CA2347247A1 - Genes for assessing cardiovascular status and compositions for use thereof - Google Patents

Abstract

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

Description

GENES FOR ASSESSING CARDIOVASCULAR STATUS
AND C.'OMFOSITIONS FOR USE THEREOF
hIELD OF THE INVENTION
The present invention relates to genetic polymorphisms and polymorphism patterns useful for assessing cardiovascular status in humans. More particularly, the invention relates to identifying and using polymorphism patterns comprising a polymorphism in the a gene encading a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ~~i-adrenoceptor to predict a treatment outcome or likelihood of developing cardiovascular disease, and to assist in diagnosis and in prescription of an effective therapeutic regimen.
BACKGROUND OF THE INVENTION
The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular physiology in mammals. Specifically, RAAS regulates salt-water homeostasis and the maintenance of vascular tone. Stimulation or inhibition of this system raises or lowers blood pressure, respectively, and disturbances in this system may be involved in the etiology of, for example, hypertension, stroke, and myocardial 2 0 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 CONFIRMATION COPY

(ACE), angiotensinogen (ACTT), type 1 angiotensin II receptor (AT1), type 2 angiotensin II
receptor (AT'2) and aldosterane synthase.
International Patent Application No. PCT/IB98/00475, filed April 1, 1998, discloses for the first time an association of polymorphism patterns in ACE, AGT, and S AT 1 genes with cardiovascular status, particularly with the ability to predict the therapeutic outcome of a particular treatment regimen.
Other genes express polypeptides involved in pathways other than RAAS
that also play a role in the regulation of cardiovascular physiology, although it is believed that prior to the present invention, no known association of polymorphism patterns in these genes with cardiovascular status has been observed or reported. Among these other regulators of cardiovascular physiology are endothelin, endothelin receptor, and ~3-adrenergir receptors 1 and. '?.
RAAS Pathway Components AGT is the specific substrate ofrenin, an aspartyl protease. The human AGT gene contains five exons and four intrans which span l3Kb (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 S' 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 2 0 sequence (6:? amino acids) and the 3'-untranslated region.
Plasma AC:~T is synthesized primarily in the liver and its expression is positively regulated by estragens, 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, 2 5 which is further processed to the active octapeptide angiotensin II by the dipeptidyl carboxypept:idase designated angiotensin-converting enzyme (ACE). Cleavage of AGT by renin is the rate-limiting step in the activation of the renin-angiotensin system.
Several epidemiological observations indicate a possible role of AGT in blood pressure regulation. A highly significant correlation between plasma AGT

3 0 concentration and blood pressure has been observed in epidemiological studies (Walker et al., J. Hypertension, 1979, 1:287-291 ). Interestingly, a number of allelic dimorphisms have been identified in the AGT gene. The frequency of at least two of them ( 174M and 235T) have been partially characterized and in certain populations shown to be WO 00/22166 _ 3 _ PCT/IB99/01678 significantly elevated in hypertensive subjects (Jeunemaitre et al., Cell, 1992, 71:169-180). In addition, a specific polymorphism, 235T, has been suggested to be directly involved in coronary atheros;clerosis (Ishigami et al., Circulation, 1995, 91:951-4).
Futhermore, the presence of A or G at position 1218 in the AGT regulatory region has been correlated with differences in in vitro transcriptional capacity for this gene (moue et.
al., J. Clin. Invest., 1997, 09:1786). However, the foregoing are studies involving only one or at mast two polymorphisms. Furthermore, the sole disclosed use is susceptibility to disease.
The human AC'E gene is also a candidate as a marker for hypertension and myocardial infarction. AC.'.E inhibitors constitute an important and effective therapeutic approach in t:he control of human hypertension (Sassaho et al., Am. J. Med., 1987, 83:227-235). In plasma and on the surface of endothelial cells, ACE converts the inactive angiotensin 1 molecule (Ang I) into active angiotensin II (Ang II) (Bottari et al., Front.
Neuroendocrinology, 1993, 14:123-171). Another ACE substrate is bradykinin, a potent vasodilator and inhibitor of smooth muscle cell proliferation, which is inactivated by ACE
(Ehlers et al., Biochemistry, 1989, 28:5311-5318; Erdos, E.G., Hypertension, 1990, 16:363-370; Johnston, C.l. Dra~~,rs (suppl. I), 1990, 39:21-31).
Levels of ACE are very stable within individuals, but differ greatly between individuals. A greater risk of myocardial infarction has been identified in a group of 2 0 subjects with an ACE polymorphism designated ACE-DD (Cambien et al., Nature, 1992, 359:641-644), and a 12-fold greater risk of myocardial infarction has been identified in a subgroup of patients having a combination of the ACE polymorphism ACE-DD and one of the AGT polymorphisms (235T) described above (Kamitani et al., Hypertension, 1994, 24:381). Recently, six AC,'.E polymorphisms were identified and characterized (Villard et al., Am. J. Human Genet., 1996, 58:1268-1278).
The vasocon<~trictive, cell growth-promoting and salt conserving actions of angiotensin :fI are mediated through binding to and activation of angiotensin receptors, of which at least two types have been cloned (,AT 1 and AT2). The type I Ang I1 receptor (AT1), a G-protein-coupled seven transmembrane domain protein, is widely distributed in 3 0 the body and mediates almost all known Ang II effects (Fyhrquist et al., J. Hum.
Hypertension, 1995, 5:519-_'i24,1.
Several polymorphisms have been identified in the AT1 receptor gene.
Initial studiea suggest that at least one of them is more frequent in hypertensive subjects WO 40/22166 _ 4 _ PCT/IB99/01678 (AT"''''C)(Bonnardeaux et al., Hypertension, 1994, 24:63-69). This polymorphism, combined with the ACE-DD polymorphism, has been shown to correlate strongly with the risk of myocardial infarction (Tiret et al., Lancet, 1994, 344:910-913).
Endothelin (FT) Regulation of Cardiovascular Physiology Endothelia is a potent vasoconstrictive peptide characterized by long lasting action. It was first discovered as a vasoeonstricting 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 preproendotlnelin, which is cleaved after removal of the signal sequence by an endopeptidase, followed by cleavage with endothelia converting enzyme (Xu et al., Cell, 1994, 78:473: Shimada et al., J. Biol. Chem., 1994, 269:18274). Analysis of the human ET gene has revealed the existence of two additional ET-like peptides expressed in various tissues, termed ET-2 and ET-3 (moue et al., Proc. Natl. Acad. Sci. USA, 1989, 86:2863).
The first endothelia was accordingly termed ET-1.
One of the important discoveries following characterization of ETs was the discovery of two ET receptors, I:TA and ETA (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.
2 0 ETA exists as a single copy gene located on human chromosome 4 (Hosoda et al., J. Biol.
Chem., 1992, 267:18797; Cyr et al., Biochem. Biophys. Res. Commun., 1991, 181:184).
ETA exists as a single copy gene located on human chromosome 13 (Arai et al., J. Biol.
Chem., 1993, 268:3463-7U), although a splice variant of ETA has been found (Shyamala et al., Cell. I',4o1. Biol. Res., 1994, 40:285-9fi). The cDNA sequence of ETA
has been deposited with GenBank with accession number S57498.
In the early s~Cage of ET research, a great number of pharnzacological studies suggested that the responses to ETs could be divided into two groups according to the pharmacological potency of the three peptides. Indeed, these two receptors, ETA and ETB, are distinct in their ligamd binding affinity and distribution in tissues and cells. ETA
has a high ai:finity to ET-1 and ET-2, but a low affinity to ET-3. ET,~ has equally potent affinities to all three endogenous ETs. ETA exists on smooth muscle and mediates vasoconstricaion. In contrast, ETE; exists on endothelium and mediates the release of relaxing factors such as nitric oxide and prostacycline. However, several reports demonstrated that ETB on some vascular smooth muscle also mediated vasoconstriction.
Cloning of the ET r~ecepton gene facilitated the development of ET-receptor antagonists, such as BE-18257B, and BQ-123 and FR139317, two derivatives of BE-18257B (See Masaki, Cardiovascular Res., 1998, 39:530). Many selective and non-selective antagonists for ETA and ETa have emerged.
Although the pathophysiological role of ET is still unclear, ET antagonists demonstrated significant beneficial effects in pathological conditions, including congestive heart failure, pulmonary hypertension, cerebrovascular spasm after subarachnoid hemorrhage, acute renal failure, and essential hypertension. ET or ET receptor knockout mice have also provided important information regarding the physiological and pathophysiological significance of ET (Masaki, satpr-a). In particular, mice with a knockout of ET 3 or the ETA, receptor genes exhibit phenotypic changes that resemble Hirschsprung's disease, a human hereditary syndrome associated with a missense mutation of the ETB gene (Pfiffenberg;er, et al., Cell, 1994, 79:1257).
Despite advances in understanding the role of the endothelin pathway in the treatment of cardiovascular diseases, questions remain. Not all experimental models of hypertension respond to end.othelin antagonists, and it remains a uncertain whether endothelin antagonists improve cardiac structure and function beyond the benefits of blood pressure reduction (Moreau., Cardiovascular Res., 1998, 39:534). Thus, there is a need in the art for a reliable and efff:ctive means for predicting whether endothelin antagonists will be effective for treating hypertension in a given individual.
-Adrenergic Receptors ,(3-Adrenoceptors) The adrenoceptors fall into three major groups, a,, az, ~3, within each of which further subtypes can be distinguished pharmacologically (Lullmann, et al. in Color Atlas of Pharmacology, Ne~h~ York, 1993). Adrenergic receptors are all G-protein linked.
They are involved in regulation of the cardiovascular system, and in the control of metabolic activity, e.g., insulin secretion and glucose release. They also mediate 3 0 constriction or relaxation of smooth muscle cells in the respiratory, gastrointestinal, and genitourinary tracts (Berne and Levy. Principles of Physiology (2"d Ed.), Mosby-Year Books, Inc., 1996, pp. 691-696).

Adrenoceptors are targets for epinephrine and norepinephrine, which are representatives of the family of monoamine neurotransmitters. Epinephrine has equally high affinity for all a- and ~i~-receptors while norepinephrine differs from epinephrine by its low affinity for (32-receptors (The Biochemical Basis of Neuropharrnacology, (7'~' Ed.) New York, 1996, pp. 226-2f2). The adrenoceptors themselves interact preferentially with three different classes of (i-proteins: GS (~3-adrenoceptors) mediating activation of adenylate cyclase, G; (a~-adrenoceptors) mediating inhibition of adenylate cyclase, and Gq (a,-adrenocc:ptors) mediating activation of phosphoiipase C (Hieble et al., J.
of Med.
Chem., 1995, 38:3415-3444;).
The pharmacological interest of adrenoceptors is mainly for the treatment of cardiovascular diseases, e.~;.. through the development of ~3-antagonists, a,-antagonists and a~-agonists to treat hypertension, but they are also considered important for the treatment of asthma ((3Z-agonists).
The (32-adren~ergic receptor is expressed on a number of cell types, e.g., bronchial smooth muscle, where its activation results in relaxation and bronchial dilatation. 'lf hese receptors are also being expressed on epithelial cells, vascular endothelium, alveolar walls., immune cells, and presynaptic nerve terminals (Liggett, Am.
J. Respir. Grit. Care. Med., 1997, 156:S 156-S 162). Cardiac cells express mainly Vii,-, but also a small fraction of ~i2-adrenoceptors (Collins et al., Biochimica et Biophysica Acta, 1993, 1172:171-174). (3,-adrenoceptors are also expressed in brain and pineal gland.
~0-Adrenoceptor Fc~r~ction The (3,- and ~3,-adrenergic receptors are coupled to a GS-protein complex, which activates adenylate c,yclase. Agonist binding to the Vii,-receptor, located in cardiac 2 5 muscle cell:, mediates increased contractility and cardiac output (Lullmann, supra).
Agonist binding to the ~3z-receptor, located in peripheral vascular arteries, mediates vasodilation by increasing the amount of CAMP, and thereby inhibiting activation of myosin kin,~se, which is necessary for smooth muscle cell constriction (ibid.) Activation of CAMP in cardiac cells by agonist binding to both (3 i- and ~3-2-adrenoceptors activates the 3 0 cAMP-dependent protein kinase (PKA) (Castellano and Bohm, Hypertension, 1997, 29:715-722). ~i-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 o1' melatonin (Collins et crl., supra).

WO 00/22166 _. ~ _ PCTlIB99/01678 The ~3,-recept:ors mediate increased conversion of glycogen to glucose (glycogenolysis) in both the liver and skeletal muscle (H. Liallmann, et al., supra), and stimulate influx of potassium into muscle cells to prevent hyperkalemia (Berne and Levy, supra).
The ~3-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 (SARK and PKA (:Hieble et al., supra). (SARK phosphorylates specific serine or threonine re:~idues in the (:."-terminal of receptors that are occupied by an agonist. The phosphorylation triggers binding of the cytostolic protein ~i-arrestin and results in the uncoupling from Gsa. PKA is activated by c:AMP and phosphorylates the (3z-adrenoceptor by a relatively slow process. The phosphorylated receptor loses the ability to activate GS
(Castellano and Bohm, supra ). Prolonged interaction of agonists with adrenoceptors generally results in receptor desensitization.
~i-Adrerroceptor Gene Structure The human (3,-adrenoceptor gene is located on the long arm of chromosome 10, the same chromosome as for the a2A-adrenoceptor gene. The coding sequence of this gene is deposited with GenEtank, 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 (Elall, 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 dons 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 2 5 motifs. Thi~~ type of receptor, reminiscent of "housekeeping genes", has been described for other G-protein coupled receptors as well (Collins et al., supra).
The human ~3~,~-adrenoceptor gene is located on the long arm of chromosome 5, the same chromosome as the a",-adrenoceptor gene. The coding sequence has been deposited with GenBank, with accession numbers M 15169, J02728, or M 16106.
The 3 0 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 eases 5' of the translation initiation codon, and it can form strong secondary structures due to high G-C; content. There are two TATA boxes (separated by roughly 10 WO 00/22166 _ 8 - PC1'/IB99/01678 bp) and a CAAT box located approximately 30 and 80 base pairs upstream, respectively, from the mRNA start region.
~3-Adrertoc~tor Gene Regulation There are some regulatory regions identified in the promoter region of the (3,-adrenoceptor gene: a cAMP response element (CRE), a consensus thyroid response element (TRIO, and a glucocorticoid response element (GRE). This is consistent with the evidence that both thyroid horn~one and corticosteroids affect adrenergic sensitivity in both heart and adipose tissue. The CRE region might have a self regulatory function, as has been sho~,vn for the (3,-adrenoceptor gene (Collins et al., supra).
There are several regulatory domains in the 5' flanking region of the X32-adrenoceptor. Among these is a CAMP-responsive element (CRE), which is recognized and stimulated by a phosphoprotein called CRE binding protein (CREB). CREB is partially under the control of PKA-dependent phosphorylation processes. This is seen as an increase in (32-adrenoceptor mRNA level in the early phase after exposure to (3-agonists.
However, the level of mRNA is decreased after prolonged exposure to agonists, probably mediated by a shortening of mRNA half life (Castellano and Bohm, supra). It has also been shown that transcription of the (32-adrenoceptor gene is upregulated by stimulation with glucocorticoids in a variety of tissues (Collins et al., supra). In the 3' flanking region 2 0 there are sequences homologous to glucocorticoid response elements. These might be responsible for the increased expression of (32 adrenoceptor observed in transfected cells after treatment with hydrocortisone (Emorine and Marullo, Proc. Natl. Acad.
Sci. USA, 1987, 84:6995-6999).
2 5 J3-Arlrenoc~tor Proteir: Structure The proposed model for ~3-adrenoceptors is like most of the G-protein binding receptors, a seven a~-helical transmembrane structure, where the seven a-helices are radially arranged around a central "pore", in which the receptor ligands bind. The (3-adrenoceptors have an extracellular glycocyiated N-terminus, and an intracellular 3 0 C-terminus. The ~3,-receptor consists of 477 amino acids; the ~3,-receptor consists of 413 amino acids.
The overall amino acid identity of human ~3,- and ~3z- adrenoceptors is only 54%. However, it is likely that the pharmacological differences between (3,-receptors and WO 00/22166 _ 9 _ PCT/IB99/01678 (3,-receptors are due to subtle changes in orientation of the primary binding sites, resulting in a slightly different binding; site rather than to specific amino acid substitutions (Hieble et a.l., J. Med. Chern. 1995, 38:3415-3444).
Site-directed mutagenesis has demonstrated that an aspartic acid residue, Asp-113, located in the third transmembrane-spanning helix, and two serine residues, Ser-204 and Ser-207, are required for full agonist binding to the biz-adrenoceptor. The ~3,-adrenoceptor contains identical amino acid residues located in corresponding positions to those shown to be important for agonist binding to the X32-adrenoceptor.
Another aspartic acid residue, Asp-7S~. located in the second a-helix of both (3-receptors is highly l0 conserved in G-protein coupled receptors (Hieble et al., supra). Ser-319 has a potential role in agonist binding to the ~3=-adrenoceptor.
Mutation of T vr-350, located in the cytoplasmic tail of the X32-receptor, interferes with coupling of the receptor to G,, (Hieble et al., supra). Also, palmitoylation of Cys-341 in the C:-terminal enables the (32-adrenoceptor to form a fourth intracytopla~;mic loop, which increases the ability of the agonist-bound receptor to mediate adenylyl cyclase stimulation (Strosberg, Preotein Science, 1993, 2:1 I98-I209).
~(3-Adrenoeeptors as Drug Targets No cause of disease can be identified in 80-90% of patients with 2 0 hypertension. They have so-called essential hypertension, which affects 5-10% of the general population, and is the most common cause of disease in developed countries ( J.
Axford, MeGdicine, Blackwell Science Ltd., I996, 10.119-10.130). Betablockers have been widely used in the treatment of hypertension. They are particularly useful for the treatment of juvenile hypertf:nsion with tachycardia and high cardiac output.
Betablockers 2 5 or beta-adrenergic Mockers ~~~ere first introduced as a treatment for essential hypertension in 1964, and are still recomnnended as first choice because the cost for betablockers is low, which improves patient compliance. They act by binding; to (3,-receptors on the cardiac smooth muscle cells, which leads to decreased cardiac output. Most betablockers are not specific ~3,-receptor antagonists but binds to ~3z-receptors as well. The binding to 3 0 Vii,-receptors gives the opposite of the desired effect though inhibition of biz-receptors leads to vasoconsi:riction. This gives a side effect with cold hands and feet because most of the ~3z-receptors are located in the peripheral vascular arteries.

WO 00/22166 -10 - PCT/iB99/016'f8 They have also been known to cause bronchospasms as well as some central nervous system side effects {nightmares, somnolence). They decrease insulin secretion, which makes thenn inappropriate to treat hypertensives with diabetes mellitus, and they can cause heart failure and peripheral artery obstructive disease (Velaseco and Rodrigues, Journal of Human Hypertension 10, Suppl. 1, S77-580, 1996).
~3-adrenoceptor agonists, such as dopamine and dobutamine are used to stimulate myocardial ~3,-adrf~noceptors in the acute management of congestive heart failure. The: act by increasing contractility and cardiac output.
A lot of differ°ent ~3z-agonists are used in the treatment of asthma. They exert their primary effect on the ~3z-adrenergic receptor of bronchial smooth muscle, resulting in relaxation and bronchial dilatation. They also protect against bronchoconstrictor challenge (Hall, supra).
Thus, there is a clear need in the art for an improved understanding of the effects of betablockers on different subjects, and to predict which patients will have a better response to treatment with betablockers.
Need for Effective Cardiovascular Status Assessment The high morbidity and mortality associated with cardiovascular disease demonstrate a need in the art for methods and compositions that allow the determination 2 o and/or prediction of the thcr;apeutic regimen that will result in the most effective treatment outcome in a patient suffering from cardiovascular disease. This includes identification of individuals who are more or less responsive to particular therapeutic regimens, including, e.g., particular drugs that are conventionally used to treat cardiovascular disease.
Furthermore, the heterogeneity in responses to cardiovascular therapies emphasizes .a need for another approach to rational drug development. In particular, populations that are identified as non-responsive to a particular therapeutic regimen can be identified for development amd testing of alternative regimens. Thus, effective treatment regimens could be developed for a larger percentage of the affected population.
In summary, there is a need to reduce or eliminate trial and error in 3 0 selecting a therapeutic regimen for a particular individual. It would be desirable instead to predict whel:her a given individual will be responsive to, e.g., a particular class of drugs or even to a particular drug or whether he/she is likely to suffer from adverse reactions or side-effects.

