EP1465998A2 - Single nucleotide polymorphisms predicting cardiovascular disease and medication efficacy - Google Patents

Single nucleotide polymorphisms predicting cardiovascular disease and medication efficacy

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Publication number
EP1465998A2
EP1465998A2 EP03729224A EP03729224A EP1465998A2 EP 1465998 A2 EP1465998 A2 EP 1465998A2 EP 03729224 A EP03729224 A EP 03729224A EP 03729224 A EP03729224 A EP 03729224A EP 1465998 A2 EP1465998 A2 EP 1465998A2
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Prior art keywords
gene
polypeptide
genes
polymoφhic
protein
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German (de)
French (fr)
Inventor
Udo Stropp
Stephan Schwers
Harald Kallabis
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Bayer AG
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Bayer Healthcare AG
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Priority to EP08006507A priority Critical patent/EP1978108A3/en
Priority to EP03729224A priority patent/EP1465998A2/en
Publication of EP1465998A2 publication Critical patent/EP1465998A2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to genetic polymorphisms useful for assessing cardiovascular risks in humans, including, but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, arterial inflammation, myocardial infarction, and stroke.
  • the present invention identifies and describes gene variations which are individually present in humans with cardiovascular disease states, relative to humans with normal, or non-cardiovascular disease states, and/or in response to medications relevant to cardiovascular disease.
  • the present invention provides methods for the identification and therapeutic use of compounds as treatments of cardiovascular disease.
  • the present invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease, and for monitoring the efficacy of compounds in clinical trials.
  • the present invention provides methods to use gene variations to predict personal medication schemes omitting adverse drug reactions. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various cardiovascular diseases, and for the identification of subjects exhibiting a predisposition to such conditions.
  • Cardiovascular disease is a major health risk throughout the industrialized world.
  • Cardiovascular diseases include but are not limited by the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, atherosclerosis, ischemic diseases ofthe heart, coronary heart disease, all kinds of atrial and ventricular a ⁇ hythmias, hypertensive vascular diseases and peripheral vascular diseases.
  • Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent ofthe underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in an perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases include stable angina, unstable angina and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyanhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation) as well as bradycardic forms of arrhythmias.
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others).
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • Atherosclerosis the most prevalent of vascular diseases, is the principal cause of heart attack, stroke, and gangrene of the extremities, and thereby the principal cause of death. Atherosclerosis is a complex disease involving many cell types and molecular factors (for a detailed review, see Ross, 1993, Nature 362: 801-809 and Lusis, A. J., Nature 407, 233-241 (2000)).
  • a protective response to insults to the endothelium and smooth muscle cells (SMCs) of the wall of the artery consists of the formation of fibrofatty and fibrous lesions or plaques, preceded and accompanied by inflammation.
  • SMCs smooth muscle cells
  • the advanced lesions of atherosclerosis may occlude the artery concerned, and result from an excessive inflammatory-fibroproliferative response to numerous different forms of insult.
  • shear stresses are thought to be responsible for the frequent occunence of atherosclerotic plaques in regions of the circulatory system where turbulent blood flow occurs, such as branch points and inegular structures.
  • the first observable event in the formation of an atherosclerotic plaque occurs when blood-borne monocytes adhere to the vascular endothelial layer and transmigrate through to the sub-endothelial space. Adjacent endothelial cells at the same time produce oxidized low density lipoprotein (LDL). These oxidized LDLs are then taken up in large amounts by the monocytes through scavenger receptors expressed on their surfaces. In contrast to the regulated pathway by which native LDL (nLDL) is taken up by nLDL specific receptors, the scavenger pathway of uptake is not regulated by the monocytes.
  • LDL low density lipoprotein
  • foam cells lipid-filled monocytes
  • endothelial and SMCs which sunound them lead to a state of chronic local inflammation which can eventually lead to smooth muscle cell proliferation and migration, and the formation of a fibrous plaque.
  • Such plaques occlude the blood vessel concerned and thus restrict the flow of blood, resulting in ischemia.
  • Ischemia is a condition characterized by a lack of oxygen supply in tissues of organs due to inadequate perfusion. Such inadequate perfusion can have number of natural causes, including atherosclerotic or restenotic lesions, anemia, or stroke, to name a few.
