MXPA99009044A

MXPA99009044A - Methods for assessing cardiovascular status and compositions for use thereof

Info

Publication number: MXPA99009044A
Application number: MXPA/A/1999/009044A
Authority: MX
Inventors: Torbjorn Norberg Leif; Kristina Andersson Maria; Harry Rutger Lindstrom Per
Original assignee: Kristina Andersson Maria; Eurona Medical Ab; Lindstroem Per Harry Rutger; Norberg Leif Torbjoern
Priority date: 1997-04-04
Filing date: 1999-10-01
Publication date: 2000-06-01

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 angiotensin converting enzyme (ACE), angiotensinogen (AGT), and/or type 1 angiotensin II receptor (AT1). The invention also provides isolated nucleic acids endoding ACE, AGT, and AT1 polymorphisms, nucleic acid probes that hybridize to polymorphic positions, kits for the prediction of cardiovascular status, and nucleic acid and peptide targets for use in identifying candidate cardiovascular drugs.

Description

METHODS TO EVALUATE THE CARDIOVASCULAR STATUS AND COMPOSITIONS TO USE THEMSELVES Field of the Invention The present invention relates to genetic polymorphisms for assessing cardiovascular status in humans. BACKGROUND OF THE INVENTION The renin-angiotensin-aldosterone system (SRAA) plays an important role in cardiovascular physiology in mammals. Specifically, RAAS regulates salt-water homeostasis and maintenance of vascular tone. The stimulation or inhibition of this system raises or lowers the blood pressure, respectively, and alterations 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, eg, the control of cell growth. The renin-angiotensin system includes at least the enzyme renin conversion, angiotensin (ACE), angiotensinogen (AGT), angiotensin II receptor type 1 (AT1) and angiotensin II receptor type 2 (AT2). AGT is the specific substrate of renin, an aspartyl protease. The human AGT gene contains five exons and four introns that expand by 13Kb (Gaillard et al., DNA 8: 87-99, 1989: Fu amizu et al., J. Biol. Chem. 265: 7576-7582, 1990). The first exon (37 bp) encodes the 5 'untranslated region of the mRNA. The second exon encodes the signal peptide and the first 252 amino acids of the mature protein. Exons 3 and 4 are shorter and code for 90 and 48 amino acids, respectively. Exon 5 contains a short coding sequence (62 amino acids) and the 3 'untranslated region. The plasma of AGT is synthesized mainly 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 2: 403-410, 1989). The separation of the amino-terminal segment of AGT by renin released a pro-hormone of dicapeptide, angiotensin-1, which is further processed to angiotensin II by active octapeptides by the dipeptidyl carboxypeptidase designated angiotensin-converting enzyme (ACE). The separation of AGT by renin is the limiting step of the regimen in the activation of the renin-angiotensin system. Several epidemiological observations indicate a possible role of AGT in the regulation of blood pressure. A highly significant correlation between plasma AGT concentration and blood pressure was observed in epidemiological studies (Waiker et al., J. Hypertension 1: 287-291, 1979). Interestingly, a number of allelic dimorphisms have been identified in the AGT gene. The frequency of at least two of them (174 M and 235T) has been partially characterized and in certain populations it has been shown to be significantly elevated in hypertensive subjects (Jeunemaitre et al., Cell 71_: 169-180, 1991). In addition, it has been suggested that a specific 235T polymorphism is directly involved in coronary atherosclerosis (Ishigami et al. Circulation 9_1_: 951-4, 1995). In addition, the presence of A or G at position 1218 in the AGT regulatory region has been correlated with differences in the in vitro transcriptional capacity for this gene (Inuoe et al., J. Clin Invest 9.9: 1786, 1997). The human ECA 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 hypertension in humans (Sassaho et al., Am. J. Med. 8_3: 227-235, 1987). In plasma and on the surface of endothelial cells, ECA converts the inactive angiotensin I molecule (Ang I) into active angiotensin II (Ang II) (Bottari et al., Front, Neuroendocrinology 1_4: 123-171, 1993). Another CE substrate is bradykinin, a potent vasodilator and inhibitor of smooth muscle cell proliferation, which is inactivated by ECA (Ehlers et al., Biochemistry 28.:5311-5318, 1989; Erdos, E.G., Hypertension ± 6: 363-370, 1990; Johnston, C.l. Drugs (suppl.1) 39: 21-31, 1990). The levels of RCTs are very stable within individuals, but they differ greatly between individuals. It has been suggested that plasma ECA levels be determined genetically as a consequence of diallel polymorphisms, located in or near the ECA gene. Prior to the present invention, no definitive association between polymorphisms and hypertension or blood pressure was demonstrated. However, a higher risk of myocardial infarction has been identified in a group of subjects with an RCT polymorphism designed by RCT-DD (Cambien et al., Nature 359: 641-644, 1992) and a 12-fold increased risk of infarction at baseline. myocardium has been identified in a subgroup of patients who have a combination of the RCT polymorphism RCT-DD and one of the AGT polymorphisms (235T) described above (Kamitani et al., Hypertension 24: 381, 1994). Recently, six RCT polymorphisms were identified and characterized (Villard et al., Am. J. Human Genet. 58: 1268-1278, 1996). The actions of vasoconstrictor conservation, cell growth promotion and Ang II salt are mediated through the binding to, and activation of, angiotensin receptors, of which at least two types have been cloned (AT1 and AT2). The Ang II receptor type 1 (AT1), a seven transmembrane domain protein coupled to the G protein, was widely distributed in the body and mediated almost all known effects of Ang II (Fyhrquist et al., J. Hum. Hypertension 5519-524, 1995).

Several polymorphisms have been identified in the AT1 receptor gene. Initial studies suggest that at least one of them is more frequent in hypertensive subjects (AT1166C) (Bonnardeaux et al., Hypertension 24_: 63-69, 1994). This polymorphism, combined with the RCT-DD polymorphism, has been shown to be strongly related to the risk of myocardial infarction (Tiret et al., Lancet 344: 910-913, 1994).