There is also ai need in the art for methods and compositions that allow the identification of individuals having a predisposition to cardiovascular disease, such as, e.g., myocardial infarction, hypertension, atherosclerosis, and stroke, to facilitate early intervention and disease prevention.
The present invention addresses these and other needs in the art by providing polymorphisms and polymotphic patterns that are characteristic of cardiovascular status, and by using these polymorphisms and patterns to prescribe or to develop more; effective treatments or to assist in diagnosis.
Citation of an .y reference in this application should not be construed as an admission that the reference His prior art to the invention. Each cited reference is incorporated herein by reference in its entirety.
SL:~MMARY OF THE INVENTION
In one aspect, the present invention provides reagents and methods for predicting whether a particular therapeutic regime (such as a specific drug, a class of drugs or any other ~:herapeutic regime, pharmacological or not) would be effective in improving a cardiovascular condition in a human individual, or would be ineffective for that purpose, or its use would be associated with adverse reactions or undesirable side-effects.
A particular advantage of the invention is that one or more polymorphic 2 o markers provide a basis for predicting the outcome of a treatment regimen.
By comparing a polymorphi;c pattern of a subject who requires treatment for a cardiovascular disorder, for example )zypertension, with a reference pattern previously established to correlate with responsivity to the treatment regimen, a physician can predict whether a treatment plan, such as administration of an ACE inhibitor, is likely or not to be effective before subjecting the subject to the 'treatment plan. For example, a comparison of the test polymotphic pattern from an individual with reference polymorphic 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 3 0 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 WO 00/22166 _ 12 _ PCT/IB99/01678 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 usc~ in the appropriate population, and decrease the number of required patients and therefore the duration and cost of clinical trials. It may also lead to identification of another subgroup which can be the target for development of another therapeutic regimen.
All of the foregoing applications within the scope of the invention can be deemed to be assessments of an individual's cardiovascular status, as the term is broadly defined below.
The foregoing methods of the invention are carried out by comparing a test polymorphic pattern established by at least one polymorphic position within a gene encoding A(:E, A'1'I, AGT, renin, aldosterone synthase, type-2 angiotensin II
receptor, endothelia receptor, and ~3-adrenoceptor with a polymorphic 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.
2 0 The polymorphic pattern preferably consists of at least two (and more preferably a~. least three) polymorphic positions, at least one of which is in the gene encoding a I>olypeptide from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, and (3-adrenoceptor, and a second polymorphism in a gene encoding a polypeptide selected from the group consisting of ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, and (3-adrenoceptor.
Additionally., the invention provides methods for assessing whether a particular individual has a genetic predisposition to a cardiovascular pathology. This aspect of thc~ invention corn~prises comparing a test polymorphic pattern established by at 3 0 least one and preferably at least two and most preferably at least three polymorphic positions within a gene encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, and (3-adrenoceptor in conjunction with one or more, and preferably two or more, other polymorphic positions in ACE, ATI, AGT, renin, WO 00/22166 _ 13 _ PCT/IB99/01678 aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or (3-adrenoceptor, with a polymorphic pattern ot~individuals exhibiting a predisposition to a cardiovascu;Lar syndrome. T'he conclusion drawn depends on whether the individual's polyymorphism 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, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin I1 receptor, endothelia receptor, and ~3-adrenoceptor, the nucleic acid comprising a polymorphism in the ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin Il receptor, endothelia receptor, and ~i-adrenoceptor gene. In preferred embodiments, the polymorphism, in combination with one or more other polymorphi.<;ms in the sequence of the same gene or a gene encoding a protein selected from the group consisting of human ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, and ~3-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 ;polymorphisms according to the invention (which are described using the numbering indicated in Table 1 below) include without limitation:
Nucleic acids encoding renin having one or more polymorphic positions in the last exon of the gene (exon l0), which is a cytosine to thymine transition that creates a 2 0 premature stop codon at position 387.
Nucleic acids encoding aldosterone synthase promoter at position -344.
Nucleic acids encoding (3-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 ~3-adrenergic receptor-1 coding region, at positions 231, 758, 1037, 1251, 14()3., and 1528, as numbered in GenBank accession number J03019. In preferred embodiments, the bases at specific positions are 231 A, 231 G, 1251 C, 1251 G, 1403 A, 1403 G, 1528 C, and 1528 A.
Nucleic acids encoding ~3-adrenergic receptor-2 regulatory region, at positions 932, 934, 987, 1006, 1120, 1221, 1541, and 1568, as numbered in GenBank accession number M 15169, ar J02728, or M 16106. In preferred embodiments, the bases WO 00/22166 _ ~ 4 _ PCT/1B99/01678 at specific positions are 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..
Nucleic acids encoding (3-adrenergic receptor-2 coding region, at positions 839, 872, 10~I5, 1284, 1316, 1846, 1891, 2032, 2068, and 2070, as numbered in GenBank accession number Y00106. In preferred embodiments, the bases at specific positions are 839 A, 839 C., 872 C, 872 G. 1045A, 1045 G, 1284 C, 1284 T, 1316 A, 1316 C, 1846 C, 1846 G, 203x! A, 2032 G, 2068 no inseu, 2068 G, 2068 C, 2070 no insert, and 2070 C.
Nucleic acids encoding endothelin-A receptor at positions 969, 1005, 1146, and 2485, as numbered in GenBank accession number S57498. In preferred embodiments, the bases at specific positions are 969 C, 969 T, 1005 A, 1005 G, 1146 A, 1146 G, 2485 T, and 2485 C.
Nucleic acids comprising polymorphisms present in other genes, which can be used in combination with a polymorphism 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 ~3-adrenoceptor to establish a polymorhisn-~ pattern, have been disclosed in International Patent Application No.
PCT/IB98/0(1475, and include:
(i) Nucleic acids encoding ACE having one or more polymorphic positions at the position in the regulatory region numbered 5106; positions in the coding region numbered 375, 582, 731, L0(i0, 2741, 3132, 3387, 3503, and 3906; and position 1451 all positions as numbered in (~enBank entry X62855. In preferred embodiments, the sequences at the polymorphic positions in the ACE regulatory region are one or more of 5106C and 5106T; and the sequences at the polymorphic positions in the coding region are one or more of 375A, 3750, 582C, 582T, 731A, 731 G, 10606, 1060A, 27416, 2741T, 3132C, 3132T, 3387T, 3387C. 35036, 3503C, 39066, and 3906A. The invention also encompasses; a nucleic acid c=ncoding a deletion of nucleotides 1451-1783 as numbered in GenBank emry X62855.
(ii) Nucleic. acids encoding ACiT having one or more polymorphic positions at positions in the regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072, 3 0 and 1204; positions in the coding region numbered 273, 912, 997, 1116, and 1174; and position 49 ass numbered in GenBank entry M24688. In preferred embodiments, the sequences at the polymorphic positions in the AGT regulatory region are one or more of 395T, 395A, 412C, 412T, 4326, 432A, 449T, 449C, 692C, 692T, 8396, 839A, 10076, 1007A, 10726, 1072A, 12040, and 1204A; the sequences at the polymorphic position in the coding region are one or more of 2730, 273T, 9120, 912T, 9976, 9970, 1 I
166, 1116A, 11740 and 1174A; and the sequence at position 49 in GenBank entry M246$8 is either A or C~.
(iii) Nucleic acids encoding ATI having one or more polymorphic positions at positions in the regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; and the position in the coding region numbered 449. In preferred embodiments, the sequences at the polymorphic positions in the AT1 regulatory region are one or more of 1427A, 1427T, 1756T, 1756A, 1853T, 18536, 2046T, 20460, 2354A, 23540, 2355~G, 23550, 241 ~~A and 241 SG; and the sequences at the polymorphic positions in the codin~; region are one or more of 4496, 4490, 678T, 6780, 1167A, 11676, 1271 A, and 1271 C.
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 polymorphic position in the gene encoding ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ~i-adrenoceptor and other polymorphic positions in the other genes, including without limitation the polymorphic positions and sequences disclosed herein. Also provided are nucleic acid probes that hybridize specifically to the identified polymorphic positions;
2 0 peptides and polypeptides comprising polymorphic positions; and polymorphism-specific antibodies, i.e., sequence-specific antibodies that bind differentially to polymorphic variants of the foregoing genes, that can be used to identify particular polymorphic variants.
In another aspect, the invention provides kits for the determination of 2 5 polymorphic patterns in an individual's genes. The kits comprise a means for detecting polymorphic sequences, including without limitation oligonucleotide probes that hybridize at or adjacent to the polymorphic positions and polymorphism-specific antibodies.
In yet another aspect, the invention provides nucleic acid and polypeptide targets for u,se in screening methods to identify candidate cardiovascular drugs. Nucleic 3 0 acid targets may be, e.gl., DMA or RNA and are preferably at least about 10, and most preferably a,t least about 15., residues in length and comprise one or more polymorphic positions in a gene encodin;s a polypeptide from the group consisting of ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and ~3-WO 00/22166 PCT/IB99/O1b78 adrenoceptor. Peptide targets are at least about 5 amino acids in length and may be as large or larger than the full-length polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
The invention in based, in part, on the discovery that polymorphisms in certain genes in the RAAS, endothelia, and ~i-adrenoceptors pathways define polymorphism patterns that c:arrelate with cardiovascular status. Most significantly, by comparing a test individual's polymorphism pattern with a reference polymorphism pattern, which is a polymorphism 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 polymorphism pattern. In particular, particular patterns correlate with responsiveness to ACE inhibitors, non-responsiveness to ACE inhibitors, and predisposition to cardiovascular diseases or dysfunctions, including myocardial infarction and stroke.
The invention provides a powerful predictive tool for clinical testing and treatment of cardiovascular disease. For clinical testing, the present invention permits smaller, more efficient clinical trials by identifying individuals who are likely to respond poorly to a treatment regimen and reducing the amount of uninterpretable data.
By 2 0 evaluating a test individual's polymorphism pattern, a physician can prescribe a prophylactic or therapeutic regimen customized to that individual's cardiac status.
Adverse responses to particular therapies can be avoided by excluding those individuals whose cardiovascular status puts them at risk for that therapy. Appropriate changes in lifestyle, including diet, environmental stress, and exercise levels can be prescribed for 2 5 individuals whose test polymorphic pattern matches a reference pattern that correlates with increased predisposition to cardiovascular disease.
Definitions "Cardiovascular status" as used herein refers to the physiological status of 3 0 an individual's cardiovascular system, as reflected in one or more status markers or indicators including genotype. Cardiovascular status shall be deemed to include without limitation not only the absence or presence of a pathology or disease in one or more components of the individual"s cardiovascular system and the individual's predisposition to developing; such a condition, but also the individual's responsivity, i.e., the ability or inability of the individual to respond (positively or negatively) to a particular prophylactic or therapeutic regimen or treatment for a cardiovascular condition, such as a drug or a class of drugs. A negative response includes one or more adverse reactions and side effects. Status markers include without limitation clinical measurements such as, e.g., blood pressure, electrocardiographic profile, differentiated blood flow analysis, and the presence of increased levels of cellular proteins associated with a cardiovascular event.
Examples of such proteins, also called diagnostic markers, which are important in cardiac events include myosin light chain, myosin heavy chain, myoglobin, troponin I, troponin T, CK-MB, etc. (see U.S. Patents No. 5,604,105 and No. 5,744,358). Status markers according to the invention are assessed using conventional methods well known in the art.
Also included in the evaluation of cardiovascular status are quantitative or qualitative changes in sl:atus markers with time, such as would be used, e.g., in the determination of an individual's response to a particular therapeutic regimen or of a predisposed individual's eventual development of a cardiovascular condition.
Examples of cardiovascular syndromes that are included in the foregoing definition of cardiovascular status include diagnosis of, or predisposition to, one or more cardiovascular syndromes, such as, e.g., hypertension, acute myocardial infarction, silent myocardial infarction, unstable angina, stroke, and atherosclerosis. It will be understood 2 0 that a diagnosis of a cardiovascular syndrome made by a medical practitioner encompasses not only clinical measurements but also medical judgment.
"Responsivit:y", as used herein, refers to the type and degree of response an individual exhibits to a particular therapeutic regimen, i.e., the effect of a treatment on an individual. Responsivity breaks down into three major categories: therapeutic effect; no 2 5 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 effecas, 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.
3 0 A "predispos,ition to develop a cardiovascular syndrome" refers to an increased likelihood, relative to the general population, to develop a cardiovascular syndrome, as defined above. A predisposition does not signify certainty, and development of the syndrome may be forestalled or prevented by prophylaxis, e.g., adopting a modified diet, exercise program, or treatment with gene therapy or pharmaceuticals.
Naturally, an advantage of the present invention is that it permits identification of individuals who are, based on their genotype, predisposed to develop a cardiovascular syndrome, and for whom prophylactic intervention can be especially important.
A "polymorphism" 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 polymorphism are "alleles". A "polymorphic position" is a predetermined nucleotide position within thc~ sequence. In some cases, genetic polymorphisms are reflected by a.n amino acid sequence variation, and thus a polymorphic position can result 1 o in location of a polymorphism in the amino acid sequence at a predetermined position in the sequence of a polypeptidc:. An individual "homozygous" for a particular polymorphism is one in which both copies of the gene contain the same sequence at the polymorphic position. An individual "heterozygous" for a particular polymorphism is one in which the two copies of the gene contain different sequences at the polymorphic position.
A "polymorpl-uism pattern" as used herein denotes a set of one or more polymorphisms, including without limitation single nucleotide polymorphisms, which may be contained in the sequence of a single gene or a plurality of genes. In the simplest case, a polymorphism pattern can consist of a single nucleotide polymbrphism in only one 2 o position of one of two alleles of an individual . However, one has to look at both copies of a gene. A polymorphism pattern that is appropriate for assessing a particular aspect of cardiovascul;~r status (e.g., predisposition to hypertension) need not contain the same number {nor identity, of course) of polymorphisms as a polymorphism pattern that would be appropriate for assessing .another aspect of cardiovascular status (e.g., responsivity to 2 5 ACE inhibitors for control o:f hypertension). A "test polymorphism pattern" as used herein is a polymorphism pattern dcaermined for a human subject of undefined cardiovascular status. A "reference polymorphism pattern" as used herein is determined from a statistically ~;ignificant correlation of patterns in a population of individuals with pre-determined cardiovascular status.
3 o "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-WO 00/22166 _ 19 _ PCT/IB99/01678 RNA and RNA-RNA hybrids., as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases and non-naturally occurring phosphoester analog bonds, such as phosphorothioates and thioesters. The term nucleic acid molecule, and in particular DNA
or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, ;inter aiia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A
"recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.
As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a eDNA molecule, or an mRNA
molecule encoding a gene, cDNA, mRNA, or other nucleic acid of interest.
Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled 2 0 oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotidc;s (one or both of which may be labeled) can be used as PCR
primers, either for cloning full length or a fragment of a gene of interest, or to detect the presence of nucleic acids encoding the gene of interest. In a further embodiment, an oligonucleotiide of the invention can form a triple helix with a double stranded sequence of 2 5 interest in a I~NA molecule. Ln still another embodiment, a library of oligonucleotides arranged on ;3 solid support, such as a silicon wafer or chip, can be used to detect various polymorphisms of interest. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring; phosphoester analog bonds, such as thioester bonds.
3 0 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 WO 00/22166 _ 2 0 _ PCT/IB99/01678 less than about SO%, preferably less than about 75%, and most preferably less than about 90%, of the cellular componewts 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 seduences 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 c:an 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 ioniic strength (see Sambrook et al., 1989, Molecular Cloning: A
Laboratory ATanual, Second Edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of SS°C, can be used, e.g., Sx SSC, 0.1% SDS, 0.25% milk, and no for~rnamide; or 30%
formamide, Sx 2 0 SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T~" e.g., 40°/~ forrrramide, with Sx or 6x SCC. High stringency hybridization conditions correspond to the highest T'm., e.g., 50% formarnide, Sx or 6x SCC.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency oi° 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 cor~rrplementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T",) of nucleic acid hybridizations decreases in the following order:
RNA:RNA, 3 0 DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tn, have been derived (see Sambrook et al., supra, 9.50-9.51).
For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et czl., supra, 11.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides: preferably at least about 15 nucleotides;
and more preferably the length is at least about 20 nucleotides.
In a specific embodiment, the term "standard hybridization conditions"
refers to a T~., of 55 °C, and utilizes conditions as set forth above.
In a preferred embodiment, the T", is 60°C; in a more preferred embodiment, the T", is 65°C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2XSSC, at 42°C.' in 50% formamide, 4XSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
A "gene" for a particular protein as used herein refers to a contiguous nucleic acid sequence corresponding to a sequence present in a genome which comprises (i) a "coding (or transcribed) region," which comprises exons (i.e., sequences encoding a polypeptide sequence, or "protein-coding" or "transcribed sequences"), introns, sequences at the junction between exons and introns, and 5' and 3' untranslated regions (uTRs); and (ii) regulatory sequences, which flank the coding region at both 5' and 3' termini. For example, the "ACE gene" as used herein encompasses the regulatory and coding regions of the human gene encoding angiotensin converting enzyme. Similarly, the "AGT
gene"
encompasses regulatory and coding regions of the human gene encoding angiotensinogen and the "ATl'i gene" encompasses regulatory and coding regions of the human gene 2 0 encoding type I angiotensin II receptor. Typically, regulatory sequences according to the invention are: located 5' (i.e., upstream) of the coding region segment. The reference sequences, obtained from tsenBank, which were used in practicing the present invention are shown in Table 1.

WO 00/22166 _ 2 2 - PCT/IB99/01678 Table 1. GenBank Accession Numbers Abbreviatiion Compared master Numbering according SEQ
to sequence sequence entry in GenBank1D

NO.

AGT RegulatoryX 15323 X 15323 122 Re ions AGT Coding M24686 (exon Protein-coding sequences123 2) from Region M24687 (exon exon 2-5 were spliced 124 3) together M24688 (exon as described in the 125 4) GenBank M24689 (exon entries. 126 S) Nucleotide I is assigned to the first nucleotide of the initiator methionine colon.

X62855 (intron 127 16) ACE RegulatoryX94359 X94359 128 Re ion ACE Coding J04144 J04144 129 Region Nucleotide 1 is assigned to the first nucleotide of the initiator methionine colon.

AT1 RegulatoryU07144 U07144 130 Re ion AT1 Coding S80239 (exon The protein-coding sequence131 3) of Region 577410 (exon 580239 was spliced to 132 5) position 288 of entry S77410.

Nucleotide 1 is assigned to the first nucleotide of the initiator methionine colon in entry S80239.

Renin M10030, X34914, X01391,133 Aldosterone D i 3752 134 s nthase 2 (3-adrenoceptorl X69168/g28421 136 Regulator/
Region (BIP) p-adrenoceptorl J03019/g17899 137 Coding Region 6 - 2 3 - PCT/IB99/Ol 678 (i-adrenoceptor2 M 15169 138 Regulatory 102728 Region (B2P) M16106 (3-adrenoceptor2 Y00106/g29370 139 Coding Region (B2R) EndothelinA 557498 140 {ETA) Receptor Codin The present inventors have surprisingly and unexpectedly discovered the existence of genetic polymorphisms within the human gene encoding ACE, ATI, AGT, renin, aldostf;rone synthase, type-2 angiotensin II receptor, endothelin receptor, or (3-adrenoceptor which, singly or in combination, can be used to assess cardiovascular status, depending on which component of cardiovascular status is under evaluation. In accordance with the invention, the polymorphic pattern of the gene, alone or in combination with other genes encoding renin, ACE, AGT, ATl, AT2, aldosterone synthase, endothelin receptor, and ~3-adrenergic receptors 1 and 2 in an individual can predict the re;sponsivity of the individual to particular therapeutic interventions and serve 2 0 as an indicator of predisposition to various forms of cardiovascular disease. The invention provides methods for assessing cardiovascular status by detecting polymorphic patterns in an individuail. The present invention also provides isolated nucleic acids derived from the gene which <:omprise these polymorphisms, including probes which hybridize specifically to polymorphic positions and primers .that amplify the region of the gene in which the 2 5 polymorphism is located; isolated polypeptides and peptides comprising polymorphic residues; and antibodies which specifically recognize ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or (3-adrenoceptor polypeptides containing one or more polymorphic amino acids.
3 0 Methods for Assessing Cardiovascular Status The present iowention provides diagnostic methods for assessing cardiovascular status in a human individual. The methods are carried out by comparing a polymorphic: position or pattern ("test polymorphic pattern") within the individual's gene encoding A(:E, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II
receptor, 3 5 endothelin receptor, or ~3-adrenoceptor with the polymorphic patterns of humans WO 00/22166 _ 24 _ PCT/IB99/01678 exhibiting a predetermined cardiovascular status ("reference polymorphic pattern"). If the cardiovascular status is the prediction of responsivity to a therapy, a single polymorphic position can provide a pattern for comparison. However, it is preferable to use more than one polymophic position for the pattern to improve the accuracy of the prediction. If the cardiovascular status is predisposition to a cardiovascular syndrome, at least two, and preferably at least three, polymorphic positions are used to make the pattern.
In addition, other polymorphisms in genes encoding angiotensin converting enzyme (ACE), angiotensinol;en (AGT), type i angiotensin II receptor (ATl), type 2 angiotensin II
receptor, renin, aldosterone s;ynthase, endothelin, receptor or (3-adrenergic receptors 1 and 2 can be useda to establish a polymorphic pattern for the individual.
For any meaningful prediction, the polymorphic pattern of the individual is identical to the polymorphic pattern of individuals who exhibit particular status markers, cardiovascular syndromes, and/or particular patterns of response to therapeutic interventions.
In one embodiment, the method involves comparing an individual's test polymorphic pattern with refi~rence polymorphic patterns of individuals who have been shown to respond positively or negatively to a particular therapeutic regimen.
Therapeutic regimen as used herein refers. to treatments aimed at the elimination or amelioration of symptoms and events associated cardiovascular disease. Such treatments include without limitation one or more of alteration in diet, lifestyle, and exercise regimen;
invasive and noninvasive surgical techniques such as atherectomy, angioplasty, and coronary bypass surgery; and pharmaceutical interventions, such as administration of ACE
inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antagonists, cardiac glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor antagonists, calcium channel Mockers, 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 polymorphic patterns associated with cardiovascular disease are also encompassed. The present inventors have discovered that particular polymorphic patterns correlate with an individual's responsivity to ACE
inhibitors (se:e, e.g., Example: 3 below). It is contemplated, for example, that patients who are candidates for a particular therapeutic regimen will be screened for polymorphic patterns that correlate with rtaponsivity to that particular regimen.

In another embodiment, the method involves comparing an individual's polymorphic pattern with polymorphic 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 (seek 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.
Identircation of Pol iy uOr~nhic Patterns In practicing the methods of the invention, an individual's polymorphic pattern can b~: established e.g-. by obtaining DNA from the individual and determining the sequence at a predetermined polymorphic 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 2 0 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 polymorphic 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 polymorphism (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.
3 0 Anal., 1996, 6: I51. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
In an alternate embodiment, biopsy tissue is obtained from a subject.
Antibodies that are capable of distinguishing between different polymorphic forms of WO 00/22166 _ 2 6 _ PCT/IB99/01678 ACE, ATI, A.GT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ~i-adrenoceptor are then applied to samples of the tissue to determine the presence or absence of a polymorphic 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 lparticular polymorphism or polymorphic pattern, and its allelic distribution (i.e., homozygosity vs. heterozygosity) is determined by comparing the values obtained from a patient with norms established from populations of patients having known l0 polymorphic patterns.
In another alternate embodiment, RNA is isolated from biopsy tissue using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al., Anal. Biochem., 1987, 162:156). The isolated RNA is then subjected to coupled reverse transcription and amplification. by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that sire specific for a selected polymorphism. 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 polymorphism. In another embodiment, RNA i;s reverse-transcribed and amplified, after which the amplified 2 0 sequences are identified by, ~e.g., direct sequencing. In still another embodiment, cDNA
obtained from the RNA can 'be cloned and sequenced to identify a polymorphism.
Establishing Re~erertce Polyn:orivhism Patterns In practicing the present invention, the distribution of polymorphic patterns 2 5 in a large nmmber of individuals exhibiting particular 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 for age, ethnic origin, and/or any other statisti~~ally or medically relevant parameters, who exhibit quantitatively or qualitatively different cardiovascular status. Correlations are achieved using any method 3 0 known in the; ari, including nominal logistic regression or standard least squares regression analysis. In this manner, it is possible to establish statistically significant correlations between panricular po(ymophic patterns and particular cardiovascular statuses.
It is further possible to establish statistically significant correlations between particular WO 00/22166 - 2 ~ - PCT/IB99/01678 polymorphic patterns and changes in cardiovascular status such as, would result, e.g., from particular treatment regimens. Thus, it is possible to correlate polymorphic patterns with responsivity to particular treatments.
A statistically significant correlation preferably has a "p" value of less than S 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 polymorphisms to be included in a reference pattern depends not only on the prevalence of a polymorphism and its predictive value for the particular use, but also on the value of the use and its requirement for accuracy of l0 prediction. The greater the predictive value of a polymorphism, the lower the need for inclusion of more than one polymorphism in the reference pattern. However, if a polymorphism 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 seleca instead two or more polymorphisms which are more 15 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 purpose.
If for examples the use for a reference pattern is prediction of response to a drug, and among the afflicted population only a 30% response to the drug is observed, the reference pataern need only permit selection of a population that improves the response 2 0 rate by 10% to provide a significant improvement in the state of the art.
On the other hand, if the use for the reference pattern is selection of subjects for a particular clinical study, the pattern should be as selective as possible and should therefore include a plurality of polymorphisms that together provide a high predictive accuracy for the intended response.
2 5 In establishing reference polymorphism patterns, it is desirable to use a defined population. For example, tissue libraries collected and maintained by state or national departments of health can provide a valuable resource, since genotypes deternlined from these samples can be matched with medical history, and particularly cardiovascular status, of the individual. Such tissue libraries are found, for example, in 30 Sweden, Iceland, Norway, a;nd Finland. As can be readily understood by one of ordinary skill in the art, specific polymorphisms may be associated with a closely linked population. However, other polymorphisms in the same gene may correlate with cardiovascular status of other genetically related populations. Thus, in addition to the WO 00/22166 _ 2 8 - PCT/tB99/01678 specific polymorphisms provided in the instant application, the invention identifies genes in which any polymorphisms can be used to establish reference and test polymorphism patterns for evaluating cardiovascular status of individuals in the population.
In a specific embodiment, DNA samples can be obtained form a well defined population, such as 2'77 Caucasian males born in Uppsala, Sweden between 1920 and 1924. In ~~ specific embodiment, such individuals are selected for the test population based on their medical history, i.e., they were either (i) healthy, with no signs of cardiovascular disease ( 100); or (ii) had suffered one of acute myocardial infarction (68), silent myocardial infarction (:34), stroke (18), stroke and acute myocardial infarction (I9), or high blood pressure at age 50 (39). DNA samples are obtained from each individual.
In a specific embodiment, DNA sequence analysis can be carried out by:
(i) amplifying; short fragments of each of the genes using polymerise chain reaction (PCR) and (ii) sequencing the amplified fragments. The sequences obtained from each individual can then be compared with th.e first known sequences, e.g., as set forth in Table 1, to identify polymorphic positions.
Conwaring Test Patterns tn Reference Patterns As noted above, the test pattern from an individual can be compared to a reference pattern established for a predetermined cardiovascular status.
Identity between 2 0 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 probabiility depends on the prevalence of the polymorphism and the statistical significance of its correlation with a cardiovascular status.
2 5 Pol~morphic Positions Polymorphic positions in the genes encoding ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, or (3-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.
3 0 DNA sequence determination may be achieved using any conventional method, including, e.g., chemicsrl or enzymatic sequencing.