  • ischemia may sometimes affect cardiovascular tissue, such as in ischemic heart disease. Ischemia may occur in any organ, however, that is suffering a lack of oxygen supply.
  • Atherosclerotic disease of epicardial coronary arteries.
  • atherosclerosis causes an absolute decrease in myocardial perfusion in the basal state or limits appropriate increases in perfusion when the demand for flow is augmented.
  • Coronary blood flow can also be limited by arterial thrombi, spasm, and, rarely, coronary emboli, as well as by ostial nanowing due to luetic aortitis.
  • Congenital abnormalities such as anomalous origin of the left anterior descending coronary artery from the pulmonary artery, may cause myocardial ischemia and infarction in infancy, but this cause is very rare in adults.
  • Myocardial ischemia can also occur if myocardial oxygen demands are abnormally increased, as in severe ventricular hypertrophy due to hypertension or aortic stenosis. The latter can be present with angina that is indistinguishable from that caused by coronary atherosclerosis.
  • two or more causes of ischemia will coexist, such as an increase in oxygen demand due to left ventricular hypertrophy and a reduction in oxygen supply secondary to coronary atherosclerosis.
  • cardiovascular disorders are pharmaceutical based medications that are not targeted to an individual's actual defect; examples include angiotensin converting enzyme (ACE) inhibitors and diuretics for hypertension, insulin supplementation for non-insulin dependent diabetes mellitus
  • ACE angiotensin converting enzyme
  • NIDDM neurodegenerative disease 2019
  • targeted treatment strategies were available it might be possible to predict the response to a particular regime of therapy and could markedly increase the effective- ness of such treatment.
  • targeted therapy requires accurate diagnostic tests for disease susceptibility, once these tests are developed the opportunity to utilize targeted therapy will become widespread. Such diagnostic tests could initially serve to identify individuals at most risk of hypertension and could allow them to make changes in lifestyle or diet that would serve as preventative measures.
  • the benefits associated by coupling the diagnostic tests with a system of targeted therapy could include the reduction in dosage of administered drugs and thus the amount of unpleasant side effects suffered by an individual. In more severe cases a diagnostic test may suggest that earlier surgical intervention would be useful in preventing a further deterioration in condition.
  • a first aspect of the invention provides a method of diagnosis of disease in an individual, said method comprising determining one, various or all genotypes in said individual ofthe genes listed in the Examples.
  • the invention provides a method of identifying an individual predisposed or susceptible to a disease, said method comprising determining one, various or all genotypes in said individual ofthe genes listed in the Examples.
  • the invention is of advantage in that it enables diagnosis of a disease or of certain disease states via genetic analysis which can yield useable results before onset of disease symptoms, or before onset of severe symptoms.
  • the invention is further of advantage in that it enables diagnosis of predisposition or susceptibility to a disease or of certain disease states via genetic analysis.
  • the invention may also be of use in confirming or conoborating the results of other diagnostic methods.
  • the diagnosis of the invention may thus suitably be used either as an isolated technique or in combination with other methods and apparatus for diagnosis, in which latter case the invention provides a further test on which a diagnosis may be assessed.
  • the present invention stems from using allelic association as a method for genotyping individuals; allowing the investigation of the molecular genetic basis for cardiovascular diseases.
  • the invention tests for the polymorphisms in the sequences of the listed genes in the Examples.
  • the invention demonstrates a link between this polymorphisms and predispositions to cardiovascular diseases by showing that allele frequencies significantly differ when individuals with "bad” serum lipids are compared to individuals with "good” serum levels.
  • the meaning of "good and bad” serum lipid levels is defined in Table 1.
  • Certain disease states would benefit, that is to say the suffering of the patient may be reduced or prevented or delayed, by administration of treatment or therapy in advance of disease appearance; this can be more reliably canied out if advance diagnosis of predisposition or susceptibility to disease can be diagnosed.
  • the present invention is based at least in part on the discovery that a specific allele of a polymorphic region of a so called "candidate gene" is associated with CVD.
  • Candidate gene as used herein includes genes that can be assigned to either normal cardiovascular function or to metabolic pathways that are related to onset and/or progression of cardiovascular diseases. As the development of cardiovascular diseases is not completely understood, the term “candidate gene” may also comprise genes with presently unknown function.
