatment of cardiovascular diseases, e.g.. through the development of β-antagonists, ,-antagonists and α2-agonists to treat hypertension, but they are also considered important for the treatment of asthma (β2-agonists).
The β2-adrenergic receptor is expressed on a number of cell types, e.g., bronchial smooth muscle, where its activation results in relaxation and bronchial dilatation. These receptors are also being expressed on epithelial cells, vascular endothelium, alveolar walls, immune cells, and presynaptic nerve terminals (Liggett, Am. J. Respir. Crit. Care. Med., 1997, 156:S156-S162). Cardiac cells express mainly β,-, but also a small fraction of β2-adrenoceptors (Collins et al, Biochimica et Biophysica Acta, 1993, 1172:171-174). β, -adrenoceptors are also expressed in brain and pineal gland.
β-Adrenoceptor Function
The β,- and β2-adrenergic receptors are coupled to a Gs-protein complex, which activates adenylate cyclase. Agonist binding to the β, -receptor, located in cardiac muscle cells, mediates increased contractility and cardiac output (Lϋllmann, supra).
Agonist binding to the β2-receptor, located in peripheral vascular arteries, mediates vasodilation by increasing the amount of cAMP, and thereby inhibiting activation of myosin kinase, which is necessary for smooth muscle cell constriction (ibid.) Activation of cAMP in cardiac cells by agonist binding to both β,- and β-2-adrenoceptors activates the cAMP-dependent protein kinase (PKA) (Castellano and Bδhm, Hypertension, 1997,
29:715-722). β-adrenoceptors also regulate the control of melatonin production in the pineal gland, by the cAMP activation of one of the enzymes (5-HT-N-acetyl transferase) involved in the synthesis of melatonin (Collins et al, supra). The β2-receptors mediate increased conversion of glycogen to glucose (glycogenolysis) in both the liver and skeletal muscle (H. Lullmann, et al, supra), and stimulate influx of potassium into muscle cells to prevent hyperkalemia (Berne and Levy, supra). The β-receptors are regulated on the protein level by desensitization. The initial desensitization process results from the 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 phosphorylates specific serine or threonine residues in the C-terminal of receptors that are occupied by an agonist. The phosphorylation triggers binding of the cytostolic protein β-arrestin and results in the uncoupling from Gsα. PKA is activated by cAMP and phosphorylates the β2-adrenoceptor by a relatively slow process. The phosphorylated receptor loses the ability to activate Gs (Castellano and Bόhm, supra). Prolonged interaction of agonists with adrenoceptors generally results in receptor desensitization.
β-Adrenoceptor Gene Structure The human β,-adrenoceptor gene is located on the long arm of chromosome 10, the same chromosome as for the α2A-adrenoceptor gene. The coding sequence of this gene is deposited with GenBank, accession number X69168. The regulatory region is also deposited with GenBank, accession number J03019. It codes for an intronless gene product of 1431 base pairs (Hall, Thorax, 1996, 51 :351-353). Both the promoter and the coding region of the gene are rich in G and C residues, which make up greater than 70%> of the bases. The promoter does not contain any paired consensus TATA box and CAAT box elements but instead clusters with an inverted CAAT box and SP, or AP-2 binding motifs. This type of receptor, reminiscent of "housekeeping genes", has been described for other G-protein coupled receptors as well (Collins et al, supra).
The human β2-adrenoceptor gene is located on the long arm of chromosome 5, the same chromosome as the I B-adrenoceptor gene. The coding sequence has been deposited with GenBank, with accession numbers Ml 5169, J02728, or Ml 6106. The regulatory region sequence is also deposited with GenBank, accession number Y00106. It codes for an intronless gene product of 1239 base pairs (Hall, supra). The promoter region is 200-300 bases 5' of the translation initiation codon, and it can form strong secondary structures due to high G-C content. There are two TATA boxes (separated by roughly 10 bp) and a CAAT box located approximately 30 and 80 base pairs upstream, respectively, from the mRNA start region.
β-Adrenoceptor Gene Regulation There are some regulatory regions identified in the promoter region of the β,-adrenoceptor gene: a cAMP response element (CRE), a consensus thyroid response element (TRE), and a glucocorticoid response element (GRE). This is consistent with the evidence that both thyroid hormone and corticosteroids affect adrenergic sensitivity in both heart and adipose tissue. The CRE region might have a self-regulatory function, as has been shown for the β2-adrenoceptor gene (Collins et al., supra).
There are several regulatory domains in the 5' flanking region of the β2-adrenoceptor. Among these is a cAMP-responsive element (CRE), which is recognized and stimulated by a phosphoprotein called CRE binding protein (CREB). CREB is partially under the control of PKA-dependent phosphorylation processes. This is seen as an increase in β2-adrenoceptor mRNA level in the early phase after exposure to β-agonists. However, the level of mRNA is decreased after prolonged exposure to agonists, probably mediated by a shortening of mRNA half-life (Castellano and Bδhm, supra). It has also been shown that transcription of the β2-adrenoceptor gene is upregulated by stimulation with glucocorticoids in a variety of tissues (Collins et al., supra). In the 3' flanking region there are sequences homologous to glucocorticoid response elements. These might be responsible for the increased expression of β2 adrenoceptor observed in transfected cells after treatment with hydrocortisone (Emorine and Marullo, Proc. Natl. Acad. Sci. USA, 1987, 84:6995-6999).
β-Adrenoceptor Protein Structure
The proposed model for β-adrenoceptors is like most of the G-protein binding receptors, a seven -helical transmembrane structure, where the seven α-helices are radially arranged around a central "pore", in which the receptor ligands bind. The β-adrenoceptors have an extracellular glycocylated N-terminus, and an intracellular C-terminus. The β,-receptor consists of 477 amino acids; the β2-receptor consists of 413 amino acids.
The overall amino acid identity of human β,- and β2- adrenoceptors is only 54%. However, it is likely that the pharmacological differences between β2-receptors and β, -receptors are due to subtle changes in orientation of the primary binding sites, resulting in a slightly different binding site rather than to specific amino acid substitutions (Hieble et al, J. Med. Chem. 1995, 38:3415-3444).
Site-directed mutagenesis has demonstrated that an aspartic acid residue, Asp-113, located in the third transmembrane-spanning helix, and two serine residues, Ser-204 and Ser-207, are required for full agonist binding to the β2-adrenoceptor. The β,-adrenoceptor contains identical amino acid residues located in corresponding positions to those shown to be important for agonist binding to the β2-adrenoceptor. Another aspartic acid residue, Asp-79, located in the second -helix of both β-receptors is highly conserved in G-protein coupled receptors (Hieble et al, supra). Ser-319 has a potential role in agonist binding to the β^-adrenoceptor.
Mutation of Tyr-350, located in the cytoplasmic tail of the β2-receptor, interferes with coupling of the receptor to Gs (Hieble et al, supra). Also, palmitoylation of Cys-341 in the C-terminal enables the β2-adrenoceptor to form a fourth intracytoplasmic loop, which increases the ability of the agonist-bound receptor to mediate adenylyl cyclase stimulation (Strosberg, Preotein Science, 1993, 2: 1198-1209).
β-Adrenoceptors as Drug Tarεets
No cause of disease can be identified in 80-90% of patients with hypertension. They have so-called essential hypertension, which affects 5-10% of the general population, and is the most common cause of disease in developed countries ( J. Axford, Medicine, Blackwell Science Ltd., 1996, 10.119-10.130). Betablockers have been widely used in the treatment of hypertension. They are particularly useful for the treatment of juvenile hypertension with tachycardia and high cardiac output. Betablockers or beta-adrenergic blockers were first introduced as a treatment for essential hypertension in 1964, and are still recommended as first choice because the cost for betablockers is low, which improves patient compliance. They act by binding to β, -receptors on the cardiac smooth muscle cells, which leads to decreased cardiac output. Most betablockers are not specific β,-receptor antagonists but binds to β2-receptors as well. The binding to β2-receptors gives the opposite of the desired effect though inhibition of β2-receptors leads to vasoconstriction. This gives a side effect with cold hands and feet because most of the β2-receptors are located in the peripheral vascular arteries. WO 00/22166 _ 1 Q _ PCT/IB99/01678
They have also been known to cause bronchospasms as well as some central nervous system side effects (nightmares, somnolence). They decrease insulin secretion, which makes them inappropriate to treat hypertensives with diabetes mellitus, and they can cause heart failure and peripheral artery obstructive disease (Velaseco and Rodrigues, Journal of Human Hypertension 10, Suppl. 1, S77-S80, 1996). β-adrenoceptor agonists, such as dopamine and dobutamine are used to stimulate myocardial β, -adrenoceptors in the acute management of congestive heart failure. They act by increasing contractility and cardiac output.
A lot of different β2-agonists are used in the treatment of asthma. They exert their primary effect on the β2-adrenergic receptor of bronchial smooth muscle, resulting in relaxation and bronchial dilatation. They also protect against bronchoconstrictor challenge (Hall, supra).
Thus, there is a clear need in the art for an improved understanding of the effects of betablockers on different subjects, and to predict which patients will have a better response to treatment with betablockers.
Need for Effective Cardiovascular Status Assessment
The high morbidity and mortality associated with cardiovascular disease demonstrate a need in the art for methods and compositions that allow the determination and/or prediction of the therapeutic regimen that will result in the most effective treatment outcome in a patient suffering from cardiovascular disease. This includes identification of individuals who are more or less responsive to particular therapeutic regimens, including, e.g., particular drugs that are conventionally used to treat cardiovascular disease.
Furthermore, the heterogeneity in responses to cardiovascular therapies emphasizes a need for another approach to rational drug development. In particular, populations that are identified as non-responsive to a particular therapeutic regimen can be identified for development and testing of alternative regimens. Thus, effective treatment regimens could be developed for a larger percentage of the affected population.
In summary, there is a need to reduce or eliminate trial and error in selecting a therapeutic regimen for a particular individual. It would be desirable instead to predict whether a given individual will be responsive to, e.g., a particular class of drugs or even to a particular drug or whether he/she is likely to suffer from adverse reactions or side-effects. There is also a need in the art for methods and compositions that allow the identification of individuals having a predisposition to cardiovascular disease, such as, e.g., myocardial infarction, hypertension, atherosclerosis, and stroke, to facilitate early intervention and disease prevention. The present invention addresses these and other needs in the art by providing polymoφhisms and polymoφhic patterns that are characteristic of cardiovascular status, and by using these polymoφhisms and patterns to prescribe or to develop more effective treatments or to assist in diagnosis.
Citation of any reference in this application should not be construed as an admission that the reference is prior art to the invention. Each cited reference is incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides reagents and methods for predicting whether a particular therapeutic regime (such as a specific drug, a class of drugs or any other therapeutic regime, pharmacological or not) would be effective in improving a cardiovascular condition in a human individual, or would be ineffective for that puφose, or its use would be associated with adverse reactions or undesirable side-effects.
A particular advantage of the invention is that one or more polymoφhic markers provide a basis for predicting the outcome of a treatment regimen. By comparing a polymoφhic pattern of a subject who requires treatment for a cardiovascular disorder, for example hypertension, with a reference pattern previously established to correlate with responsivity to the treatment regimen, a physician can predict whether a treatment plan, such as administration of an ACE inhibitor, is likely or not to be effective before subjecting the subject to the treatment plan. For example, a comparison of the test polymoφhic pattern from an individual with reference polymoφhic patterns of individuals exhibiting differing responses to a particular therapeutic intervention can be used to predict the type or degree of responsivity of the individual to such intervention. The present invention thus represents a significant breakthrough in treating cardiovascular pathologies in that it reduces or eliminates trial and error in selecting a treatment for a particular individual cardiovascular patient.