WO 00/22166 _ 2 9 - PCT/IB99/01678 The polymorphic positions of genes for use in the invention include without limitation those listed below, whose numbering corresponds to the GenBank sequences listed in Tablc: 1.
(i) ACE: pasitions in the regulatory region (designated ACR) numbered 5106, 5349, and 5496; positions in the coding region (designated ACE) numbered 375, 582, 731, 1060. 1215, 2193, 2328, 2741, 3132, 3387, 3503, and 3906; and position 1451 as numbered in GenBank entry X62855.
(ii) AGT: positions in the regulatory region (designated AGR) numbered 39:5, 412, 432, 449, 692, 839, 1007, 1072, 1204, and 1218; positions in the coding region (designated A<~ T) numbered 273, 620, 803, 912, 997, 1116, and 1174; and position 49 as numbered in GenBank entry M24688.
(iii) AT1: positions in the regulatory region (designated ATR) numbered 1427, 1756, 1853, 2046, 2354, 2355, and 2415; and positions in the coding region (designated ~~T 1 ) numbered 449, 678, 1167, and 1271.
(iv) Renin: A mutant renin gene in familial elevation of prorenin, a point mutation in the last exon of the gene (exon 10), has been identified (Villard et al., J.
Biol. Chem., 1994, 269:30307-12). A cytosine to thymine transition creates a premature stop codon a~: position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus.
2 0 (v) Aldosl:erone synthase: A position in the promoter region of aldosterone ~~ynthase, position -344 (with the initiation codon starting at 1) has been reported by (:ambien et al. at the International Meeting on Hypertension held in Amsterdam in June 1998.
~3-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 BRI) numbered 231, 758, 1037, 1251, 1403, and 1528.
~3-adrenergic receptor-2, positions in the regulatory region (designated B2P) numbered 9_f2, 934, 987, 1006. 1120, 1221, 1541, and 1568; and positions in the coding region (desil;nated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, 3 0 and 2070.
Endothelin receptor type A coding region (designated ETA) numbered 969, 1005, 1 146, and 2485.

WO 00/22166 _ 3 ~ - PCT/IB99/01678 In preferred embodiments, the base at each of the above polymorphic positions is one of:
(i) ACE Regulatory Region: 51060, 5106T, 5349A, 5349T, 5496T, and 54960;
(ii) ACE Coding Region: 375A, 3750, 5820, 582T, 731A, 7316, 10606, 1060A, 12150, 1215'1,, 21936, 2193A, 2328A, 23286, 27416, 2741T, 31320, 3132T, 3387T, 33870, 35036, 35030, 39066, and 3906A; and a deletion of nucleotides 1451-1783 as numbered in Gf.nBank entry X62855;
(iii) AGT Regulatory Region: 395T, 395A, 4120, 412T, 4326, 432A, 449T, 4490, fi92C, 692T, 8396, 839A, 10076, 1007A, 10726, 1072A, 12040, 1204A, 1218A, 12186;
(iv) AGT Coding Region: 2730, 273T, 6200, 620T, 803T, 8030, 9120, 912T, 9976, !~97C, I 1166, 1116A, 11740, and 1174A; and A or G at position 49 in GenBank entry M24688;
(v) ATI Regulatory Region: 1427A, 1427T, 1756T, 1756A, 1853T, 18536, 2046'0, 20460, 2354A, 23540, 23556, 23550, 2415A and 24156; and (vi) ATl Coding Region: 4496, 4490, 678T, 6780, 1167A, 11676, 1271 A, and 1271 C.
(vii) ~3-adrenergic receptor-I regulatory region: 2238 G, 2238 A, 2577 C, 2 0 2257 T, 2757 A, and 2757 G.
(viii) ~i-adrenergic receptor-I coding region: 231 A, 231 G, 758 C, 758 T, 1251 C, 1251 G, 1403 A, 14013 G, 1528 C, and 1528 A.
{ix) ~3-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 2 5 I 568 T.
(x) (3-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, no insert, 2068 G, 2068 C, 2070 no insert, and 2070 C.
(xi) Endothelin receptor type A: 969 C, 969 T, 1005 A, 1005 G, 1146 3 0 A, 1146 G, 2485 T, and 2485 C.
An individual may be homozygous or heterozygous for a particular polymorphic position (see, e.g., Table 6 below).

WO 00/22166 _ 31 _ PCT/IB99/01678 Non-limiting examples of polymorphic patterns comprising one or more polymorphism in ACE, AGT, and/or AT1 genes according to the invention include the following, which were correlated with an increased incidence of clinical signs of cardiovascular disease:
ACR 5349 A/'f, AGR 1218 A; ACR 5496 C, AGR 1204 A/C; ACR 5496 C/T, AGR 1218 A, AGT 620 C/T; ACE 2193 A, AGR 1204 C, ACE 2328 G; ACE 2193 A, AGR 1204 A/C; ACE 338'7 T, AGR 1218 A; ACE 3387 T, AGT 620 C/T; AGR 1204 A/C, AT 1 678 C/T; AGR I 204 A/C, AT 1 127 I A/C; ACE 1215 C, AGR 1204 A/C;
AGR
1204 A/C, AT 1 I I 67 A, ACE 3906 A/G; AGR 1204 A, AGT 620 C, AT 1 1271 A, AT

1167 A, AGR. 395 A/T; AGR 1204 A/C, AGT 620 C/T, ATl 1271 A/C, AT1 1167 A, AGR 395 T; AGR 1204 A/C, AGT 620 C/T, ATl 1271 A/C, ATl 1167 A/G, AGR 395 T;
AGR 1204 A., AT1 678 C, AT l 1167 A, AGR 395 A/T; AGR 1204 A/C, ATl 678 C/T, ATI 1167 A, AGR 395 T; AGT 620 C/T, AT'1 1271 A/C, AT1 1167 A, AGR 395 T; AGT
620 C/T, AT1. 1271 A/C, AT1 1167 A/G, AC~R 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, ATI 678 C/T; AT1 116'7 A, AGR 395 T; ACE 2193 A, AGR 1218 A, AGT 803 A;
ACE 2193 A., AGT 620 C/T; ACE 2328 G, AGT 620 C/T; ACE 3387 T, AGR 1204 AIC;
ACE 2193 A., ACE 2328 G, AGR 1204 C; ACE 2193 A/G, AGR 1072 G/G, AT1 1167 A/A. Additional polymorphism patterns are shown in the Tables in Examples 4 and 5, 2 0 below.
Polvntorphistn Patterns Correlated With ACE Inhibitor Responsiveness The following; table lists a set of polymorphism patterns that have been found to correlate with responsiveness to AC:E inhibitor treatment:
2 5 Response to ACE-Inhibitor Treatment Position Genotype Position Genotype Position Genotype ACE:2193: A/G AGR:1072: G/G AT1:1167: A/A

ACE:2193: A/G AGR:1072: G/G ACE:1060: G/G

ATR:2354: A/A AT1:678: C/T AT1:1167: A/A

3 ACR:5496: ClT AGR:1204: A/A AGR:839: GlG

ACR:5496: C/T AGR:1204: A/A AGT:620: C/C

ACR:5496: C/T ACE:1060: G/G AGR:449: C/T

Position Genotype Position Genotype Position Genotype ACR:5496: C/T ACE:1215: C/T AGR:1204: A/A

ACR:5496: C/T ACE:3906: A/G AGR:1218: A/G

ACR:5496: C/T AGR:449: C/T AGR:839: G/G

ACR:5496: C/T AGR:449: C/T AT 1:1271:A/A

ACR:5496: C/T AGR:449: ClT AGR:1072: G/G

ACR:5496: C/T AGR:1072: G/G AGR:1204: A/A

ACE:1060: G/G AGR:449: C/T AGT:620: C/C

ACE:1060: G/G AGR:1007: G/G AT1:678: C/T

ACE:121 C/T ACE:3906: A/G AGR:1218: A/G
S:

AGR:449: C/T AGR:1204: A/A AGT:620: C/C

AGR:449: C/T AGR:1204: A/A AGT:1116: G/G

AGR:449: C/T AGR:839: G/G AGT:620: C/C

AGR:449: C/T AGT:620: C/C AGT:1116: G/G

AGR:449: C/T AGT:1116: G/G AT1:1271: A/A

AGR:449: C/T AGR:1072: G/G AGT:620: C/C

AGR:1007: G/C AGR:1072: G/G AT1:678: C/T

AGR:1072: G/G AGR:1204: A/A AGT:1116: G/G

ACR:5496: C/T AGR:1204: A/A AT1:1167: A/A

AGR:1007. G/G ' AGR:1204: A/A AGT:1116: G/G

Po~ornhisnt Patterns of Correlated With ACE Inhibitor Non-responsiveness The followin;~ table lists a set of polymorphism patterns that have been found to correlate with non-responsiveness to ACE inhibitor treatment:
Non-Response to ACE-Inhibitor Treatment 2 Position Genotype Position ~ Genotype Position Genotype ACE:1060: G/G AGR:1204: A/C AGT:620: C/C

ACE:1215: C/T ACE:2193: A/G AGR:1204 A/C

ACE:2193_ A/G ATR:2046: C/T I AT1:678:I C/C

WO 00/22166 _ 3 3 - PCT/IB99/0167t3 ~

Position ' Position Genotype Position Genotype 1 Genotype 2 2 3 3 ACE:2193: A/G AT 1:678: C/C AT 1:1271:A/C

Polymorphisr» Patterns Correlated Witlr Predisposition to MI
The following table lists a set of polymorphism patterns that have been found to come;late with predisposition to myocardial infarction:
Predisposition to MI
Position Genotype Position Genotype Position Genotype ACE:2193: A/A AGR:1204: A/C - -AGR:449: T/T AGR:1204: A/C AT1:1271: A/C

ACE:2193: A/(1 AGR:620: C/T AGR:1116 G/G

ACE:2193: A/G AGR:449: T/T AT1:1271: A/C

ACR:5349: A/A . AGR:449: T/T AT1:1271: A/C

ACE:2193: A/A AGR:620: C/T AT1:1271: A/A

~'ol rv rtorphis»: Patterns Correlated With Predisposition to Stroke The following; table lists a set of polymorphism patterns that have been found to correlate with predisposition to stroke:
Predisposition to Stroke Position Genotype Position Genotype Position Genotype 2 ACE:2193: A/A AGR:395: A/T - -AGR:1007: A/G AGR:1072: GIG AT1:1167: A/A

AGR:395: A/T AGR:1072: G/G AGR:1218: A/G

Polyvnorplris»r Patterns in ~3-Adre»ergic Receptor Genes 2 5 The followins, table lists a set of polymorphism patterns that have been found in (3-adrenergic receptor genes:
Positions can-ying genetic variation in the Beta adrenergic receptors I and 2.

2238 C/A. 231 A/G 839 A/G 932 2757 A/G 2068 no insert, G/G, C/G
2070 no insert /C

B1P: Beta adrenergic receptor 1, regulatory promoter region.
B1R: Beta adrenergic receptor 1, coding region.
B2P: Beta adnenergic receptor 2, regulatory promoter region.
B2R: Beta adrenergic receptor 2, coding region.
A number of dlifferent polymorphisms have been identified in the type 2 ~i-adrenoceptor. All of these differed from the wild type sequence by a single base change.
Four of the p~~lymorphisms alter the amino acid sequence of the receptor protein (Hall, Thorax, 1996, 51:351-353). 'The amino acid sequence modifications are described in 2 0 greater detail below:
Argl6~Gly: 'Che G1y16 variant undergoes an enhanced agonist-promoted down regulation as compared to wild type but the coupling to adenylyl cyclase and agonist binding are maintained (Ligg;ett, Am. J. Respir. Crit. Care Med., 1997, I56:S
156-5162).
G1n27~Glu: 'the G1u27 variant displays very little agonist-promoted 2 5 downregulation and the coupling to adenylyl cyclase and agonist binding are maintained (id. ).
Va134~Met: Met34 is very rare. No altering of receptor function has been found (id.).

Thr164~I1e: Uncommon (about 5%). The Ilel64 variant shows depressed coupling to adenylyl cyclase and decreased affinities for agonists with hydroxyl groups on their ~i-carbons, such as epinephrine, norepinephrine, and isoproterenol compared to wild type (id.).
The polymorphism at nucleic acid 523 (CGG~AGG) might be linked with one of the other functional polymorphisms (id.).
There are no differences in frequency of these polymorphisms between the normal group and those with asthma but they have been correlated to differences in response to treatment with a~:onists in asthma, e.g., the G1y16 variant undergoes an enhanced agonist-promoted downregulation compared to wild type (id.).
Pol ryyo_rphisnr Patterns irr Ertdotl:elirr Receptor Type A Gene The following table lists a set of polymorphism patterns that have been found in the coding region of the endothelin receptor type A gene:
Position 969 Position 1005 Position l 146 Position 2485 C/C'. A/A A/A T/T

C/T A/G A/G T/C

T/T G/G

Isolated Polymorphic Nucleic Acids Vectors Probes & Primers and Arravs Vectors for Expression o~'Polyniorpl:ic Variants The present invention provides isolated nucleic acids comprising the polymorphic; positions described above for the human genes encoding ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and (3-2 5 adrenoceptor; vectors comprising the nucleic acids; and transformed host cells comprising the vectors. The invention a~l5o provides probes which are useful for detecting these polymorphisms.
The nucleic acids encoding a gene comprising a polymorphism that is useful for dc;termining cardiovascular status of an individual is particularly valuable for 3 0 screening, whether by direct screening of the nucleic acid with the polymorphism, or by screening the polypeptide expressed by that nucleic acid.

WO 00/22166 _ 3 ~ - PCT/IB99/01678 In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA, are used. Such techniques are well known arid 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); D.1~'.9 Cloning: A Practical Approach, Volumes I and II, 1985 (D.N. Glover ed.); Oligonucleotide Synthesis, 1984, (M.L. Gait ed.); Natcleic Acid Hybridization., 1985, (Hames and Higgins); Ausubel et al., Current Protocols in Molecular Biology, 1997 (John fViley and Sons); and Methods in Enzymology Vol.

and Vol. 155 (,Wu and Grossman, and Wu, eds., respectively).
Insertion of nucleic acids (typically DNAs) comprising the sequences of the present invention into a vector is easily accomplished when the termini of both the DNAs and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to nnodify the termini of the DNAs and/or vector by digesting back single-stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerise.
Alternatively, .any site desired may be produced, e.g., by ligating nucleotide sequences (linkers) onto the termini. Such linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated by 2 0 the use of the polymerise chain reaction (PCR). See, e.g., Saiki et al., Science, 1988, 239:48. The cleaved vector and the DNA fragments may also be modified if required by homopolyme:ric tailing.
The nucleic acids may be isolated directly from cells or may be chemically synthesized using known methods. Alternatively, the polymerise chain reaction (PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genom~ic 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 incorporation into a given vector for recombinant expression.
3 0 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, S-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 pU(~ 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, CaClz 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 bc: effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation., etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source ~of recombinantly produced ACE-, AGT-, or AT1-derived 2 0 peptides and polypeptides.
Nucleic acids encoding ACE-, AGT-, or AT1-derived gene sequences may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell and thereby effect homologous recombination at the site of an endogenous l;ene 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.
Oligorrucleotides The nucleic acids of the present invention find use as probes for the 3 0 detection of genetic polymoiphisms, as primers for the expression of polymorphisms, 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 by and most WO 00/22166 - 3 8 _ PCT/IB99/01678 preferably 17-25 by in length., which hybridize at high stringency to one or more of the gene-derived polymorphic sequences disclosed herein or to a sequence immediately adjacent to a polymorphic position. Furthermore, in some embodiments a full-length gene sequence may be used as a probe. In one series of embodiments, the probes span the polymorphic positions in the genes disclosed above. In another series of embodiments, the probes correspond to sequences immediately adjacent to the polymorphic positions.
The oligonuclcotide nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. PNAs are also included. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
2 0 Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
PCR amplification of gene segments that contain a polymorphism provides a powerful tool for detecting the polymorphism. The oligonucleotides of the invention can also be used as PCR primers to amplify gene segments containing a polymorphism of interest. The; amplified segment can be evaluated for the presence or absence of a polymorphism by restriction endonuclease activity, SSCP, or by direct sequencing. In another embodiment, the primer is specific for a polymorphic sequence on the gene. If the polymorphism is present, the primer can hybridize and DNA will be produced by PCR.
However, if the polymorphism is absent, the primer will not hybridize, and no DNA will be produced. Thus, PCR can be used to directly evaluate whether a polymorphism is 3 o present or absent.
Molecular library arrays of oligonucleotides (including oligonucleotides with modifications as described above) are another powerful tool for rapidly assessing whether one: or more polymorphisms are present in a ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, or ~3-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.
Polvmorphic Polvpeptides and Polymorphism-Specific Antibodies The present invention encompasses isolated peptides and polypeptides encoded by all or a portion of a gene encoding a polypeptide selected from the group consisting of .ACE, ATI, AG'C, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelia receptor, and ~i-ad:renoceptor, comprising polymorphic positions disclosed above. In one; preferred embodiment, the peptides and polypeptides are useful screening targets to identify cardiovascular drugs. In another preferred embodiment, the peptides and polypeptides are capable of eliciting antibodies in a suitable host animal that react specifically with a polypeptide comprising the polymorphic position and distinguish it from other polypeptides having a different amino acid sequence at that position.
Polypeptides according to the invention are preferably at least five or more residues in length, preferably at least fifteen residues. Methods for obtaining these polypeptides are described below. Many conventional techniques in protein biochemistry and immunology are used. Such techniques are well known and are explained in Immunochemical Methods in C:'ell and Molecular Biology, 1987 (Mayer and Waler, eds;
2 0 Academic Press, London); Scopes, Protein Purifccation: Principles and Practice, Second Edition 1987 (Springer-Verla.g, N.Y.) and Handbook of Experimental Immunology, Volumes I-IV 1986 (Weir and Blackwell eds.).
Nucleic acids comprising protein-coding sequences can be used to direct the recombinant expression o~f ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endoo.helin receptor, or ~i-adrenoceptor-derived polypeptides in intact cells or in cell-free translation systems. The known genetic code, tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The polypeptides may be isolated from. human cells, or from heterologous organisms or cells (including, but not 3 0 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 lVlerrifield, J. Am. Chem. Soc., 1963, 85:2149.
Methods for polvpeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrerut distribution. For some purposes, it is preferable to produce the polypeptide 1 o in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such a.s. but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ~3-adrenoceptor or against peptides derived therefrom, can be used as purification reagents.
Other purification methods are possible.
The present invention also encompasses derivatives and homologues of the polypeptides. For some purposes, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent 2 0 molecules, i.e., function-conservative variants. For example, one or more amino acid residues witb.in 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 F>olypeptides may be modified by, for example, phosphoryla~:ion, 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 3 0 recognize the polymorphic positions of the invention and distinguish a peptide or polypeptide containing a particular polymorphism from one that contains a different sequence at that position. Such polymorphic position-specific antibodies according to the present invention include polvclonal and monoclonal antibodies. The antibodies may be elicited in an animal host by irnmunizatian with ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin Il. receptor, endothelin receptor, or ~3-adrenoceptor-derived immunogenic components or may be formed by in vitro immunization of immune cells.
The immunogenic components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems. The antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA.
Alternatively, the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains. The antibodies include hybrid antibodies (i.e., containing two sets of heavy chairi/light chain combinations, each of which recognizes a different antigen), l0 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 c>f a second heavy chain). Also included are Fab fragments, including Fab" and F(ab)z fragments of antibodies. Methods for the production of all of the above types of antibodies and derivatives are well-known in the art and are discussed in more detail below. For example, techniques for producing and processing polyclonal antisera are disclosed in Mayer and Walker, Tmmunochemical Methods ire Cell and Molecular Biology, 1987 (Academic Press, London). The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as 2 0 direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., Schreier et al., Hybridoma Techniques, 1980;
U.S. Patent Nos. 4,341,7Ei1; 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 ATl-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 Ar;~tibody Purification, 1989 (Amicon Division, W.R. Grace & Co.) General 3 0 protein purification methods are described in Protein Purification:
Principles and Practice, R.>i:. Scopes, Ed., 1987 (Springer-Verlag, New York, NY).
Methods for determining the immunogenic capability of the disclosed sequences and the characteristics of the resulting sequence-specific antibodies and immune WO 00/22166 _ 4 2 _ PCT/IB99/01678 cells are well-known in the arl:. For example, antibodies elicited in response to a peptide comprising a particular polymorphic sequence can be tested for their ability to specifically recognize that polymorphic sequence, i.e., to bind differentially to a peptide or polypeptide comprising the polymorphic sequence and thus distinguish it from a similar peptide or polypeptide containing a different sequence at the same position.
Diagnostic Methods and Kits The present invention provides kits for the determination of the sequence at a polymorphic position or positions within the ACE, ATl, AGT, renin, aldosterone synthase, type-2 angiotensin l~l receptor, endothelin receptor, or (3-adrenoceptor gene in an individual, in combination with determination of the sequence at polymorphism positions of other genes. The kits comprise a means for determining the sequence at the polymorphic positions, and nuay optionally include data for analysis of polymorphic patterns. The means for sequence determination may comprise suitable nucleic acid-based and immunol ogical reagents I;see below). Preferably, the kits also comprise suitable buffers, control reagents where appropriate, and directions for determining the sequence at a polymorphic position. The kits may also comprise data far correlation of particular polymorphic patterns with desirable treatment regimens or other indicators.
2 0 Nucleic.4cid-Based Diagnostic Methods and Kits The invention provides nucleic acid-based methods for detecting polymorphic patterns in a biological sample. The sequence at particular polymorphic positions in the genes is determined using any suitable means known in the art, including without limitation hybridization with polymorphism-specific probes and direct 2 5 sequencing.
The present invention also provides kits suitable for nucleic acid-based diagnostic applications. In one embodiment, diagnostic kits include the following components:
(i) Probe DNA: The probe DNA may be pre-labelled; alternatively, 3 0 the probe DNA may be unlabelled and the ingredients for labelling may be included in the kit in separate containers; and (ii) Hybridization reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
In another embodiment, diagnostic kits include:
(i) Sequence determination primers: Sequencing primers may be pre-labelled oer may contain an affinity purification or attachment moiety;
and (ii) Sequence determination reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular sequencing protocol. In one preferred embodiment, the kit comprises a panel of sequencing primers, whose sequences correspond to sequences adjacent to the polymorphic positions.
Arttiboy~-Based Diagnostic Metl:ods and Kits The invention .also provides antibody-based methods for detecting polymorphic patterns in a biological sample. The methods comprise the steps of (i) 1 S contacting a sample with one or more antibody preparations, wherein each of the antibody preparations is specific for a particular polymorphic 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 auitable means known in the art, wherein the detection of a complex indicates 2 0 the presence of the particular polymorphic form in the sample.
Typically, immunoassays use either a labelled antibody or a labelled antigenic component (e.g., that competes with the antigen in the sample for binding to the antibody). Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signals from the 2 5 probe are als~~ known, such as. for example, those that utilize biotin and avidin, and enzyme-labe:Lled immunoassays, such as ELISA assays.
The present in~~~ention also provides kits suitable for antibody-based diagnostic applications. Diagnostic kits typically include one or more of the following components:
30 (i) Polymorph~i.sm-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 WO 00/22166 - 4 4 ~ PCT/IB99/OI678 {ii) Reaction components: The kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.
The kits referred to above may include instructions for conducting the test.
Furthermore, in preferred embodiments, the diagnostic kits are adaptable to high-throughput and/or automated operation.
Drug Targets and Screenine Methods According to tlhe present invention, nucleotide sequences derived from the gene encoding; a polymorphic form ofACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or (3-adrenoceptor, and peptide sequences derived from that polymorphic 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 v~ithout limitation (i) isolated nucleic acids derived from the gene encoding ACE, AT1, AcJT, renin, aldosterone synthase, type-2 angiotensin II
receptor, endothelin receptor, or ~i-adrenoceptor and (ii) isolated peptides and polypeptides derived from ACE, ATI, AG'T, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, or ~3-adre;noceptor polypeptides, each of which comprises one or more polymorphic positions.
In vitro screenir:g methods In one series of embodiments, an isolated nucleic acid comprising one or more polymorphic positions its 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 polymorphic 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 polymorphic position;
(ii) contacting the nucleic acids with a multiplicity of test compounds under conditions appropriate for binding; and 3 0 (iii) identifying those compounds that bind selectively to either the first or second nucleic acid sequence:.
Selective binding as used herein refers to any measurable difference in any parameter of binding, such as, e.g., binding affinity, binding capacity, ete.