  • Candidate genes falling into this group include but are not limited by genes of the cholesterol pathway, apolipoproteins and their modifiying factors.
  • Ischemic diseases of the heart and in particular myocardial infarction may be caused by a thrombotic occlusion.
  • Genes falling into this group include all genes of the coagulation cascade and their regulatory elements.
  • Atherosclerosis Complications of atherosclerosis are the most common causes of death in Western societies.
  • atherosclerosis can be considered to be a form of chronic inflammation resulting from interaction modified lipoproteins, monocyte-derived macrophages,T cells, and the normal cellular elements of the arterial wall. This inflammatory process can ultimately lead to the development of complex lesions, or plaques, that protrude into the arterial lumen. Finally plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction and stroke (Glass et al., Cell 2001, 104:503-516).
  • hypertension is an independent risk factor for CVD
  • genes that are involved in the regulation of systolic and diastolic blood pressure affect an individuals risk for CVD (Safer, Cun Opin Cardiol 2000, 15:258-263).
  • hypertension and diabetes appear to be interdependent, since hypertension is approximately twice as frequent in patients with diabetes compared with patients without the disease.
  • recent data suggest that hypertensive persons are more predisposed to the development of diabetes than are normotensive persons (Sowers et al., Hypertension 2001, 37:1053-1059).
  • the present invention is based at least in part on the discovery of polymo ⁇ hisms, that lie in genomic regions of unknown physiological function.
  • CA SNPs CVD associated SNPs
  • CA genes CVD associated genes
  • CA SNPs in the following genes:
  • VLDL very low density lipoprotein
  • HLP III type III hyperlipoproteinemia
  • Apolipoprotein e receptor 2 (low density lipoprotein receptor-related protein 8
  • This gene encodes an apolipoprotein E receptor, a member of the low density lipoprotein receptor (LDLR) family.
  • Apolipoprotein E is a small lipophilic plasma protein and a component of lipoproteins such as chylomicron remnants, very low density lipoprotein (VLDL), and high density lipoprotein (HDL).
  • VLDL very low density lipoprotein
  • HDL high density lipoprotein
  • the apolipoprotein E receptor is involved in cellular recognition and internalization of these lipoproteins.
  • Alternative splicing generates three transcript variants for this gene; additional variants have been described, but their full length nature has not been determined.
  • Diazepam binding inhibitor (acyl-CoA-binding protein); binds and induces medium- chain acyl-CoA ester synthesis
  • Non-muscle myosin heavy chain 9 motor protein that provides force for muscle contraction, cytokinesis and phagocytosis; contains an ATPase head domain and a rod-like tail domain
  • Apolipoprotein A-I is the major protein component of high density lipoprotein (HDL) in the plasma. Synthesized in the liver and small intestine, it consists of two identical chains of 77 amino acids; an 18-amino acid signal peptide is removed co- translationally and a 6-amino acid propeptide is cleaved post-translationally. Variation in the latter step, in addition to modifications leading to so-called isoforms, is responsible for some of the polymo ⁇ hism observed.
  • APOA1 is a cofactor for lecithin cholesterolacyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters.
  • LCAT lecithin cholesterolacyltransferase
  • the APOA1, APOC3 and APOA4 genes are closely linked in both rat and human genomes.
  • the A-I and A-IV genes are transcribed from the same strand, while the C-III gene is transcribed convergently in relation to A-I.
  • Defects in the apolipoprotein A-l gene are associated with HDL deficiency and Tangier disease.
  • Apolipoprotein C-III is a very low density lipoprotein (VLDL) protein.
  • APOC3 inhibits lipoprotein lipase and hepatic lipase; it is thought to delay catabolism of triglyceride-rich particles.
  • the APOA1, APOC3 and APOA4 genes are closely linked in both rat and human genomes.
  • the A-I and A-IV genes are transcribed from the same strand, while the A-l and C-III genes are convergently transcribed.
  • An increase in apoC-III levels induces the development of hypertriglyceridemia.
  • Apolipoprotein B is the main apolipoprotein of chylomicrons and low density lipoproteins (LDL).