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 positive treatment produced in a patient suffering from cardiovascular disease. This includes the identification of individuals who are more or less susceptible to particular therapeutic regimens, including, e.g., particular drugs that are conventionally used to treat cardiovascular disease. There is also a need in the art for methods and compositions that allow for the identification of individuals who have a predisposition to cardiovascular disease, such as, for example, myocardial infarction, hypertension, atheroclerosis and stroke, to facilitate early intervention and prevention of the illness. SUMMARY OF THE INVENTION The present invention provides methods for evaluating cardiovascular status in a human individual. Cardiovascular status is the physiological state of the cardiovascular system as reflected in one or more state markers. The state markers include without limitation the clinical parameters, such as, for example, blood pressure, electrocardiographic profile. as well as diagnoses of the cardiovascular state made by the expert doctors, such as, for example, acute myocardial infarction, inactive myocardial infarction, apoplexy and atherosclerosis. Changes in markers of status over time. The methods of the invention are carried out by the steps of: (i) determining the sequence of one or more polymorphic positions within one or more of the genes encoding the angiotensin converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor type 1 (AT1) in the individual to establish a polymorphic pattern for the individual; and (ii) comparing the polymorphic pattern established in (i) with the polymorphic patterns of individuals exhibiting predetermined markers of cardiovascular status. The polymorphic pattern of the individual is preferably highly similar and, more preferably, is identical to the polymorphic pattern of individuals exhibiting particular state markers, cardiovascular syndromes and / or particular patterns of response to therapeutic interventions. For example, a comparison of the polymorphic pattern of an individual with the polymorphic patterns of individuals exhibiting different responses to a particular therapeutic intervention can be used to predict the degree of responsibility of the individual to said intervention. In a similar manner, the methods of the invention can be used to predict the predisposition to different cardiovascular syndromes. The invention also provides isolated nucleic acids encoding ECA, AGT. and AT1 in an individual, each of which comprises at least one polymorphic position. In preferred embodiments, the polymorphic position, either alone or in combination with other polymorphic positions in the sequence of human ACE, AGT or AT1, or in one or more of other human genes, is predicted from a particular level of response to a given treatment and / or indicates a predisposition to one or more clinical syndromes associated with cardiovascular disease Nucleic acids isolated according to the invention (which are described using the numeration indicated in Table 1 below) include without limitation (i) Nucleic acids encoding ECA having one or more po-morphic 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 numbered at the entry of Genbank X62855 In preferred embodiments, the sequences at the polymorphic positions in the regulatory region of ECA are one or more of 5106C and 5106T, and the sequences in the polymorphic 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 at the entry of Genbank X62855 (n) Nucleic acids encoding AGT having one or more polymorphic positions in the positions in the regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072 and 1204; the positions in the coding region 273, 912, 997, 1116 and 1174; and position 49 numbered as the entry of Genbank M24688. In preferred embodiments, the sequences at the polymorphic positions in the AGT regulatory region are one or more of 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 692C, 692T, 839G, 839A, 1007A, 1072G, 1072A , 1204C and 1204A; the sequences at the polymorphic position in the coding region are one or more of 273C, 273T, 912C, 912T, 997G, 997C, 1116G, 116A, 1174C and 1174A; and the sequence at position 49 at the Genbank entry M24688 either A or G. (iii) Nucleic acids encoding AT1 having one or more polymorphic positions at the 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 polymorphic positions in the AT1 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 polymorphic positions in the coding region are one or more of 449G, 449C, 678T, 678C, 1167A, 1167G, 1271A and 1271C. The invention also encompasses targets of isolated nucleic acid sequences wherein each sequence in the library comprises one or more polymorphic positions in the genes encoding human ECA, AGT, or AT1, including without limitation the polymorphic positions and sequences described in I presented. Nucleic acid probes that hybridize specifically to the identified polymorphic positions are also provided; peptides and polypeptides comprising polymorphic positions; and antibodies specific for polymorphism, i.e., antibodies specific for sequences that differentially bind to polymorphic variants of the ECA, AGT, or AT1 polypeptides and, preferably, can be used to identify particular polymorphic variants. In still another aspect, the invention provides equipment for the determination of polymorphic patterns in the genes of ACE, AGT and / or AT1 of an individual. The kits comprise a means for detecting polymorphic sequences, including without limitation oligonucleotide probes that hybridize to, or are adjacent to, the polymorphic positions and to the polymorphism-specific antibodies. In yet another aspect, the invention provides nucleic acid targets and polypeptides for use in screening methods to identify candidate cardiovascular drugs. The nucleic acid targets may be, eg, DNA or RNA and preferably at least about 10, and even more preferably at least about 15 in length and comprise one or more polymorphic positions. The peptide targets are at least about 5 amino acids in length and can be as long or longer than full-length ACE, AGT, or AT1 polypeptides.

DETAILED DESCRIPTION OF THE INVENTION All patents, patent applications, publications and other materials cited herein are incorporated herein by reference in their entirety. In the case of inconsistencies, it is intended to control the present description, including definitions. Definitions 1. A "polymorphism" as used herein, denotes a variation in the sequence of a gene in an individual. A "polymorphic position" is a predetermined nucleotide position within the sequence of a gene or a predetermined amino acid position in the sequence of a polypeptide in which a polymorphism is located. An individual "homozygote" for a particular polymorphism is one in which both copies of the gene contain the same sequence at the polymorphic position. An individual "heterozygote" for a particular polymorphism is one in which the two copies of the gene contain different sequences in the polymorphic position 2. A "polymorphism pattern" as used herein, denotes a set of one or more polymorphisms, which may be contained in the sequence of a single or a plurality of genes A polymorphism pattern may comprise polymorphisms of nucleotides or amino acids 3. "Nucleic acid" or "polynucleotides" as used herein refers to polymers containing purine and pyrimidine of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.Nucleic acids include, without limitation, single-stranded or double-stranded molecule, ie, DNA-DNA hybrids, DNA- RNA and RNA-RNA, as well as "protein nucleic acids" (ANP) formed by conjugating bases to a structure of the amino acid base. eicos that contain modified bases. 4. An "isolated" nucleic acid or polypeptide, as used herein, refers to a nucleic acid or polypeptide that is removed from its original environment (e.g., its natural environment if present in nature). An isolated nucleic acid or polypeptide contains less than about 50%, preferably less than about 75% and even more preferably less than about 90%, of the cellular components with which it was originally associated. 5. A nucleic acid sequence or polypeptide, i.e. "derived from" a designated sequence, refers to a sequence corresponding to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are homologous or complementary to the sequence. 6. A "probe" refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target nucleic acid due to the complementarity of at least one sequence in the probe with a sequence in the nucleic acid White. 7. Nucleic acids can be "hybridized" to one another when at least one strand of the nucleic acid can anneal another strand of nucleic acid under defined restriction conditions. The restriction of hybridization was determined, eg, by a) the temperature at which the hybridization and / or washing was carried out, and b) the ionic strength and polarity (eg, formamide) of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two nucleic acids contain substantially complementary sequences; depending on the restriction of hybridization, however, inequalities can be tolerated. The appropriate restriction for hybridizing nucleic acids depends on the length of nucleic acids and the degree of compiementarity, the variables well known in the art, for example, "high restriction" as used herein, refers to hybridization and / or washed at 68 ° C in 0.2XSSC, at 42 ° C in 50% formamide, 4XSSC, or under conditions that give hybridization levels equivalent to those observed under either of these two conditions. 8. A "gene" of a particular protein as used herein, refers to a nucleic acid sequence contiguous with a sequence present in a genome which comprises (i) a "coding region", which comprises exons ( that is, sequences encoding a polypeptide sequence or "protein coding sequences"), bystanders and sequences at the junction between exons and introns; and (ii) regulatory sequences, which flank the coding region at both 5 'and 3' ends. For example, the "ECA gene" as used herein encompasses the regulatory and coding regions and the human gene encoding the angiotensin-converting enzyme. Similarly, the "AGT gene" encompasses the regulatory and coding regions of the human gene encoding angiotensinogen and the "AT1 gene" encompasses the regulatory regions and encodes the angiotensin II receptor type 1 human gene coding. Normally, the regulatory sequences according to the invention are located 5 '(ie, upstream) of the coding segment. The reference sequences, obtained from Genbank, which were used to practice the present invention are shown in Table 1.