WO 00/22166 _ 4 5 _ PCT/IB99/01678 In another series of embodiments, an isolated peptide or polypeptide comprising one or more polytnorphic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner. T'he screening methods involve:
(i) providing a first peptide or polypeptide containing a particular sequence at a polymorphic position and. a second peptide or polypeptide whose sequence is identical to the first peptide or polypeptide except for a different sequence at the same polymorphic position;
(ii) contacting the polypeptides with a multiplicity of test compounds under conditions appropriate for binding; and (iii) identifying those compounds that bind selectively to one of the nucleic acid sequences.
In preferred embodiments, high-throughput screening protocols are used to survey a large; number of test compounds for their ability to bind the genes or peptides disclosed above in a sequencf:-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 2 0 Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI).
Alternatively., libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily rr~odified through conventional chemical, physical, and 2 5 biochemical means.
In vivo screening methods Intact cells or whole animals expressing polymorphic variants of a gene 30 encoding ACE, ATI, AGT, renin, aldosterone synthase, type-2 angiotensin II
receptor, endothelin receptor, or ~3-adrenoceptor can be used in screening methods to identify candidate cardiovascular drugs.

WO 00/22166 _ 4 6 _ PCT/IB99/01678 In one series of embodiments, a permanent cell line is established from an individual exhibiting a particular polymorphic pattern. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are programmed to express a gene comprising one or more poIymorphic sequences by introduction of appropriate DNA. Identilacation of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to particular polymorphic variants of the gene; (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of the gene; and (iii ) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions the gene.
In another series of embodiments, transgenic animals are created in which (i) a human ACE, ATI, AGT, renin, aldosterone synthase, ype-2 angiotensin II
receptor, endothelia receptor, or (3-adrenoceptor gene having different sequences at particular polymorphic positions are stably inserted into the genome of the transgenic animal; and7or (ii) the endogenous genes are inactivated and replaced with human genes having different sequences at particular polyrr~orphic positions. See, e.g., Coffman, Semin.
Nephrol., 1997, 17:404; Esthe;r et al., Lab. Invest., 1996, 74:953; Murakami et al., Blood Press. Suppl., 1996, 2:36. Such animals can be treated with candidate compounds and monitored for one 2 0 or more clinical markers of cardiovascular status.
Furthermore, populations that are not amenable to an established treatment for a cardiov<~scular disease or disorder can be selected for testing of alternative treatments. Moreover, treatments that are not as effective in the general population, but that are highly effective in the selected population, may be identified that otherwise would 2 5 be overlooked. This is an especially powerful advantage of the present invention, since it eliminates some of the randomness associated with clinical trials.
The following are intended as non-limiting examples of the invention.
Example 1: Methods for :identification of Pol~rmorphic Positions in Human Genes 3 0 Encoding AC.'E, AGT, and AT1 The following; studies were performed to identify polymorphic residues within the genes encoding human ACE, AGT, and ATl.

WO 00/22166 _ 4 ~ _ PCT/IB99/01678 DNA samples were obtained from 277 individuals. The individuals were Caucasian males born 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 frorr~ each individuall.
DNA sequence analysis 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 known ACE, AGT, and ATI genomic sequences (see Table I).
(i) Amplification: PCR reactions employed the primers shown in Table 2 below.

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i Where indicated, the primers were modified in one of the following ways:
(i) a biotin molecule was conjugated to the 5' terminus of the indicated sequence (B); (ii) a sequence of nucleotides derived from M 13, 5'-CAGGAAACAGCTATGACT-3' (SEQ ID
N0:120), was added at the 5' terminus of the indicated sequence (MT); or (iii) the sequence 5'-AGTCACGACGTTGTAAAACGACGGCCAGT-3' (SEQ ID N0:121) was added at the 5' terminus of the indicated sequence {T = Tail). Nucleotides were numbered according to the GenBank sequences listed in Table 1 where indicated. When the sequences involved were not publicly available, the numbering was as in the following examples: The designation "i~-~.: 1-200" indicates that the primer sequence is located within the sequence extending; 200 by upstream of, and including, the nucleotide immediately upstream of the first coding nucleotide of exon 4. Similarly, the designation "i+4: 1-200" :indicates that th~~ primer sequence is located within the sequence extending from the nucleotide that is located immediately downstream of the last coding nucleotide of exon 4 downstream for 200 bp. In each case, the specific location of the primer sequence is indicated in Table 2 in the column marked "Nucleotides".
The reaction components used for PCR are described in Table 3, and the reaction conditions for PCR are described Table 4, below.

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w < a WO 00/22166 _ 6 6 - PCT/IB99/01678 All of the PCR. products (except fragments ACEDI, AT1-spec. 1 and AT1-spec. 2) were subjected to solid phase sequencing according to the protocol commercially available from Pharmacia Biotech. The sequencing reactions are performed with a sequencing primer having a complementary sequence to the "Tail" sequence previously described in Table 2. The nucleotide sequence of the sequencing primer was 5'-CGACGTTGTAAAACGAC~GGCCAGT-3' (SEQ ID N0:122), and the primer was fluorescently labeled with a C'.y-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 ALFexpreasT"~ system cornmerciaily 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 AT1-1271 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 desiignated 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 Jposition. If the shorter PCR product was present in both reactions, the individual is a Jheterozygote 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 2'i ACE, AGT, and AT1. The polymorphic positions, the variant nucleotides found at each of the positions.. and the PCR fragment in which the polymorphism 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.
Polymorphisms that resulted in alternate amino acids in ACE, AGT, or AT1 are also indicated. As used herein below, the designations "AGR" , "ACR", and "ATR"
refer to the regulatory regions of the human AGT, ACE, and AT 1 genes, respectively;
and the designations "AGT", "ACE", and "AT 1 ", refer to the coding regions of the AGT, ACE, and AT I genes.

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U _ E~ E-a WO 00/22166 - ~ 1 _ PCT/IB99/01678 A subset of these polymorphic positions were further analyzed in an additional 187 individuals. 'table 7 shows the polymorphic positions, the sequence at these positions,, and the genotype frequencies for each position in a population of 277 as described in ExampIc I above.
Table 7 - F'olvmorphic Positions and Frequencies Gene Position Genetic variationFre uenc er cent) AGR 692 C(:-CT-TT 78-21-I

ACR 5496 TT-TC-C:C 38-43-19 WO 00/22166 _ ~ 2 _ PCT/IB99/01678 Gene Position Genetic variationFre uency ( er cent Example 2: Correlation of Polymorphism Patterns with Cardiovascular Disease The polymorphic 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). Polymorphic patterns, i.e., combinations of sequences at particular polymorphic positions, that show a statistically significant correlation with one or more of these markers are shown below.
Table 8 - Polymorphism Patterns ACR 5349 A/'L'. AGR 1218 A
Healthy MI ~ Stroke High BP Total (n) # of events 3 7 3 5 17 within rou 3 5.8 8.1 12.8 ACR 5496 C. .AGR 1204 A/C;
i Heaithv MI Stroke High BP Total (n) # of events 2 7 3 2 13 within rou 2 5.8 8.1 5.1 ACR 5496 C/'C. AGR 1218 A. AGT 620 C/T
Healthy MI Stroke High BP Total (n) # of events4 13 I 3 21 withiin 4 10.8 2.7 7.7 rou WO 00/22166 _ 7 ~ - PCT/IB99/01678 ACE 2193 A, AGR 1204 C, ACE; 2328 G
Healthy MI Stroke High BP Total (n) # of events0 11 3 3 16 within 0 9.2 8. I 7.7 rou ACE 2193 A, AGR 1204 A/C
Healthy MI Stroke High BP Total (n) # of events1 1 0 1 3 within 1 0.8 0 2.6 rou ACE 3387 T, AGR 1218 A
Healthy MI Stroke High BP Total (n) # of events2 4 1 3 10 within 2 3.3 2.7 7.7 rou ACE 3387 T, A.GT 620 C/T
Healthy MI Stroke High BP Total (n) # of events1 10 3 2 15 i I
within 1 8.3 8.1 5.1 rou AGR 1204 A/C'., ATI 678 C/T
Healthy MI Stroke High Total ~ (n) # of events5 23 5 6 37 within 5 19.2 13.5 15.4 rou AGR 1204 A/C, AT1 1271 A/C
Healthy MI Stroke High BP Total (n) # of events3 17 3 4 26 within 3 14.2 8.1 10.3 rou WO 00/22166 _ ~ 4 _ PCT/IB99/01678 ACE 1215 C, AGR 1204 A/C
Healthy MI Stroke High BP Total (n) # of events 3 13 5 6 25 within 3 10.8 13.5 15.4 rou AGR 1204 A/C. AT1 I 167 A. ACE 3906 A/G
Healthy MI Stroke High BP Total (n) # of events 0 5 1 0 6 within 0 4.2 2.7 0 rou AGR 1204 A, AGT 620 C, A'T1 1271 A, ATl 1167 A, AGR 395 A/T
Healthy MI Stroke High BP Total (n) # of events1 4 5 3 11 within 1 3.3 I3.5 7.7 rou AGR 1204 A/C. AGT 620 C/T, .AT1 1271 A/C, AT1 1167 A. AGR 395 T
Healthy MI Stroke High BP Total (n) # of events 3 13 3 2 20 within 3 10.8 8.1 5.1 rou AGR 1204 A/C:, AGT 620 C/T, AT1 1271 A/C, ATI 1167 A/G, AGR 395 T
Healthy MI Stroke High BP Total (n) # of events 0 2 0 1 3 within 0 1.7 0 2.6 rou WO 00/22166 _ ~ 5 _ PCT/IB99/01678 Summary of the three previous polymorphic patterns (which involve the same polymorphic positions):
Healthy MI Stroke High BP Total (n) # of events4 19 $ 6 34 /. within 4 15.8 21.6 15.4 rou AGR 1204 A, ATI 678 C, AT1 1167 A, AGR 395 A/T
Healthy MI Stroke High BP Total (n) October 12, # of events1 2 2 1 S

within 1 1.7 5.4 2.6 rou AGR 204 A/C, AT1 1 678 C/T, A, AGR 395 T

1-Iealthy M1 Stroke High Total BP (n) # of events 3 I 8 5 4 28 within rou 3 15.0 13.5 10.3 Summary of the two previous oolvmorc~hic patterns:
Healthy MI Stroke High Total BP {n) # of events 4 2U 7 S 33 within =ou a- ~ 16.7 18.9 12.8 -AGT 620 C/T, AT 1 1271 A/C, AT I I 167 A, AGR 395 T
I
Healthy MI Stroke High Total BP (n) # of events Z 8 1 2 I 3 within rou 2 6.7 2.7 5.1 AGT 620 C/T, AT I l 271 A/C, AT I l 167 A/G, AGR 395 T
He<~lthy MI Stroke High Total BP (n) # of events U 2 0 1 3 within rou 0 1.7 0 2.6 WO 00/22166 _ ~ 6 _ PCT/IB99/01678 AGT 620 C, A'r 1 127 I A, AT 1 I 167 A, AGR 395 A/T
Healthy MI Stroke High Total BP (n) # of evf;ntsl 4 5 3 11 within rou 1 3.3 13.5 7.7 Summary of the three previous polvrnomhic patterns:
Healthy MI Stroke High Total BP (n) # of events 3 14 6 6 27 within rou 3 11.7 16.2 15.4 AGT 620 C. A'T1 678 A. ATI 1167 A, AGR 395 A/T
Healthy MI Stroke High Total BP (n) # of events 1 2 2 1 S

within rou 1 1.7 5.4 2.6 AGT 620 C/T. AT1 678 C/T: A'f l 1167 A. AGR 395 T
Healthy MI Stroke High Total BP (n) # of events 3 1 S 4 4 24 within rou 3 12.5 10.8 10.3 Summary of the two previous polvmomhic patterns:
Healthy MI Stroke High Total BP (n) # of events 4 17 6 5 29 within groupI 4 ~ 14.2 16.2 I 12.9 I

ACE 2193 A, .AGR 1218 A, AGT 803 A
Healthy MI Stroke High Total BP (n) # of events 2 5 1 3 11 within rou 2 4.2 2.7 7.7 WO 00/22166 _ ~ ~ _ PCT/IB99/01678 ACE 2193 A, AGT 620 C/T
Healthy MI Stroke High Total BP (n) # of events 1 11 3 2 16 within rou 1 9.2 8.1 5.1 ACE 2328 G, AGT 620 C/T
Healthy MI Stroke High Total BP (n) # of ev<:nts1 11 3 2 I 6 within rou 1 9.2 8.1 5.1 ACE 3387 T, AGR 1204 A/C
Healthy MI Stroke High BP Total (n) # of events 0 10 3 3 15 within rou 0 8.3 8.1 7.7 Example 3: Correlation Between a Specific Polymorphism Pattern and Treatment Response The following study was undertaken to define polymorphic patterns in the human ACE, AGT, and/or A'C 1 genes that predict the efficacy of treatments for S cardiovascular disease.
Two groups oil hypertensive patients were studied, 41 in the first group and 20 in the second group. The groups were analyzed independently and in combination.
The patients in this population were each treated with one of the following five ACE inhibitors: Captopril, Trandolapril, Lisinopril, Fosinopril, or Enalapril. The effect of the drugs on mean arterial blood pressure was quantified. Mean arterial blood pressure was defined as 2/3 of the diastolic blood pressure + 1/3 of systolic blood pressure.
The individuals were also categorized as "high responders," i.e., those exhibiting a decrease of more than 16 mm Hg during treatment with an ACE inhibitor drug, and "low responders," i.e., those not exhibiting a decrease of more than 16 mm Hg.
1-'i One particula~~ polymorphic pattern, ACE 2193 A/G, AGR 1072 G/G, AT1 1167 A/A, which was present in 51% of the first study population, discriminated between high responders and low responders. In the second group of 20 patients, the pattern was WO 00/22166 _ ~ g _ PCT/IB99/01678 less prevalent {25%), but the correlation with lowered blood pressure remained statistically significant. Individuals having; this polymorphic pattern (designated "1"
below) experienced a larger decrease in blood pressure than those lacking this polymorphic pattern (designated "0" below).
Table 9 - Response to Treatment Correlates with Polymorphic Pattern Polymorphic PatternObservations Mean (mm Hg) S.D.

Chan a in B.P.

0 36 -11.4 8.6 1 25 -18.1 9.7 Furthermore, t:he distribution of high responders and low responders (as defined above') was as follows:
Table 10 - Responder Status Correlates with Polymorphic Pattern Pol morphic Pattern Low res onder % Hi h res onder 0 80.1 19.4 1 24.0 76.0 Taken together, the results from the two groups indicate that the presence of this polymorphic pattern correlates with an incremental decrease of 6.4-7.3 mm Hg relative to individuals not having this polymorphic pattern.
The prevalence of this polymorphic pattern was 41 % in this hypertensive population. 'l'his suggests that testing for this polymorphic pattern in hypertensive patients, followed by prescribing ACE inhibitors only to those patients having this polymorphic pattern, could increase the response rate from 43% (in a hypertensive population in general) to 76°~o in hypertensive population selected according to the methods of the invention.
If even one polymorphism was absent from the pattern, the high resolution 2'i of the variable and therefore the predictive value would be lost.

WO 00/22166 _ ~ 9 - PCT/IB99/01678 Example 4: Correlation Between a Specific Polymorphism Pattern and Treatment Response or Predisposition to a Cardiovascular Syndrome In accordance vrith the methods disclosed in the above examples, additional correlations of polymorphic patterns with either responsiveness to ACE
inhibitors, non-responsiveness to ACE inhibitors, predisposition to myocardial infarction, or predisposition to stroke were derived. These correlations are presented in the following table:

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~1 d WO 00/22166 _ 8 3 - PCT/IB99/01678 Polymorphic patterns, i.e., combinations of sequences at particular polymorphic positions, that show a statistically significant correlation with myocardial infarction and stroke are shown below:
Table 12 - Correlation with MI
MI (n=120) Healthy (n=100) Genetic Signature Prevalence MI Healthy P-value ACE:2193 ~~A 5.0% 100% 0% 0.001 ANP:1204 ~~C

AGR:449 'TT

AGR:1204 ,AC 8.2% 89% 11% 0.002 AT 1:1271 ,AC

ACE:2193 AG

AGT:620 CT 4.5% 100% 0% 0.002 AGT:1167 GG

ACE:2193 AG

AGR:449 TT 1.7.7% 74% 26% 0.007 AT1:1271 AC

ACR:5349 AA

AGR:449 TT 114.5% 75% 25% 0.013 AT1:1271 AC

ACE:2193 AA

AGT:620 CT 3.6% 100% 0% 0.009 AT1:1271 AA

- 8 4 _ PCT/IB99/01678 Stroke (n=37) Healthy (n=100) Table 13 - Correlation With Stroke Genetic Signature Prevalence Stroke Healthy P-value ACE:2193 AA 2.9% 100% 0% 0.005 AGR, 395 AT

AGR;.1007 AG

AGR,1072 GG 9.5% 85% 15% 0.000008 AT1:1167 AA

AGR;.395 AT

AGR:1072 GG 4.4% 83% 17% 0.006 AGR:1218 AG

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, .and are provided for description.
Various patents, patent applications, methodologies, and publications are cited herein, the disclosures of which are incorporated by reference in their entireties.
Example 5: Correlation Between a Specific Polymorphism Pattern and High Blood Pressure Treatment with a ~i-Blocker Using the techniques described in Examples 1 and 2, we investigated poiymorphisms in the ADBP 1~ and ADBR2 genes (regulator/promoter regions and coding regions). The source material was the same as described above.
At the outset, we identified a set of key polymorphisms. Some of these are known; others are new. The are summarized in Table 14.

Table 14 -Polymorphic Positions in the Genes Encoding the Beta Adrenergic Receptors 1 and 2 Gene:Location Genotypes B 1 P:223 8 A/A A/G G/G

B 1 P:2577 C/C C/T T/T

B 1 P:2757 A/A A/G G/G

B I 8:231 A/A A/G G/G

B 18:758 C/C C/T T/T

B 1 R:1251 C/C C/G G/G

B 18:1403 A/A A/G G/G

B 18:1528 A/A AJC C/C

B2P:934 A/A A/G G/G

B2P:987 C/C C/G G/G

B2P:1006 A/A A/G G/G

B2P:1120 C/C C/G G/G

B2P:1221 C/C C/T T/T

B2P:1541 C/C C/T T/T

B2P:1568 C/C C/T T/T

B2R:839 A/A A/G G/G

B2R:872 C/C C/G G/G

B2R:1045 A/A A/G G/G

828:1284 C/C C/T T/T

828:1316 A/A A/C C/C

828:1846 C/C C/G G/G

2~~ 828:2032 A/A A/G G/G

828:2068 no insertion insertion insertion G/G C/G

828:2070 no insertion insertion C/C

Abbreviations used for the df.notation of the genes:

WO 00/22166 _ $ 6 _ PCT/IB99/01678 B 1 P (A.DBR 1 ) - putative promoter region of the gene encoding the beta adrenergic receptor 1 B 1 R (A.DBR 1 ) - the protein coding region of the gene encoding the beta adrenergic receptor 1 B2P (A.DBR2) - putative promoter region of the gene encoding the beta adrenergic receptor 2 B2R (~~DBR2) - the protein coding region of the gene encoding the beta adrenergic receptor 2 These positions, one or a combination of several, can be used to predict a good response to anti-hypertensive treatment with Beta-blockers.
Indeed, we identified various polymorphisms/polymorphic patterns that can be used in the prediction of high response to anti-hypertensive treatment with beta-blockers. These are shown in Table 15.
Table 15 - Polymorphisms Predictive of ~3-Blocker Responsitivity GS Positions - PrevalenceP-value Comments genotypes ('%) GS B2R~ 1846 - 7.7 0.0049 Patients show a 8.3 mmHg I C/C DBP

reduction compared to the group lacking this genotype.

GS2 B2R:1045 - 6.6 (1.0105 Patients will show a A/A 8.1 mmHg DBP

reduction compared to the group lacking this genotype.

GS3 B2R:1316 - 6.6 0.0105 Patients that will show A/A a 8.1 mmHg DBP reduction compared to the group lacking this genotype.

Not only positions in the beta adrenergic receptors, but also positions in the renin-angiotensin- aldosterone-system can be used to predict response to anti-hypertensive treatment with beta- blockers (.ree Table 16). The numbering give according to the numberings in GenBank as df:fmed in Table l, supra.
Tabte R6 - RAAS Polymorphism Associated with ~i-Blocker Responsitivity GS Positions - Prevalence Comments Genotypes (%) GS l ATR:2046 - C/C.'10 Patients will show more OSB + that 10-mmHg AT1:678 - T/T DBP (diastolic blood pressure reduction compared to the group lacking this genotype.

WO 00/22166 _ 8 ~ _ PCT/IB99/01678 The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all sizes and all molecular weight or molecular mars values are approximate, and are provided for description.
Patents, patent applications, procedures, and publications cited throughout this application are incorporated herein by reference in their entireties.