  • the protein occurs in the plasma in 2 main isoforms, apoB-48 and apoB-100. The first is synthesized exclusively by the gut, the second by the liver.
  • the intestinal (B-48) and hepatic (B-100) forms of apoB are coded by a single gene and by a single mRNA transcript larger than 16 kb.
  • the 2 proteins share a common amino terminal sequence.
  • the precursor has 4,563 amino acids
  • the mature apoB-100 has 4,536 amino acid residues.
  • circulating B-48 is homologous over its entire length (estimated to be between 2,130 and 2,144 amino acid residues) with the amino-terminal portion of B-100 and contains no sequence from the carboxyl end of B-100. From structural studies, it is thought that apoB-48 represents the amino-terminal 47% of apoB-100 and that the carboxyl terminus of apoB-48 is in the vicinity of residue 2151 of apoB-100.
  • Apolipoprotein B-48 may be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C-to-U change in the codon CAA encoding
  • LIM domains are highly conserved cysteine-rich structures containing 2 zinc fingers. Although zinc fingers usually function by binding to DNA or RNA, the LIM motif probably mediates protein-protein interactions.
  • LIM kinase- 1 and LIM kinase-2 belong to a small subfamily with a unique combination of 2 N- terminal LIM motifs and a C-terminal protein kinase domain.
  • LIMKl is likely to be a component of an intracellular signaling pathway and may be involved in brain development. LIMKl hemizygosity is implicated in the impaired visuospatial constructive cognition of Williams syndrome. Two splice variant have been identified.
  • Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurofransmitters, and steroid hormones.
  • the phenol sulfotransferase STP2 maps to chromosome 16 (Dooley and Huang, 1996) and preferentially catalyzes the sulfonation of 'simple' planar phenols.
  • the low density lipoprotein receptor (LDLR) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands.
  • Low density lipoprotein (LDL) is normally bound at the cell membrane and taken into the cell ending up in lysosomes where the protein is degraded and the cholesterol is made available for repression of microsomal enzyme 3-hydroxy-3-methylglutaryl coen- zyme A (HMG CoA) reductase, the rate-limiting step in cholesterol synthesis.
  • HMG CoA 3-hydroxy-3-methylglutaryl coen- zyme A
  • a reciprocal stimulation of cholesterol ester synthesis takes place. Mutations in the LDL receptor (LDLR) gene cause the autosomal dominant disorder, familial hypercholesterolemia.
  • ATP-binding cassette sub-family B (MDR/TAP), member 1
  • the membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and infra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance.
  • the protein encoded by this gene is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs. This protein also functions as a transporter in the blood-brain barrier.
  • Annexin VI belongs to a family of calcium-dependent membrane and phospholipid binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways.
  • the annexin VI gene is approximately 60 kbp long and contains 26 exons. It encodes a protein of about 68 kDa that consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to human annexins I and II sequences, each of which contain four such repeats. Exon 21 of annexin VI is alternatively spliced, giving rise to two isoforms that differ by a 6-amino acid insertion at the start of the seventh repeat. Annexin VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis.
  • Protein C inhibitor (PCI-B) Protein C inhibitor
  • Protein C inhibitor may be a serine protease inhibitor; member ofthe se ⁇ in family of serine protease inhibitors.
  • Subunit of NADH-ubiquinone oxidoreductase (complex I); transports electrons from NADH to ubiquinone.
  • Lysosomal alpha-glucosidase (acid maltase) This gene encodes acid alpha-glucosidase, which is essential for the degradation of glycogen to glucose in lysosomes. Different forms of acid alpha-glucosidase are obtained by proteolytic processing. Defects in this gene are the cause of glycogen storage disease ⁇ , also known as Pompe's disease, which is an autosomal recessive disorder with a broad clinical spectrum.
  • Interleukin 6 induces the maturation of B cells into immunoglobulin-secreting cells.
  • fibrinogen a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains.
  • fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots.
  • various cleavage products of fibrinogen and fibrin regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types. Mutations in this gene lead to several disorders, including dysfibrinogenemia, hypofibrinogenemia and thrombo- philia. Alternative splicing results in two isoforms, varying in the carboxy-terminus.