An additional advantage of the invention derives from the ability to eliminate subjects from clinical trials who are predictably non-responsive, or at risk for an adverse response, to a particular treatment regimen. Furthermore, adverse results in an early trial can be evaluated to identify polymorphic patterns, so that the adverse results can be correlated with a sub-population of the test population permitting exclusion of such sub-population from the treatment group. The invention may thus ensure that a beneficial drug can be approved for use in the appropriate population, and decrease the number of required patients and therefore the duration and cost of clinical trials. It may also lead to identification of another subgroup which can be the target for development of another therapeutic regimen.
All of the foregoing applications within the scope of the invention can be deemed to be assessments of an individual's cardiovascular status, as the term is broadly defined below.
The foregoing methods of the invention are carried out by comparing a test polymoφhic pattern established by at least one polymoφhic position within a gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor with a polymoφhic pattern of a population of individuals exhibiting a predetermined responsivity to the regimen (reference pattern). If the test pattern matches the reference pattern, there is a statistically significant probability that the individual has the same cardiovascular status as that correlated with the reference pattern. The polymoφhic pattern preferably consists of at least two (and more preferably at least three) polymoφhic positions, at least one of which is in the gene encoding a polypeptide from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor, and a second polymoφhism in a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor.
Additionally, the invention provides methods for assessing whether a particular individual has a genetic predisposition to a cardiovascular pathology. This aspect of the invention comprises comparing a test polymoφhic pattern established by at least one and preferably at least two and most preferably at least three polymoφhic positions within a gene encoding ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor in conjunction with one or more, and preferably two or more, other polymoφhic positions in ACE, ATI , AGT, renin, WO 00/22166 _ χ 3 _ PCT/IB99/01678
aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β- adrenoceptor, with a polymoφhic pattern of individuals exhibiting a predisposition to a cardiovascular syndrome. The conclusion drawn depends on whether the individual's polyymoφhism pattern matches the reference pattern. The invention also provides an isolated nucleic acid having a sequence corresponding to part or all of the gene encoding ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor, the nucleic acid comprising a polymoφhism in the ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor gene. In preferred embodiments, the polymoφhism, in combination with one or more other polymoφhisms in the sequence of the same gene or a gene encoding a protein selected from the group consisting of human ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor, is predictive of a particular type or level of responsivity to a given treatment, or indicates a predisposition to one or more clinical syndromes associated with cardiovascular disease, or both. The isolated polymoφhisms according to the invention (which are described using the numbering indicated in Table 1 below) include without limitation:
Nucleic acids encoding renin having one or more polymoφhic positions in the last exon of the gene (exon 10), which is a cytosine to thymine transition that creates a premature stop codon at position 387.
Nucleic acids encoding aldosterone synthase promoter at position -344. Nucleic acids encoding β-adrenergic receptor- 1 regulator region, at positions 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724, and 2757, as numbered in GenBank accession number X69168. In preferred embodiments, the bases at specific positions are 2238 G, 2238 A, 2577 C, 2257 T, 2757 A, and 2757 G.
Nucleic acids encoding β-adrenergic receptor- 1 coding region, at positions 231, 758, 1037, 1251, 1403, and 1528, as numbered in GenBank accession number J03019. In preferred embodiments, the bases at specific positions are 231 A, 231 G, 1251 C, 1251 G, 1403 A, 1403 G, 1528 C, and 1528 A. Nucleic acids encoding β-adrenergic receptor-2 regulatory region, at positions 932, 934, 987, 1006. 1 120, 1221, 1541, and 1568, as numbered in GenBank accession number M 15169, or J02728, or M 16106. In preferred embodiments, the bases at specific positions are 934 A. 934 G, 987 C, 987 G, 1006 A, 1006 G, 1 120 C, 1 120 G, 1221 C, 1221 T, 1541 C, 1541 T, 1568 C, and 1568 T..
Nucleic acids encoding β-adrenergic receptor-2 coding region, at positions 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070, as numbered in GenBank accession number Y00106. In preferred embodiments, the bases at specific positions are 839 A, 839 G, 872 C, 872 G. 1045A, 1045 G, 1284 C, 1284 T, 1316 A, 1316 C, 1846 C, 1846 G, 2032 A, 2032 G, 2068 no insert, 2068 G, 2068 C, 2070 no insert, and 2070 C.
Nucleic acids encoding endothelin-A receptor at positions 969, 1005, 1 146, and 2485, as numbered in GenBank accession number S57498. In preferred embodiments, the bases at specific positions are 969 C, 969 T, 1005 A, 1005 G, 1146 A, 1146 G, 2485 T, and 2485 C.
Nucleic acids comprising polymoφhisms present in other genes, which can be used in combination with a polymoφhism from a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor to establish a polymorhism pattern, have been disclosed in International Patent Application No. PCT/IB98/00475, and include:
(i) Nucleic acids encoding ACE having one or more polymoφhic positions at the position in the regulatory region numbered 5106; positions in the coding region numbered 375, 582, 731, 1060. 2741, 3132, 3387, 3503, and 3906; and position 1451 all positions as numbered in GenBank entry X62855. In preferred embodiments, the sequences at the polymoφhic positions in the ACE regulatory region are one or more of 5106C and 5106T; and the sequences at the polymoφhic positions in the coding region are one or more of 375A, 375C, 582C, 582T, 731A, 731G, 1060G, 1060A, 2741G, 2741T, 3132C, 3132T, 3387T, 3387C. 3503G, 3503C, 3906G, and 3906A. The invention also encompasses a nucleic acid encoding a deletion of nucleotides 1451-1783 as numbered in GenBank entry X62855.
(ii) Nucleic acids encoding AGT having one or more polymoφhic positions at positions in the regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072, and 1204; positions in the coding region numbered 273, 912, 997, 11 16, and 1 174; and position 49 as numbered in GenBank entry M24688. In preferred embodiments, the sequences at the polymoφhic positions in the AGT regulatory region are one or more of 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 692C, 692T, 839G, 839A, 1007G, 1007A, 1072G, 1072A, 1204C, and 1204A; the sequences at the polymoφhic position in the coding region are one or more of 273C, 273T, 912C, 912T, 997G, 997C, 1116G, 1 116A, 1 174C and 1174A; and the sequence at position 49 in GenBank entry M24688 is either A or G. (iii) Nucleic acids encoding ATI having one or more polymoφhic positions at positions in the regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; and the position in the coding region numbered 449. In preferred embodiments, the sequences at the polymoφhic positions in the ATI regulatory region are one or more of 1427A, 1427T, 1756T, 1756A, 1853T, 1853G, 2046T, 2046C, 2354A, 2354C, 2355G, 2355C, 2415A and 2415G; and the sequences at the polymoφhic positions in the coding region are one or more of 449G, 449C, 678T, 678C, 1167A, 1167G, 1271 A, and 1271C.
The invention also encompasses libraries of isolated nucleic acid sequences, such as arrays on a solid surface, wherein each sequence in the library comprises a polymoφhic position in the gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β- adrenoceptor and other polymoφhic positions in the other genes, including without limitation the polymoφhic positions and sequences disclosed herein. Also provided are nucleic acid probes that hybridize specifically to the identified polymoφhic positions; peptides and polypeptides comprising polymoφhic positions; and polymoφhism-specific antibodies, i.e., sequence-specific antibodies that bind differentially to polymoφhic variants of the foregoing genes, that can be used to identify particular polymoφhic variants.
In another aspect, the invention provides kits for the determination of polymoφhic patterns in an individual's genes. The kits comprise a means for detecting polymoφhic sequences, including without limitation oUgonucleotide probes that hybridize at or adjacent to the polymoφhic positions and polymoφhism-specific antibodies.
In yet another aspect, the invention provides nucleic acid and polypeptide targets for use in screening methods to identify candidate cardiovascular drugs. Nucleic acid targets may be, e.g., DNA or RNA and are preferably at least about 10, and most preferably at least about 15, residues in length and comprise one or more polymoφhic positions in a gene encoding a polypeptide from the group consisting of ACE, ATI , 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 polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
The invention in based, in part, on the discovery that polymoφhisms in certain genes in the RAAS, endothelin, and β-adrenoceptors pathways define polymoφhism patterns that correlate with cardiovascular status. Most significantly, by comparing a test individual's polymoφhism pattern with a reference polymoφhism pattern, which is a polymoφhism pattern from a population of individuals with known cardiovascular status, one is able to predict whether the test individual has an increased likelihood to share the same cardiovascular status as that correlated with the reference polymoφhism pattern. In particular, particular patterns correlate with responsiveness to ACE inhibitors, non-responsiveness to ACE inhibitors, and predisposition to cardiovascular diseases or dysfunctions, including myocardial infarction and stroke.
The invention provides a powerful predictive tool for clinical testing and treatment of cardiovascular disease. For clinical testing, the present invention permits smaller, more efficient clinical trials by identifying individuals who are likely to respond poorly to a treatment regimen and reducing the amount of uninteφretable data. By evaluating a test individual's polymoφhism pattern, a physician can prescribe a prophylactic or therapeutic regimen customized to that individual's cardiac status. Adverse responses to particular therapies can be avoided by excluding those individuals whose cardiovascular status puts them at risk for that therapy. Appropriate changes in lifestyle, including diet, environmental stress, and exercise levels can be prescribed for individuals whose test polymoφhic pattern matches a reference pattern that correlates with increased predisposition to cardiovascular disease.
Definitions
"Cardiovascular status" as used herein refers to the physiological status of an individual's cardiovascular system, as reflected in one or more status markers or indicators including genotype. Cardiovascular status shall be deemed to include without limitation not only the absence or presence of a pathology or disease in one or more components of the individual's cardiovascular system and the individual's predisposition to developing such a condition, but also the individual's responsivity, i.e., the ability or inability of the individual to respond (positively or negatively) to a particular prophylactic or therapeutic regimen or treatment for a cardiovascular condition, such as a drug or a class of drugs. A negative response includes one or more adverse reactions and side effects. Status markers include without limitation clinical measurements such as, e.g., blood pressure, electrocardiographic profile, differentiated blood flow analysis, and the presence of increased levels of cellular proteins associated with a cardiovascular event. Examples of such proteins, also called diagnostic markers, which are important in cardiac events include myosin light chain, myosin heavy chain, myoglobin, troponin I, troponin T, CK-MB, etc. (see U.S. Patents No. 5,604,105 and No. 5,744,358). Status markers according to the invention are assessed using conventional methods well known in the art. Also included in the evaluation of cardiovascular status are quantitative or qualitative changes in status markers with time, such as would be used, e.g., in the determination of an individual's response to a particular therapeutic regimen or of a predisposed individual's eventual development of a cardiovascular condition.
Examples of cardiovascular syndromes that are included in the foregoing definition of cardiovascular status include diagnosis of, or predisposition to, one or more cardiovascular syndromes, such as, e.g., hypertension, acute myocardial infarction, silent myocardial infarction, unstable angina, stroke, and atherosclerosis. It will be understood that a diagnosis of a cardiovascular syndrome made by a medical practitioner encompasses not only clinical measurements but also medical judgment.