Table 1 The inventors of the present surprisingly and unexpectedly have discovered the existence of genetic polymorphisms within human genes encoding ACE, AGT, and AT1 which, alone or in combination, can be used to assess cardiovascular status. According to the invention, the polymorphic pattern of the sequences ECA, AGT and / or AT1 in an individual can predict the response 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 evaluating cardiovascular status by detecting polymorphic patterns in an individual. The present invention also provides isolated nucleic acids derived from genes ECA, AGT and AT1 comprising these polymorphisms, including probes that specifically hybridize to polymorphic positions; polypeptides and isolated peptides comprising polymorphic residues; and to antibodies that specifically recognize the ECA, AGT, or AT1 polypeptides that contain one or more polymorphic amino acids. Methods for Evaluating Cardiovascular Status The present invention provides diagnostic methods for evaluating cardiovascular status in a human individual. Cardiovascular status as used herein refers to the physiological state of an individual cardiovascular system as reflected by one or more markers or indicators. The state markers include, without limitation, clinical measurements such as, eg, blood pressure electrocardiographic profile and differential blood flow analysis. The state markers, according to the invention, include diagnoses of one or more cardiovascular syndromes, such as, for example, hypertension, acute myocardial infarction, inactive myocardial infarction, apoplexy and atheromalerosis. It will be understood that a diagnosis of a cardiovascular syndrome made by a medical practitioner encompasses clinical measurements and medical judgment. State markers according to the invention are evaluated using conventional methods well known in the art. Also included in the assessment of cardiovascular status are quantitative or qualitative changes in the markers of status over time, so that they could be used, e.g., to determine the response of an individual to a therapeutic regimen. The methods were carried out by the steps of: (i) determining the sequence of one or more polymorphic positions within one or more of the genes encoding the angiotensin converting enzyme (ACE), angiotensinogen (AGT) or receptor angiotensin II type 1 (AT1) in the individual to establish a polymorphic pattern for the individual; and (ii) compare the polymorphic pattern established in (i) with the patterns of human beings that exhibit different markers of cardiovascular status. The polymorphic pattern of the individual is preferably highly similar and, more preferably, is identical to the polymorphic pattern of individuals exhibiting particular state markers, cardiovascular syndromes and / or particular patterns of response to therapeutic interventions. The therapeutic patterns may also include polymorphic positions in other genes shown, in combination with one or more polymorphic positions in ECA, AGT or AT1, to correlate with the presence of state markers. In one embodiment, the method involves comparing a polymorphic pattern of an individual with polymorphic patterns of individuals that have been shown to respond positively or negatively to a particular therapeutic regimen. The therapeutic regimen as used herein, refers to treatments assisted in the elimination or reduction of symptoms and events associated with cardiovascular disease. Such treatments include, without limitation, one or more of alteration in diet, lifestyle and exercise regimen; invasive and non-invasive 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 , midazole receptor blockers, endothelial receptor blockers and organic nitrites. Interventions are also covered with yet unknown pharmaceutical agents whose activity correlates with the particular polymorphic patterns associated with cardiovascular disease. The present inventors have discovered that particular pohmorphic patterns correlate with an individual's responsibility to ACE inhibitors (see, for example, the following Example 3) It is contemplated, for example, that patients who are candidates for a particular therapeutic regimen they were screened for po-morphic patterns that correlate with the response to the particular regimen. In a preferred embodiment, the presence or absence of an individual of a polymorphic pattern comprising ACE2193 A / G, AGR 1072 G / G and AT1 A / A (see more below) is determined to identify the response of an individual to ACE inhibitors. In another embodiment, the method involves comparing a polymorphic pattern of individuals with polymorphic patterns of individuals exhibiting or having exhibited one or more markers of cardiovascular disease, such as g, high blood pressure, abnormal electrocardiographic profile, myocardial infarction, stroke or atherosclerosis (see, for Example, Example 2 below) To practice the methods of the invention, a polymorphic pattern of the individual has been established by obtaining DNA from the individual and determining the sequence at the predetermined polymorphic positions in ECA, AGT and AT1 as described above. DNA can be obtained from any cellular source Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, urinary epithelial cells, fetal cells or any cells present in tissue obtained by biopsy. Cells can also be obtained from bodily fluids, including without limitation, blood, saliva, sweat, urine, cerebrospinal fluid, stool and tissue exudates at the site of infection or inflammation. The DNA is extracted from the cell source or body fluid using any of the numerous methods that are normal in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source. The determination of the DNA sequence extracted at polymorphic positions in the ECA, AGT and / or AT1 genes is achieved by any means known in the art, including but not limited to, direct sequencing, hybridization with allele-specific oligonucleotides, specific PCR for alleles, ligase PCR, HOT separation, denaturing gradient gel electrophoresis (DDGE) and single-strand conformal polymorph (SSCP). Direct sequencing can be achieved by any method including, without limitation, chemical sequencing, using the axam-Gilbert method; by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; and sequencing using a fragment-based technology. See, for example, Little and others, Genet. Anal. 6,151, 1996. Preferably, the DNA of a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. In an alternative embodiment, the biopsy tissue is obtained from a subject. Antibodies that are capable of distinguishing between different polymorphic forms of ECA, AGT and / or AT1 and then applied to tissue samples to determine the presence or absence of a polymorphic form specified by the antibody. The antibodies can be polyclonal or monoclonal, preferably monoclonal. The measurement of the specific antibody that binds to the cells can be achieved by any known method, e.g., quantitative flow cytometry or enzyme-linked or fluorescence-linked immunoassay. The presence or absence of a particular polymorphism or polymorphic pattern, and its allelic distribution (ie, homozygosity against heterozygosity) is determined by comparing values obtained from a patient with established norms of patient populations that have known polymorphic patterns. In an alternative embodiment, the RNA is isolated from the biopsy tissue using normal methods well known to those skilled in the art such as extraction of guanidinium thiocyanate-phenol-chloroform (Chomocyznski et al., 1987, Anal. Biochem., 162: 156 ). The isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (TI-PCR), using specific oligonucleotide primers. The conditions for collecting the primer are chosen in order to ensure specific reverse transcription and amplification; therefore, the appearance of an amplification product diagnoses the presence of particular alleles. In another embodiment, the RNA is reverse transcribed and amplified, after which the amplified sequences are identified by, eg, direct sequencing. To practice the present invention, the distribution of polymorphic patterns in a large number of individuals exhibiting particular markers of cardiovascular status is determined by any of the methods described above and compared with the distribution of polymorphic patterns in patients that have been matched by age. , ethnic origin and / or other statistically and medically relevant parameters, which exhibit quantitatively or qualitatively different state markers. The correlations are achieved using any method known in the art, including nominal logistic regression or regression analysis of normal minimum squares. In this way, it is possible to establish statistically significant correlations between particular polymorphic patterns and particular cardiovascular states. It is also possible to establish statistically significant correlations between particular polymorphic patterns and changes in cardiovascular status, such as could result, e.g., from particular treatment regimens. In this way, it is possible to correlate the polymorphic patterns with the response to particular treatments. Polymorphic Positions in Genes that Code ECA. AGT and AT1 The polymorphic positions in the genes encoding ACE, AGT and AT1 that are encompassed by the invention, are identified by determining the DNA sequence of all or part of the genes ECA, AGT and / or AT1 in a multiplicity of individuals in a population. DNA sequence determination can be achieved using any conventional method, including, eg, chemical or enzymatic sequencing. The polymorphic positions of the invention include without limitation those listed below, which numbering corresponds to the Genbank sequences listed in Table 1. (i) RCT: positions in the regulatory region (designated ACR) numbered 5106, 5349 and 5496; positions in the coding region (designated RCTs) numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503 and 3906; and position 1451 as it is numbered at the entrance of Genbank X62855. (ii) AGT: positions in the regulatory region (designated AGR) numbered 395, 412, 432, 449, 692, 939, 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 it was numbered at the entrance of Genbank M24688. (iii) AT1: positions in the regulatory region (designated ATR) numbered 1427, 1756, 1853, 2046, 2354. 2355 and 2415; and the positions in the coding region (designated AT1) numbered 449, 678, 1167 and 1271. In the preferred embodiments, the sequence in each of the above polymorphic positions is one of: (i) ECA Regulatory Region: 5106C. 5106T, 5349A, 5349T, 5496T and 5496C; (ii) ECA Coding Region: 375A, 375C, 582C, 582T, 731A, 731G, 1060G, 1060A, 1215C, 1215T, 2193G, 2193A, 2328A, 2328A, 2328G, 2741G, 2741T, 31.32C, 3132T, 3387T, 3387C, 3503G, 3503C, 3906G and 3906A; and a deletion of nucleotides 1451-1783 as numbered in the entry of Genbank X62855; (iií) 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, 1116G, 1116A, 1174C and 1174A; and A or G at position 49 at the entrance of Genbank M24688; (v) AT1 Regulatory Region: 1427A, 1427T, 1756T, 1756A, 1853T, 1853G, 2046T, 2046C, 2354A, 2354C, 2355G, 2355C, 2415A and 2415G; and (vi) Coding Region of AT1: 449G, 449C, 678T, 678C, 1167A, 1167G, 1271A and 1271C. An individual can be homozygous or heterozygous for a particular polymorphic position (see, for example, Table 6 below). Non-limiting examples of polymorphic patterns comprising one or more polymorphisms in the ACE, AGT and / or genes AT1 according to the invention include the following, which correlated an increasing 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; ECA 2193 A, AGR 1204 C, ECA 2328 G; ECA 2193 A, AGR 1204 A / C; ECA 3387 T, AGR 1218 A; ECA 3387 T, AGT 620 C / T; AGR 1204 A / C, AT1 678 C / T; AGR 1204 A / C, AT1 1271 A / C; ECA 1215 C, AGR 1204 A / C; AGR 1204 A / C, AT1 1167 A, ECA 3906 A / G; AGR 1204 A, AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T, AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T; AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T; AGR 1204 A, AT1 678 C, AT1 1167 A, AGR 395 A / T; AGR 1204 A / C, AT1 678 C / T, AT1 1167 A, AGR 395 T; AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T; AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T; AGT 620 C, AT1 678 A, AT1 1167 A, AGR 395 A / T; AGT 620 C / T, AT1 678 C / T; AT1 1167 A, AGR 395 T; ECA 2193 A, AGR 1218 A, AGT 803 A; ECA 2193 A, AGT 620 C / T; ECA 2328 G, AGT 620 C / T; ECA 3387 T, AGR 1204 A / C; ECA 2193 A, ECA 2328 G, AGR 1204 C; and ECA 2193 A / G, AGR 1072 G / G, AT1 1167 A / A. Isolated Polymorphic Nucleic Acids, Probes and Vectors The present invention provides isolated nucleic acids comprising the polymorphic portions described above for the human ECA, AGT and AT1 genes; vectors comprising the nucleic acids; and transformed host cells comprising the vectors. The invention also provides probes that are useful for detecting these polymorphisms. To practice the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. Said techniques are well known and are fully explained in, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratoy Manual. Second Edition, 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. Gaid 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). The insertion into a vector of nucleic acids (usually the DNAs) comprising the sequences of the present invention is easily accomplished when the terminations of both DNAs and the vector comprise a compatible restriction site. If this could be used, it may be necessary to modify the DNA and / or vector endings by digesting surplus single-stranded DNA generated by the restriction endonuclease separation in order to produce blunt ends or to achieve the same result by filling it in the terminations of a single strand with an appropriate DNA polymerase. Alternatively, any desired site can be produced, e.g., by ligating the nucleotide (crosslinker) sequences on the terminations. Said interleavers may comprise specific oligonucleotide sequences that define the desired restriction sites. Restriction sites can also be generated by the use of polymerase chain reaction (PCR). See, for example Saiki et al., 1988, Science 239: 48. The separated vector and the DNA fragments can also be modified if required by the homopolymeric configuration. The nucleic acids can be isolated directly from cells or can be synthesized chemically using known methods. Alternatively, the polymerase