  • Paraoxonase/arylesterase 2 possibly functions in protecting low density lipoprotein against oxidative modification; member of a family that hydrolyzes toxic organo- phosphates.
  • Defensins are a family of microbicidal and cytotoxic peptides thought to be involved in host defense. They are abundant in the granules of neutrophils and also found in the epithelia of mucosal surfaces such as those of the intestine, respiratory tract, urinary tract, and vagina. Members of the defensin family are highly similar in protein sequence and distinguished by a conserved cysteine motif. Several alpha defensin genes appear to be clustered on chromosome 8. The protein encoded by this gene, defensin, alpha 6, is highly expressed in the secretory granules of Paneth cells ofthe small intestine, and likely plays a role in host defense of human bowel.
  • MYH7 encodes the cardiac muscle beta (or slow) isoform of myosin. Changes in the relative abundance of MYH7 and MYH6 (the alpha, or fast, isoform of cardiac myosin heavy chain) conelate with the contractile velocity of cardiac muscle. Mutations in MYH7 are associated with familial hypertrophic cardiomyopathy.
  • Endothelial leukocyte adhesion molecule 1 (ELAM-1) The endothelial leukocyte adhesion molecule- 1 is expressed by cytokine-stimulated endothelial cells. It is thought to be responsible for the accumulation of blood leukocytes at sites of inflammation by mediating the adhesion of cells to the vascular lining. It exhibits structural features such as the presence of lectin- and EGF-like domains followed by short consensus repeat (SCR) domains that contain 6 conserved cysteine residues. These proteins are part of the selectin family of cell adhesion molecules. This gene is present in single copy in the human genome and contains 14 exons spanning about 13 kb of DNA. Adhesion molecules participate in the interaction between leukocytes and the endothelium and appear to be involved in the pathogenesis of atherosclerosis.
  • SCR short consensus repeat
  • LAMB2 Lamin B2
  • Lamin B2 is a member of a family of structural nuclear envelope proteins.
  • Estrogen receptor 2 (ER beta) Estrogen receptor beta 2 is a transcriptional activator involved in regulation of reproduction; exists in five isoforms.
  • This gene encodes tissue-type plasminogen activator, a secreted serine protease which converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme.
  • Tissue-type plasminogen activator is synthesized as a single chain which is cleaved by plasmin to a two chain disulfide linked protein. This enzyme plays a role in cell migration and tissue remodeling. Increased enzymatic activity causes hyper- fibrinolysis, which manifests as excessive bleeding; decreased activity leads to hypofibrinolysis which can result in thrombosis or embolism. Alternative splicing of this gene produces three transcripts.
  • Laminins a family of extracellular matrix glycoproteins, are the major non- collagenous constituent of basement membranes. They have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. Many of the effects of laminin are mediated through interactions with cell surface receptors. These receptors include members of the integrin family, as well as non-integrin laminin-binding proteins. This gene encodes a high-affinity, non-integrin family, laminin receptor 1. This receptor has been variously called 67 kD laminin receptor, 37 kD laminin receptor precursor (37LRP) and p40 ribosome-associated protein.
  • laminin receptor 1 The amino acid sequence of laminin receptor 1 is highly conserved through evolution, suggesting a key biological function. It has been observed that the level of the laminin receptor transcript is higher in colon carcinoma tissue and lung cancer cell line than their normal counte ⁇ arts. Also, there is a conelation between the upregulation of this polypeptide in cancer cells and their invasive and metastatic phenotype. Multiple copies of this gene exist, however, most of them are pseudogenes thought to have arisen from retropositional events.
  • Integrin, beta 3 platelet glycoprotein Ilia, antigen CD61
  • the ITGB3 protein product is the integrin beta chain beta 3.
  • Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. A given chain may combine with multiple partners resulting in different integrins. Integrin beta 3 is found along with the alpha lib chain in platelets. Integrins are known to participate in cell adhesion as well as cell-surface mediated signalling.
  • MCL1 Myeloid cell differentiation protein
  • Mcll is essential for preimplantation development and implantation, and suggests that it has a function beyond regulating apoptosis.
  • TRIP1 Tumor necrosis factor type 1 receptor associated protein
  • TRAP is a highly conserved member of a class of molecular chaperones with multiple functions. It is involved in the maturation of a subset of proteins involved in signal transduction.