"Responsivity", as used herein, refers to the type and degree of response an individual exhibits to a particular therapeutic regimen, i.e., the effect of a treatment on an individual. Responsivity breaks down into three major categories: therapeutic effect; no effect; and adverse effect. Naturally, there can be differing degrees of a therapeutic effect, e.g., between full elimination and partial elimination of symptomology. In addition, adverse effects, or side effects, may be observed even though the treatment is beneficial, i.e., therapeutically effective. Indeed, the present invention may permit identification of individuals with complex responsivity traits or patterns. A "predisposition to develop a cardiovascular syndrome" refers to an increased likelihood, relative to the general population, to develop a cardiovascular syndrome, as defined above. A predisposition does not signify certainty, and development of the syndrome may be forestalled or prevented by prophylaxis, e.g., adopting a modified diet, exercise program, or treatment with gene therapy or pharmaceuticals. Naturally, an advantage of the present invention is that it permits identification of individuals who are, based on their genotype, predisposed to develop a cardiovascular syndrome, and for whom prophylactic intervention can be especially important. A "polymoφhism" as used herein denotes a variation in the nucleotide sequence of a gene in an individual. Genes that have different nucleotide sequences as a result of a polymoφhism are "alleles". A "polymoφhic position" is a predetermined nucleotide position within the sequence. In some cases, genetic polymoφhisms are reflected by an amino acid sequence variation, and thus a polymoφhic position can result in location of a polymoφhism in the amino acid sequence at a predetermined position in the sequence of a polypeptide. An individual "homozygous" for a particular polymoφhism is one in which both copies of the gene contain the same sequence at the polymoφhic position. An individual "heterozygous" for a particular polymoφhism is one in which the two copies of the gene contain different sequences at the polymoφhic position.
A "polymoφhism pattern" as used herein denotes a set of one or more polymoφhisms, including without limitation single nucleotide polymoφhisms, which may be contained in the sequence of a single gene or a plurality of genes. In the simplest case, a polymoφhism pattern can consist of a single nucleotide polymbφhism in only one position of one of two alleles of an individual . However, one has to look at both copies of a gene. A polymoφhism pattern that is appropriate for assessing a particular aspect of cardiovascular status (e.g., predisposition to hypertension) need not contain the same number (nor identity, of course) of polymoφhisms as a polymoφhism pattern that would be appropriate for assessing another aspect of cardiovascular status (e.g., responsivity to ACE inhibitors for control of hypertension). A "test polymoφhism pattern" as used herein is a polymoφhism pattern determined for a human subject of undefined cardiovascular status. A "reference polymoφhism pattern" as used herein is determined from a statistically significant correlation of patterns in a population of individuals with predetermined cardiovascular status. "Nucleic acid" or "polynucleotide" as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. Nucleic acids include without limitation single- and double-stranded molecules, i.e., DNA-DNA, DNA- RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases and non-naturally occurring phosphoester analog bonds, such as phosphorothioates and thioesters. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.
As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, cDNA, mRNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a gene of interest, or to detect the presence of nucleic acids encoding the gene of interest. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a double stranded sequence of interest in a DNA molecule. In still another embodiment, a library of oligonucleotides arranged on a solid support, such as a silicon wafer or chip, can be used to detect various polymoφhisms of interest. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds. An "isolated" nucleic acid or polypeptide as used herein refers to a nucleic acid or polypeptide that is removed from its original environment (for example, its natural environment if it is naturally occurring). 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 that is "derived from" a designated sequence refers to a sequence that corresponds to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are identical to or complementary to the sequence.
A "probe" refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target nucleic acid due to complementarity of at least one sequence in the probe with a sequence in the target nucleic acid. Generally, a probe is labeled so it can be detected after hybridization.
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press: 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, low stringency hybridization conditions, corresponding to a Tm of 55 °C, can be used, e.g., 5x SSC, 0.1%> SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5%) SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40%) formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50%> formamide, 5x or 6x SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al, supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, supra, 1 1.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides: preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
In a specific embodiment, the term "standard hybridization conditions" refers to a Tm of 55 °C, and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60°C; in a more preferred embodiment, the Tm is 65 °C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2XSSC, at 42°C in 50% formamide, 4XSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions. A "gene" for a particular protein as used herein refers to a contiguous nucleic acid sequence corresponding to a sequence present in a genome which comprises (i) a "coding (or transcribed) region," which comprises exons (i.e., sequences encoding a polypeptide sequence, or "protein-coding" or "transcribed sequences"), introns, sequences at the junction between exons and introns, and 5' and 3' untranslated regions (uTRs); and (ii) regulatory sequences, which flank the coding region at both 5' and 3' termini. For example, the "ACE gene" as used herein encompasses the regulatory and coding regions of the human gene encoding angiotensin converting enzyme. Similarly, the "AGT gene" encompasses regulatory and coding regions of the human gene encoding angiotensinogen and the "ATI gene" encompasses regulatory and coding regions of the human gene encoding type I angiotensin II receptor. Typically, regulatory sequences according to the invention are located 5' (i.e., upstream) of the coding region segment. The reference sequences, obtained from GenBank, which were used in practicing the present invention are shown in Table 1.
Table 1. GenBank Accession Numbers
The present inventors have suφrisingly and unexpectedly discovered the existence of genetic polymoφhisms within the human gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β- adrenoceptor which, singly or in combination, can be used to assess cardiovascular status, depending on which component of cardiovascular status is under evaluation. In accordance with the invention, the polymoφhic pattern of the gene, alone or in combination with other genes encoding renin, ACE, AGT, ATI, AT2, aldosterone synthase, endothelin receptor, and β-adrenergic receptors 1 and 2 in an individual can predict the responsivity of the individual to particular therapeutic interventions and serve as an indicator of predisposition to various forms of cardiovascular disease. The invention provides methods for assessing cardiovascular status by detecting polymoφhic patterns in an individual. The present invention also provides isolated nucleic acids derived from the gene which comprise these polymoφhisms, including probes which hybridize specifically to polymoφhic positions and primers that amplify the region of the gene in which the polymoφhism is located; isolated polypeptides and peptides comprising polymoφhic residues; and antibodies which specifically recognize ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor polypeptides containing one or more polymoφhic amino acids.
Methods for Assessing Cardiovascular Status
The present invention provides diagnostic methods for assessing cardiovascular status in a human individual. The methods are carried out by comparing a polymoφhic position or pattern ("test polymoφhic pattern") within the individual's gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor with the polymoφhic patterns of humans exhibiting a predetermined cardiovascular status ("reference polymoφhic pattern"). If the cardiovascular status is the prediction of responsivity to a therapy, a single polymoφhic position can provide a pattern for comparison. However, it is preferable to use more than one polymoφhic position for the pattern to improve the accuracy of the prediction. If the cardiovascular status is predisposition to a cardiovascular syndrome, at least two, and preferably at least three, polymoφhic positions are used to make the pattern. In addition, other polymoφhisms in genes encoding angiotensin converting enzyme (ACE), angiotensinogen (AGT), type 1 angiotensin II receptor (ATI), type 2 angiotensin II receptor, renin, aldosterone synthase, endothelin, receptor or β-adrenergic receptors 1 and 2 can be used to establish a polymoφhic pattern for the individual.
For any meaningful prediction, the polymoφhic pattern of the individual is identical to the polymoφhic pattern of individuals who exhibit particular status markers, cardiovascular syndromes, and/or particular patterns of response to therapeutic interventions. In one embodiment, the method involves comparing an individual's test polymoφhic pattern with reference polymoφhic patterns of individuals who have been shown to respond positively or negatively to a particular therapeutic regimen. Therapeutic regimen as used herein refers to treatments aimed at the elimination or amelioration of symptoms and events associated cardiovascular disease. Such treatments include without limitation one or more of alteration in diet, lifestyle, and exercise regimen; invasive and noninvasive surgical techniques such as atherectomy, angioplasty, and coronary bypass surgery; and pharmaceutical interventions, such as administration of ACE inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antagonists, cardiac glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor antagonists, calcium channel blockers, HMG-CoA reductase inhibitors, imidazoline receptor blockers, endothelin receptor blockers, and organic nitrites. Interventions with pharmaceutical agents not yet known whose activity correlates with particular polymoφhic patterns associated with cardiovascular disease are also encompassed. The present inventors have discovered that particular polymoφhic patterns correlate with an individual's responsivity to ACE inhibitors (see, e.g., Example 3 below). It is contemplated, for example, that patients who are candidates for a particular therapeutic regimen will be screened for polymoφhic patterns that correlate with responsivity to that particular regimen. In another embodiment, the method involves comparing an individual's polymoφhic pattern with polymoφhic patterns of individuals who exhibit or have exhibited one or more markers of cardiovascular disease, such as, e.g., high blood pressure, abnormal electrocardiographic profile, myocardial infarction, unstable angina, stroke, or atherosclerosis (see. e.g., Example 2 below) and drawing analogous conclusions as to the individual's responsivity to therapy, predisposition to developing a syndrome, etc., as detailed above.
Identification of Polymorphic Patterns In practicing the methods of the invention, an individual's polymoφhic pattern can be established e.g. by obtaining DNA from the individual and determining the sequence at a predetermined polymoφhic position or positions in a gene, or more than one gene.
The DNA may be obtained from any cell source. Non-limiting examples of cell sources available in clinical practice include without limitation blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Cells may also be obtained from body fluids, including without limitation blood, saliva, sweat, urine, cerebrospinal fluid, feces, and tissue exudates at the site of infection or inflammation. DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source. Determination of the sequence of the extracted DNA at polymoφhic positions is achieved by any means known in the art, including but not limited to direct sequencing, hybridization with allele-specific oligonucleotides, allele-specific PCR, ligase-PCR, HOT cleavage, denaturing gradient gel electrophoresis (DGGE), and single- stranded conformational polymoφhism (SSCP). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam- Gilbert method; by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; and sequencing using a chip-based technology. See, e.g., Little et al, Genet. Anal., 1996, 6: 151. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
In an alternate embodiment, biopsy tissue is obtained from a subject. Antibodies that are capable of distinguishing between different polymoφhic forms of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor are then applied to samples of the tissue to determine the presence or absence of a polymoφhic form specified by the antibody. The antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, or enzyme-linked or fluorescence-linked immunoassay. The presence or absence of a particular polymoφhism or polymoφhic pattern, and its allelic distribution (i.e., homozygosity vs. heterozygosity) is determined by comparing the values obtained from a patient with norms established from populations of patients having known polymoφhic patterns.