chain reaction (PCR) method can be used to produce the nucleic acids of the invention. The chemically synthesized threads or the genomic material as patterns. The 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 appropriate, to facilitate incorporation into a given vector for recombinant expression. The nucleic acids of the present invention can be flanked by native ECA, AGT, or AT1 gene sequences or can be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, regions that they do not code 5 'and 3'. The nucleic acids can also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, ": encapsulation", substitution of one or more of the nucleotides present in nature with an analogue, modifications of internucleotides such as, for example, those with uncharged bonds (e.g. , methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.,) and with charged linkers (eg, phosphorothioates, phosphorodithioates, etc.). The nucleic acids may contain one or more covalently linked portions, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (v. ., acridine, psoralen, etc.), chelants (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The ANPs are also included. The nucleic acid can be derivatized by the formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate ligature. In addition, the nucleic acid sequences of the present invention can also be modified with a tag capable of providing a detectable signal, either directly or indirectly. Illustrative labels include radioisotopes, fluorescent molecules, biotin, and the like. The invention also provides nucleic acid vectors comprising the sequences of genes derived from AGT and AT1 described or derivatives or fragments thereof. A large number of vectors, including plasmid and fungal vectors, have been described for replication and / or deletion in a variety of eukaryotic and prokaryotic hosts, and can be used for gene therapy as well as for the simple cloning or expression of proteins. Non-limiting examples of any vector include, without limitation, pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wl) or pRSET or pREP (Invitrogen, San Diego, CA) and many appropriate host cells, using methods described above. in the present or in some way 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 can be transformed / transfected / infected as appropriate, by any method including electroporation, CaCI2 mediated DNA uptake, fungal or viral infection, microinjection, microprojection or other established methods. Suitable host cells included bacteria, archaebacteria, fungi, especially yeast and plant and animal cells, especially mammalian cells. A large number of regulatory regions of transcription initiation and termination have been isolated and shown to be effective in the transcription and translation of heterologous proteins in several hosts. Examples of these regions, isolation methods, manner of handling, etc., are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced ECA, AGT or AT1 derived peptides and polypeptides. Nucleic acids that encode sequences of genes derived from ECA, AGT or AT1 can also be introduced into cells by recombination events. For example, said sequence can be introduced into a cell and thus carry out the homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene. Other methods based on recombination such as non-homologous recombinations or deletion of endogenous genes by homologous recombination can also be used. The nucleic acids of the present invention are used as probes for the detection of genetic polymorphisms and as standards for the recombinant production of peptides or polypeptides derived from normal ECA, AGT or AT1 or variants. Probes according to the present invention comprise without limitation isolated nucleic acids of about 10-10 bp, preferably 15-76 bp and more preferably 17-25 bp in length, which hybridize at a high restriction to one or more of the polymorphic sequences derived from genes of ACE, AGT, or AT1 described herein or to a sequence immediately adjacent to a polymorphic position. In addition, in some embodiments a full length gene sequence can be used as a probe. In a series of modalities, the probes expand the polymorphic positions in the ECA, AGT or AT1 genes described above. In another series of modalities, the probes correspond to sequences immediately adjacent to the polymorphic positions. Polypeptides of ACE, AGT and AT1 Polymorphs and Antibodies Specific to Polymorphisms The present invention encompasses isolated peptides and polypeptides that encode ECA. AGT, AT1 comprising polymorphic positions described above. In a preferred embodiment, the peptides and polypeptides are useful screening targets to identify cardiovascular drugs. In other preferred embodiments, the peptides and polypeptides are capable of producing antibodies in a suitable host animal that specifically reacts with a polypeptide comprising the polymorphic position and distinguishes it from other polypeptides having a sequence different from the position. The polypeptides according to the present invention preferably have five or more residues in length, preferably at least fifteen residues. The methods for obtaining said polypeptides are described below. Many conventional techniques in the biochemistry of protein and immunology are used. These techniques are well known and are explained in Immunochemical Methods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press, London); Scopes, 1987, Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.) and Handbook of Experimental Immunology, 1986, Volumes I - 1 V (Weir and Blackwell eds). Nucleic acids comprising protein coding sequences can be used to direct the recombinant expression of polypeptides derived from ECA, AGT or AT1 in intact cells or free translation systems. The known genetic code, specified if desired for the most efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The polypeptides can be isolated from human cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insects, plants and mammalian cells) in which appropriate protein coding sequences have been introduced and expressed. In addition, the polypeptides may be part of the recombinant fusion proteins. The peptides and polypeptides may be chemically synthesized by commercially available automatic methods, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Polypeptides are preferentially prepared by solid phase peptide synthesis as described by Merpfield, 1963, J Am Chem Soc 852149 Methods for the purification of polypeptides are well known in the art, including without limitation, preparative disk gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography and countercurrent distribution For some purposes, it is preferably to produce the pohpeptide 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 a solid phase matrix. Alternatively, antibodies raised against ECA, AGT or AT1 or against peptides derived therefrom, can be used as purification reagents. Other purification methods are possible. The present invention also encompasses derivatives and homologs of the polypeptides. For some purposes, nucleic acid sequences encode peptides that can be altered by substitutions, additions, or deletions that provide functionally equivalent molecules, ie, conservative variants of function. For example, one or more amino acid residues within the sequence can be substituted by other amino acids of similar properties such as, for example, positively charged amino acids (arginine, lysine and histidine); negatively charged amino acids (aspartate and glutamate); neutral polar amino acids; and non-polar amino acids. The isolated polypeptides can be modified, for example, by phosphorylation, sulfonation, acylation or other protein modifications. They can 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 invention also encompasses antibodies that specifically recognize the polymorphic positions of the invention and distinguish a peptide or polypeptide containing a particular polymorphism from one that contains a sequence different from this position. Said antibodies specific for the polymorphic position according to the present invention, include polyclonal antibodies and monoclonal antibodies. Antibodies can be produced in a host animal, by immunization with immunogenic components derived from ECA, AGT or AT1 or they can be formed or in vitro immunization of immune cells. The immunogenic components used to give the antibodies can be isolated from human cells or produced in recombinant systems. The antibodies can also be produced in recombinant systems programmed with DNA encoding appropriate antibodies. Alternatively, the antibodies can be constituted by biochemical reconstitution of purified heavy and light chains. Antibodies include hybrid antibodies, (ie, containing two groups of heavy chain / light chain combinations, each of which recognizes a different antigen), chimeric antibodies (ie, in which either heavy chains, light chains or both, are fusion proteins) and univalent antibodies (ie comprised of a heavy chain / light chain complex linked to the constant region of a second heavy chain). Also included are Fab fragments, including Fab 'fragments and F (ab) 2 antibodies. The methods for the production of all the above types of antibodies and derivative are well known in the art and are discussed in more detail below. For example, techniques for producing and processing polyclonal antisera are described in Mayer and Walek, 1987, Immunochemical Methods in Cell and Molecular Biology, (Academic Press, London). The general methodology for forming monoclonal antibodies by hybridomas is well known. The immortal antibody production 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, for example, Schreier et al., 1980, Hybridoma Techniques; Patents of E.U.A nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491,631; and 4,493,890. Monoclonal antibody panels produced against epitopes derived from CE, AGT, or AT1 can be screened for various properties; that is, for isotype affinity, epitope, etc. The antibodies of this invention can be purified by standard methods, including but not limited to preparative gel gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange, and partition chromatography and countercurrent distribution. Purification methods for antibodies are described, e.g., in he 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., 1987, Springer-Verlag, New York, NY. Methods for determining the immunogenic capacity of the sequences described and the characteristics of the antibodies specific for resulting sequence and immune cell are well known in the art. For example, antibodies produced in response to a peptide comprising the particular polymorphic sequence can be tested for their ability to specifically recognize the polymorphic sequence, i.e. to differentially bind to a peptide or polypeptide comprising the polymorphic sequence and thereby distinguish it from a similar peptide or polypeptide that contains a different sequence in the same position. Methods and Diagnostic Equipment The present invention provides equipment for the determination of the sequence in the polymorphic positions within the genes ECA, AGT and AT1 in an individual. The equipment comprises a means to determine the sequence in one or more polymorphic positions and optionally may include data for the analysis of polymorphic patterns. The mean for the determination of the sequence may comprise reagents based on suitable nucleic and immunological acids (see below). Preferably, the kits can also comprise suitable pH buffer solutions, control reagents where appropriate and directions to determine the sequence in a polymorphic position. The teams can also understand data for the correlation of particular polymorphic patterns with suitable treatment regimes or other Indicators. Nucleic acid-based diagnostic methods and equipment The invention provides nucleic acid-based methods for detecting polymorphic patterns in a biological sample. The sequence at particular polymorphic positions in the genes encoding ACE, AGT, and / or AT1 is determined using suitable means known in the art, including without limitation, hybridization with specific probes for polymorphism and direct sequencing. The present invention also provides suitable equipment for diagnostic applications based on nucleic acids. In one modality, the diagnostic equipment includes different components: (i) DNA probe: The DNA probe can be previously labeled; alternatively, the DNA probe may be unlabelled and the labeling ingredients may be included in the separate container kit; and (ii) Hybridization reagents: The equipment may also contain other reagents appropriately packed materials required for the particular hybridization protocol, including solid phase matrices, if applicable and are normal. In another embodiment, diagnostic kits include: (i) Sequence determination initiators: Sequence primers may be pre-marked or may contain an affinity purification or binding portion: and (ii) Sequence determination reagents: The kit it may also contain other appropriately packaged reagents and materials required for the particular sequencing protocol. In a preferred embodiment, the kit comprises a panel of sequencing primers, whose sequences correspond to the sequences adjacent to the following polymorphic positions: ECA 2193 A / G, AGR 1072 G / G, AT1 1167 A / A; as well as a means to detect the presence of each polymorphic sequence. Antibody-Based Diagnostic Methods and Equipment The invention also provides antibody-based methods for detecting polymorphic patterns in a biological sample. The methods comprise the steps of: (i) contacting a sample with one or more antibody preparations, wherein each antibody preparation is specific for a particular polymorphic form of ACE, AGT or AT1, under conditions in which it can be form a stable antigen-antibody complex between the antibody and antigen 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 polymorphic form in the sample. Typically, the immunoassay utilizes a labeled antibody or a labeled antigenic component (e.g., that competes with the antigen in the sample to bind to the antibody). Suitable labels include, without limitation, enzyme-based, fluorescent, chemiluminescent, radioactive, or dye-based molecules. Analyzes that amplify signals for the probe are also known, such as, for example, those using biotin and avidin and enzyme-labeled immunoassays, such as ELISA analysis.