  • Xanthine dehydrogenase belongs to the group of molybdenum-containing hydroxyl- ases involved in the oxidative metabolism of purines.
  • the enzyme is a homodimer.
  • Xanthine dehydrogenase can be converted to xanthine oxidase by reversible sulfhydryl oxidation or by ineversible proteolytic modification. Defects in xanthine dehydrogenase cause xanthinuria, may contribute to adult respiratory stress syndrome, and may potentiate influenza infection through an oxygen metabolite- dependent mechanism.
  • Stromal cell-derived factor 1 is a lymphocyte chemoattractant that signals through the receptor CXCR4.
  • the protein encoded by this gene is a smooth muscle myosin belonging to the myosin heavy chain family.
  • the gene product is a subunit of a hexameric protein that consists of 2 heavy chain subunits and 2 pairs of non-identical light chain subunits. It functions as a major contractile protein, converting chemical energy into mechanical energy through the hydrolysis of ATP.
  • the gene encoding a human ortholog of rat NUDEl is transcribed from the reverse strand of MYHl l gene, and its 3' end overlaps with that of the latter.
  • chromosome 16 [inv(16)(pl3q22)] produces a chimeric transcript consisting of the first 165 residues from the N terminus of core-binding factor beta in a fusion with the C-terminal portion of the smooth muscle myosin heavy chain.
  • This chromosomal rea ⁇ angement is associated with acute myeloid leukemia of the M4Eo subtype.
  • Alternative splicing generates isoforms that are differentially expressed, with ratios changing during muscle cell maturation. Additional splice variants have been described but their full- length nature has not been determined.
  • the scaffolding protein encoded by this gene is the main component of the caveolae plasma membranes found in most cell types.
  • the protein links integrin subunits to the tyrosine kinase FYN, an initiating step in coupling integrins to the Ras-ERK pathway and promoting cell cycle progression.
  • the gene is a tumor suppressor gene candidate and a negative regulator of the Ras-p42/44 MAP kinase cascade.
  • CAV1 and CAV2 are located next to each other on chromosome 7 and express colocalizing proteins that form a stable hetero-oligomeric complex. By using alternative initiation codons in the same reading frame, two isoforms (alpha and beta) are encoded by a single transcript from this gene.
  • AP-2 transcription factors have been detected previously in embryonic renal tissues. It was shown that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl- X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia.
  • TGFB2 Transforming growth factor beta 2
  • Transforming growth factor-beta 2 (glioblastoma-derived T cell suppressor factor); suppresses IL2 - dependent growth of T cells and is a member of a family of cytokines that transmits signals through transmembrane serine/threonine kinases.
  • Tumor necrosis factor alpha-induced protein 2 (TNFAIP2, B94)
  • vascular endothelium Secreted by vascular endothelium, expression is induced by tumor necrosis factor alpha, interleukin-1 beta, and lipopolysaccharide.
  • Protein phosphatase IA (formerly 2C), magnesium-dependent, alpha isoform Magnesium- or manganese-dependent alpha protein phosphatase IA; regulates cell stress responses.
  • Osteonidogen (nidogen 2) ATP-binding cassette, sub-family A (ABC1), member 1
  • ABC ATP-binding cassette
  • This protein is a member of the ABC1 subfamily.
  • Members of the ABC1 subfamily comprise the only major ABC subfamily found exclusively in multicellular eukaryotes. With cholesterol as its substrate, this protein functions as a cholesteral efflux pump in the cellular lipid removal pathway. Mutations in this gene have been associated with Tangier's disease and familial high-density lipoprotein deficiency.
  • This gene was identified based on its homology to the gene encoding the RING3 protein, a serine/threonine kinase. The gene localizes to 9q34, a region which contains several major histocompatibility complex (MHC) genes. The function ofthe encoded protein is not known.
  • MHC major histocompatibility complex
  • PPA gamma Peroxisome proliferator activated receptor-gamma
  • Peroxisome proliferator activated receptor-gamma regulates adipocyte and macrophage gene expression and differentiation.
  • Tumor necrosis factor beta (Lymphotoxin alpha, LTA)
  • Lymphotoxin alpha a member of the tumor necrosis factor family, is a cytokine produced by lymphocytes.