In another alternate embodiment, RNA is isolated from biopsy tissue using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al, Anal. Biochem., 1987, 162:156). The isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a selected polymoφhism. Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic of the presence of a particular polymoφhism. In another embodiment, RNA is reverse-transcribed and amplified, after which the amplified sequences are identified by, e.g., direct sequencing. In still another embodiment, cDNA obtained from the RNA can be cloned and sequenced to identify a polymoφhism.
Establishing Reference Polymorphism Patterns
In practicing the present invention, the distribution of polymoφhic patterns in a large number of individuals exhibiting particular cardiovascular status is determined by any of the methods described above, and compared with the distribution of polymoφhic patterns in patients that have been matched for age, ethnic origin, and/or any other statistically or medically relevant parameters, who exhibit quantitatively or qualitatively different cardiovascular status. Correlations are achieved using any method known in the art, including nominal logistic regression or standard least squares regression analysis. In this manner, it is possible to establish statistically significant correlations between particular polymoφhic patterns and particular cardiovascular statuses. It is further possible to establish statistically significant correlations between particular polymoφhic patterns and changes in cardiovascular status such as, would result, e.g., from particular treatment regimens. Thus, it is possible to correlate polymoφhic patterns with responsivity to particular treatments.
A statistically significant correlation preferably has a "p" value of less than or equal to 0.05. Any standard statistical method can be used to calculate these values, such as the normal Student's T Test, or Fischer's Exact Test.
The identity and number of polymoφhisms to be included in a reference pattern depends not only on the prevalence of a polymoφhism and its predictive value for the particular use, but also on the value of the use and its requirement for accuracy of prediction. The greater the predictive value of a polymoφhism, the lower the need for inclusion of more than one polymoφhism in the reference pattern. However, if a polymoφhism is very rare, then its absence from an individual's pattern might provide no indication as to whether the individual has a particular status. Under these circumstances, it might be advisable to select instead two or more polymoφhisms which are more prevalent. Even if none of them has a high predictive value on its own, the presence of both (or all three) of them might be sufficiently predictive for the particular puφose.
If for example the use for a reference pattern is prediction of response to a drug, and among the afflicted population only a 30%> response to the drug is observed, the reference pattern need only permit selection of a population that improves the response rate by 10% to provide a significant improvement in the state of the art. On the other hand, if the use for the reference pattern is selection of subjects for a particular clinical study, the pattern should be as selective as possible and should therefore include a plurality of polymoφhisms that together provide a high predictive accuracy for the intended response. In establishing reference polymoφhism patterns, it is desirable to use a defined population. For example, tissue libraries collected and maintained by state or national departments of health can provide a valuable resource, since genotypes determined from these samples can be matched with medical history, and particularly cardiovascular status, of the individual. Such tissue libraries are found, for example, in Sweden, Iceland, Norway, and Finland. As can be readily understood by one of ordinary skill in the art, specific polymoφhisms may be associated with a closely linked population. However, other polymoφhisms in the same gene may correlate with cardiovascular status of other genetically related populations. Thus, in addition to the specific polymoφhisms provided in the instant application, the invention identifies genes in which any polymoφhisms can be used to establish reference and test polymoφhism patterns for evaluating cardiovascular status of individuals in the population.
In a specific embodiment, DNA samples can be obtained form a well defined population, such as 277 Caucasian males born in Uppsala, Sweden between 1920 and 1924. In a specific embodiment, such individuals are selected for the test population based on their medical history, i.e., they were either (i) healthy, with no signs of cardiovascular disease (100); or (ii) had suffered one of acute myocardial infarction (68), silent myocardial infarction (34), stroke (18), stroke and acute myocardial infarction (19), or high blood pressure at age 50 (39). DNA samples are obtained from each individual. In a specific embodiment, DNA sequence analysis can be carried out by: (i) amplifying short fragments of each of the genes using polymerase chain reaction (PCR) and (ii) sequencing the amplified fragments. The sequences obtained from each individual can then be compared with the first known sequences, e.g., as set forth in Table 1, to identify polymoφhic positions.
Comparing Test Patterns to Reference Patterns
As noted above, the test pattern from an individual can be compared to a reference pattern established for a predetermined cardiovascular status. Identity between the test pattern and the reference pattern means that the tested individual has a probability of having the same cardiovascular status as that represented by the reference pattern. As discussed above, this probability depends on the prevalence of the polymoφhism and the statistical significance of its correlation with a cardiovascular status.
Polymorphic Positions
Polymoφhic positions in the genes encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β- adrenoceptor which are encompassed by the invention can be identified by determining the DNA sequence of all or part of the gene in a multiplicity of individuals in a population. DNA sequence determination may be achieved using any conventional method, including, e.g., chemical or enzymatic sequencing. The polymoφhic positions of genes for use in the invention include without limitation those listed below, whose numbering corresponds to the GenBank sequences listed in Table 1.
(i) ACE: positions in the regulatory region (designated ACR) numbered 5106, 5349, and 5496; positions in the coding region (designated ACE) numbered 375, 582, 731 , 1060. 1215, 2193, 2328, 2741, 3132, 3387, 3503, and 3906; and position 1451 as numbered in GenBank entry X62855.
(ii) AGT: positions in the regulatory region (designated AGR) numbered 395, 412, 432, 449. 692, 839, 1007, 1072, 1204, and 1218; positions in the coding region (designated AGT) numbered 273, 620, 803, 912, 997, 1116, and 1174; and position 49 as numbered in GenBank entry M24688.
(iii) ATI : positions in the regulatory region (designated ATR) numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; and positions in the coding region (designated ATI) numbered 449, 678, 1167, and 1271. (iv) Renin: A mutant renin gene in familial elevation of prorenin, a point mutation in the last exon of the gene (exon 10), has been identified (Villard et al, J. Biol. Chem., 1994, 269:30307-12). A cytosine to thymine transition creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus. (v) Aldosterone synthase: A position in the promoter region of aldosterone synthase, position -344 (with the initiation codon starting at 1) has been reported by Cambien et al. at the International Meeting on Hypertension held in Amsterdam in June 1998. β-adrenergic receptor- 1, positions in the regulatory region (designated BP1) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724, and 2757; and positions in the coding region (designated BR1) numbered 231, 758, 1037, 1251, 1403, and 1528. β-adrenergic receptor-2, positions in the regulatory region (designated B2P) numbered 932, 934, 987, 1006. 1120, 1221, 1541, and 1568; and positions in the coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070.
Endothelin receptor type A coding region (designated ETA) numbered 969, 1005, 1 146, and 2485. In preferred embodiments, the base at each of the above polymoφhic positions is one of:
(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, 2193G, 2193A, 2328A, 2328G, 2741G, 2741T, 3132C, 3132T, 3387T, 3387C, 3503G, 3503C, 3906G, and 3906A; and a deletion of nucleotides 1451-1783 as numbered in GenBank entry X62855;
(iii) AGT Regulatory Region: 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 692C, 692T, 839G, 839A, 1007G, 1007A, 1072G, 1072A, 1204C, 1204A, 1218A, 1218G;
(iv) AGT Coding Region: 273C, 273T, 620C, 620T, 803T, 803C, 912C, 912T, 997G, 997C, 11 16G, 1116A, 1174C, and 1174 A; and A or G at position 49 in GenBank entry M24688; (v) _ ATI Regulatory Region: 1427A, 1427T, 1756T, 1756A, 1853T,
1853G, 2046T, 2046C, 2354A, 2354C, 2355G, 2355C, 2415A and 2415G; and '
(vi) ATI Coding Region: 449G, 449C, 678T, 678C, 1167A, 1167G, 1271A, and 1271C.
(vii) β-adrenergic receptor- 1 regulatory region: 2238 G, 2238 A, 2577 C, 2257 T, 2757 A, and 2757 G.
(viii) β-adrenergic receptor-1 coding region: 231 A, 231 G, 758 C, 758 T, 1251 C, 1251 G, 1403 A, 1403 G, 1528 C, and 1528 A.
(ix) β-adrenergic receptor-2 regulatory region: 934 A, 934 G, 987 C, 987 G, 1006 A, 1006 G, 1120 C, 1120 G, 1221 C, 1221 T, 1541 C, 1541 T, 1568 C, and 1568 T.
(x) β-adrenergic receptor-2 coding region: 839 A, 839 G, 872 C, 872 G, 1045A, 1045 G, 1284 C, 1284 T, 1316 A, 1316 C, 1846 C, 1846 G, 2032 A, 2032 G, 2068 no insert, 2068 G, 2068 C, 2070 no insert, and 2070 C.
(xi) Endothelin receptor type A: 969 C, 969 T, 1005 A, 1005 G, 1 146 A, 1 146 G, 2485 T, and 2485 C.
An individual may be homozygous or heterozygous for a particular polymoφhic position (see, e.g., Table 6 below). Non-limiting examples of polymoφhic patterns comprising one or more polymoφhism in ACE, AGT, and/or ATI genes according to the invention include the following, which were correlated with an increased incidence of clinical signs of cardiovascular disease: ACR 5349 A/T, AGR 1218 A; ACR 5496 C, AGR 1204 A/C; ACR 5496
C/T, AGR 1218 A, AGT 620 C/T; ACE 2193 A, AGR 1204 C, ACE 2328 G; ACE 2193 A, AGR 1204 A/C; ACE 3387 T, AGR 1218 A; ACE 3387 T, AGT 620 C/T; AGR 1204 A/C, ATI 678 C/T; AGR 1204 A/C, ATI 1271 A C; ACE 1215 C, AGR 1204 A C; AGR 1204 A/C, ATI 1167 A, ACE 3906 A/G; AGR 1204 A, AGT 620 C, ATI 1271 A, ATI 1167 A, AGR 395 A T; AGR 1204 A C, AGT 620 C/T, ATI 1271 A/C, ATI 1 167 A,
AGR 395 T; AGR 1204 A/C, AGT 620 C/T, ATI 1271 A/C, ATI 1167 A G, AGR 395 T; AGR 1204 A, ATI 678 C, ATI 1 167 A, AGR 395 A/T; AGR 1204 A C, ATI 678 C/T, ATI 1167 A, AGR 395 T; AGT 620 C/T, ATI 1271 A/C, ATI 1167 A, AGR 395 T; AGT 620 C/T, ATI 1271 A C, ATI 1167 A/G, AGR 395 T; AGT 620 C, ATI 1271 A, ATI 1167 A, AGR 395 A T; AGT 620 C, ATI 678 A, ATI 1167 A, AGR 395 A/T; AGT 620 C/T, ATI 678 C/T; ATI 1167 A, AGR 395 T; ACE 2193 A, AGR 1218 A, AGT 803 A; ACE 2193 A, AGT 620 C/T; ACE 2328 G, AGT 620 C/T; ACE 3387 T, AGR 1204 A/C; ACE 2193 A, ACE 2328 G, AGR 1204 C; ACE 2193 A/G, AGR 1072 G/G, ATI 1167 A/A. Additional polymoφhism patterns are shown in the Tables in Examples 4 and 5, below.
Polymorphism Patterns Correlated With ACE Inhibitor Responsiveness
The following table lists a set of polymoφhism patterns that have been found to correlate with responsiveness to ACE inhibitor treatment: Response to ACE-Inhibitor Treatment
The following table lists a set of polymoφhism patterns that have been found to correlate with non-responsiveness to ACE inhibitor treatment:
Non-Response to ACE-Inhibitor Treatment
Polymorphism Patterns Correlated With Predisposition to MI
The following table lists a set of polymoφhism patterns that have been found to correlate with predisposition to myocardial infarction:
Predisposition to MI
Polymorphism Patterns Correlated With Predisposition to Stroke
The following table lists a set of polymoφhism patterns that have been found to correlate with predisposition to stroke:
Predisposition to Stroke
Polymorphism Patterns in β-Adrenergic Receptor Genes The following table lists a set of polymoφhism patterns that have been found in β-adrenergic receptor genes:
Positions carrying genetic variation in the Beta adrenergic receptors 1 and
2.
B1P: Beta adrenergic receptor 1, regulatory promoter region. B1R: Beta adrenergic receptor 1, coding region. B2P: Beta adrenergic receptor 2, regulatory promoter region. B2R: Beta adrenergic receptor 2, coding region.
A number of different polymoφhisms have been identified in the type 2 β- adrenoceptor. All of these differed from the wild type sequence by a single base change. Four of the polymoφhisms alter the amino acid sequence of the receptor protein (Hall, Thorax, 1996, 51:351-353). The amino acid sequence modifications are described in greater detail below:
Argl6-*Gly: The Glylό variant undergoes an enhanced agonist-promoted down regulation as compared to wild type but the coupling to adenylyl cyclase and agonist binding are maintained (Liggett, Am. J. Respir. Crit. Care Med., 1997, 156:S 156-S162). Gln27→Glu: The Glu27 variant displays very little agonist-promoted downregulation and the coupling to adenylyl cyclase and agonist binding are maintained (id.).