The present invention also provides suitable equipment for antibody-based diagnostic applications. Diagnostic equipment typically includes one or more of the following components: (i) Antibodies specific for polymorphism: Antibodies may be pre-labeled; alternatively, the antibody may be unlabelled and the labeling ingredients may be included in the kit in separate containers, or a labeled secondary antibody is provided, and (ii) Reaction components: The kit may also contain other appropriately packaged reagents and required materials for the particular immunoassay protocol, including solid-phase matrices, if applicable and standards. The equipment referred to above may include instructions to carry out the test. Furthermore, in the preferred embodiments, the diagnostic equipment can be adapted to the operation of a high number of steps and / or automatic. Targets and Drug Screening Methods According to the present invention, the nucleotide sequences derived from genes encoding ECA, AGT, and AT1 and peptide sequences derived from the ACE, AGT, and AT1 polypeptides, particularly those containing a more polymorphic sequences. they comprise useful targets for identifying cardiovascular drugs, ie, 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 ECA, AGT and AT1 coding genes and (ii) isolated peptides and polypeptides derived from ECA, AGT and AT1 polypeptides, each of which comprises a or more polymorphic positions. In vitro Screening Methods: In a number of embodiments, an isolated nucleic acid comprising one or more polymorphic positions was tested in vitro for its ability to bind to the test compounds in a sequence-specific manner. The methods comprise: (i) providing a first nucleic acid containing a particular sequence in a polymorphic position and a second nucleic acid whose sequence is identical to that of the first nucleic acid except for a different sequence in the same polymorphic position. (ii) contacting the nucleic acids with a multiplicity of test compounds under conditions appropriate for binding; and (ii) identifying compounds that selectively bind to the first or second nucleic acid sequence. Selective binding as used herein refers to any measurable difference in any binding parameter, such as, for example, binding affinity, binding capacity, etc.

