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  • 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.

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