Val34-Met: Met34 is very rare. No altering of receptor function has been found (id.). Thrl64→Ile: Uncommon (about 5%). The He 164 variant shows depressed coupling to adenylyl cyclase and decreased affinities for agonists with hydroxyl groups on their β-carbons, such as epinephrine, norepinephrine, and isoproterenol compared to wild type (id.).
The polymoφhism at nucleic acid 523 (CGG→AGG) might be linked with one of the other functional polymoφhisms (id.).
There are no differences in frequency of these polymoφhisms between the normal group and those with asthma but they have been correlated to differences in response to treatment with agonists in asthma, e.g., the Glyl6 variant undergoes an enhanced agonist-promoted downregulation compared to wild type (id.).
Polymorphism Patterns in Endothelin Receptor Type A Gene
The following table lists a set of polymoφhism patterns that have been found in the coding region of the endothelin receptor type A gene:
Isolated Polymorphic Nucleic Acids, Vectors, Probes & Primers and Arrays
Vectors for Expression of Polymorphic Variants
The present invention provides isolated nucleic acids comprising the polymoφhic positions described above for the human genes encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β- adrenoceptor; vectors comprising the nucleic acids; and transformed host cells comprising the vectors. The invention also provides probes which are useful for detecting these polymoφhisms.
The nucleic acids encoding a gene comprising a polymoφhism that is useful for determining cardiovascular status of an individual is particularly valuable for screening, whether by direct screening of the nucleic acid with the polymoφhism, or by screening the polypeptide expressed by that nucleic acid. In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA, are used. Such techniques are well known and are explained fully in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, 1989 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D.N. Glover ed.); Oligonucleotide Synthesis, 1984, (M.L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Ausubel et al, Current Protocols in Molecular Biology, 1997 (John Wiley and Sons); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively). Insertion of nucleic acids (typically DNAs) comprising the sequences of the present invention into a vector is easily accomplished when the termini of both the DNAs and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini of the DNAs and/or vector by digesting back single- stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerase.
Alternatively, any site desired may be produced, e.g., by ligating nucleotide sequences (linkers) onto the termini. Such linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated by the use of the polymerase chain reaction (PCR). See, e.g., Saiki et al., Science, 1988, 239:48. The cleaved vector and the DNA fragments may also be modified if required by homopolymeric tailing.
The nucleic acids may be isolated directly from cells or may be chemically synthesized using known methods. Alternatively, the polymerase chain reaction (PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates. Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate incoφoration into a given vector for recombinant expression. The nucleic acids of the present invention may be flanked by native gene sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like. The invention also provides nucleic acid vectors comprising the disclosed genes or derivatives or fragments thereof. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple cloning or protein expression. Non-limiting examples of suitable vectors include without limitation pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP (Invitrogen, San Diego, CA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The particular choice of vector/host is not critical to the practice of the invention. Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl2 mediated DNA uptake, calcium phosphate precipitation, fungal or viral infection, lipofection, microinjection, microprojectile, or other established methods. Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced ACE-, AGT-, or ATI -derived peptides and polypeptides.
Nucleic acids encoding ACE-, AGT-, or ATI-derived gene sequences may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene. Other recombination-based methods such as nonhomologous recombinations or deletion of endogenous genes by homologous recombination may also be used.
Oligonucleotides
The nucleic acids of the present invention find use as probes for the detection of genetic polymoφhisms, as primers for the expression of polymoφhisms, or in molecular library arrays for high throughput screening.
Probes in accordance with the present invention comprise without limitation isolated nucleic acids of about 10 - 100 bp, preferably 15-75 bp and most preferably 17-25 bp in length, which hybridize at high stringency to one or more of the gene-derived polymoφhic sequences disclosed herein or to a sequence immediately adjacent to a polymoφhic position. Furthermore, in some embodiments a full-length gene sequence may be used as a probe. In one series of embodiments, the probes span the polymoφhic positions in the genes disclosed above. In another series of embodiments, the probes correspond to sequences immediately adjacent to the polymoφhic positions.
The oligonucleotide nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. PNAs are also included. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
PCR amplification of gene segments that contain a polymoφhism provides a powerful tool for detecting the polymoφhism. The oligonucleotides of the invention can also be used as PCR primers to amplify gene segments containing a polymoφhism of interest. The amplified segment can be evaluated for the presence or absence of a polymoφhism by restriction endonuclease activity, SSCP, or by direct sequencing. In another embodiment, the primer is specific for a polymoφhic sequence on the gene. If the polymoφhism is present, the primer can hybridize and DNA will be produced by PCR. However, if the polymoφhism is absent, the primer will not hybridize, and no DNA will be produced. Thus, PCR can be used to directly evaluate whether a polymoφhism is present or absent.
Molecular library arrays of oligonucleotides (including oligonucleotides with modifications as described above) are another powerful tool for rapidly assessing whether one or more polymoφhisms are present in a ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor gene, preferably in combination with other genes. Molecular library arrays are disclosed in US Patents No. 5,677,195, No. 5,599,695, No. 5,545,531, and No. 5,510,270.
Polymorphic Polypeptides and Polymorphism-Specific Antibodies
The present invention encompasses isolated peptides and polypeptides encoded by all or a portion of a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor, comprising polymoφhic positions disclosed above. In one preferred embodiment, the peptides and polypeptides are useful screening targets to identify cardiovascular drugs. In another preferred embodiment, the peptides and polypeptides are capable of eliciting antibodies in a suitable host animal that react specifically with a polypeptide comprising the polymoφhic position and distinguish it from other polypeptides having a different amino acid sequence at that position. Polypeptides according to the invention are preferably at least five or more residues in length, preferably at least fifteen residues. Methods for obtaining these polypeptides are described below. Many conventional techniques in protein biochemistry and immunology are used. Such techniques are well known and are explained in Immunochemical Methods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press, London); Scopes, Protein Purification: Principles and Practice, Second Edition 1987 (Springer- Verlag, N.Y.) and Handbook of Experimental Immunology, Volumes I-IV 1986 (Weir and Blackwell eds.).
Nucleic acids comprising protein-coding sequences can be used to direct the recombinant expression of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor-derived polypeptides in intact cells or in cell-free translation systems. The known genetic code, tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The polypeptides may be isolated from human cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an appropriate protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins. Peptides and polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis as described by Merrifield, J. Am. Chem. Soc, 1963, 85:2149.
Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some puφoses, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as. but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor or against peptides derived therefrom, can be used as purification reagents. Other purification methods are possible.
The present invention also encompasses derivatives and homologues of the polypeptides. For some puφoses, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e., function-conservative variants. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids. The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to. radioisotopes and fluorescent compounds.
The present invention also encompasses antibodies that specifically recognize the polymoφhic positions of the invention and distinguish a peptide or polypeptide containing a particular polymoφhism from one that contains a different sequence at that position. Such polymoφhic position-specific antibodies according to the present invention include polyclonal and monoclonal antibodies. The antibodies may be elicited in an animal host by immunization with ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor-derived immunogenic components or may be formed by in vitro immunization of immune cells. The immunogenic components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems. The antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA. Alternatively, the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains. The antibodies include hybrid antibodies (i.e., containing two sets of heavy chain/light chain combinations, each of which recognizes a different antigen), chimeric antibodies (i.e., in which either the heavy chains, light chains, or both, are fusion proteins), and univalent antibodies (i.e., comprised of a heavy chain/light chain complex bound to the constant region of a second heavy chain). Also included are Fab fragments, including Fab' 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 discussed in more detail below. For example, techniques for producing and processing polyclonal antisera are disclosed in Mayer and Walker, Immunochemical Methods in Cell and Molecular Biology, 1987 (Academic Press, London). The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with
Epstein-Barr virus. See, e.g., Schreier et al, Hybridoma Techniques, 1980; U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491 ,632; and 4,493,890. Panels of monoclonal antibodies produced against ACE, AGT, or ATI-derived epitopes can be screened for various properties; i.e. for isotype, epitope affinity, etc. The antibodies of this invention can be purified by standard methods, including but not limited to preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. Purification methods for antibodies are disclosed, e.g., in The Art of Antibody Purification, 1989 (Amicon Division, W.R. Grace & Co.) General protein purification methods are described in Protein Purification: Principles and
Practice, R.K. Scopes, Ed., 1 87 ( Springer- Verlag, New York, NY).
Methods for determining the immunogenic capability of the disclosed sequences and the characteristics of the resulting sequence-specific antibodies and immune cells are well-known in the art. For example, antibodies elicited in response to a peptide comprising a particular polymoφhic sequence can be tested for their ability to specifically recognize that polymoφhic sequence, i.e., to bind differentially to a peptide or polypeptide comprising the polymoφhic sequence and thus distinguish it from a similar peptide or polypeptide containing a different sequence at the same position.
Diagnostic Methods and Kits
The present invention provides kits for the determination of the sequence at a polymoφhic position or positions within the ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor gene in an individual, in combination with determination of the sequence at polymoφhism positions of other genes. The kits comprise a means for determining the sequence at the polymoφhic positions, and may optionally include data for analysis of polymoφhic patterns. The means for sequence determination may comprise suitable nucleic acid-based and immunological reagents (see below). Preferably, the kits also comprise suitable buffers, control reagents where appropriate, and directions for determining the sequence at a polymoφhic position. The kits may also comprise data for correlation of particular polymoφhic patterns with desirable treatment regimens or other indicators.
Nucleic Acid-Based Diagnostic Methods and Kits
The invention provides nucleic acid-based methods for detecting polymoφhic patterns in a biological sample. The sequence at particular polymoφhic positions in the genes is determined using any suitable means known in the art, including without limitation hybridization with polymoφhism-specific probes and direct sequencing.
The present invention also provides kits suitable for nucleic acid-based diagnostic applications. In one embodiment, diagnostic kits include the following components:
(i) Probe DNA: The probe DNA may be pre-labelled; alternatively, the probe DNA may be unlabelled and the ingredients for labelling may be included in the kit in separate containers; and (ii) Hybridization reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
In another embodiment, diagnostic kits include: (i) Sequence determination primers: Sequencing primers may be pre-labelled or may contain an affinity purification or attachment moiety; and
(ii) Sequence determination reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular sequencing protocol. In one preferred embodiment, the kit comprises a panel of sequencing primers, whose sequences correspond to sequences adjacent to the polymoφhic positions.
Antibody-Based Diagnostic Methods and Kits
The invention also provides antibody-based methods for detecting polymoφhic patterns in a biological sample. The methods comprise the steps of: (i) contacting a sample with one or more antibody preparations, wherein each of the antibody preparations is specific for a particular polymoφhic form of the gene under conditions in which a stable antigen-antibody complex can form between the antibody and antigenic components in the sample; and (ii) detecting any antigen-antibody complex formed in step (i) using any suitable means known in the art, wherein the detection of a complex indicates the presence of the particular polymoφhic form in the sample.
Typically, immunoassays use either a labelled antibody or a labelled antigenic component (e.g., that competes with the antigen in the sample for binding to the antibody). Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labelled immunoassays, such as ELISA assays.
The present invention also provides kits suitable for antibody-based diagnostic applications. Diagnostic kits typically include one or more of the following components: (i) Polymorphism-specific antibodies: The antibodies may be pre-labelled; alternatively, the antibody may be unlabelled and the ingredients for labelling may be included in the kit in separate containers, or a secondary, labelled antibody is provided; and (ii) Reaction components: The kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.
The kits referred to above may include instructions for conducting the test. Furthermore, in preferred embodiments, the diagnostic kits are adaptable to high- throughput and/or automated operation.
Drug Targets and Screening Methods
According to the present invention, nucleotide sequences derived from the gene encoding a polymoφhic form of ACE, ATI, AGT, renin, aldosterone synthase, type- 2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor, and peptide sequences derived from that polymoφhic for, are useful targets to identify cardiovascular drugs, i.e., compounds that are effective in treating one or more clinical symptoms of cardiovascular disease. Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor and (ii) isolated peptides and polypeptides derived from ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor polypeptides, each of which comprises one or more polymoφhic positions.
In vitro screening methods In one series of embodiments, an isolated nucleic acid comprising one or more polymoφhic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner. The methods comprise: (i) providing a first nucleic acid containing a particular sequence at a polymoφhic position and a second nucleic acid whose sequence is identical to that of the first nucleic acid except for a different sequence at the same polymoφhic position;
(ii) contacting the nucleic acids with a multiplicity of test compounds under conditions appropriate for binding; and (iii) identifying those compounds that bind selectively to either the first or second nucleic acid sequence.
Selective binding as used herein refers to any measurable difference in any parameter of binding, such as, e.g., binding affinity, binding capacity, etc. In another series of embodiments, an isolated peptide or polypeptide comprising one or more polymoφhic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner. The screening methods involve:
(i) providing a first peptide or polypeptide containing a particular sequence at a polymoφhic position and a second peptide or polypeptide whose sequence is identical to the first peptide or polypeptide except for a different sequence at the same polymoφhic position;
(ii) contacting the polypeptides with a multiplicity of test compounds under conditions appropriate for binding; and (iii) identifying those compounds that bind selectively to one of the nucleic acid sequences.
In preferred embodiments, high-throughput screening protocols are used to survey a large number of test compounds for their ability to bind the genes or peptides disclosed above in a sequence-specific manner. Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (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 from e.g. Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
In vivo screening methods
Intact cells or whole animals expressing polymoφhic variants of a gene encoding ACE, ATI , AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor can be used in screening methods to identify candidate cardiovascular drugs. In one series of embodiments, a permanent cell line is established from an individual exhibiting a particular polymoφhic pattern. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are programmed to express a gene comprising one or more polymoφhic sequences by introduction of appropriate DNA. Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to particular polymoφhic variants of the gene; (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of the gene; and (iii) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions the gene.
In another series of embodiments, transgenic animals are created in which (i) a human ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or β-adrenoceptor gene having different sequences at particular polymoφhic positions are stably inserted into the genome of the transgenic animal; and/or (ii) the endogenous genes are inactivated and replaced with human genes having different sequences at particular polymoφhic positions. See, e.g., Coffman, Semin. Nephrol., 1997, 17:404; Esther et al, Lab. Invest., 1996, 74:953; Murakami et al, Blood Press. Suppl., 1996, 2:36. Such animals can be treated with candidate compounds and monitored for one or more clinical markers of cardiovascular status.
Furthermore, populations that are not amenable to an established treatment for a cardiovascular disease or disorder can be selected for testing of alternative treatments. Moreover, treatments that are not as effective in the general population, but that are highly effective in the selected population, may be identified that otherwise would be overlooked. This is an especially powerful advantage of the present invention, since it eliminates some of the randomness associated with clinical trials.
The following are intended as non-limiting examples of the invention.
Example 1 : Methods for Identification of Polymorphic Positions in Human Genes Encoding ACE. AGT. and ATI
The following studies were performed to identify polymoφhic residues within the genes encoding human ACE, AGT, and ATI. DNA samples were obtained from 277 individuals. The individuals were Caucasian males bom in Uppsala, Sweden between 1920 and 1924. Individuals were selected for the test population based on their medical history, i.e., they were either (i) healthy, with no signs of cardiovascular disease (100); or (ii) had suffered one of acute myocardial infarction (68), silent myocardial infarction (34), stroke (18), stroke and acute myocardial infarction (19), or high blood pressure at age 50 (39). DNA samples were obtained from each individual.
DNA sequence analysis was carried out by: (i) amplifying short fragments of each of the ACE, AGT, and ATI genes using polymerase chain reaction (PCR) and (ii) sequencing the amplified fragments. The sequences obtained from each individual were then compared with known ACE, AGT, and ATI genomic sequences (see Table 1).
(i) Amplification: PCR reactions employed the primers shown in Table 2 below.
Table 2 - PCR Primers
Where indicated, the primers were modified in one of the following ways: (i) a biotin molecule was conjugated to the 5' terminus of the indicated sequence (B); (ii) a sequence of nucleotides derived from Ml 3, 5'-CAGGAAACAGCTATGACT-3' (SEQ ID NO: 120), was added at the 5' terminus of the indicated sequence (MT); or (iii) the sequence 5'-AGTCACGACGTTGTAAAACGACGGCCAGT-3' (SEQ ID NO: 121 ) was added at the 5' terminus of the indicated sequence (T = Tail). Nucleotides were numbered according to the GenBank sequences listed in Table 1 where indicated. When the sequences involved were not publicly available, the numbering was as in the following examples: The designation "i-4: 1-200" indicates that the primer sequence is located within the sequence extending 200 bp upstream of, and including, the nucleotide immediately upstream of the first coding nucleotide of exon 4. Similarly, the designation "i+4: 1-200" indicates that the primer sequence is located within the sequence extending from the nucleotide that is located immediately downstream of the last coding nucleotide of exon 4 downstream for 200 bp. In each case, the specific location of the primer sequence is indicated in Table 2 in the column marked "Nucleotides".
The reaction components used for PCR are described in Table 3, and the reaction conditions for PCR are described Table 4, below.
Table 3 -PCR Components
All temperatures are given in degrees Celsius.
*) indicates the default initial temperatures (°C) and times of the program. **\ indicates the default temperature (°C) of the program. indicates the default number of cycles of the program, referring to the section of the PCR program where three different temperatures are employed.
Any differences are indicated in "Modifications" in Table 5 below.
The amplified fragments are described in Table 5 below with respect to the primers and PCR reaction conditions used for amplification.
Table 5 - Amplified Fragments
All of the PCR products (except fragments ACEDI, ATI-spec. 1 and ATI- spec. 2) were subjected to solid phase sequencing according to the protocol commercially available from Pharmacia Biotech. The sequencing reactions are performed with a sequencing primer having a complementary sequence to the "Tail" sequence previously 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 on the 5 '-nucleotide. The positions carrying a genetic variation were identified by determination of the nucleotide sequence by the use of the ALFexpress™ system commercially available from Pharmacia Biotech. The detection of the fragment ACEDI was performed by analyzing the sizes of the amplified fragments by gel electrophoresis, where the presence of a shorter PCR product (192 base pairs) indicated the D-allele and a longer PCR product (479 base pairs) indicated the I-allele. The presence of both bands indicated a heterozygote for the two alleles. The detection of the allele-specific reaction of position ATI- 1271 was performed by separately running two parallel PCR reactions on the same sample and comparing the sizes of the amplified fragments. A PCR product of 501 base pairs should always be present as a control in both parallel runs, whereas the presence of a PCR product of 378 base pairs in the reaction designated ATI -spec. 1 indicated the presence of an A in this position. The presence of a PCR product of 378 base pairs in the reaction designated AT1- spec. 2 indicated a C in this position. If the shorter PCR product was present in both reactions, the individual is a heterozygote for A and C.
Results The analysis described above resulted in the identification of polymorphic positions within the regulatory and coding/intron segments of the human genes encoding ACE, AGT, and ATI . The polymoφhic positions, the variant nucleotides found at each of the positions, and the PCR fragment in which the polymoφhism was identified are shown in Table 6 below. Also shown are the frequencies of each genotype in a population of 90 individuals, expressed as the percent of the study population having that genotype. Polymoφhisms that resulted in alternate amino acids in ACE, AGT, or ATI are also indicated. As used herein below, the designations "AGR" , "ACR", and "ATR" refer to the regulatory regions of the human AGT, ACE, and ATI genes, respectively; and the designations "AGT", "ACE", and "ATI ", refer to the coding regions of the AGT, ACE, and ATI genes. Table 6 - Polymorphisms Found in ACE, AGT, and ATI Genes
A subset of these polymoφhic positions were further analyzed in an additional 187 individuals. Table 7 shows the polymoφhic positions, the sequence at these positions, and the genotype frequencies for each position in a population of 277 as described in Example 1 above.
Table 7 - Pol mor hic Positions and Fre uencies
Example 2: Correlation of Polymorphism Patterns with Cardiovascular Disease
The polymoφhic positions identified as in Example 1 were correlated with the following markers of cardiovascular status present in the study population: healthy (100 individuals evaluated); myocardial infarction (MI) (120 individuals); stroke (37 individuals); and high blood pressure (BP) (39 individuals). Polymoφhic patterns, i.e., combinations of sequences at particular polymoφhic positions, that show a statistically significant correlation with one or more of these markers are shown below.
Table 8 - Polymorphism Patterns
ACR 5496 C/T, AGR 1218 A, AGT 620 C/T
ACE 2193 A, AGR 1204 C, ACE 2328 G
Healthy MI Stroke High BP Total (n)
# of events 0 1 1 3 3 16
% within 0 9.2 8.1 7.7 group
ACE 2193 A, AGR 1204 A/C
Healthy MI Stroke High BP Total (n)
# of events 1 1 0 1 3
% within 1 0.8 0 2.6 group
ACE 3387 T, AGR 1218 A