  • LTA is highly inducible, secreted, and exists as homo- trimeric molecule. LTA forms heterotrimers with lymphotoxin-beta which anchors lymphotoxin-alpha to the cell surface. LTA mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. LTA is also involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis.
  • Site-specific DNA binding protein involved in transcriptional regulation and signal transduction.
  • S-adenosyl methionine transferase catalyzes the formation of S-adenosylmethionine from methionine and ATP. Both the beta and alpha isoforms may be encoded by one gene. Methionine adenosyltransferase deficiency is known to be caused by recessive as well as dominant mutations, the latter identified in autosomal dominant persistant hypermethioninemia.
  • Coagulation factor III thromboplastin, tissue factor
  • This gene encodes coagulation factor III which is a cell surface glycoprotein.
  • This factor enables cells to initiate the blood coagulation cascades, and it functions as the high-affinity receptor for the coagulation factor VII.
  • the resulting complex provides a catalytic event that is responsible for initiation ofthe coagulation protease cascades by specific limited proteolysis.
  • this factor is a potent initiator that is fully functional when expressed on cell surfaces.
  • extracellular, transmembrane, and cytoplasmic This protein is the only one in the coagulation pathway for which a congenital deficiency has not been described.
  • Palmitoylated membrane protein 2 is a member of a family of membrane-associated proteins termed MAGUKs (membrane-associated guanylate kinase homologs).
  • Palmitoylated membrane protein 2 contains a conserved sequence, called the SH3 (src homology 3) motif, found in several other proteins that associate with the cytoskeleton and are suspected to play important roles in signal transduction.
  • SH3 src homology 3
  • CSF2RB Granulocyte-macrophage colony stimulating factor 2 receptor, beta chain
  • CSF2RB is a common beta chain of the high affinity receptor for IL-3, IL-5 and CSF. Defective CSF2RB has been reported to be associated with protein alveolar proteinosis
  • Coagulation factor II Prothrombin, F2
  • Coagulation factor II is proteolytically cleaved to form thrombin in the first step of the coagulation cascade which ultimately results in the stemming of blood loss.
  • F2 also plays a role in maintaining vascular integrity during development and postnatal life. Mutations in F2 leads to various forms of thrombosis and dysprothrombinemia.
  • LPL Lipoprotein lipase
  • LPL encodes lipoprotein lipase, which is expressed in heart, muscle, and adipose tissue. LPL functions as a homodimer, and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Severe mutations that cause LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.
  • This gene encodes the beta C chain of inhibin, a member of the TGF-beta super- family. This subunit forms heterodimers with beta A and beta B subunits. Inhibins and activins, also members of the TGF-beta superfamily, are hormones with opposing actions and are involved in hypothalamic, pituitary, and gonadal hormone secretion, as well as growth and differentiation of various cell types.
  • This gene encodes a protein that possesses three distinct enzymatic activities, 5,10- methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclo- hydrolase and 10-formyltetrahydrofolate synthetase. Each of these activities catalyzes one of three sequential reactions in the interconversion of 1 -carbon derivatives of tetrahydrofolate, which are substrates for methionine, thymidylate, and de novo purine syntheses.
  • the trifunctional enzymatic activities are confened by two major domains, an aminoterminal portion containing the dehydrogenase and cyclohydrolase activities and a larger synthetase domain.
  • PACAP gene for pituitary adenylate cyclase activating polypeptide This gene encodes adenylate cyclase activating polypeptide 1. Mediated by adenylate cyclase activating polypeptide 1 receptors, this polypeptide stimulates adenylate cyclase and subsequently increases the cAMP level in target cells. Adenylate cyclase activating polypeptide 1 is not only a hypophysiotropic hormone, but also functions as a neurotransmitter and neuromodulator. In addition, it plays a role in paracrine and autocrine regulation of certain types of cells. This gene is composed of five exons.
  • Exons 1 and 2 encode the 5' UTR and signal peptide, respectively; exon 4 encodes an adenylate cyclase activating polypeptide 1 -related peptide; and exon 5 encodes the mature peptide and 3' UTR.