SEQUENCE LISTING
<110> Eurona Medical AB
Norberg, 7~eif Torbjorn Andersson; Maria Kristina Lindstrom, Per Harry Rutger <120> GENES FUR ASSESSING CARDIOVASCULAR
STATUS AND COMPOSITONS FOR USE THEREOF
<130> 1248/1E973--'.'S1 <140> TBA
<141>
<160> 140 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 18 <212> DNA
<213> "Artifici<31 Sequence"
<400> 1 tgcgtgcttc agaagtcc lg <210> 2 <211> 19 <212> DNA
<213> "Artificial Sequence"
<400> 2 ccagggaggt gaagaaatc 19 <210> 3 <211> 19 <212> DNA
<213> "Artificia.? Sequence"
<400> 3 agccaggcag taatgacct 19 <210> 4 <211> 18 <212> DNA
<213> "Artificial Sequence"
<400> 4 gcccactott cccttatg 18 <210> 5 <211> 18 <212> DNA
<2J_3> "Artificial sequence"
<400> 5 tgccctgaca gacagagc 18 <27.0> 6 <2J.1> 16 <2J'.2> DNA
<2:.3> "Artificia.'_ Sequence"
<400> 6 gccctggtc~t gcctgt 16 <2:L0> 7 <2:L1> 18 < 2 .L 2 > DNA

<2:L3> "ArtificialSequence"

<4t)0> 7 tgcctggat:a tgtgttgc 18 <2:L0> 8 <2:L1> 18 < 2 :L 2 > DNA

<2:L3> "Artificial.Sequence"

<400> 8 tgcctggata tgtgttgc 18 <2:10> 9 <2:11> 16 < 2 12 > DNA

<213> "Artific.i<~l=equence"

<430> 9 gccctcgc~~t ctcact 16 <210> 10 <211> 18 <212> DNA
<213> "Artificia'~ Sequence"
<400> 10 tcccctctcc ctgtacct 18 <210> 11 <211> 18 <212> DNA
<213> "Artificia~! Sequence"
<400> 11 gtgctggggt agggtaga 18 <210> 12 _7_ <2).1> 16 <27.2> DNA
<27.3> "Artificial Sequence"
<400> 12 tccccctgac ctggct 16 <27.0> 13 <27.1> 16 < 2 ). 2 > DNA
<2).3> "Artificial Sequence"
<400> 13 ggggcacccLt gatgtt 16 <2:.0> 14 <2:.1> 16 <2:~2> DNA
<2:L3> "Artificial Sequence"
<400> 14 ggggcaccclt gatgtt <2:L0> 15 <2:L1> 17 < 2.L 2 > DNA
<2:L3> "Artificial. Sequence"
<400> 15 gccagagcct ttggttt 17 <2:L0> 16 <2:L1> 19 <2:12> DNA
<2:13> "Artificie~l. Sequence"
<400> 16 tggaagagc:c gacttacag 19 <2:L0> 17 <2:11> 17 < 2 :L 2 > DNA
<2:L3> "Artificial Sequence"
<400> 17 tcccagagc3c aaagagg 17 <2:10> 18 <211> 19 <212> DNA
<213> "Artifici<~l Sequence"
<400> 18 gtttctactg cggcttcat:. 19 <21.0> 19 <21.1> 19 <21.2> DNA
<21.3> "Artificial Sequence"
<4C~0> 19 gtttctact.g cggcttcat 19 <21.0> 2b <21.1> 19 <21.2> DNA
<21.3> "Artificial Sequence"
<400> 20 tcctggaac~a gggagtttc 19 <21.0> 21 <21.1> 19 <21.2> DNA
<21.3> "Artificial Sequence"
<4C10> 21 gcaggatgag agcaacaac 19 <21.0> 22 <21.1> 24 < 21_ 2 > DNA
<21.3> "Artificial Sequence"
<400> 22 ctggagacc:a ctcccatcct ttct 24 <21_0> 23 <21.1> 25 <212> DNA
<213> "Artificial Sequence"
<400> 23 gatgtggcc:a tcacattcgt cagat 25 <2~_0> 24 <2u1> 18 < 2 :L 2 > DNA
<2:L3> "Artificia.l Sequence"
<400> 24 cttccgtgc~g actcatgt 18 <2:L0> 25 <2:L1> 17 < 2 :L 2 > DNA
<2:L3> "Artificial Sequence"
<400> 25 tgcaccgt<3a ggctcta 17 <210> 26 <211> 18 <212> DNA
<213> "Artificial. Sequence"
<400> 26 gcccaatagg aggaagca lg <210> 27 <211> 19 <212> DNA

<213> "Artificia__Sequence"

<400> 27 cccaccccat ctccaagaa 19 <210> 28 <211> 19 <212> DNA

<213> "Artificial Sequence"

<400> 28 tccctgatgg gctgctctc 19 <210> 29 <211> 19 <212> DNA

<213> "Artificial Sequence"

<400> 29 caaggccctc aaccaactc 1g <210> 30 <211> 20 <212> DNA
<213> "Artificial Sequence"
<400> 30 ttcccacaaa agctccagtg 20 <210> 31 <211> 20 <212> DNA
<213> "Artificial Sequence"
<400> 31 ggctcaaaat ggcaagtgtt 20 <210> 32.
<211> 20 <212> DNA
<213> "Artificial Sequence"
<400> 32 gggccatgtc cttctgactc 20 <21.0> 33 <21.1> 19 <21.2> DNA
<21.3> "Artificial Sequence"
<400> 33 cagcctggag gggttaaga 19 <27.0> 34 <27.1> 20 < 2 7. 2 > DNA

<27.3> "Artificia~ 5ecruenee"

<4C10> 34 cccttctgag cgagctgagt 20 <27.0> 35 <21.1> 21 <27.2> DNA

<213> "Artificial Sequence"

<400> 35 ggccatgtt:g agctacttca 21 a <27L0> 36 <2:L1> 20 <2:L2> DNA

<2~3> "Artificial Sequence"

<400> 36 cctccagcct tgggtcttaa 20 <2_LO> 37 <2:L1> 20 <2:L2> DNA

<2:L3> "Artificial Sequence"

<400> 37 ttcccatccc agtctctggt 20 <2:L0> 38 <2:L1> 19 < 2 :L 2 > DNA

<2:L3> "Artifici~~lSequence"

<400> 38 ggcagcctc3g ttgatgagt 19 <2:10> 39 <2:11> 23 <2:12> DNA

<2:13> "Artificial Sequence"

<400> 39 attccagctc tgaaattctcga 23 t WO 00/2216b PCT/IB99/01678 <i 10> 40 <211> 18 < i 12 > DNA
<113> "Artificial Sequence"
<9:00> 40 gagcccct:cc agcacctc 18 <110> 41 <'l.ll> 17 < <:12 > DNA
c~:13> "Artifir_ial Sequence"
<9:00> 41 acccgagcct gcccacc 17 <110> 42 <'s'.11 > 18 < a:12 > DNA
<i!13> "Artificial Sequence"
<9:00> 42 ggtcggg<a g ggaagatc '18 <'1.10> 43 <211> 18 <:! 12 > DNA

<<!13> "ArtificialSequence"

c9~00> 43 tcggctct:gc cccttctc 18 <210> 44 <i 11> 21 <:!12> DNA

c:!13> "ArtificialSequence"

<5:00> 44 gccctttc:tc cagcttcctc 21 t <:!i0> 45 <211> 17 < :! 12 > DNA

<:!13> "ArtificialSequence"

<<l00> 45 cggcggcagc agcaaca 17 <10> 46 c:!11> 1.8 <:!12> DNA

<213> "Artificial Sequence"

<<t00> 46 gagcccct:cc agcacctc 18 _7_ WO 00/22166 PCT/IB99/01b78 <210> 47 <211> 17 <212> DNA
<213> "Artificial Sequence"
<400> 47 acccgagcct gcccacc 17 <210> 48 <211> 18 <212> DNA
<213> "Artificia?. Sequence"
<400> 48 ggtcgggctg ggaagatc 18 <210> 49 <211> 18 <212> DNA

<213> "Artifici<~l Sequence"

<400> 49 tcggctctgc cccttctc 18 <210> 50 <211> 21 <212> DNA

<213> "Artificial Sequence"

<400> 50 gccctttcac cagcttcctc 21 t:

<210> 51 <211> 17 <212> DNA

<213> "Artifici<~~LSequence"

<400> 51 cggcggcagc agcaaca 17 <210> 52 <211> 23 <212> DNA

<213> "Artificial Sequence"

<400> 52 atggcactta aaggtcagt:t 23 aat <210> 53 <211> 19 <212> DNA

<213> "Artifici;s:1Sequence"

<400> 53 tacggaagcc caagaagtt: 19 _g_ <210> 54 <211> 18 <212> DNA
<213> "Artificial Sequence"
<400> 54 ctccccaacg gctgtctt lg <210> 55 <211> 21 <212> DNA
<213> "Artific:iai ~~quence"
<400> 55 agcagcaaca tccagttctg t 21 <210> 56 <211> 19 <21.2> DNA
<213> "Artificial Sequence"
<4C0> 56 tcccacgct.c tctggactt 19 <21.0> 57 <21.1> 26 <21.2> DNA
<21.3> "Artificial Sequence"
<400> 57 ctgatctcag ctacacatgg atacta 26 <27.0> 58 <21.1> 20 <27.2> DNA
<21.3> "Artificial Sequence"
<4C10> 58 cctgtcttqg gtgactcttc 20 <27.0> 59 <27_1> 20 <27_2> DNA
<27.3> "Artific:ial Sequence"
<400> 59 ttctgggct:a aatggtgaca 20 <210> 60 <27_1> 21 < 2 J. 2 > DNA
<27_3> "Artificial Sequence"
<400> 60 cttgtcttc:g gtgtcaagtt t 21 _g_ WO 00/22166 PC'T/IB99/01678 «;lo> 61 <211> 18 < e'! 12 > DNA
<213> "Artificial Sequence"
<9:00> 61 gggagcct:tg gaccacac lg <<!10> 62 <<!11> 20 <212> DNA

<<!13> "Artificia~ Sequence"

<~00> 62 agcctgcatg aacctgtcaa. 20 <:!10> 63 <211> 17 <:!12> DNA

<:!13> "Artifici.a._Sequence"

<400> 63 tggtgggc:gt gttcaca 17 <'.'.10 > 6 4 <11> 17 <:>.12 > DNA

<:?13> "Artificial Sequence"

<!l00> 64 gccagagc:ca gcagaga 17 <10> 65 < 211> 7.8 < 212 > DNA

<?13> "Artificial Sequence"

<~~00> 65 ccacattcca ggggagac 18 <210> 66 <211> 20 <212> DNA

<;213> "Artificial Sequence"

<~400> 66 cctgtct!:.gg gtgactcttc: 20 <210> 67 <211> 18 <212> DNA

<:213> "Artific:Wl Sequence"

<400> 67 ccacatt~ca ggggagac: 18 WO 00/221b6 PCT/IB99/01678 <210> 68 <211> 21 <212> DNA

<213> "Artificia_L Sequence"

<400> 68 gtcccttcag tgccctaata 21 c, <210> 69 <211> 2:1 <212> DNA

<213> "Artifici<~l Sequence"

<400> 69 acagccagat tgaaagacac 21 a <210> 70 <211> 22 <212> DNA

<213> "Artifici<~:~Sequence"

<400> 70 aaccctttta ctggtcatgt 22 ga <210> 71 <211> 19 <212> DNA

<213> "Artificial Sequence"

<400> 71 cgctcatggg atgtgtgac 19 <210> 72 <211> 23 <212> DNA

<213> "Artificia~y Sequence"

<400> 72 tgttttcccc agtgtctatta 23 ag <210> 73 <211> 21 <212> DNA

<213> "Artifici<~i Sequence"

<400> 7:3 gcagggtcga gttacacatt 21 t.

<210> 74 <211> 21 <212> DNA

<213> "Artifici<3i Sequence"

<400> 74 cctcaggctg tcacacacct 2i a <2:L0> 75 <2:L1> 21 <2:L2> DNA
<2:L3> "Artificial Sequence"
<400> 75 cggcttacca tctgctgtag t 21 <2:L0> 76 <2:L1> 21.
< 2 :L 2 > DNA
<213> "Artificial Sequence"
<400> 76 ctccttgaac ctgcttgtgt 21 t <2:L0> 77 <2:L1> 22 <2:L2> DNA

<2:L3> "Artificial Sequence"

<41)0> 7?

gcattgaa<~g atgtgctgtt ct <2:L0> 7$

<2:L1> 25 < 2 :L 2 > DNA

<2:L3> "Artificia.lSequence"

<400> 78 taacgact~~c aaaagcaagttac 25 ct <2:10> 79 <2:11> 19 <2:12> DNA

<2:13> "Artificial Sequence"

<400> 79 agagggca~~g ggagagtct <2:10> 80 <2:11> 18 <2:12> DNA

<213> "Artificial Sequence"

<400> 80 ggcagcaggg tcagaagt 18 <210> 81 <211> 20 <212> DNA

<213> "Artificial Sequence"

<4D0> 81 gctggaga~~g agggttacat 20 <210> 82 <2:L1> 21 < 2 :L 2 > DNA
<2;L3> "Artificial Sequence"
<4I)0> 82 tgcaaacttc ggtaaatgtg 21 t <2:L0> 83 <2:L1> 19 <2:L2> DNA

<2:L3> "Artifici~:.~.eauence"

<41)0> 83 cagaacaacg gcagcttct. lg <2:L0> 84 <2:L1> 24 < 2:L 2 > DNA

<2:L3> "Artificial.Sequence"

<400> 84 actggctg~~c ttatgcttttct 24 ta <2:10> 85 <2:11> 22 <2:12> DNA

<2:13> "Artificial Sequence"

<400> 85 gggttgaavt ttgggactca 22 t:a <2,10> 86 <211> 20 <212> DNA

<213> "Artificial Sequence"

<400> 86 gccagttt~~c cagctataat 20 <210> 87 <211> 23 <212> DNA

<213> "Artificial Sequence"

<400> 87 tgatgcctag ttgaatcaata 23 ac <210> 88 <211> 23 <212> DNA

<213> "Artifici<zw Sequence"

<400> 88 gaaggcttat gaaattcagaa 23 <ag _13_ <2:10> 89 <2:11> 22 <2:12> DNA
<2:13> "Artifici<~1 Sequence"
<400> 89 aaagtcggtt cagtccacat as 22 <2:10> 90 <2:11> 22 <212> DNA

<2:13> "Artificia~~ Sequence"

<400> 90 aaacagcttg gtggtgatag tic 22 ' <210> 91 <211> 20 <212> DNA

<213> "Artifici<~l Sequence"

<4D0> 91 gcaggtgact ttggctacaa 20 <210> 92 <211> 23 <212> DNA

<213> "Artificial Sequence"

<400> 92 aggaaaca~~g aaacccagt:a tat 23 <210> 93 <211> 24 <212> DNA

<213> "Artificial Sequence"

<400> 93 cctgtacgct agtgtgtttc tact 24 <210> 94 <211> 20 <212> DNA

<213> "Artificial Sequence"

<400> 94 ctggattccc caccaaatat 20 <230> 95 <211> 23 <212> DNA
<213> "Artificial Sequence"
<400> 95 tgctccttct ttcacaaaat tac 23 <210> 96 <211> 26 <212> DNA
<213> "Artificial Sequence"
<400> 96 cttccgttat tatgtgtgat attagt 26 <210> 97 <211> 23 <212> DNA
<213> "Artificial Sequence"
<400> 97 gcatgtacct aaaaagtcct gtc 23 <210> 98 <211> 22 <212> DNA
<213> "Artificial Sequence"
<400> 98 attggcatat ccatcacctt as 22 <210> 99 <211> 22 <212> DNA
<213> "Artifi<~ial Sequence"
<400> 99 gatctcccaa ctcatgctat ga 22 <210> 100 <211> 22 <212> DNA
<213> "Artificial Sequence"
<400> 100 attggattca atttgcctac at 22 <210> 101 <211> 25 <212> DNA
<213> "Artificial Sequence"
<400> 101 tttggtaata cagttgtgga tcata 25 <210> 102 <211> 20 <212> DNA
<213> "Artificial Sequence"
<900> 102 tgcaacttgg gtagcatgtc 20 -IS-<2,L0> 103 <211> 21 <212> DNA
<213> "Artificia.l Sequence"
<4U0> 103 agtcgtcc~~g tgtcaactat c 21 <2,10> 104 <211> 23 <212> DNA

<213> "Artifici~~l Sequence"

<4U0> 104 cgttgtct~c cgttattatg tgt 23 <210> 105 <211> 25 <212> DNA

<213> "Artificial Sequence"

<400> 1U5 ttattgcatg tacctaaaaa gtgta 25 <210> lU6 <211> 25 <212> DNA

<213> "Artificial Sequence"

<400> 106 gcattcatat aaagatcaaa t.cagt 25 <210> 107 <211> 23 <212> DNA

<213> "Artificial. Sequence"

<400> 107 caccctgata acaaaaccag ata 23 <210> 108 <211> 21 <212> DNA

<213> "Artificial. Sequence"

<400> lU8 ctttctggca tcaacctcac t: 21 <210> 109 <211> 25 <212> DNA

<213> "Artifi<~i<al Sequence"

<400> 109 acttttaagg acgaattaga gaact 25 <27.0> 110 <27.1> 22 <212> DNA
<27.3> "Artificial Sequence"
<400> 110 gtccacccta gaatttcata ac 22 <27_0> 111 <27_1> 18 < 2 7.2 > DNA
<27_3> "Artificial S~auence"
<400> 111 cccaacctc:c tccctctc lg <210> 112 <211> 19 <2J.2> DNA
<27_3> "Artificial Sequence"
<400> 112 gctcgctct:c cctcacgac 19 <210> 113 <211> 17 <27.2> DNA
<27.3> "Artificial Sequence"
<400> 113 tccagccgca ccccatc 17 <27.0> 114 <27.1> 19 <27_2> DNA
<27_3> "Artificial Sequence"
<400> 114 gctcgctct:c cctcacgac 19 <27.0> 115 <2:.1> 17 <2=.2> DNA
<27.3> "Artificial Sequence"
<400> 115 tccagccgca ccccatc 17 <210> 116 <2=L1> 22 <2:.2> DNA
<2=.3> "Artific:ial Sequence"
<400> 116 gcccctcac~a taatgtaagc tc 22 _17_ <2:L0> 117 <2:L1> 21 < 2 :L 2 > DNA
<2:L3> "Artificial Sequence"
<400> 117 aaccggcacg aaaactttac t. 21 <2:L0> 11.8 <2:L1> 22 < 2 :L 2 > DNA
<2:L3> "Artificia:Sequence"
<400> 118 gcacttcact accaaatgag ca 22 <2:10> 119 <2:11> 2?.
<2:12> DNA
<2:13> "Artificial Sequence"
<400> 119 gcacttca~~t accaaatgag cc 22 <210> 120 <211> 18 <212> DNA

<213> "Artifici;sl Sequence"

<400> 120 caggaaacag ctatgact 18 <210> 121 <211> 29 <212> DNA

<213> "Artificial Sequence"

<400> 121 agtcacgacg ttgtaaaacg acggccagt 29 <210> 122 <211> 24 <212> DNA

<213> "Artificial Sequence"

<400> 122 cgacgttgta aaacgacggc cagt 24 <210> 123 <211> 1278 <212> DNA

<213> Homo sapien <400> 123 ccagacaagt gatttttgag gagtccctat ctataggaac aaagtaatta 60 aaaaaatgta _18_ tttcagaatttacaggcccatgtgagatatgatttttttaaatgaagatttagagtaatg120 ggtaaaaaagaggtatttgtgtgtttgttgattgttcagtcagtgaatgtacagcttctg180 cctcatatccaggcaccatctcttcctgctctttgttgttaaatgttccattcctgggta240 atttcatgtctgccatcgtggatatgccgtggctccttgaacctgcttgtgttgaagcag300 gatcttccttcctgtcccttc:agtgccctaataccatgtatttaaggctggacacatcac360 cactcccaacctgcctcar_ccactgcgtcacttgtgatcactggcttctggcgactctca420 ccaaggtctctgtcatgcact:gta actacaaaagcaagtcttacctataggaaa480 ataacg ataagaattataacccttttactggtcatgtgaaacttaccatttgcaatttgtacagca540 taaacacagaacagcacatct:ttcaatgcctgcatcctgaaggcattttgtttgtgtctt600 tcaatctggctgtgctattgt:tggtgtttaacagtctccccagctacactggaaacttcc660 agaaggcacttttcacttgct:t<;tgtgttttccccagtgtctattagaggcctttgcaca720 gggtaggctctttggagcagctgaaggtcacacatcccatgagcgggcagcagggtcaga780 agtggcccccgtgttgcctaagcaagacatcccctgccctctgccctctgcacctccgg840 c cctgcatgtccctgtggcctcttgggggtacatctcccggggctgggtcagaaggcctgg900 gtggttggcctcaggctgt:cacacacctagggagatgctcccgtttctgggaaccttggc960 cccgactcctgcaaacttcgc;taaatgt:gtaactcgaccctgcaccggctcactctgttc1020 agcagtgaaactctgcatcg<~tcactaagacttcctggaagaggtcccagcgtgagtgtc1080 gcttctggcatctgtcctt:ct:ggccagcctgt:ggtctggccaagtgatgtaaccctcctc1140 tccagcctgtgcacaggcagc:ct:gggaacagc:tccatccccacccctcagctataaatag1200 ggcctcgtgacccggcca<;gggaagaagctgccgttgttctgggtactacagcagaaggt1260 aagccgggggccccctca 1278 <210> 124 <211> 1347 <212> DNA
<213> Homo sapien <400> 124 gtccatctcctcatctcctcttctcataaggacacaggtcatattagatcagggctcacc60 ctcatggcctcattttaacttaatcatctctttaaagatcctgtctccaaataatggtca120 cattctaggtcctggggtttaggacttcaacacgggcattatggccgttggggaggtagg180 acataattcagctgatattgtgcattttgcacttggatcatgtagatattttccatggag240 ctttgaatccatttcttcttttttttgtagacatgaatgatttattctgggctaaatggt300 gacaggaatattgagacaatgaaagatctggttagatggcacttaaaggtcagttaataa360 ccacctttcaccctttgcaaaatgatatttcaggtatgcggaagcgagcaccccagtctg420 agatggctcctgccggtgtgagcctgagggccaccatcctctgcctcctggcctgggctg480 gcctggctgcaggtgaccgggtgtacatacaccccttccacctcgtcatccacaatgaga540 gtacctgtgagcagctggcaaaggccaatgccgggaagcccaaagaccccaccttcatac600 ctgctccaattcaggccaagacatcccctgtggatgaaaaggccctacaggaccagctgg660 tgctagtcgctgcaaaacttgacaccgaagacaagttgagggccgcaatggtcgggatgc720 tggccaacttcttgggcttccgtatatatggcatgcacagtgagctatggggcgtggtcc780 atggggccaccgtcctctccccaacggctgtctttggcaccctggcctctctctatctgg840 gagccttggaccacacagctgacaggct:acaggcaatcctgggtgttccttggaaggaca900 agaactgcacctcccggct.ggatgcgcacaaggtcctgtctgccctgcaggctgtacagg960 gcctgctagt ggcccagggcagggctgatagccaggcccagctgctgctgtccacggtgg1020 tgggcgtgttcacagccccaggcctgcacctgaagcagccgtttgtgcagggcctggctc1080 tctatacccctgtggtcctcccacgctctctggacttcacagaactggatgttgctgctg1140 agaagattgacaggttcatgcaggctgtgacaggatggaagactggctgctccctgatgg1200 gagccagtgtggacagcaccctggctttcaacacctacgtccacttccaaggtaaggcaa1260 acctctctgctggctctggccctaggacttagtatccatgtgtagctgagatcagccagt1320 caggccttggagatgggcagggggcag 1347 <210> 125 <211> 377 <212> DNA