In another series of embodiments, an isolated peptide or polypeptide comprising one or more polymorphic positions is tested in vitro for its ability to bind to the test compounds in a sequence specific manner. Screening methods involve: (i) providing a first peptide or polypeptide that contains a particular sequence in a polymorphic position and a second peptide or polypeptide whose sequence is identical to the first peptide or polypeptide except for a different sequence in the same polymorphic position; (ii) contacting the polypeptides with a multiplicity of test compounds under conditions appropriate for binding; Y (ii) identify the compounds that selectively bind to one of the nucleic acid sequences. Preferred embodiments, screening protocols of high pass content are used to screen a large number of test compounds for their ability to bind to the genes or peptides described above in a sequence-specific manner. The test compounds are screened from large banks of synthetic or natural compounds. Numerous means are currently used for the random and targeted synthesis of compounds based on saccharides, peptides. and nucleic acids. Synthetic compound banks are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). Alternatively, libraries and natural compounds in the form of bacteria, fungi, plants and animal extracts are available from eg, Pan Laboratories (Bothell, WA) or MycoSearch (NC) or can be easily produced. Additionally, natural banks are produced synthetically are compounds that are easily modified through conventional chemical, physical and biochemical means. In vivo screening methods: Intact cells or whole animals expressing polymorphic variants of genes encoding ACE, AGT and / or AT1 can be used screening methods to identify candidate cardiovascular drugs. In a series of modalities, a permanent cell line is established for an individual that exhibits a particular polymorphic pattern. Alternatively, the cells (including without limitation cells of mammals, insects, yeast or bacteria) are programmed to express a gene comprising one or more polymorphic sequences by the introduction of an appropriate DNA. The identification of candidate compounds can be achieved using any suitable analysis, including without limitation (i) analysis to measure the selective binding of the test compounds to particular polymorphic variants of ECA, AGT, or AT1; (I) analysis to measure the ability of at least one compound to modify (i.e., inhibit or increase) the measurable amount or function of ECA, AGT or AT1; and (iii) analysis that measures the ability of a compound to modify (i.e., inhibit or increase) the transcriptional activity of sequences derived from the promoter (ie regulatory) regions of ACE, AGT or AT1 genes. In another set of embodiments, the transgenic animals created in which (i) one or more genes of HSA, AGT, or AT1 having different sequences and particular polymorphic positions are stably inserted into the genome of the transgenic animal; and / or (ii) the endogenous EC, AGT and / or AT1 genes are inactivated and replaced with the human ECA, AGT and / or AT1 genes having different sequences at polymorphic positions. See, for example Coffman, Semin, Nephrol. 1_7: 404, 1997; Esther et al., Lab. Invst. 74/953, 1996; Murakami and others, Blood Press. Suppl. 2.36, 1996. Said animals can be treated with candidate compounds and monitored for one or more clinical markers of cardiovascular status. The following are intended to be non-limiting examples of the invention. Example 1: Methods for the Identification of Polymorphic Positions in Human Genes Coding for ECA, AGT and AT1 The following studies were carried out to identify polymorphic residues within the genes encoding ECA, AGT and AT1. DNA samples were obtained from 277 individuals. The individuals were Caucasian men born in Uppsala, Sweden between 1920 and 1924. Individuals were selected for the test population based on their medical history, that is, they were (i) healthy, without signs and cardiovascular disease (1010); or (ii) had suffered from acute myocardial infarction (68), inactive myocardial infarction (34), stroke (18), stroke and acute myocardial infarction (19) or high blood pressure at the age of 50 ( 39). DNA samples were obtained from each individual. In DNA sequence analysis it was carried out by: (i) amplifying short fragments of each of the ACE, AGT and AT1 genes using polymerase chain reaction (PCR) and (ii) sequencing the amplified fragments. The sequences obtained from each individual were then compared with the genomic sequences of ECA, AGT and AT1 (see Table 1). (i) Amplification: the PCR reactions used the primers shown in the following Table 2. r Oí O n n Table 2 or on I heard r to n in in or in ) on or in 00 r in O in in eo in or in in or When indicated, the primers are modified into one of the following: (i) a biotin molecule was conjugated to the 5 'end of the indicated sequence (B); (ii) a nucleotide sequence derived from M13, 5'-CAGGAAACAGCTATGACT-3 ', is added to the 5' end of the indicated sequence (MT); or (ii) the sequence 5'-AGTCACGACGTTGTAAAACGACGGCCAGT-3 'was added to the 5'-position of the indicated sequence (T = Tail). The nucleotides were listed according to the Genbank sequences listed in Table 1 where indicated. When the sequences involved are not publicly available, the numbering was as in the following examples: the designation "i-4: 1-200" indicates that the sequence of the primer is located within the sequence extending 200 bp upstream of the first nucleotide of exon coding 4. Similarly, the designation "+ 4: 1-200" indicates that the initiator sequence is located within the sequence extending from the nucleotide, i.e. located immediately downstream of at least one nucleotide encoding exon 4 downstream for 200 bp. In each case, the specific location of the initiator sequence is indicated in Table 2 in the column marked "Nucleotides". The reaction components used for PCR are described in Table 3 below: ro rO in or in Table 3 in r » in or in in in or in in ^ ro in or in in. in rO in O in The reaction conditions used for PCR are described in Table 4 below. Table 4 in or in in r in or in in All temperatures are given in degrees Celsius. *) indicates the initial temperatures (° C) and default program times **) indicates the temperature (° C) of the default program ***) indicates the number of cycles of the default program, referring to the section of the CPR program where three different temperatures are used. in oo Any of the 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 by the amplification. Table 5 in O in or in in cr K) in or in O) or ro ro in or in c » ro ro in or in to All PCR products (except the ACEDI, AT1-spec.1 and AT1-spec.2 fragments) were subjected to solid phase sequencing according to the commercially available protocol of Pharmacia Biotech. The sequencing reactions were carried out with a sequencing primer having a sequence complementary to the "Tail" sequence previously described in Table 2. The nucleotide sequence of the sequencing primer was 5'-CGACGTTGTAAAACGACGGCCAGT-3 ', and the initiator was fluorescently labeled with a Cy-5 molecule on nucleotide 50. The positions were carried out in a genetic variation, were identified by nucleotide sequence determination by using the ALFexpress ™ system commercially available from Pharmacia Biotech. Detection of the ACEDI fragment was carried out by analyzing the sizes of the fragments amplified by gel electrophoresis, where the presence of a short PCR product (192 base pairs) indicated the D allele and a longer PCR product ( 479 base pairs) indicated the I allele. The presence of the bands indicated a heterozygote for the two alleles. Detection of the allele-specific reaction at position AT1-1271 was carried out separately by operating the PCR reactions of the two alleles on the same sample and comparing the sizes of the amplified fragments. A PCR product of 501 base pairs could be present as a control in both parallel operations. While the presence of a CPR product of 337 base pairs in the reaction designated AT1-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 short PCR product is present in both reactions, the individual is a heterozygote for A and C. Results: The results described above resulted in the identification of polymorphic positions within the regulatory segments and encoding / introns segments of human genes encoding ACE, AGT and AT1. The polymorphic positions have been found, the nucleotides of variants in each of the positions and the PCR fragment in which the polymorphism was identified, is shown in Table 6 below. The frequencies of each genotype are also shown in a population of 90 individuals. Expressed as the percentage of the study of the population that have the genotype. Polymorphisms that result in alternate amino acids in ACE, AGT and AT1 are also indicated. As used hereinafter, the designations "AGR", "ACR" and "ATR" refer to regulatory regions of the AGT, ECA and AT1 genes, respectively; and the designations "AGT", "ECA" and "AT1", refer to the coding regions of the AGT, ECA and AT1 genes. ro ro en o en en Table 6 05 I heard ro ro in or in ro ro in or in s > oo- A subgroup of these polymorphic positions were further analyzed in 187 additional individuals. Table 7 shows the polymorphic positions, the sequences in these positions and the genotype frequencies for each position in a population of 277 as described in Example 1 above. Table 7 Example 2: Correlation of Polymorphic Patterns with Cardiovascular Disease The polymorphic positions identified as in Example 1 correlate with the following markers of cardiovascular status present in the population study: myocardial infarction (Ml); apoplexy; and high blood pressure. Polymorphic patterns, ie, combinations of sequences at particular polymorphic positions, which show a statistically significant correlation with one or more of these markers are shown below. 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 ECA 2193 A, AGR 1204 C, ECA 2326 G ECA 2193 A, AGR 1204 A / C ECA 3387 T, AGR 1218 A ECA 3387 T, AGT 620 C / T AGR 1204 A / C, AT1 678 C / T AGR 1204 A / C, AT1 1271 A / C ECA 1215 C, AGR 1204 A / C AGR 1204 A / C, AT1 1167 A , ECA 3906 A / G AGR 1204 A, AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T Summary of the three previous polymorphic patterns (involving the same polymorphic positions AGR 1204 A, AT1 678 C, AT1 1167 A, AGR 395 A / T AGR 1204 A / C, AT1 678 C / T, AT1 1167 A, AGR 395 T Summary of the two previous polymorphic patterns AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T AGT 620 C / T , AT1 1271 A / C, AT1 1167 A / G, AGR 395 T AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T Summary of the three previous polymorphic patterns AGT 620 C, AT1 678 A, AT1 1167 A, AGR 395 A / T AGT 620 C / T, AT1 678 C / T; AT1 1167 A, AGR 395 T Summary of the two previous polymorphic patterns: ECA 2193 A, AGR 1218 A, AGT 803 A ECA 2193 A, AGT 620 C / T ECA 2328 G, AGT 620 C / T ECA 3387 T, AGR 1204 A / C Example 3: Correlation Between a Specific Polymorphic Pattern and Treatment Response The following study was carried out to define the polymorphic patterns in the human ACE, AGT and / or AT1 genes to 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. Patients in this population were treated with one of the following five ACE inhibitors: Captopril, Trandolapril, Lisinopirl, Fosinopril or Enalapriil. The effect of the drugs on the arterial blood pressure medium was quantified. The arterial blood pressure medium was defined as 2/3 of the diastolic blood pressure + 1/3 systolic blood pressure. Individuals were also categorized as "high responders," that is, those who exhibited a decrease of more than 16 mm Hg during treatment with an ACE-inhibiting drug and "low responders," that is, those who do not exhibit a decrease elsewhere. of 16 mm Hg. A particular polymorphic pattern, ECA 2193 A / G, AGR 1072 G / G, AT1 1167 A / A, which is present in 51% of the first study of the 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 lower blood pressure was evident. Individuals with this polymorphic pattern (designated "1" right away) experienced a greater decrease in blood pressure than those with this polymorphic pattern (designated "0" right away).