Healthy MI Stroke High BP Total (n)
# of events 2 4 1 3 10
% within 2 3.3 2.7 7.7 group
ACE 3387 T, AGT 620 C/T
Healthy MI Stroke High BP Total (n)
# of events 1 10 3 2 15
% within 1 8.3 8.1 5.1 group
AGR 1204 A/C, ATI 678 C/T
Healthy MI Stroke • High Total (n)
# of events 5 23 5 6 37
% within 5 19.2 13.5 15.4 group
AGP . 1204 A/C, ATI 1271 A/C
Healthy MI Stroke High BP Total (n)
# of events 3 17 3 4 26
% within 3 14.2 8.1 10.3 group ACE 1215 C, AGR 1204 A/C
AGR 1204 A/C, ATI 1 167 A, ACE 3906 A/G
AGR 1204 A, AGT 620 C, ATI 1271 A, ATI 1 167 A, AGR 395 A/T
AGR 1204 A/C, AGT 620 C/T, ATI 1271 A/C, ATI 1167 A, AGR 395 T
AGR 1204 A/C, AGT 620 C/T, ATI 1271 A/C, ATI 1 167 A/G, AGR 395 T
Summary of the three previous polymoφhic patterns (which involve the same polymoφhic positions):
AGR 1204 A, ATI 678 C, ATI 1 167 A, AGR 395 A/T
AGR 1204 A/C, ATI 678 C/T, ATI 1 167 A, AGR 395 T
Summar of the two revious ol mo hic atterns:
AGT 620 C/T, ATI 1271 A/C, ATI 1 167 A, AGR 395 T
AGT 620 C/T, ATI 1271 A/C, ATI 1 167 A/G, AGR 395 T
AGT 620 C, ATI 1271 A, ATI 1 167 A, AGR 395 A/T
ACE 2193 A, AGT 620 C/T
Example 3: Correlation Between a Specific Polymorphism Pattern and Treatment Response
The following study was undertaken to define polymoφhic patterns in the human ACE, AGT, and/or ATI genes that predict the efficacy of treatments for cardiovascular disease.
Two groups of hypertensive patients were studied, 41 in the first group and 20 in the second group. The groups were analyzed independently and in combination.
The patients in this population were each treated with one of the following five ACE inhibitors: Captopril, Trandolapril, Lisinopril, Fosinopril, or Enalapril. The effect of the drugs on mean arterial blood pressure was quantified. Mean arterial blood pressure was defined as 2/3 of the diastolic blood pressure + 1/3 of systolic blood pressure. The individuals were also categorized as "high responders," i.e., those exhibiting a decrease of more than 16 mm Hg during treatment with an ACE inhibitor drug, and "low responders," i.e., those not exhibiting a decrease of more than 16 mm Hg.
One particular polymoφhic pattern, ACE 2193 A/G, AGR 1072 G/G, ATI 1 167 A/A, which was present in 51% of the first study population, discriminated between high responders and low responders. In the second group of 20 patients, the pattern was less prevalent (25%), but the correlation with lowered blood pressure remained statistically significant. Individuals having this polymoφhic pattern (designated "1 " below) experienced a larger decrease in blood pressure than those lacking this polymoφhic pattern (designated "0" below).
Table 9 - Response to Treatment Correlates with Polymorphic Pattern
Furthermore, the distribution of high responders and low responders (as defined above) was as follows:
Table 10 - Responder Status Correlates with Polymorphic Pattern
Taken together, the results from the two groups indicate that the presence of this polymoφhic pattern correlates with an incremental decrease of 6.4-7.3 mm Hg relative to individuals not having this polymoφhic pattern.
The prevalence of this polymoφhic pattern was 41% in this hypertensive population. This suggests that testing for this polymoφhic pattern in hypertensive patients, followed by prescribing ACE inhibitors only to those patients having this polymoφhic pattern, could increase the response rate from 43% (in a hypertensive population in general) to 76% in hypertensive population selected according to the methods of the invention.
If even one polymoφhism was absent from the pattern, the high resolution of the variable and therefore the predictive value would be lost. Example 4: Correlation Between a Specific Polymorphism Pattern and Treatment Response or Predisposition to a Cardiovascular Syndrome
In accordance with the methods disclosed in the above examples, additional correlations of polymoφhic patterns with either responsiveness to ACE inhibitors, non- responsiveness to ACE inhibitors, predisposition to myocardial infarction, or predisposition to stroke were derived. These correlations are presented in the following table:
Polymoφhic patterns, i.e., combinations of sequences at particular polymoφhic positions, that show a statistically significant correlation with myocardial infarction and stroke are shown below:
Table 12 - Correlation with MI MI (n=120) Healthy (n= 100)
Genetic Signature Prevalence MI Healthy P-value
Table 13 - Correlation With Stroke
Stroke (n=37) Healthy (n=100)
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that 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 description.
Various patents, patent applications, methodologies, and publications are cited herein, the disclosures of which are incoφorated by reference in their entireties.
Example 5: Correlation Between a Specific Polymorphism Pattern and High Blood Pressure Treatment with a β-Blocker
Using the techniques described in Examples 1 and 2, we investigated polymoφhisms in the ADBP 1 and ADBR2 genes (regulator/promoter regions and coding regions). The source material was the same as described above.
At the outset, we identified a set of key polymoφhisms. Some of these are known; others are new. The are summarized in Table 14. Table 14 -Polymorphic Positions in the Genes Encoding the Beta Adrenergic Receptors 1 and 2
Abbreviations used for the denotation of the genes: BlP (ADBRl) putative promoter region of the gene encoding the beta adrenergic receptor 1 BlR (ADBRl) the protein coding region of the gene encoding the beta adrenergic receptor 1 B2P (ADBR2) putative promoter region of the gene encoding the beta adrenergic receptor 2 B2R (ADBR2) the protein coding region of the gene encoding the beta adrenergic receptor 2
These positions, one or a combination of several, can be used to predict a good response to anti-hypertensive treatment with Beta-blockers.
Indeed, we identified various polymoφhisms/polymoφhic patterns that can be used in the prediction of high response to anti-hypertensive treatment with beta-blockers. These are shown in Table 15.
Table 15 - Polymorphisms Predictive of β-Blocker Responsitivity
Not only positions in the beta adrenergic receptors, but also positions in the renin-angiotensin- aldosterone-system can be used to predict response to anti-hypertensive treatment with beta- blockers (see Table 16). The numbering give according to the numberings in GenBank as defined in Table 1, supra.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all sizes and all molecular weight or molecular mass values are approximate, and are provided for description.
Patents, patent applications, procedures, and publications cited throughout this application are incoφorated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A method for assessing cardiovascular status in an individual to be tested comprising comparing (a) a test polymoφhic pattern comprising at least one polymoφhic position within a gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor of the individual, with (b) a reference polymoφhic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status; and concluding whether the individual possesses the cardiovascular status based on whether the test pattern matches the reference pattern.
2. The method according to claim 1, wherein the polymoφhic pattern of (a) includes a polymoφhic position within a second gene encoding a polypeptide selected from the group consisting of ACE, ATI, and AGT.
3. The method according to claims 1 or 2, wherein the predetermined cardiovascular status is predisposition to a cardiovascular syndrome.
4. The method according to 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 polymoφhisms.
6. The method according to claim 5, wherein the reference pattern comprises at least three polymoφhisms.
7. The method according to claims 1 or 2, wherein the predetermined cardiovascular status is a response to a cardiovascular treatment regimen.
8. The method according to claim 7, wherein the treatment regimen comprises administering a cardiovascular drug selected from the group consisting of an ACE inhibitor, an angiotensin II receptor antagonist, a diuretic, an alpha-adrenoreceptor antagonist, a cardiac glycoside, a phosphodiesterase inhibitor, a beta-adrenoreceptor antagonist, a calcium channel blocker, a HMG-CoA reductase inhibitor, an imidizoline receptor blocker, an endothelin receptor blocker, and an organic nitrite.
9. The method according to claim 1, wherein the polymoφhic position of the gene is selected from the group consisting of a position in the renin coding region comprising a cytosine to thymine transition in exon 10 that creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus; a position in a promoter region of aldosterone synthase numbered -344 (with the initiation codon starting at 1); positions in the regulatory region of β-adrenergic receptor-1 gene (designated B1P) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724, and 2757; positions in the β-adrenergic receptor-1 coding region (designated B1R) numbered 231, 758,1037, 1251, 1403, and 1528; positions in the β-adrenergic receptor-2 gene regulatory region (designated B2P) numbered 932, 934, 987, 1006, 1120, 1221, 1541, and 1568; positions in the β-adrenergic receptor-2 gene coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070; positions in the endothelin receptor type A coding region (designated ETA) numbered 969, 1005, 1146, and 2485; and combinations of any of the foregoing.
10. The method according to claim 2, wherein the polymoφhic position of the second gene is selected from the group consisting of positions in the ACE regulatory region numbered 5106, 5349, and 5496; positions in the ACE coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503, and 3906; position 1451 in the ACE gene as numbered in GenBank entry X62855; positions in the AGT regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072, 1204, and 1218; positions in the AGT coding region numbered 273, 620, 803, 912, 997, 11 16, and 1 174; position 49 in the AGT gene as numbered in GenBank entry M24688; positions in the ATI regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; and positions in the ATI coding region numbered 449, 678, 1 167, and 1271.
1 1. 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, wherein the nucleic acid comprises a polymoφhic position and wherein the polymoφhic position is a position in a polymoφhic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status.
12. A probe which hybridizes at high stringency to a polymoφhic position as defined in claim 11.
13. A library of nucleic acids comprising a nucleic acid which comprises
(a) a polymoφhic position of a gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor, wherein the polymoφhic position is a position in a polymoφhic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status, and
(b) nucleic acids comprising other polymoφhic positions of human genes that are indicative of cardiovascular status of an individual, wherein the human genes are selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor.
14. The library of claim 13 wherein the nucleic acids are oligonucleotides.
15. A kit for assessing cardiovascular status comprising
(a) sequence determination primers and
(b) sequence determination reagents, wherein the primers are selected from the group consisting of primers that hybridize to or immediately adjacent to (i) a polymoφhic position in a gene gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β- adrenoceptor, and (ii) a second polymoφhic position in a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and β-adrenoceptor.
16. The kit of claim 15, wherein the second polymoφhic position is selected from the group consisting of positions in the ACE regulatory region numbered 5106, 5349, and 5496; positions in the ACE coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503, and 3906; position 1451 in the ACE gene as numbered in GenBank entry X62855; positions in the AGT regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072, 1204, and 1218; positions in the AGT coding region numbered 273, 620, 803, 912, 997, 1116, and 1 174; position 49 in the AGT gene as numbered in GenBank entry M24688; positions in the ATI regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; positions in the ATI coding region numbered 449, 678, 1167, and 1271 ; a position in the renin coding region comprising a cytosine to thymine transition in exon 10 that creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus; a position in a promoter region of aldosterone synthase numbered -344 (with the initiation codon starting at 1); positions in the regulatory region of β-adrenergic receptor-1 gene (designated B1P) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724, and 2757; positions in the β-adrenergic receptor-1 gene coding region (designated B1R) numbered 231, 758, 1037, 1251 , 1403, and 1528; positions in the β-adrenergic receptor-2 regulatory region (designated B2P) numbered 932, 934, 987, 1006, 1120, 1221, 1541, and 1568; positions in the β-adrenergic receptor-2 gene coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070; positions in the endothelin receptor type A coding region (designated ETA) numbered 969, 1005, 1 146, and 2485; and combinations of any of the foregoing.
EP99947799A 1998-10-14 1999-10-13 Genes for assessing cardiovascular status and compositions for use thereof Withdrawn EP1121462A2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US10430298P 1998-10-14 1998-10-14
US10428698P 1998-10-14 1998-10-14
US104302P 1998-10-14
US104286P 1998-10-14
PCT/IB1999/001678 WO2000022166A2 (en) 1998-10-14 1999-10-13 Genes for assessing cardiovascular status and compositions for use thereof