<213> Homo sapien <400> 125 cctgcccctgtcttgggtgactcttccctccctgtctcctgtctgatttcagggaagatg60 aagggcttctccctgctggccgagccccaggagttctgggtggacaacagcacctcagtg120 tctgttcccatgctctctggcatgggcaccttccagcactggagtgacatccaggacaac180 ttctcggtgactgaagtgcccttcactgagagcgcctgcctgctgctgatccagcctcac240 tatgcctctgacctggacaaggtggagggtctcactttccagcaaaactccctcaactgg300 atgaagaaactgtctcccc:ggtagagccctcccggtctcccctggaatgtgggagccaca360 ctctcctgacccaggct 377 <210> 126 <211> 273 <212> DNA
<213> Homo sapie>_n <400> 126 cctctgggagagccctcactgtgtggcctggagccttcctaactgtgcatcatctcccca60 ggaccatccacctgaccatgccccaactggtgctgcaaggatcttatgacctgcaggacc120 tgctcgcccaggctgagctgcccgccattctgcacaccgagctgaacctgcaaaaattga180 gcaatgaccgcatcagggt.gggggaggtatttaccttccttgcctacctggtccattgca240 caggtgagcatgattaaggaaaagagctatggt ' 273 <210> 127 <211> 945 <212> DNA
<213> Homo sapie~n <400> 127 agcccaccgccggccctctagccctcacgaccctgggtcacccatgcgccctcagaatga60 tcctgatcctgatgtctggtcctttgcaggtgctgaacagcattttttttgagcttgaag120 cggatgagagagagcccac:agagtctacccaacagcttaacaagcctgaggtcttggagg180 tgaccctgaaccgcccattcctgtttgctgtgtatgatcaaagcgccactgccctgcact240 tcctgggccgcgtggccaacccgctgagcacagcatgaggccagggccccagaacacagt300 gcctggcaaggcctctgcccctggcctttgaggcaaaggccagcagcagataacaacccc360 ggacaaatcagcgatgtgtcacccccagtctcccaccttttcttctaatgagtcgacttt420 gagctggaaagcagccgtttctccttggtctaagtgtgctgcatggagtgagcagtagaa480 gcctgcagcggcacaaatgcacctcccagtttgctgggtttattttagagaatgggggtg540 gggaggcaagaaccagtgtttagcgcgggactactgttccaaaaagaattccaaccgacc600 agcttgtttgtgaaacaaaaaagtgttcccttttcaagttgagaacaaaaattgggtttt660 aaaattaa.agtatacattt.ttgcattgccttcggtttgtatttagtgtcttgaatgtaag720 aacatgacctccgtgtag;:gtctgtaataccttagttttttccacagatgcttgtgattt780 ttgaacaa.tacgtgaaagatgcaagcacctgaatttctgtttgaatgcggaacaatagct840 ggttatttctcccttgtgt.tagtaataaacgtcttgccacactaagcctccaaatttact900 ctttatta.gacgccaacagatgtatacattcagccagatagactg 945 «;10> 128 «;11> 1856 <112> DNA
<<:13> Homo sapien <9:00> 128 gtgagagctc atgtgcaggc tgagtgagag gcgagggctg ggactggcat ggggcccggg 60 ggtgctg<~gt gagagcacag .agttgggctc ccctcgctct tggggtcagc gtgcccagga 120 aatgccctat cttgttttcc acgagggggg cttctctgcc cactgagagc cggcacctac 180 ttcataccatgccccgatcagctgcccctccctcagaaccgccctctgcttaagggtgtc240 cactctctcctgtcctctctccatgccgcccctcagagcagcgggatctcaaagttatat300 ttcatgggcttggactccaaatggggggaactcggggacactagctccccccggcctcct360 ttcgtgaccctgcccttgact~cctcaccttc:tctgtctttcctgagcccctctcccagc420 atgtgactgataaggaaattcragtcacacagcccctgaaagcgccagactagaacctgag480 cctctgattcctctcacttc<:ctcccctaccctgccacttcctactggatagaagtagac540 agctcttg.actgtcctctttt_ctccccactggctggtccttcttagccccagcccgtttg600 aaagagctcacccccgacacaaggacccgca<:acagatacctcccagctccctctcaacc660 caccctttccagggttggaga acttgaggcat=aaacattcttccatgaggaatctccacc720 cagaaatg~ggtcr_ttctggcc_cccagcccagctcccacattagaacaatgacaaatagaa780 ggggaaatggaa<aataaacaqaagaaacggttttcccaggacagggtttggcctacaagt840 tgtggatgtgggtacccat:.gccaagtgtgaggggaggctggccgggtgtggtggctcatg900 ctctaatcccagcactttqgciaggccaaggtgagtagatcacttgaggccgggagtttga960 gaccagcctggccaacatqg~gaaaccccatc:tgtactaaaaatacaaaagttagctggg1020 cgtggtggtagatgcctgt.agtcccagctacttgggaggctgaggcatgagaatcgcttg1080 agcccagccagggcaatacacscaagaccccgt:ctctacaaataaaatacaaaaaattagt1140 tggatgtggtggtgcatgcctgtagtcctagctgctagggaggctgagatggaaggattg1200 cttgagcctgggaggtcaaggctgcagtgagc:cgagatggcgccactgcactccagcctg1260 ggcaacagagtgagaccct_gr_ctcagaaaga<~aaaaaaaaaaaaaggagaggagagagac1320 tcaagcacgcccctcacaggactgctgaggccctgcaggtgtctgcagcatgtgcccagg1380 ccggggactctgtaagccacr_gctggagaccactcccatcctttctcccatttctctaga1440 cctgctgcctatacagtcacr_ttttttttttr_tttgagacggagtctcgctctgtcgccc1500 aggctggagtgcagtggcggc~atctcggctr_actgcaacgtcrgcctcccgggttcacgc1560 cattctcctgcctcagcctcc:caagtagctgggaccacagcgr_ccgccactacgcccggc1620 taattttttgtatttttagtagagacggggtttcaccgttttagccgggatggtctcgat1680 ctcctgacctcgtgatccgcc:cgcctcggcctcccaaagtgctgggattacaggcgtgat1740 acagtcacttttatgtggtttcgccaatttta~ttccagctctgaaattctctgagctccc1800 cttacaagcagaggtgagc.:t<aagggctggagctcaagccattcaaccccctaccag 1856 <210> 129 <211> 4020 <212> DNA
<213> Homo sapier <400> 129 gccgagcaccgcgcaccgcgtcatgggggccgcctcgggccgccgggggccggggctgct60 gctgccgctgccgctgctgttgctgctgccgccgcagcccgccctggcgttggaccccgg120 gctgcagcccggcaacttttctgctgacgaggccggggcgcagctcttcgcgcagagcta180 caactccagcgccgaacaggtgctgttccagagcgtggccgccagctgggcgcacgacac240 caacatcaccgcggagaatgcaaggcgccaggaggaagcagccctgctcagccaggagtt300 tgcggaggcctggggccagaaggccaaggagctgtatgaaccgatctggcagaacttcac360 ggacccgcagctgcgcaggatcatcggagctgtgcgaaccctgggctctgccaacctgcc420 cctggctaagcggcagcagtacaacgccctgctaagcaacatgagcaggatctactccac480 cgccaaggtctgcctccccaacaagactgccacctgctggtccctggacccagatctcac540 caacatcctggcttcctcr3caaagctacgcc,stgctcctgtttgcctgggagggctggca600 caacgctgcgggcatcccr~c~gaaaccgctgtacgaggatttcactgccctcagcaatga660 agcctacaagcaggacggctvcacagacacgggggcctactggcgctcctggtacaactc720 ccccaccttcgaggacgat:cwggaacacctctaccaacagctagagcccctctacctgaa780 cctccatgccttcgtccgcc~,cgcactgcatcgccgatacggagacagatacatcaacct840 caggggacccatccctgcr:c.atctgctgggagacatgtgggcccagagctgggaaaacat900 ctacgacatggtggtgccttt..ccagacaagcccaacctcgatgtcaccagtactatgct960 gcagcagggctggaacgcca~gcacatgttccgggtggcagaggagttcttcacctccct1020 ggagctctcccccatgcctcccgagttctgggaagggtcgatgctggagaagccggccga1080 cgggcgggaagtggtgtgc:cacgcctcggcttgggacttctacaacaggaaagacttcag1140 gatcaagcagtgcacacggg~cacgatggaccagctctccacagtgcaccatgagatggg1200 ccatatacagtactacctgcagtacaaggatctgcccgtctccctgcgtcggggggccaa1260 ccccggcttccatgaggccattggggacgtgctggcgctctcggtctccactcctgaaca1320 tctgcacaaaatcggcctgctggaccgtgtcaccaatgacacggaaagtgacatcaatta1380 cttgctaaaaatggcactggaaaaaattgccttcctgccctttggctacttggtggacca1440 gtggcgctggggggtctttagtgggcgtacccccccttcccgctacaacttcgactggtg1500 gtatcttcgaaccaagtatcaggggatctgtcctcctgttacccgaaacgaaacccactt1560 tgatgctggagctaagttt:catgttccaaatgtgacaccatacatcaggtactttgtgag1620 ttttgtcctgcagttccagtt:ccatgaagccc:tgtgcaaggaggcaggctatgagggccc1680 actgcacc;~gtgtgacatrtaccggtccaccaaggcaggggccaagctccggaaggtgct1740 gcaggctggctcctccaggccctggcaggaggtgctgaaggacatggtcggcttagatgc1800 cctggatg~~ccagccgctgct:caagtacttcc:agccagtcacccagtggctgcaggagca1860 gaaccagc~3gaacggcgac3gtcctgggctggcccgagtaccagtggcacccgccgttgcc1920 tgacaact,~cccggagggcatagacctggtgactgatgaggctgaggccagcaagtttgt1980 ggaggaat~~tgaccggacatcccaggtggtgtggaacgagtatgccgaggccaactggaa2040 ctacaaca~~caacatcace.acagagaccagcaagattctgctgcagaagaacatgcaaat2100 agccaacc,scaccctgaagt<acggcacccaggccaggaagtttgatgtgaaccagttgca2160 gaacaccactatcaagcggatcataaagaaggttcaggacctagaacgggcagcgctgcc2220 tgcccaggagctygaggagtacaacaagatcctgttggatatggaaaccacctacagcgt2280 ggccactgtgtgccacccgaatggcagctgcctgcagctcgagccagatctgacgaatgt2340 gatggccacatcccggaaatatgaagacctgttatgggcatgggagggctggcgagacaa2400 ggcggggagagccatcctccagttttacccgaaatacgtggaactcat~aaccaggctgc2460 ccggctcaatggctatgtagatgcaggggactcgtggaggtctatgtacgagacaccatc2520 cctggagcaagacctggagcggctcttccaggagctgcagccactctacctcaacctgca2580 tgcctacgtgcgccgggccctgcaccgtcactacggggcccagcacatcaacctggaggg2640 gcccattc~~tgctcacctgct:ggggaacatgtgggcgcagacctggtccaacatctatga2700 cttggtggtgcccttcccttcagccccctcgatggacaccacagaggctatgctaaagca2760 gggctgga~~gcccaggaggatgtttaaggaggctgatgatttcttcacctccctggggct2820 gctgcccgtgcctcctgagtt:ctggaacaagt:cgatgctggagaagccaaccgacgggcg2880 ggaggtggtctgccacgcctcggcctgggact:tctacaacggcaaggacttccggatcaa2940 gcagtgcaccaccgtgaactt:ggaggacctggtggtggcccaccacgaaatgggccacat3000 ccagtatttcatgcagtacaaagacttacctgtggccttgagggagggtgccaaccccgg3060 cttccatg,~ggccattggggacgtgctagccctctcagtgtctacgcccaagcacctgca3120 cagtctcaacctgctgagcagtgagggtggcagcgacgagcatgacatcaactttctgat3180 gaagatggcccttgacaagat:cgcctttatccccttcagctacctcgtcgatcagtggcg3240 ctggagggtatttgatggaagcatcaccaaggagaactataaccaggagtggtggagcct3300 caggctgaagtaccagggcctctgccccccagtgcccaggactcaaggtgactttgaccc3360 aggggccaagttccacattcr_ttctagcgtgccttacatcaggtactttgtcagcttcat3420 catccagttccagttccac::gaggcactgtgccaggcagctggccacacgggccccctgca3480 caagtgtgacatctaccagtr_caaggaggccgggcagcgcctggcgaccgccatgaagct3540 gggcttca~3taggccgtggccggaagccatgcagctgatcacgggccagcccaacatgag3600 cgcctcggccatgttgagct<~cttcaagccgctgctggactggctccgcacggagaacga3660 gctgcatggggagaagctgggctggccgcagt:acaactggacgccgaactccgctcgctc3720 agaagggcccctcccagacagcggccgcgtcagcttcctgggcctggacctggatgcgca3780 gcaggcccgcgtgggccagtc3gctgctgctcttcctgggcatcgccctgctggtagccac3840 cctgggcctcagccagcggctcttcagcatccgccaccgcagcctccaccggcactccca3900 cgggccccagttcggctccgaggtggagctgagacactcctgaggtgacccggctgggtc3960 ggccctgcccaagggcctc:cc:accagagactgggatgggaacactggtgggcagctgagg4020 <210> 130 <211> 2720 <212> DNA.
<213> Homo sapien <400> 130 aagcttgctg gggttttgat <agagattttg tttaacctgt agatcatttg aagattaatg 60 ccattgtaacgatattaaatctttcaatccaagaacatggaatgtcattccatttattta120 ggtctaccttatttcaacaattctttttgtttgttttcagactacaagttttagatcctt180 ttgttaaatttatttcttagggttttttttgttttgttttgttttgttggttggttggtt240 tgttttgagatggagtctcactctgtcacccaggctggagtgcagtggcacaatctcagc300 tcacagcaacctctacctcct:gggttcaagcgattattctgcctcagcctcctcctcctg360 agtagctggaactacaggcatgcaccaccacgcctggccttttttttttttttttctttt420 gcatttttagtagagacagggtttcacgatgttggccagcctggtctcgaatccctgacc480 ttgtgattcacccacctcqgcct:cccaaagtgctgagattacaggagtgagccactacac540 caggtcattt cttgatatttt=tactcttttgatctatagtaagtaaaattgtttttatct600 ttgaatttttaaatttttaac:acagttcaaatcagtgtgtctgatttcatctccttctct660 aacaaaccagggtgccagaac:tqcttcagtttctctgccttctctttgtctatgatgact720 aatgtatgaaggtatctgctc~catcaaactttaaacttcacattatccttatttctcttg780 accttgacagatctggcat:ct:tttcacctggtcgtaagcagaaagtccttgatctcctta840 actttttgaggcatggcagcatgtgaggcagggagaggacacagacccacacagcaagtg900 gtgagaagccaacagtggaat-_tc~ttttcttaattccatttgttgattgtttattgctagt960 gtatagaaatacaactgat:tttt:gtatattgatcttgtattctaaaaacttgctcaactt1020 gtttcttagttctaatagttaattaattgattccttagggctttttaatacaagatcatg1080 tcatctacaaatagaaattgtattactttctttctaatctggatgccatttatctttttt1140 tcttgtccaattgccctcact:agaacctttagtacaaagttaaatagaaatgggaagact1200 agacattttgtcttgttccagatcttagacataaaaacgttgtcttccgttattatgtgt1260 gatattagttaagttaagttt:ttcataaataaacttcacagtttgaggaagttcctattc1320 ctaatttgttgagtgttagcatgaaaaagtgttgaattttgtccaagagtttttaaaaat1380 ttttttagacaatcatgtaggctttgtccattttttacttctttaaatttattttatttg1440 atacacaatagatgtacactttttaggtacatgcaataatttaatgcccctcactataaa1500 ttcggagctgcctcctcgccqatgattccagcgcctgacagccaggaccccaggcagcag1560 cgagtgacaggactttttaggtacatgcaataatttaatgcattcatataaagatcaaat1620 cagtgcaattggcatatccat:caccttaaatatttgtctttttcttcatgctagaaacat1680 tcaagttattttctcctagctactctgaaatatacaatagattactgtaaactacagtca1740 ccctactcacctatctaacat-taattgatttttggtaaactaatctaatcttgctttctg1800 gcatcaacctcacttgaccat=ggtgtatagtccctttcatatgttattggattcaatttg1860 cctacattttgttgagaattr_t~atctatactcttaagaaatattgatctgtagtctcgt1920 gatgtctttatctggtttt~gto atcagggtgatactggcctcatagcatgagttgggaga1980 tcatccttactcttctatttt:tt~ggaagagtttgtgaagaattgatattatttcttcttt2040 aaatatttattgggtttttaaaatacatttttaaaatgcaacr_tgggtagcatgtccaat2100 aggaacaaatgagtgtccacccttgaatttcataaccctcggaattaatccatgtaatct2160 atgatccacaactgtattaccaaagttcgagttactcataggaaagagaaagaagttctc2220 taattcgtccttaaaagtttt~ccaagttcagaaaaaaaaaatgttgaagaacacgaactc2280 ccgcaggaaatgatactcctgtacccccagctcgctctccctcacgacccctcgctaggc2340 ggggttcgggaccaggtgaa<:gctgatctgatagttgacacgggacgactgtggcatcat2400 ccttgctgccgtcaatatcccgagagggaggaggttgggccgggagggtctccggggcgg2460 ggcggaggaggagggaatgcaaaacagagcctcgtccccggaacccaagaagcagcaacg2520 cccctcactataaattcggagctgcctc:ctcgccaatgattccagcgcctgacagccagg2580 accccaggcagcagcgagtg<~caggacgtctggaccggcgcgccgctagcagctctgccg2640 ggccgcggcggtgatcgat:ggggagcggctggagcggacccagcgagtgagggcgcacag2700 ccgggacgccgaggcggcgg 2720 <210> 131 <211> 480 <212> DNA
<213> Homo sapien <400> 131 gaattctcag agctggcgaa acagtctggt ccaagcagcc tctcagcagt gcctttcagc 60 ctcccctctc tgagtctttc c:accccttgc tggtacttta gtttcttcca cttctagcac 120 cacgtgtagt ttcccaatt:t <:tcttacc:ca aatttgctca cagggaaaaa aataaattaa 180 _?~_ attagccat:t tacaccacag tgtgaactta ataacaccaa caaaagttcc aaagctctag 240 ggtctcatag cacctccaga tccatgatct cattcggtgt ttccaacaat gttttgcacc 300 aaactggaca catgcttgct acttcatcat cctcatcgtg aacattatta ttattatcat 360 cattttccag atgaagaaaa tgaatcacaa gtcaactgac agtccaaagg ctccacagct 420 cagaggagc~t aaatcatgtg cttaattcag aacttttggc tcccatcact atgctcttcc 480 <2:L0> 132 <2:L1> 2268 <212> DNA
<2:L3> Homo sapien <400> 132 accccaggcagcagcgagtgacaggacgtctggaccggcgcgccgctagcagctctgccg60 ggccgcggcggtgatcgatggggagcggctcgagcggacccagcgagtgagggcgcacag120 ccgggacgccgaggcggcgggcgggagacccgcaccagcgcagccggccctcggcgggac180 gtgacgcac3cgcccggggcgcgggtttgatatttgacaaattgatctaaaatggctgggt240 ttttatctc~aataactcactgatgccatcccagaaagtcggcaccaggtgtatttgatat300 agtgtttgc:aacaaattcgacccaggtgatcaaaatgattctcaactcttctactgaaga360 tggtattaaaagaatccaagatgattgtcccaaagctggaaggcataattacatatttgt420 catgattccaactttatacagtatcatctttgtggtgggaatatttggaaacagcttggt480 ggtgatagtcatttacttttatatgaagctgaagactgtggccagtgt~tttcttttgaa540 tttagcact_ggctgacttatgctttttactgactttgccactatgggctgtctacacagc600 tatggaat<3ccgctggccctttggcaattacctatgtaagattgcttcagccagcgtcag660 tttcaaccl~gtacgctagtgt.gtttctactcacgtgtctcagcattgatcgatacctggc720 tattgttcacccaatgaagtcccgccttcgacgcacaatgcttgtagccaaagtcacctg780 catcatcatttggctgctggcaggcttggccagtttgccagctataatccatcgaaatgt840 atttttcattgagaacacr_aatattacagtttgtgctttccattatgagtcccaaaattc900 aacccttccgatagggctgggcctgaccaaaaatatactgggtttcctgtttccttttct960 gatcattcttacaagttatactcttatttggaaggccctaaagaaggcttatgaaattca1020 gaagaaca~~accaagaaatgatgatatttttaagataattatggcaattgtgcttttctt1080 tttcttttcctggattccccaccaaatattcacttttctggatgtattgattcaactagg1140 catcatac~3tgactgtagaat.tgcagatattgtggacacggccatgcctatcaccatttg1200 tatagctt~~ttttaacaattgcctgaatcctcttttttatggctttctggggaaaaaatt1260 taaaagat~~ttttctccagcttctaaaa.tatattcccccaaaagccaaatcccactcaaa1320 cctttcaa~~aaaaatgagcacgctttcctaccgcccctcagataatgtaagctcatccac1380 caagaagc~~tgcaccatgt.tttgaggttgagtgacatgttcgaaacctgtccataaagta1440 attttgtg;~aagaaggagcaagagaacattcctctgcagcacttcactaccaaatgagca1500 ttagctacttttcagaattgaaggagaaaatgcattatgtggactgaaccgacttttcta1560 aagctctg;aacaaaagctttt:ct:ttccttttgcaacaagacaaagcaaagccacattttg1620 cattagac~~gatgacggctgca cgaagaacaatgtcagaaactcgatgaatgtgttgatt1680 tgagaaattttactgacagaaat.gcaatctccctagcctgcttttgtcctgttatttttt1740 atttccac~ataaaggtatttagaatatattaaatcgttagaggagcaacaggagatgaga1800 gttccagattgttctgtcc:agtttccaaagggcagtaaagttttcgtgccggttttcagc1860 tattagca,actgtgctacacttgcacctggtactgcacattttgtacaaagatatgctaa1920 gcagtagtcgtcaagttgcagatctttttgtgaaattcaacctgtgtcttataggtttac1980 actgccaaaacaatgcccgtaagatggcttatttgtataatggtgttactaaagtcacat2040 ataaaagttaaar_tacttgtaaaggtgctgcactggtcccaagtagtagtgtcctcctag2100 tatattagtttgatttaat:at:ctgagaagtgtatatagtttgtggtaaaaagattatata2160 tcataaagtatgccttcctgttt:aaaaaaagtatatattctacacatatatatatatgta2220 tatctatatctctaaactgct:gttaatt:gattaaaatctggcaaagtt 2268 <210> 133 <211> 826 <212> DNA
<213> Homo sapic:n <400> 133 gatctacc<:accttggcctcccaaagtgctgggacaggtgtgagccaccatgcctggccc60 ctctactcttataattaaaccagctgttgcttttcctgccaagaaaccagtcatgaagat120 tcacccatc~ttctagatgggaaaactgggctgtagctgggagaggccagtagggacaaa 180 c gccaaagtt:aatatagagaatggagcttccagggtataggggttgggtctgggctaggga240 gctggaaa<:ctaggttttacgcttgtcccagttttgatgttagccctgacagtgctgttt300 ctcatcagc:ctctgcctgctccaggggtcacagggccaagccagatagagggctgctagc360 gtcactgg<~cacaagattgctttcccacagctgtccttcctccagcccctctgctcccca420 tccggaaa<:ctgggtaccc:ttcacccacctagctctgtcccgcagtgagatttattgctg480 actgccctc~ccatctaccccagggtaataaat_cagggcaaagcagaattgaatcacccc 54G
c atgcatggagtgtataaaaggggaagggctaagggagccacagaacctcagtggatctca600 gagagagcc:ccagactgagggaagcatggatggatggagaaggatgcctcgctggggact660 gctgctgct:gctctggggctcctgtacctttggtctcccgacagacaccaccacctttaa720 acggtaatt:ggtaactcaggcagagaaggggtgggcaggggtgtaggttcccaccttccc780 aacaccctc~gcttttccacatgcggtgtcattcagtccttacgatc 826 <2:~0> 134 <2:L1> 6910 < 2 :L 2 > DNA
<213> Homo sapien <400>