In addition, the distribution of high responders and low responders (as defined above) was as follows: Taken together, the results of the two groups indicate that the presence of this polymorphic pattern correlates with an increased decrease of 6.4-7.3 mm Hg in relation to individuals who do not have this polymorphic pattern. The prevalence of this polymorphic pattern was 41% of this hypertensive population. This suggests that the testing of this polymorphic pattern in hypertensive patients, followed by the prescription of ACE inhibitors only in those patients who have this polymorphic pattern, could increase the response rate of 43% (in a hypertensive population in general) to 76% in hypertensive population selected according to the methods of the invention.

Claims

CLAIMS 1. A method for evaluating the response to a treatment regimen of a cardiovascular syndrome in a human individual, which method comprises comparing a polymorphic pattern established at one or more polymorphic positions within one or more of the genes ECA, AGT or AT1 of said individual with a polymorphic pattern of the same polymorphic positions of humans that have a known response to the cardiovascular treatment regimen.
2. A method according to claim 1, wherein the treatment regimen is an alteration in diet, life and / or exercise; an invasive or non-invasive surgical technique; or a pharmaceutical intervention.
3. A method according to claim 1, wherein the syndrome is selected from the group consisting of myocardial infarction, hypertension, atheroclerosis and stroke.
4. A method according to claim 2, wherein the treatment regimen is a pharmaceutical intervention.
A method according to claim 4, wherein the treatment regimen comprises administering a cardiovascular drug selected from the group consisting of ACE inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antagonists, cardiac glycosides, inhibitors of phosphodiesterase, beta-adrenoreceptor antagonists, calcium channel blockers, HMG-CoA reductase inhibitors, midizoline receptor blockers, endothere receptor blockers and organic nitrites.
6. A method according to claim 5, wherein the polymorphic positions comprise ECA 2193, AGR 1072 and AT1 1167.
7. A method according to claim 6, wherein the polymorphic pattern comprises ECA 2193 A / G, AGR 1072 G / G and AT1 1167 A / A.
A method according to claim 1, for predicting the response of an individual suffering from a cardiovascular syndrome to treatment with an ACE inhibitor, which method comprises comparing the established polymorphic pattern by determining the sequence of (a) the gene of ECA at position 2193 in the coding region; (b) the AGT gene at position 1072 in the regulatory region; and (c) the AT1 gene at position 1167 in the coding region with the same polymorphic patterns of humans that exhibit different responses to the ACE inhibitor.
9. A method according to claim 8, wherein the polymorphic pattern comprises ECA 2193 A / G, AGR 1072 G / G, AT1 1167 A / A.
A method according to claim 1, wherein the polymorphic position is selected from the group consisting of positions in the ECA regulatory region numbered 5160, 5349 and 5496; positions in the ECA coding region numbered 376, 582, 731. 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503 and 3906; position 1451 in the ECA gene as it is at the entrance of Genbank X62866; 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 1174; position 49 in the AGT gene as it is numbered at the entrance of Genbank M24688; positions in the AT1 regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2356 and 2415; positions in the AT1 coding region numbered 449, 678, 1167 and 1271, and combinations of any of the foregoing.
11. A method according to claim 10, wherein the polymorphic standards are selected from the group consisting of: ACR 5349 A / T, AGR 1218 A; ECA 5496 C, AGR 1204 A / C; ACR 5496 C / T, AGR 1218 A, AGT 620 C / T; ECA 2193 A, AGR 1204 C, ECA 2328 G; ECA 2193 A, AGR 1204 A / C; ECA 3387 T, AGR 1218 A; ECA 3387 T, AGT 620 C / T; AGR 1204 A / C, AT1 678 C / T; AGR 1204 A / C, AT1 1271 A / C; ECA 1215 C, AGR 1204 A / C; AGR 1204 A / C, AT1 1167 A, ECA 3906 A / G; AGR 1204 A, AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T, AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T; AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T; AGR 1204 A, AT1 678 C, AT1 1167 A, AGR 395 A / T; AGR 1204 A / C, AT1 678 C / T, AT1 1167 A, AGR 395 T; AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T; AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T; AGT 620 C, AT1 678 A, AT1 1167 A, AGR 395 A / T; AGT 620 C / T, AT1 678 C / T; AT1 1167 A, AGR 395 T; ECA 2193 A, AGR 1218 A, AGT 803 A; ECA 2193 A, AGT 620 C / T; ECA 2328 G, AGT 620 C / T; ECA 3387 T, AGR 1204 A / C; ECA 2193 A, ECA 2328 G, AGR 1204 C; and ECA 2193 A / G, AGR 1072 G / G, AT1 1167 A / A.
12. An isolated nucleic acid encoding ECA in an individual, wherein the nucleic acid comprises a polymorphic position and wherein the presence of the polymorphic position, either alone or in combination with other polymorphic positions in the human ECA sequence or in one or more of other human genes, predicts the response of an individual to a treatment regimen of cardiovascular syndrome.
13. An isolated nucleic acid encoding AGT in an individual, wherein the nucleic acid comprises a polymorphic position and wherein the presence of the polymorphic position, either alone or in combination with other polymorphic positions in the human AGT sequence or in one or more of other human genes, predicts the response of an individual to a treatment regimen of cardiovascular syndrome.
14. An isolated huicellic acid encoding AT1 in an individual, wherein the nucleic acid comprises a polymorphic position and wherein the presence of the polymorphic position, either alone or in combination with other polymorphic positions in the sequence of AT1 human or in one or more of other human genes, predicts the response of an individual to a treatment regimen of cardiovascular syndrome.
15. An isolated nucleic acid derived from the human gene encoding the angiotensin converting enzyme (ACE), wherein the nucleic acid comprises a polymorphic position selected from the group consisting of a position in the regulatory region numbered 5106; positions in the coding region numbered 375, 582, 731, 1060, 2741, 3132, 3387, 3503 and 3906; and combinations of any of the foregoing.
16. The nucleic acid according to claim 15, wherein the sequence at the polymorphic position in the regulatory region is selected from the group consisting of 5106C and 5106T; and the sequence at the polymorphic position in the coding region is selected from the group consisting of 357A, 375C, 682C, 582T, 731A, 731G, 1060G, 1060A, 2741G, 2741T, 3132C, 3232T, 3387T, 3387C, 3503G, 3503C , 3906G, 3906A, as it is numbered at the entrance of Genbank X62855.
17. A probe that hybridizes with high restriction in the polymorphic position as defined in claim 15. 1d.
An isolated nucleic acid comprising a human gene encoding angiotensinogen (ANG), wherein the nucleic acid comprises a polymorphic position selected from the group consisting of positions in the regulatory region numbered 395, 413, 432, 449, 692, 639, 1007 and 1204; positions in the coding region numbered 273, 912, 997, 1116, 1174 and position 49 at the entrance of Genbank M24688; and combinations of any of the same.
19. A nucleic acid according to claim 18, wherein the sequence at the polymorphic position in the regulatory region is selected from the group consisting of 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 692T, 839G, 839A , 1007G, 1007A, 1072G, 1072A, 1204C and 104A; and the sequence to the polymorphic position in the coding region are selected from the group consisting of 273C, 273T, 912C, 912T, 997G, 997C, 1116G, 1116A, 1174C and 1174A and A or G at position 49 at the input of Genbank M24688.
20. A probe that hybridizes with high restriction at the polymorphic position as defined in claim 18.