Publications (1)

Publication Number Publication Date
EP1121462A2 true EP1121462A2 (en) 2001-08-08

Family

ID=26801367

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99947799A Withdrawn EP1121462A2 (en) 1998-10-14 1999-10-13 Genes for assessing cardiovascular status and compositions for use thereof

Country Status (7)

Country Link
EP (1) EP1121462A2 (en)
JP (1) JP2002527079A (en)
AU (1) AU6116399A (en)
CA (1) CA2347247A1 (en)
IL (1) IL142548A0 (en)
NO (1) NO20011847L (en)
WO (1) WO2000022166A2 (en)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19938390A1 (en) * 1999-08-05 2001-03-08 Max Delbrueck Centrum New sequence variants of the human ßl adrenoceptor gene and their use
WO2002000933A2 (en) * 2000-06-23 2002-01-03 Interleukin Genetics, Inc. Screening assays for identifying modulators of the inflammatory or immune responses
WO2002061131A2 (en) * 2000-12-04 2002-08-08 Bristol-Myers Squibb Company Human single nucleotide polymorphisms
DE10065666B4 (en) * 2000-12-29 2008-10-02 Wiesner, Rudolf, Prof. Dr. DNA chip for the causal diagnosis of hypertension
WO2002063045A1 (en) * 2001-02-02 2002-08-15 Genaissance Pharmaceuticals, Inc. Drug target isogenes: polymorphisms in the angiotensin receptor 2 gene
EP1394267A1 (en) * 2002-08-19 2004-03-03 Bayer HealthCare AG Single nucleotide polymorphisms predictive for cardiovascular disease, adverse drug reactions, and drug efficacy
JP4502570B2 (en) * 2002-09-24 2010-07-14 株式会社東洋紡ジーンアナリシス IgA nephropathy diagnosis using genetic polymorphism analysis and IgA nephropathy diagnosis kit
CA2581086C (en) 2004-09-14 2023-11-07 The Regents Of The University Of Colorado, A Body Corporate Method for treatment with bucindolol based on genetic targeting
AU2006211092A1 (en) 2005-02-02 2006-08-10 Royal College Of Surgeons In Ireland Pharmacogenomics of blood pressure lowering agents
US20100240760A1 (en) * 2005-03-22 2010-09-23 Jessie Gu Biomarkers for Efficacy of Aliskiren as a Hypertensive Agent
WO2007008604A2 (en) * 2005-07-08 2007-01-18 Bristol-Myers Squibb Company Single nucleotide polymorphisms associated with dose-dependent edema and methods of use thereof
EP2307572B1 (en) 2008-07-03 2016-08-31 Mor Research Applications Ltd. Diagnostic polymorphisms for cardiac disease

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6549186A (en) * 1985-10-24 1987-05-19 Biotechnology Research Partners Limited Blood tests diagnostic for hypertension
AU1728088A (en) * 1987-04-30 1988-12-02 Biotechnology Research Partners Limited Hypertension-related blood tests useful as genetic markers
US5580722A (en) * 1989-07-18 1996-12-03 Oncogene Science, Inc. Methods of determining chemicals that modulate transcriptionally expression of genes associated with cardiovascular disease
US6197505B1 (en) * 1997-04-04 2001-03-06 Pyrosequencing Ab Methods for assessing cardiovascular status and compositions for use thereof
WO1999037761A1 (en) * 1997-12-30 1999-07-29 Max-Delbrück-Centrum für Molekulare Medizin Novel sequence variants of the human beta2-adrenergic receptor gene and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0022166A3 *

Also Published As

Publication number Publication date
JP2002527079A (en) 2002-08-27
CA2347247A1 (en) 2000-04-20
WO2000022166A3 (en) 2000-09-14
NO20011847L (en) 2001-06-14
AU6116399A (en) 2000-05-01
WO2000022166A2 (en) 2000-04-20
IL142548A0 (en) 2002-03-10
NO20011847D0 (en) 2001-04-10

Similar Documents

Publication Publication Date Title
WO2013006857A1 (en) Genetic alterations associated with autism and the autistic phenotype and methods of use thereof for the diagnosis and treatment of autism
US8114592B2 (en) Genetic markers associated with age-related macular degeneration, methods of detection and uses thereof
US20040132021A1 (en) Osteolevin gene polymorphisms
AU741750B2 (en) Methods for assessing cardiovascular status and compositions for use thereof
EP1121462A2 (en) Genes for assessing cardiovascular status and compositions for use thereof
EP1448587B1 (en) Noonan syndrome gene
WO2003072828A2 (en) Fcgammariib polymorphisms detection in system lupus erythematosus
US20050233321A1 (en) Identification of novel polymorphic sites in the human mglur8 gene and uses thereof
AU754923B2 (en) Method to determine predisposition to hypertension
JP5424519B2 (en) Parkin gene mutations, compositions, methods and uses
Dumont et al. Structure of primate and rodent orthologs of the prostate cancer susceptibility gene ELAC2
JP4657857B2 (en) Diagnosis and prevention of metabolic syndrome
US9909182B1 (en) Methods for identifying subjects susceptible to charcot-marie-tooth neuropathy type 1C
AU2001239837B2 (en) Methods and composition for diagnosing and treating pseudoxanthoma elasticum and related conditions
WO2000056922A2 (en) Genetic polymorphism and polymorphic pattern for assessing disease status, and compositions for use thereof
US20050123923A1 (en) Human obesity susceptibility gene and uses thereof
US20040018497A1 (en) Human obesity LIPIN3 polynucleotide and polypeptide sequences and methods of use thereof
EP1264841A1 (en) DNA encoding a mutant peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1), detection methods and test kits therefor
Arngrimsson PREDISPOSITION TO HYPERTENSION
MXPA99009044A (en) Methods for assessing cardiovascular status and compositions for use thereof

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010502

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: JONSSON, LENA

Inventor name: LINDSTROM, PER, HARRY, RUTGER

Inventor name: ANDERSSON, MARIA, KRISTINA

Inventor name: NORBERG, LEIF, TORBJORN

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: PYROSEQUENCING AB

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: BIOTAGE AB

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MEADOWLAND BUSINESS PARTNERS AB

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080725