ggatcctgcaaggagggatacaaattacatacatttgtcaaaacccacagcatgttgacc60 accaggaggagaccccatgtg:actccaggaccctggttgataacaacgtatcgagattcc120 tcacatggaaccagtgcgctcctgtggtggagggtgtacctgtgtcagggcagggggtac180 gtggacattt:tctgcagtttttgatcaattttgcaatgaactaaatctgtggtataaaaa240 taaagtctat=taaaagaatccaaggctccctctcatctcacgataagataaagtccccat300 ccattttact:cctctcagccr_~tggagaaaggagaggccaggtcccaccaccttccaccag360 catggacccc:cagtccagaccccacgccttttctcagcatcctcagaccagcaggacttg420 cagcaatggc~gaattaggcacctgacttctccttcatctacctttggctgggggcctcca480 gccttgacct=tcgctctgaga.gtctcaggcaggtccagagccagttctcccatgacgtga540 tatgtttcc<igagcaggttcctgggtgagataaaaggatttgggctgaacagggtggagg, gagcattggaatggcactcagggcaaaggcagaggtgtgcgtggcagcgccctggctgtc660 cctgcaaagc~gcacgggcactgggcactagagccgctcgggcccctaggacggtgctgcc720 gtttgaagc<:atgccccagca.tccaggcaacaggtggctgaggctgctgcagatctggag780 ggagcagggt:tatgagcacctgcacctggagatgcaccagaccttccaggagctggggcc840 cattttcagc~taaagccctccctggccctcgctgggaacacccagatccctgcccctgct900 gcccaggacc:ctgccaggcac~tcagcactgccattcccagcaggtcccggcactctgcat960 cctttggagc~atggggaagga.gtgcagcacatgctggtctgtggtgctgccagggcaggg1020 gatagtgca<~agaaaaccccagctcactgcagagagggcaggactcagaagcactaaagt1080 tgaaaggttc:cagggagccagrcaggagggctttagctgtgaagccgctaatccaggagca1140 gggagggtg<~acaggagacactttggattgggactgcagggtggggccacgagggacatg1200 accccgtcc<~gcagggcctcctgcttggccccacaggtacaacttgggaggaccacgcat1260 ggtgtgtgtc3atgctgccggaggatgtggagaagctgcaacaggtggacagcctgcatcc1320 ctgcaggatc~atcctggagccctgggtggcctacagacaacatcgtgggcacaaatgtgg1380 cgtgttcttc~ttgtaagcggcgagttgggagctgagagctgggagcagggtgggcagcct1440 gggtgtaggc~gggaggcgac~a.gaggtaggacccaaaagcacatctgccctgggcccctgt1500 ggtgggcagt=gagggtgagc~.cccggcccagaggacggccatcctgtggggtcgcgtctg1560 cactgtgggt:tggggaagcacrggcggtggtggagaaatgggcacgggcacctctgcagag1620 aagacgcag<igcaatgagcccttctgtgtagtgagaacccgctctgcaccaacctcggcg1680 gctgctttct=cttgcggtctcrgggactgtccttcccataggtcagaaaactgaggccctg1740 agaaggggac:ttccactggcrcaggtcacaggctgagtgctgagcctggtgttcgccggg1800 gccgcagcct:ccctcagggccrctcagggtccctgcagtcctggcaaaccttcctgatggg1860 gacaatccgc~ggcaggagg<aggtggggacgcaggtggctggtggttccgttgttctcag1920 aagcaaggc<jcaaggtgggc;c:ggttgatggcactggggaggatgtttcctggcccgtgga1980 gagggtggc<~cctggtcaggt:gggcagggagaggctgatgcttggagtcggtcacctgca2040 gggatgttgtcattaggacgggggaaggactggatgaggatgtcacagtggtgacagccc2100 ccactccatggtaggaagggaacgctattgggaatagtggggtttaggtaaaagggcacc2160 cgtgggtcggggccttcactc3aggctggcctat:agatgacatctgggagagagtcaggac2220 ccaggaaggcaggtccaggaggctgggtgcgcataatggaaggaaggggagcgctcctgt2280 ctgtgtgtgvgtcttgcatctgtgcacatgctgtgtgtttctctgtacctgcattgcaca2340 tgtgtagtgtgtgcacgtgtcgtgtgtgaatgtatgtgtggtgtgtgtgcacaagtgtct2400 gtgtgtgtgcatgtgcaggtgccggcatgggtgtagtgtttgtgcacacatgcacatgcg2460 tctcttcac~acatggtgttg;~ggtcttgcatgggcgcacgtgtgcatgtgcatcttctgc2520 ctgtcatcactgtcaacagc~:cacagcayccagctggacataaataaaggagttttgcag2580 gaatgtggctgacaggggaa;attcctccc:caccat.tccctgggggcatccatggagcccc2640 cacgcactctggctgtgggt:;~ggatggcatgaagcacaaagcttggtttctgtcctgcag2700 aagatatag.atgcttcacar3agacagcagagcagatgccccagaggcactgtgcccaggg2760 cggggaagggtggggagga<:~~agggcagccaggggctctcccctcaggacactgtgtgggt2820 gaggtgggcaaagcttgacaacaggggtcacctcctttcttggagaaaagccctaccctg2880 ttactacagggagggcccgc:~atgggtgaggtggtgccagacttgggtcgccaggtcccgg2940 gaatgacctcagttaccctgtcagcacctgtgggcagaagctaccatctcatccctgctt3000 agacctgagtggcctttgcc:cagcacctggaggccgctctgagaaaaggctgcagctcga3060 acacaaacaggcagcttct<~ccagggcccccagtcagctccctgcaggccgattcccctt3120 ggggacaaggaggatgggat:acgggtcagggcctgtgtcttgctggggcggcctcacaag3180 ctctgccctggcctctgtaggaatgggcctgaatggcgcttcaaccgattgcggctgaac3240 ccagatgtgctgtcgcccaaggccgtgcagaggttcctcccgatggtggatgcagtggcc3300 agggacttctcccaggccct:gaagaagaaggtgctgcagaacgcccgggggagcctgacc3360 ctggacgtccagcccagcatcttccactacaccatagaaggtgtgggccatgcgggaagg3420 tccagccccagagaccctggagtggccagggatggggatggaggactgaagggagtgtgg3480 ggaggcagccaggaggcctggggctgccttgtgctcagcagtgcatcctccccgcagcca3540 gcaacttagctctttttggagagcggctgggcctggttggccacagccccagttctgcca3600 gcctgaacttcctccatgcc::ctggaggtcatgttcaaatccaccgtccagctcatgttca3660 tgcccaggagcctgtctcgctggatcagccccaaggtgtggaaggagcactttgaggcct3720 gggactgcatcttccagtac:ggtgaggccagggacccgggcagtgctatggggaagggac3780 accatgggggcccaatttctccttctccaccacccagtggggaatggaggccacagggag3840 gggtcggggattcctcaccttcctgccggggagattggtgcgaggctggggctgggctgg3900 gctgatccggagaatttgggatgagagcagggagatttgggtgtcggggcagtctgggca3960 ggaggaggacactgaaggatgcttcccagcaccaagatctgagggctgtcccctgctccc4020 tggacaggtgacaactgtatccagaaaatctaccaggaactggccttcaaccgccctcaa4080 cactacacaggcatcgtggcggagctcctgttgaaggcggaactgtcactagaagccatc4140 aaggccaactctatggaactcactgcagggagcgtggacacggtcaggccagcaaccagc4200 cccacccagagagggtgatgccaagcctgcctcccaggcactgcctgccaatgccacacg4260 gcacccacgttccccatcce:caggctacaggccccacatttctgttgccctcagccttcc4320 ccctcctttgttaagggatgagatttgcaggggaggggaaatgtgagctccccctcacat4380 gagactgagtttgcagttar_ctgtgtggggatccatgctccaggctggaagaaagttgga4440 tgaggccctggacacacagcagctctgtccccactggaaagctctgggtgtacaaggaga4500 aggagggttgagaggcagctggaggactccactgggcacccttcccagtgtgcccggtca4560 ccttgggccagaaatgtagatgcatgggagggcagggttgtggggaagacagcagcacag4620 gctccagccagtgcagaggggcctgtgggtgcacagtggggagaactcaatggaagcaga4680 gggagctggggctccagaactccctggatgatgctgaggtgtggccccctgccctaatgg4740 tggctgtga.gaacccgccctgaagaggctgcaggggacctgggccttggtggagatgggg4800 gtcaccttt.ccctgaagaagtcagggaatctggcccaagtggtcatcaaggtttcagatc4860 .cggggtccc:agggctctgtttttgctcagggcatggatgtctccacccctcagagggagg4920 ttgtcctgc~gaggggtgtcccgggggctgagtcctcctgtgcaaggtctgaccctgcaga4980 catggcttcagtagacagcgtttcccttgctgatgacgctctttgagctggctcggaacc5040 ccgacgtgc:agcagatcctgcgccaggagagcctggccgccgcagccagcatcagtgaac5100 atccccagaaggcaaccaccgagctgcccttgctgcgggcggccctcaaggagaccttga5160 ggtgggtgcaggctgaggcctccctgtggccctggccccctgctggagagcagcccccac5220 tgggtggtc~gcagacagaatctggggctgataaacagcgtcacccagcagcccattcccc5280 WO 00/22166 PCT/IB99/0167$
tgcacctgctcttcctccccctcaaggacagggagctcttcttcctctggaatccctctt5340 caacgccctggggattaacgtggggcatgtccttctgcgctcggggctgcttaagttagg5400 ggaggtttc~gctgggctcagcaggtgcaaggaagcacttcctacgacctgggcttcccat5460 ggatctgggacctctgcggggtcttcggtaggaagggtgctgagagcacagggagcccca5520 tccagctgaggaccctttctgtggatgcccccacctccaggctctaccctgtgggtctgt5580 ttttggagcgagtggtgagctcagacttggtgcttcagaactaccacatcccagctgggg5640 tgagtgagccccacacccctcgagctgagaacctccctccccagtcattccctgatccct5700 gctctgcaccgtccgcagacattggtacaggttttcctctactcgctgggtcgcaatgcc5760 gccttgttcccgaggcctgagcggtataatccccagcgctggctagacatcaggggctcc5820 ggcaggaacttccaccacgtgccctttggctt~ggcatgcgccagtgcctcgggcggcgc5880 ctggcagag~gcagagatgct:gctgctgctgcaccacgtaagcaggcctgggggcgggggc5940 gggacctgggcagcagaggcgggacctgcacactgggggcggggcttgcatggtgtgatt6000 gacacctgggaacagtggatggggccttggttggttgaggtcggcgtgaccagggaggat6060 ctgtgctgagcaagacagggtaggatctgggtgaggttgcttctaaacattgaaatgggg6120 actagggga.gtggggtggagcctgtacagaataatggggcttgggcaagacctgggcagg6180 attcagtctgggcctggtc:-.:gcaaggtggggctggtcagaaatgggataggttggggccc6240 aggctgctgctcccccttcagcataattgttgcacctgggacgatgggaggaagctgccc6300 caggtccatgggctactgac:caggccagatggaaacccagcctctgtcctaggtgctgaa6360 gcacttcctggtggagacactaactcaagaggacataaagatggtctacagcttcatatt6420 gaggcctgocacgtcccccctcctcactttcagagcgattaactagtcttgcatctgcac6480 ccagggtcccagcctggccaccagcttccctctgcctgaccccaggccacctgtcttctc6540 tcccacgtg~cacagcttccc:gagtcacccctctgtccagccagctcctgcacaaatggaa6600 ctccccagggcctccaggactggggcttgccaggcttgtcaaatagcaaggccagggcac6660 agctggaga.cgatcttgctg~gcagggcctggccttgtccccagccccacctggccccttc6720 tccagcaagcagtgccctctggacagcttgactctactcctcccagcgctggctccaggc6780 tcctcatga.ggccatgcaag~ggtgctgtgattttgtcccttgccttcctgcctagtctca6840 catgtccctgtccctctcgccctggccagggcctctgtgcagacagtgtcagagtcatta6900 agcgggatcc 6910 <210> 135 <211> 2476 <212> DNA
<213> Homo sapien <400>

gaattcttgttttacaagccatggctctgtttcttaatgttttctataatcactcacttt60 ttttttgcttttgacaaacattcaaaatgctaatgattcaaggatgtcctcagctctgta120 tgtgttctaagagttctatgttttttctccacagaaggcataagaactaggagctgctga180 catttcaatatgaagggcaactccacccttgccactactagcaaaaacattaccagcggt240 cttcacttcgggcttgtgaacatctctggcaacaatgagtctaccttgaactgttcacag300 aaaccatcagataagcatttagatgcaattcctattctttactacattatatttgtaatt360 ggatttctggtcaatattgtcgtggttacactgttttgttgtcaaaagggtcctaaaaag420 gtttctag~catatacatct~tcaacctcgctgtggctgatttactccttttggctactctt480 cctctato~ggcaacctatt:attcttatagatatgactggctctttggacctgtgatgtgc540 aaagtttttggttctttt<~ttaccctgaacatgtttgcaagcattttttttatcacctgc600 atgagtgttgataggtaccaatctgtcatctacccctttctgtctcaaagaagaaatccc660 tggcaagc:atcttatatagt.tccccttgtttggtgtatggcctgtttgtcctcattgcca720 acatttta.ttttcgagacgtcagaaccattgaatacttaggagtgaatgcttgcattatg780 gctttcccacctgagaaatatgcccaatggccagctgggattgccttaatgaaaaatatc840 cttggttttattatccctttaatattcatagcaacatgctattttggaattagaaaacac900 ttactgaa.gacgaatagctatgggaagaacaggataacccgtgaccaagtcctgaagatg960 gcagctgctgttgttctggccttcatcatttgctggcttcccttccatgttctgaccttc1020 ctggatgctctggcctgga~tgggtgtcattaatagctgcgaagttatagcagtcattgac1080 ctggcacttccttttgccatcctcttgggattcaccaacagctgcgttaatccgtttctg1140 tattgttttgttggaaaccggttccaacagaagctccgcagtgtgtttagggttccaatt1200 acttggctccaagggaaaagagagagtatgtcttgccggaaaagcagttctcttagagaa1260 atggagacctttgtgtctta<~acgtgagagcaaaatgcatgtaatcaacatggctacttg1320 ctttgaggctcaccagaattatttttaagtggctttaataaaataataaaatttccccta1380 atcttttctgaatcttctgaaaccaaatgtaactatgttttatcgtccagtgacttr_cag1440 gaattgcccattgtttttctgatatgtttgtacaagattttcattggtgagacatattta1500 caacctagaagtaactggtg<ata tatctcaaattgtaattaataatagattgtgaataat1560 gatttggggattcagatttctctttgaaacatgcttgtgtttcttagtggggttttatat1620 ccatttttatcaggatttccr_cttgaaccagaaccagtctttcaactcattgcatcattt1680 acaagacaacattgtaagagagatgagr_acttctaagttgagtatattataatagattag1740 tactggattattcaggctttaggcatatgctt~tttaaaaacgctataaattatattcct1800 cttgcatttcacttgagtggaggtttatagttaatctataactacatattgaatagggct1860 aggaatatagattaaatcatactcctatgctttagcttatttttacagttatagaaagca1920 agatgtactataacatagaattgcaatctataatatttgtgtgttcactaaactctgaat1980 aagcactttttaaaaaactttctactcattttaatgattgtttaaaggtttctattttct2040 ctgatacttttttgaaatcagtaaacactgtgtattgttgtaaaatgtaaaggtcacttt2100 tcacatccttgactttttagatgtgctgctttgatatataggacattgatttgattttta2160 ttattaatgctttggttctgggttgtttcctaaaatatctgggtggcttaaaaaaaactc2220 tttaacttgtaataaacccttaactggr_ataggaaatggtatccagaatggaattttgct2280 acatgggc;tctgggtggggg~~aaagagacccagtcaattacatgtttggtaccaagaaag2340 gaacctgtcagggcagtaca,stgtgactttgaaaatatataccgtgggggtagttttacc2400 ctatatct.ataaacactgtttgttccagaatctgtatgattctatggagctattttaaac2460 caattgca~ggtctaga 2476 «;10> 136 <i:ll> 3100 < i:12 > DNA
«:13> Homo sapien <9:00> 136 gaattcct:gagatccctgtactggtactcctgctagaataaccttctggtttcgagggtt60 tataattt:aatgtattatacagaaattcggagaaacttgacgtatctctgaatgagcaga120 gaaaaaaagtttcaggaaagttttccaacattccaatgtcacgtgtgtttatatatatac180 tgtgtacc~gcaaagttactggcaatctggcttctgccctgaacctctacgccaagttctg240 gccttatc:gacacagaaaagcaatgccttccacccttcgggggcatttaaggttgacaca300 ctctattc;cggtaacaccacttgacagaaagatggcaaggaaaagcctcttccccacatt360 ccctatgc;ctcaccctgcagctcagcactcagaactcctttcctgggcagcgtcaatttc420 aacttctt:ccaatcccttggctattttgagactgccccttgcgggtccagaaggaagcca480 ggaggccc~gggcagtggctcatgcctctatccagcactttgggaggccaaggtgggtgga540 tcacctgagctcaggagttcgagacgagcctaccaacatggtaaaacgccatctccacta600 aaaatacaaaattagccgtgtgtggcggcacacgcctgtaatcccagctacttgacaggc660 tgaggcac~gagaatcacttgaacccgggaggccagaggttgcagtgggccaagatcacgc720 actgaact=ccagcctgggcaacaaaagtgaaactctgtctcaaaaaaataaaaagaaaga780 cgccaggagtctcaggctttcactgacttggataacttggggtgtagtctccttcctccc840 ctttctaeacgctcttggtttcctggccactcctcctaacctccccaagttggaaaatcag900 ctgataaatcatgggcagggggtgaataaacaaatcactccccagttttacatactacat960 atgatgct:gaggtttgggcctaaagcaaattcatatgtttgcatttatgtaagagaaagt1020 ggttaaa<actggttgattttatataaacttgaaaaacattactttttgcttttgacctgg1080 acagctct=ggaagtcggt:ctctccttgcaaggtaaggatatgagcttggtcatattgatg1140 accatcaacgaacatgatgttcgttacgggcgctgttcttattccatgctagaaggacca1200 gtgagcattatataacattttcatgcacccttacataaacaattagaagctgtttactcc1260 tcaaggactcaattagagattttcttaatttttttatttttttttttatttcaggcctga1320 gctgagg<~cact.acaggagccaaattagagaagggggcttgcagtcctgccatattgcat1380 attcatg<3ctttcttgtccrctcacccccagtataatcctttttgccaggttaacaactc1440 _?g_ actaatttcaaagcaattcatttgccaactcaaaaaaaaaaaaaaaaaaaaaaacccaca1500 gttccaactttaaattgcttttggattttaatcctctggggtatttatatgtttccttgg1560 ctcatccctccaaaactaatctcagagttttgggaatctgggaacttgggcaaaggggga1620 aaccaggcacgaagaccaaggatctgaaatcgcagttcattacgtcagcataaactgaca1680 gtacctttttgtttgtattgacccagctcaaattataaaatcacatgagtaataaaacac1740 aaaatacaggtgttatcattgaaaagtctaaaatgtaattaaacgcgtttttccccctcg1800 cggtggtacgtttactctgtatctcaacgtttctgtctccctaagcctctcctttcatat1860 catgagcatacatttttgcatcatgctcacacgttcattagctaggactgaggagtgtgt1920 acgattccaaagggccctcagcgttagctgttagatgcacaaaccttcgcttcctttcca1980 catctactccactcgaggttcaacagaggatcttgaagaacagcgccagagagcattctt2040 gacagatgcgcgcttggctccagcaaccgctctgctgggaagtttcttctaaccactaac2100 accacctccaatcccccaagctgtcacgacgcatgctggctgggtcccgtcttgacgggg2160 gaagggttttacacacaccctgctaggctgccccacatcacaaccaagctcgcagggcaa2220 actcctcca.agcctggcgg<icaagctgtcccaggcgctctggcgcttcctgaacaccaag2280 gtcccctcc:cccgtcaagggagctagcgctctgttccgcagagaaccccggaactgcagg2340 tcgagggga.tgcggggggagcgggggcgcaggagggagccgagtgctggaggcaaacggg2400 gcgcaggagcgggtgcgggaggcaaacggggcgcaggagtgggtgcgggaggcaaacggg2460 cgcaggagt.gggtgcgggagcgagtgggggctcaggaggggtcgggcccgggggagccag2520 gcgcggaagggggcgcgggggaacagggaccaggaaccagcgggcgcaggaaggggtgcg2580 tccgaaggocacccgcgggcgcacgggaggcactagctacgcgatcagctcgggactctca2640 ggagccgct.caattgccaacgggaggggggtgcggggagttggaggtgggggagcagacc2700 agacggggc~cgtgcctttgcccgcattggctgcaggagcctgacgcgaggccccgggggt2760 tggcttggc~gagtgggagcggggtggggtgggtgctgggtgccggagctgcgggcccggc2820 gcgctcaga~aacatgctgaagtcccggcggctcttccagcagcaggcagcggctccagca2880 gcagcaggc:aggcaggcaggcaggcaggcaggcagcggcagcgacagcgctcggctctgc2940 gggaaaacc~ggcccggcgcccatgctccggccccgcggggcggctgccctgacccggccg3000 cgacctccctctgcgcaccacgccgcccgggcttctggggtgttccccaaccacggccca3060 gccctgccacaccccccgcccccggcctccgcagctcggc 3100 <27_0> 137 <27.1> 1723 <27_2> DNA
<27_3> Homo sapien <4C>0> 137 tgctacccc~cgcccgggcttctggggtgttccccaaccacggcccagccctgccacaccc60 cccgccccc:ggcctccgcagctcggcatgggcgcgggggtgctcgtcctgggcgcctccg120 agcccggtaacctgtcgtcggccgcaccgctccccgacggcgcggccaccgcggcgcggc180 tgctggtgc:ccgcgtcgccgcccgcctcgttgctgcctcccgccagcgaaagccccgagc240 cgctgtctc:agcagtggacagcgggcatgggtctgctgatggcgctcatcgtgctgctca300 tcgtggcgc~gcaatgtgctggtgatcgtggccatcgccaagacgccgcggctgcagacgc360 tcaccaaccacttcatcatgtccctggccagcgccgacctggtcatggggctgctggtgg420 tgccgttcc~gggccaccatcgtggtgtggggccgctgggagtacggctccttcttctgcg480 agctgtggacctcagtggacgtgctgtgcgtgacggccagcatcgagaccctgtgtgtca540 ttgccctgc~accgctacctcgccatcacctcgcccttccgctaccagagcctgctgacgc600 gcgcgcgg<7cgcggggcct:cgtgtgcaccgr_gtgggccatctcggccctggtgtccttcc660 tgcccatccacatgcactggtggcgggcggagagcgacgaggcgcgccgctgctacaacg720 accccaagt:gctgcgactt:cctcaccaaccgggcctacgccatcgcctcgtccgtagtct780 ccttctacc7t gcccctgtgcatcatggccttcgtgtacctgcgggtgttccgcgaggccc840 agaagcag<3tgaagaagatcaacagctgcgagcgccgtttcctcggcggcccagcgcggc900 cgccctcgccctcgccctcgcccgtccccgcgcccgcgccgccgcccggacccccgcgcc960 ccgccgccc~ccgccgccaccaccccgctggccaacgggcgtgcgggtaagcggcggccct1020 cgcgcctcc~tggccctacgcgagcagaaggcgctcaagacgctgggcatcatcatgggcg1080 tcttcacgctctgctggctgcccttcttcctggccaacgtggtgaaggccttccaccgcg1140 agctggtgcccgaccgcct~cttcgtcttcttcaactggctgggctacgccaactcggcct1200 tcaaccccatcatctactgccgcagccccgacttccgcaaggccttccagggactgctct1260 gctgcgcgcgcagggctgcccgccggcgccacgcgacccacggagaccggccgcgcgcct1320 cgggctgtctggcccggcccggacccccgccatcgcccggggccgcctcggacgacgacg1380 acgacgatgtcgtcggggccacgccgcccgcgcgcctgctggagccctgggccggctgca1440 acggcggggcggcggcggacagcgactcgagcctggacgagccgtgccgccccggcttcg1500 cctcggaatccaaggtgtagggcccggcgcggggcgcggactccgggcacggcttcccag1560 gggaacgaggagatctgtgtttacttaagaccgatagcaggtgaactcgaagcccacaat1620 cctcgtctgaatcatccgaggcaaagagaaaagccacggaccgttgcacaaaaaggaaag1680 tttgggaagggatgggagagtggcttgctgatgttccttgttg 1723 <210> 138 <211> 3451 <212> DNA
<213> Homo sapi~°_n <400> 138 cccgggttcaagagattctcctgtctcagcctcccgagtagctgggactacaggtacgtg60 ccaccacacctggctaatttttgtatttttagtagagacaagagttacaccatattggcc120 aggatcttttgctttctatagcttcaaaatgttcttaatgttaagacattcttaatactc180 tgaaccatatgaatttgccattttggtaagtcacagacgccagatggtggcaatttcaca240 tggcacaacccgaaagattaacaaactatccagcagatgaaaggattttttttagtttca300 ttgggtttactgaagaaattgtttgaattctcattgcatctccagttcaacagataatga360 gtgagtgatgccacactctcaagagttaaaaacaaaacaacaaaaaaattaaaacaaaag420 cacacaactttctctctctgtcccaaaatacatacttgcatacccccgctccagataaaa480 tccaaagggtaaaactgtcttcatgcctgcaaattcctaaggagggcacctaaagtactt540 gacagcgagtgtgctgaggaaatcggcagctgttgaagtcacctcctgtgctcttgccaa600 atgtttgaaagggaatacactgggttaccgggtgtatgttgggaggggagcattatcagt660 gctcgggtgaggcaagttcggagtacccagatggagacatccgtgtctgtgtcgctctgg720 atgcctccaagccagcgtgtgtttactttctgtgtgtgtcaccatgtctttgtgcttctg780 ggtgcttctgtgtttgtttctggccgcgtttctgtgttggacaggggtgactttgtgccg840 gatggcttctgtgtgagagcgcgcgcgagtgtgcatgtcggtgagctgggagggtgtgtc900 tcagtgtctatggctgtggttcggtataagtctgagcatgtctgccagggtgtatttgtg960 cctgtatcrtgcgtgcctcggtgggcactctcgtttccttccgaatgtggggcagtgccgg1020 tgtgctgccctctgccttgagacctcaagccgcgcaggcgcccagggcaggcaggtagcg1080 gccacaga~agagccaaaagctcccgggttggctggtaaggacaccacctccagctttagc1140 cctctggcrgccagccagggtagccgggaagcagtggtggcccgccctccagggagcagtt1200 gggccccc~cccgggccagccccaggagaaggagggcgaggggaggggagggaaaggggag1260 gagtgcct:cgccccttcgcggctgccggcgtgccattggccgaaagttcccgtacgtcac1320 ggcgaggc~cagttcccctaaagtcctgtgcacataacgggcagaacgcactgcgaagcgg1380 cttcttcagagcacgggctggaactggcaggcaccgcgagcccctagcacccgacaagct1440 gagtgtgc:aggacgagtccccaccacacccacaccacagccgctgaatgaggcttccagg1500 cgtccgct:cgcggcccgcagagccccgccgtgggtccgcccgctgaggcgcccccagcca1560 gtgcgctt:acctgccagactgcgcgccatggggcaacccgggaacggcagcgccttcttg1620 ctggcacc:caatagaagccatgcgccggaccacgacgtcacgcagcaaagggacgaggtg1680 tgggtggt:gggcatgggcatcgtcatgtctctcatcgtcctggccatcgtgtttggcaat1740 gtgctggt:catcacagccattgccaagttcgagcgtctgcagacggtcaccaactacttc1800 atcacttc:actggcctgtgctgatctggtcatgggcctggcagtggtgccctttggggcc1860 gcccatat:tcttatgaaaatgtggacttttggcaacttctggtgcgagttttggacttcc1920 attgatgt:gctgtgcgtcacggccagcattgagaccctgtgcgtgatcgcagtggatcgc1980 tactttgccattacttcacctttcaagtaccagagcctgctgaccaagaataaggcccgg2040 gtgatcatatgatggtgtcrgattgtgtcaggccttacctccttcttgcccattcagatg2100 c cactggt<accgggccacccaccaggaagccatcaactgctatgccaatgagacctgctgt2160 gacttctt:cacgaaccaagc:ctatgccattgcctcttccatcgtgtccttctacgttccc2220 ctggtgat:catggtcttcgt:ctactccagggtctttcaggaggccaaaaggcagctccag2280 aagattgacaaatctgaggqccgcttccatgtccagaaccttagccaggtggagcaggat2340 gggcggacqgggcatggactccgcagatcttccaagttctgcttgaaggagcacaaagcc2400 ctcaagacc~ttaggcatcatcatgggcactttcaccctctgctggctgcccttcttcatc2460 gttaacatt:gtgcatgtgatccaggataacctcatccgtaaggaagtttacatcctccta2520 aattggataggctatgtcaattctggtttcaatccccttatctactgccggagcccagat2580 ttcaggatt:gccttccaggagcttctgtgcctgcgcaggtcttctttgaaggcctatggg2640 aatggctacaccagcaacggcaacacaggggagcagagtggatatcacgtggaacaggag2700 aaagaaaat:aaactgctgtgtgaagacctcccaggcacggaagactttgtgggccatcaa2760 ggtactgtc~cctagcgataacattgattcacaagggaggaattgtagtacaaatgactca2820 ctgctgtaaagcagtttttctacttttaaagacccccccccccccaacagaacactaaac2880 agactattt:aacttgagggr_aataaacttagaataaaattgtaaaaattgtatagagata2940 tgcagaaggaagggcatccttctgccttttttatttttttaagctgtaaaaagagagaaa3000 acttatttc~agtgattatt.tgttatttgtacagttcagttcctctttgcatggaatttgt3060 aagtttatc~tctaaagagctttagtcctagaggacctgagtctgctatattttcatgact3120 tttccatgt=atctacctcactattcaagtattaggggtaatatattgctgctggtaattt3180 gtatctgaaggagattttccttcctacacccttggacttgaggattttgagtatctcgga3240 cctttcagctgtgaacatggactcttcccccactcctcttatttgctcacacggggtatt3300 ttaggcagggatttgaggagcagcttcagttgttttcccgagcaaaggtctaaagtttac3360 agtaaataaaatgtttgaccatgccttcattgcacctgtttgtccaaaaccccttgactg3420 gagtgctgttgcctcccccactggaaaccgc 3451 <2:L0> 139 <2:L1> 2305 < 2:L 2 > DNA
<2:L3> Homo sapie:n <400> 139 gaattcatgccgcgtttctgtgttggacaggggtgactttgtgccggatggcttctgtgt60 gagagcgcgcgcgagtgtgcatgtcggtgagctgggagggtgtgtctcagtgtctatggc120 tgtggttcggtataagtctaagcatgtctgccagggtgtatttgtgcctgtatgtgcgtg180 cctcggtgggcactctcgt.ttccttccgaatgtggggcagtgccggtgtgctgccctctg240 ccttgagacctcaagccgcgcaggcgcccagggcaggcaggtagcggccacagaagagcc300 aaaagctc~~cgggttggctggtaagcacaccacctccagctttagccctctggggccagc360 cagggtag~~cgggaagcaytggtggcccgccctccagggagcagttgggccccgcccggg420 ccagcctc;aggagaaggagggcgaggggaggggagggaaaggggaggagtgcctcgcccc480 ttcgcggctgccggcgtgccattggccgaaagttcccgtacgtcacggcgagggcagttc540 ccctaaagtcctgtgcacataacgggcagaacgcactgcgaagcggcttcttcagagcac600 gggctgga.actggcaggcaccgc:gagcccctagcacccgacaagctgagtgtgcaggacg660 agtccccaccacacccacaccacagccgctgaatgaggcttccaggcgtccgctcgcggc720 ccgcagag~cccgccgtgggt:ccgcctgctgaggcgcccccagccagtgcgcttacctgc780 cagactgcgcgccatggggcaacccgggaacggcagcgccttcttgctggcacccaatag840 aagccatgcgccggaccacgacgtcacgcagcaaagggacgaggtgtgggtggtgggcat900 gggcatcgtcatgtctctcatcgtcctggccatcgtgtttggcaatgtgctggtcatcac960 agccattgccaagttcgaycytctgcagacggtcaccaactacttcatcacttcactggc1020 ctgtgctgatctggtcatggycctggcagtggtgccctttggggccgcccatattcttat1080 gaaaatgtggacttttggcaacttctggtgcgagttttggacttccattgatgtgctgtg1140 cgtcacggccagcattgagac:cctgtgcgtgatcgcagtggatcgctactttgccattac1200 ttcacctttcaagtaccayagcctgctgaccaagaataaggcccgggtgatcattctgat1260 ggtgtggattgtgtcaggcct:tacctccttcttgcccattcagatgcactggtaccgggc1320 cacccaccaggaagccatc:aactgctatgccaatgagacctgctgtgacttcttcacgaa1380 ccaagcctatgccattgcctcttccatcgtgtccttctacgttcccctggtgatcatggt1440 cttcgtctactccagggtcat:tcaggaggccaaaaggcagctccagaagattgacaaatc1500 tgagggccgcttccatgtcc<agaaccttagccaggtggagcaggatgggcggacggggca1560 tggactccgcagatcttccaagttctgcttgaaggagcacaaagccctcaagacgttagg1620 catcatcatgggcactttcac~cctctgcaggctgcccttcttcatcgttaacattgtgca1680 tgtgatccaggataacctcatccgtaac~gaagtttacatcctcctaaattggataggcta1740 tgtcaattctggtttcaatccccttatctactgccggagcccagatttcaggattgcctt1800 ccaggagcttctgtgcctgcgcaggtcttctttgaaggcctatgggaatggctactccag1860 caacggcaacacaggggagcagagtggatatcacgtggaacaggagaaagaaaataaact1920 gctgtgtgaagacctcccaggcacggaagactttgtgggccatcaaggtactgtgcctag1980 cgataacattgattcacaagggaggaattgtagtacaaatgactcactgctgtaaagcag2040 tttttctacttttaaagacccccccccccaacagaacactaaacagactatttaacttga2100 gggtaataaacttagaataaaattgtaaaattgtatagagatatgcagaaggaagggcat2160 ccttctgccttttttatttttttaagctgtaaaaagagagaaaacttatttgagtgatta2220 tttgttatttgtacagttcagttcctctttg~~atggaatttgtaagtttatgtctaaaga2280 gctttagtcctagaggacctgagtc 2305 <210> 140 <211> 4105 <212> DNA
<213> Homo sapia_n <400> 140 gaattcgcggccgcctcttgcggtcccagagtggagtggaaggtctggagctttgggagg60 agacgggg~aggacagactggaggcgtgttcctccggagttttctttttcgtgcgagccct120 cgcgcgcg~cgtacagtcatcccgctggtctgacgattgtggagaggcggtggagaggctt180 catccatcccacccggtcgtcgccggggattggggtcccagcgacacctccccgggagaa240 gcagtgcccaggaagttttctgaagccggggaagctgtgcagccgaagccgccgccgcgc300 cggagcccgggacaccggccaccctccgcgccacccaccctcgctttctccggcttcctc360 tggcccaggcgccgcgcgga~~ccggcagctgtctgcgcacgccgagctccacggtgaaaa420 aaaaagtgaaggtgtaaaagcagcacaagtgcaataagagatatttcctcaaatttgcct480 caagatgctaaaccctttgcctcagggcatccttttggctggcactggttggatgtgtaat540 cagtgata~atcctgagagatacagcacaaatctaagcaatcatgtggatgatttcaccac600 ttttcgtctgcacagagctcagcttcctggttaccactcatcaacccactaatttggtcct660 acccagcaatggctcaatgcacaactattgcccacagcagactaaaattacttcagcttt720 caaatacattaacactgtgatatcttgtactattttcatcgtgggaatggtggggaatgc780 aactctgcacaggatcatttaccagaacaaatgtatgaggaatggccccaacgcgctgat840 agccagtcatgcccttggagaccttatctatgtggtcattgatctccctatcaatgtatt900 taagctgcagctgggcgctggccttttgatcacaatgactttggcgtatttctttgcaa960 g gctgttcc:cctttttgcagaagtcctcggtggggatcaccgtcctcaacctctgcgctct1020 tagtgttctacaggtacagagcagttgcctcctggagtcgtgttcagggaattgggattcc1080 tttggtaactgccattgaaattgtctccatctggatcctgtcctttatcctggccattcc1140 tgaagcgattggcttcgtcatggtaccctttgaatataggggtgaacagcataaaacctg1200 tatgctcaatgccacatcaaaattcatggagttctaccaagatgtaaaggactggtggct1260 cttcgggtatatttctgtatgcccttggtgtgcactgcgatcttctacaccctcatgac1320 c ttgtgagatgttgaacagaannaatggcagcttgagaattgccctcagtgaacatcttaa1380 gcagcgtc:gagaagtggcaaaaacagttttctgcttggttgtaatttttgctctttgctg1440 gttccctcatcacttaagc~cgtatattgaagaaaactgtgtataacgaaatggacaagaa1500 ccgatgtc~aattacttagtttcttactgctcatggattacatcggtattaacttggcaac1560 catgaatt:catgtataaaccccatagctctgtattttgtgagcaagaaatttaaaaattg1620 tttccagt:catgcctctgctgctgctgttaccagtccaaaagtctgatgacctcggtccc1680 catgaacqgaacaagcatccagtggaagaaccacgatcaaaacaaccacaacacagaccg1740 gagcagccataaggacagcatgaactgaccacccttagaagcactcctcggtactcccat1800 aatcctct:cggagaaaaaaatcacaaggcaactgtgactccgggaatctcttctctgatc1860 cttcttc<ataattcactcccacacccaagaagaaatgctttccaaaaccgcaaggtaga1920 ctggtttatccacccacaacatctacgaatcgtacttctttaattgatctaatttacata1980 ttctgcgtgttgtattcagcactaaaaaatggtgggagctgggggagaatgaagactgtt2040 aaatgaa;~ccagaaggatatttactacttttgcatgaaaatagagctttcaagtacatgg2100 ctagcttttatggcagttctggtgaatgttcaatgggaactggtcaccatgaaactttag2160 agattaacgacaagattttctactttttttaagtgattttttgtccttcagccaaacaca2220 atatgggctcaggtcacttttatttgaaatgtcatttggtgccagtattttttaactgca2280 WO 00/22166 PCT/IB99/O1b78 taatagcctaacatgattatttgaacttatttacacatagtttgaaaaaaaaaagacaaa2340 aatagtattcaggtgagcaattagattagtattttccacgtcactatttatttttttaaa2400 acacaaattctaaagctacaacaaatactacaggcccttaaagcacagtctgatgacaca2460 tttggcagtttaatagatgttactcaaagaattttttaagaactgtattttattttttaa2520 atggtgttttattacaagggaccttgaacatgttttgtatgttaaattcaaaagtaatgc2580 ttcaatcagatagttctttttcacaagttcaatactgtttttcatgtaaattttgtatga2640 aaaatcaatgtcaagtaccaaaatgttaatgtatgtgtcatttaactctgcctgagactt2700 tcagtgcactgtatatagaagtctaaaacacacctaagagaaaaagatcgaatttttcag2760 atgattcgc~aaattttcattcaggtatttgtaatagtgacatatatatgtatatacatat2820 cacctcctattctcttaatttttgttaaaatgttaactggcagtaagtcttttttgatca2880 ttcccttttccatataggaaacataattttgaagtggccagatgagtttatcatgtcagt2940 gaaaaataattacccacaaatgccaccagtaacttaacgattcttcacttcttggggttt300C