21. An isolated nucleic acid comprising a human gene encoding the angiotensin II receptor type 1 (AT1) wherein the nucleic acid it comprises a polymorphic position selected from the group consisting of the positions in the regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355 and 2415; a position in the coding / intron region numbered 449; and combinations of the above.
22. A nucleic acid according to claim 21, wherein the sequence at the polymorphic position in the regulatory region is selected from the group consisting of 1427A, 1472T, 1756T, 1756A, 1853T, 1853G, 2046T, 2046C, 2364A, 2354C , 2365G, 2365C, 2416A and 2415G; and the sequence at the polymorphic position in the coding / intron region is selected from the group consisting of 449G and 449C.
23. A probe that hybridizes with high restriction in the polymorphic position as defined in claim 21.
24. A nucleic acid library, each of which comprises one or more polymorphic positions within the human ECA gene, wherein the positions polymorphic are selected from the group consisting of positions in the regulatory region of RCT numbered 5106, 5349 and 5496; and the positions in the ECA coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503 and 306 in the ECA gene as numbered in the Genbank entry X62855.
25. A bank according to claim 24, wherein the sequence at the polymorphic position in the regulatory region is selected from the group consisting of 5106C, 5106T, 5349A, 5349T, 5496T and 5496C; and the sequence in polymorphic position in the coding region is selected from the group consisting of 375A, 375C, 582C, 582T, 731A, 731G, 1060G, 1060A, 1215C, 1215T, 2193G, 2193A, 2328A, 2328G, 2741G, 2741T, 3132C, 3132T, 3387T, 3387C, 3603G, 3906F, 3906A and a deletion of nucleotides 1461-1783 as numbered in the Genbank entry X62855.
26. A nucleic acid library, wherein each of which comprises one or more polymorphic positions within the human AGT gene, wherein the polymorphic position is selected from the group consisting of positions in the regulatory region numbered 395, 412, 432 , 449. 692, 839, 1007, 1072, 1204 and 1218; positions in the coding region numbered 273, 620, 803, 912, 997, 1116 and 1174; and position 49 in the AGT gene as it is numbered at the entrance of Genbank M2468d.
27. A bank according to claim 26, wherein the sequence in the polymorphic position in the regulatory region is selected from the group consisting of 395T, 395A, 412C, 412T, 432G, 432A, 449T, 449C, 592C, 692T, 839G, 339A, 1007G, 1007A, 1072G, 1072A, 1204C, 1204A, 1218A, 1218G; and the sequence in polymorphic position in the coding region is selected from the group consisting of 273C, 273T, 620C, 620T, 803T, 803C, 912C, 912T, 997G, 997C, 1116G, 1116A, 1174C, 1174A and A or G in position 49 at the Genbank entrance M24688. 2d.
A nucleic acid library, each of which comprises one or more polymorphic positions within the human AT1 gene, wherein the polymorphic position is selected from the group consisting of positions in the regulatory region numbered 1427, 1756, 1853, 2046 , 2354, 2356 and 2415; and the positions in coding region numbered 449, 678, 1167 and 1271.
29. A bench according to claim 28, wherein the sequence in the polymorphic position in the regulatory region is selected from the group consisting of 1472A, 1427T , 1756T, 1766A, 1853T, 1853T, 1853G, 2046T, 2046C, 2354A, 2354C, 2355G, 2355C, 2415A and 2415G; and the sequence at the polymorphic position in the coding region is selected from the group consisting of 449G, 449C, 678T, 678C, 1167A, 1167G, 1217A and 1271C.
30. A bank of polymorphic patterns in the human ACE, AGT and / or AT1 genes, comprising a member selected from the group consisting of: ACR 5349 A / T, AGR 1218 A; ECA 5496 C, AGR 1204 A / C; ACR 5496 C / T, AGR 1218 A, AGT 620 C / T; ECA 2193 A, AGR 1204 C, ECA 2328 G; ECA 2193 A, AGR 1204 A / C; ECA 3387 T, AGR 1218 A; ECA 3387 T, AGT 620 C / T; AGR 1204 A / C, AT1 678 C / T; AGR 1204 A / C, AT1 1271 A / C; ECA 1215 C, AGR 1204 A / C; AGR 1204 A / C, AT1 1167 A, ECA 3906 A / G; AGR 1204 A, AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T, AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T; AGR 1204 A / C, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T; AGR 1204 A, AT1 678 C, AT1 1167 A, AGR 395 A / T; AGR 1204 A / C, AT1 678 C / T, AT1 1167 A, AGR 395 T; AGT 620 C / T, AT1 1271 A / C, AT1 1167 A, AGR 395 T, AGT 620 C / T, AT1 1271 A / C, AT1 1167 A / G, AGR 395 T; AGT 620 C, AT1 1271 A, AT1 1167 A, AGR 395 A / T; AGT 620 C, AT1 678 A, AT1 1167 A, AGR 395 A / T; AGT 620 C / T, AT1 678 C / T; AT1 1167 A, AGR 395 T; ECA 2193 A, AGR 1218 A, AGT 803 A; ECA 2193 A, AGT 620 C / T; ECA 2328 G, AGT 620 C / T; ECA 3387 T, AGR 1204 A / C; ECA 2193 A, ECA 2328 G, AGR 1204 C; and ECA 2193 A / G, AGR 1072 G / G, AT1 1167 A / A.
31. A blank bank of cardiovascular drugs, each of the targets comprising an isolated peptide comprising one or more polymorphic positions in the ECA polypeptide sequence, wherein the polymorphic positions are encoded by nucleotides selected from the group consisting of nucleotide positions in the ECA coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503, and 3906.
32. A blank bank of cardiovascular drugs, each of the targets comprising an isolated peptide comprising one or more positions in the AGT polypeptide, wherein the poiimorphic positions encoded by nucleotides are selected from the group consisting of nucleotide positions numbered 273, 620, 803, 912, 997, 116 and 1174.
33 A blank bank of cardiovascular drugs, each target comprising an isolated peptide comprising one or more polymorphic positions in the AT1 polypeptide. , wherein the polymorphic positions are encoded by nucleotides selected from the group consisting of nucleotide positions numbered 449, 678, 1167 and 1271.
34. A device for evaluating the response of an individual to a treatment regimen for a cardiovascular syndrome, said equipment comprising: (i) sequence determination primers and (ii) sequence determination reagents, wherein the primers are selected from the group consisting of primers that hybridize at the polymorphic positions in human ACE, AGT or AT1 genes; and primers that hybridize immediately adjacent to the polymorphic positions in ECA genes. AGT or AT1, where the polymorphic positions predict the response of an individual to a cardiovascular treatment regimen of a cardiovascular syndrome.
35. A kit according to claim 34, wherein the polymorphic positions are selected from the group consisting of positions in the regulatory region of ECA numbered 5106, 5349 and 5496; the positions in the ECA coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503 and 306; position 1451 in the ECA gene as it is numbered at the entrance of Genbank 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, d03, 912, 997, 116, and 1174; position 59 in the AGT gene as it is numbered at the Genbank entry M24688; positions in the AT1 regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355 and 2415; positions in the AT1 coding region numbered 449, 678, 1167 and 1271; and combinations of any of the above.
36. A team to evaluate the cardiovascular state, said equipment comprising one or more specific antibodies for a polymorphic position within the polypeptides of human ACE, AGT or AT1.
37. A kit according to claim 36, wherein the polymorphic positions are encoded by a nucleotide selected from the group consisting of nucleotide positions in the coding region numbered ECA 375, 582, 731, 1060, 1215, 2193, 2328 , 2741, 3132, 3387, 3503 and 3906; nucleotide positions in the AGT coding region numbered 273, 620, 803, 912, 997, 1116, and 1174; positions in the coding region AT1 449, 678, 1167, and 1271 and combinations of any of the foregoing. r - «» »95 SUMMARY The present invention provides methods for assessing cardiovascular status in an individual, comprising determining the sequence in one or more polymorphic positions within the human genes encoding the angiotensin converting enzyme (ACE), angiotensinogen (AGT) and / or angiotensin II receptor type 1 (AT1). The invention also provides isolated nucleic acids encoding polymorphisms of ECA, AGT, and AT1, nucleic acid probes that hybridize to polymorphic positions, 10 to predict the cardiovascular status and targets of nucleic acids and peptides for use in the identification of candidate cardiovascular drugs.