tcagtatgaacctaactccccaccccaacatctccctcccacattgtcaccatttcaaag3060 ggcccacagtgacttttgctgggcattttcccagatgtttacagactgtgagtacagcag3120 aaaatcttttact:agtgtgtgtgtgtatatatataaacaattgtaaatttcttttagccc3180 atttttct<~gactgtctctgtggaatatatttgtgtgtgtgatatatgcatgtgtgtgat3240 ggtatgtatggatttaatctaatctaataattgtgccccgcagttgtgccaaagtgcata3300 gtctgagctaaaatctaggtgattgttcatcatgacaacctgcctcagtccattttaacc3360 tgtagcaaccttctgcattcataaatcttgtaatcatgttaccattacaaatgggatata3420 agaggcagcgtgaaagcagatgagctgtggactagcaatatagggttttgtttggttggt3480 tggtttgataaagcagtatttggggtcatattgtttcctgtgctggagcaaaagtcatta3540 cactttgaagtattatattgttcttatcctcaattcaatgtggtgatgaaattgccaggt3600 tgtctgat~~tttctttcagacttcgccagacagattgctgataataaattaggtaagata3660 atttgttg<3gccatattttaggacaggtaaaataacatcaggttccagttgcttgaattg3720 caaggctaagaagtactgcccttttgtgtgttagcagtcaaatctattattccactggcg3780 catcatatgcagtgatatatgcctataatataagccataggttcacaccattttgtttag3840 acaattgtctttttttcaagatgctttgtttctttcatatgaaaaaaatgcattttataa3900 attcagaaagtcatagatttctgaaggcgtcaacgtgcattttatttatggactggtaag3960 taactgtg~~tttactagcaggaatatttccaatttctacctttactacatcttttcaaca4020 agtaactt~:.gtagaaatgagccagaagccaaggccctgagttggcagtggcccataagtg4080 taaaataa~aagtttacagaaacctt 4105

Claims (22)

Claims

1. A method for assessing cardiovascular status in an individual to be tested comprising comparing (a) a test polymorphic pattern comprising at least one polymorphic position within a gene encoding a .beta.-adrenoceptor polypeptide, of the individual, with (b) a reference polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status; and concluding whether the individual possesses the cardiovascular status based on whether the test pattern matches the reference pattern, wherein said polymorphic portion is selected from positions in the regulatory region of .beta.-adrenergic receptor-1 gene (designated B1P) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724 and 2757; positions in the .beta.-adrenergic receptor-1 coding region (designated B1R) numbered 231, 758, 1037, 1251, 1403, and 1528; positions in the .beta.-adrenergic receptor-2 gene regulatory region (designated B2P) numbered 932, 934, 987, 1006, 1120, 1221, 1541 and 1568;
positions. in the .beta.-adrenergic receptor-2 gene coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032; 2068, and 2070; and combinations of any of the foregoing.

2. A method for assessing cardiovascular status in an individual to be tested comprising comparing (a) a test polymorphic pattern comprising at least one polymorphic position within a gene encoding a endothelia receptor polypeptide, of the individual, with (b) a reference polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status; and concluding whether the individual possesses the cardiovascular status based on whether the test pattern matches the reference pattern.

3. A method as claimed in claim 2, wherein said polymorphic position is selected from positions in the endothelin receptor type A coding region (designated ETA) numbered 969, 1005, 1146 and 2485; and combinations of any of the foregoing.

4. A method for assessing cardiovascular status in an individual to be tested comprising comparing (a) a test polymorphic pattern comprising at least one polymorphic position within a gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and .beta.-adrenoceptor of the individual, with (b) a reference polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status; and concluding whether the individual possesses the cardiovascular status based on whether the test pattern matches the reference pattern, wherein the polymorphic pattern of (a) includes a polymorphic position within a second gene encoding a polypeptide selected from the group consisting of ACE, AT1 and AGT.

5. The method according to any one of claims 1 to 4, wherein the predetermined cardiovascular status is predisposition to a cardiovascular syndrome.

6. The method according to claim 5, wherein the cardiovascular syndrome is selected from the group consisting of myocardial infarction, unstable angina, hypertension, atherosclerosis and stoke.

7. The method according to claim 6, wherein the reference pattern comprises at least three polymorphisms.

8. A method for assessing responsivity of an individual to a cardiovascular treatment regimen, comprising comparing (a) a test polymorphic pattern comprising at least one polymorphic position within a gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and .beta.-adrenoceptor of the individual, with (b) a reference polymorphic pattern derived from a population of individuals exhibiting a predetermined response to a cardiovascular treatment regimen; and concluding whether the individual possesses the said predetermined responsivity based on whether the test pattern matches the reference pattern.

9. The method according to claim 8, wherein the polymorphic pattern of (a) includes a polymorphic position within a second gene encoding a polypeptide selected from the group consisting of ACE, ATl and AGT.

10. The method according to claim 8 or claim 9, wherein the treatment regimen comprises administering a cardiovascular drug selected from the group consisting of an ACE inhibitor, an angiotensin II receptor antagonist, a diuretic, an alpha-adrenoreceptor antagonist, a cardiac glycoside, a phosphodiesterase inhibitor, a beta-adrenoreceptor antagonist, a calcium channel blocker, a HMG-CoA reductase inhibitor, an imidizoline receptor blocker, an endothelin receptor blocker, and an organic nitrite.

11. The method according to any one of claims 8 to 10, wherein the reference pattern comprises at least two polymorphisms.

12. The method according to any one of claims a to 10, wherein the reference pattern comprises at least three polymorphisms.

13. The method according to any one of claims 4 to 12, wherein the polymorphic position of the gene in (a) is selected from the group consisting of a position in the renin coding region comprising a cytosine to thymine transition in exon 10 that creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus; a position in a promoter region of aldosterone synthase numbered -344 (with the initiation codon starting at 1); positions in the regulatory region of .beta.-adrenergic receptor-1 gene (designated B1P) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724 and 2757;
positions in the .beta.-adrenergic receptor-1 coding region (designated B1R) numbered 231, 758, 1037, 1251, 1403, and 1528; positions in the .beta.-adrenergic receptor-2 gene regulatory region (designated B2P) numbered 932, 934, 987, 1006, 1120, 1221, 1541 and 1568; positions in the 13-adrenergic receptor-2 gene coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068, and 2070; positions in the endothelin receptor type A coding region (designated ETA) numbered 969, 1005, 1146 and 2485; and combinations of any of the foregoing.

14. The method according to any one of claims 4 to 7 and 9 to 13, wherein the polymorphic position of the second gene is selected from the group consisting of positions in the ACE regulatory region numbered 5106, 5349 and 5496; positions in the ACE coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503 and 3906; position 1451 in the ACE gene as numbered in GenBank entry X62855; positions in the AGT regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072, 1204 and 1218; positions in the AGT
coding region numbered 273, 620, 803, 912, 997, 1116 and 1174; position 49 in the AGT gene as numbered in GenBank entry M24688; positions in the AT1 regulatory region numbered 1427, 1756,, 1853, 2046, 2354, 2355 and 2415;
and positions in the. AT1 coding region numbered 449, 678, 1167 and 1271.

15. An isolated nucleic acid encoding an endothelia receptor polypeptide, in an individual, wherein the nucleic acid comprises a polymorphic position and wherein the polymorphic position is a position in a polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status.

16. An isolated nucleic acid according to claim 15, wherein said polymorphic position is selected from positions in the endothelia receptor type A coding region (designated ET A) numbered 969, 1005, 1146 and 2485; and combinations of any of the foregoing.

17. An isolated nucleic acid encoding an .beta.-adrenoceptor polypeptide, in an individual, wherein the nucleic acid comprise, a polymorphic position and wherein the polymorphic position is a position in a polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status, wherein said polymorphic position is selected from positions; in the regulatory region of .beta.-adrenergic receptor-1 gene (designated BlP) numbered 2238, 2440, 2493, 2502, 2577, 2.585, 2693, 2724 and 2757; positions in the .beta.-adrenergic receptor-1 coding region (designated B1R) numbered 231, 758, 1037, 1251, 1403, and 1528;
positions in the .beta.-adrenergic receptor-2 gene regulatory region (designated B2P) numbered 932, 934, 987, 1006, 1120, 1221, 1541 and 1568; positions in the 13-adrenergic receptor-2 gene coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1846, 1891, 2032, 2068 and 2070 and combination of any of the foregoing.

18. A probe which hybridizes at high stringency to a polymorphic position as defined in any one of claims 15 to 17.

19. A library of nucleic acids comprising a nucleic acid which comprises (a) a polymorphic position of a gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and .beta.-adrenoceptor, wherein the polymorphic position is a position in a polymorphic pattern derived from a population of individuals exhibiting a predetermined cardiovascular status, and (b) nucleic acids comprising other polymorphic positions of human genes that are indicative of cardiovascular status of an individual, wherein the human genes are selected from the group consisting of:
ACE, ATl, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and .beta.-adrenoceptor.

20. The library of claim 19 wherein the nucleic acids are oligonucleotides.

21. A kit for assessing cardiovascular status comprising (a) sequence determination primers and (b) sequence determination reagents, wherein the primers are selected from the group consisting of primers that hybridize to or immediately adjacent to (i) a polymorphic position in a gene encoding a polypeptide selected from the group consisting of renin, aldosterone synthase, type-2 angiotensin II
receptor, endothelin receptor, and .beta.-adrenoceptor, and (ii) a second polymorphic position in a gene encoding a polypeptide selected from the group consisting of ACE, AT1, AGT, renin, aldosterone synthase, type-2 angiotensin II receptor, endothelin receptor, and .beta.-adrenoceptor.

22. The kit of claim 21 wherein the second polymorphic position is selected from the group consisting of positions in the ACE regulatory region numbered 5106, 5349, and. 5496; positions in the ACE coding region numbered 375, 582, 731, 1060, 1215, 2193, 2328, 2741, 3132, 3387, 3503 and 3906; position 1451 in the ACE gene as numbered in GenBank entry X62855; positions in the AGT regulatory region numbered 395, 412, 432, 449, 692, 839, 1007, 1072, 1204 and 1218; positions in the AGT
coding region numbered 273, 620, 803, 912, 997, 1116 and 1174; position 49 in the AGT gene as numbered in GenBank entry M24688; positions in the AT1 regulatory region numbered 1427, 1756, 1853, 2046, 2354, 2355 and 2415;
position: in the AT1 coding region numbered 449, 678, 1167 and 1271; a position in the renin coding region comprising a cytosine to thymine transition in exon 10 that creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus; a position in a promoter region of aldosterone synthase numbered -344 (with the initiation codon starting at 1); positions in the regulatory region of .beta.-adrenergic receptor-1 gene (designated B1P) numbered 2238, 2440, 2493, 2502, 2577, 2585, 2693, 2724 and 2757; positions in the .beta.-adrenergic receptor-1 gene coding region (designated B1R) numbered 231, 758, 1037, 1251, 1403 and 1528; positions in the .beta.-adrenergic receptor-2 regulatory region (designated B2P) Numbered 932, 934, 987, 1006, 1120, 1221, 1541 and 1568;
positions in the .beta.-adrenergic receptor-2 gene coding region (designated B2R) numbered 839, 872, 1045, 1284, 1316, 1845, 1891, 2D32, 2068, and 2070; positions in the endothelin receptor type A coding region (designated ET A) numbered 969, 1005, 1146 and 2485; and combinations of any of the foregoing.