  • This gene encodes three different mature peptides, including two isotypes: a shorter form and a longer form.
  • Cytochrome P450 subfamily IIC (mephenytoin 4-hydroxylase), polypeptide 8
  • This gene encodes a member of the cytochrome P450 superfamily of enzymes.
  • the cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and its expression is induced by phenobarbital.
  • the enzyme is known to metabolize many xenobiotics, including the anticonvulsive drug mephenytoin, benzo(a)pyrene, 7-ethyoxycoumarin, and the anti- cancer drug taxol. Two transcript variants for this gene have been described; it is thought that the longer form does not encode an active cytochrome P450 since its protein product lacks the heme binding site. This gene is located within a cluster of cytochrome P450 genes on chromosome 10q24. Leucocyte adhesion receptor, L-selectin (SELL)
  • SELL is a cell surface component that is a member of a family of adhesion/homing receptors which play important roles in leukocyte-endothelial cell interactions.
  • the molecule is composed of multiple domains: one homologous to lectins, one to epidermal growth factor, and two to the consensus repeat units found in C3/C4 binding proteins.
  • Calmodulin binds Ca2+, regulates proteins and enzymes in a Ca2+-dependent manner
  • WISP1 is a member ofthe connective tissue growth factor family
  • Ribophorin I Ribophorin I; subunit of oligosaccharyltransferase that binds ribosomes.
  • Nonsyndromic hearing impairment protein DFNA5
  • Hearing impairment is a heterogeneous condition with over 40 loci described.
  • the protein encoded by this gene is expressed in fetal cochlea, however, its function is not known.
  • Nonsyndromic hearing impairment is associated with a mutation in this gene.
  • Cytochrome P450 2E1 (CYP2E1)
  • This gene encodes a member of the cytochrome P450 superfamily of enzymes.
  • the cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and is induced by ethanol, the diabetic state, and starvation.
  • the enzyme metabolizes both endogenous substrates, such as ethanol, acetone, and acetal, as well as exogenous substrates including benzene, carbon tetrachloride, ethylene glycol, and nitrosamines which are premutagens found in cigarette smoke. Due to its many substrates, this enzyme may be involved in such varied processes as gluconeogenesis, hepatic cinhosis, diabetes, and cancer.
  • Retinoid X receptors and retinoic acid receptors (RARs) are nuclear receptors that mediate the biological effects of retinoids by their involvement in retinoic acid-mediated gene activation. These receptors exert their action by binding, as homodimers or heterodimers, to specific sequences in the promoters of target genes and regulating their transcription.
  • the protein encoded by this gene is a member of the steroid and thyroid hormone receptor superfamily of transcriptional regulators.
  • Peroxisome proliferator-activated receptor delta is a member of the steroid hormone receptor superfamily
  • ADCA autosomal dominant cerebellar ataxias
  • ADCA is caused by the expansion of the CAG repeats, producing an elongated polyglutamine tract in the conesponding protein.
  • the expanded repeats are variable in size and unstable, usually increasing in size when transmitted to successive generations.
  • the function of the ataxins is not known.
  • the SCA1 locus has been mapped to chromosome 6, and it has been determined that the diseased allele contains 41-81 CAG repeats, compared to 6-39 in the normal allele.
  • Adducin 1 (alpha) Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). Alpha- and gamma-adducins are ubiquitously expressed. In contrast, beta- adducin is expressed at high levels in brain and hematopoietic tissues. Adducin is a heterodimeric protein that consists of related subunits, alpha and beta, which are produced from distinct genes but share a similar structure. Alpha- and beta-adducin include a protease-resistant N-terminal region and a protease-sensitive, hydrophilic
  • Adducin binds with high affinity to Ca(2+)/calmodulin and is a substrate for protein kinases A and C.
  • Zinc finger protein 202 Zinc-finger protein 202; may repress genes involved in lipid metabolism; contains zinc fingers
  • mevalonate pyrophosphate decarboxylase catalyzes the conversion of mevalonate pyrophosphate into isopentenyl pyrophosphate in one ofthe early steps in cholesterol biosynthesis. It decarboxylates and dehydrates its substrate while hydrolyzing ATP.
  • Integrin, beta 2 (ITGB2, lymphocyte function-associated antigen 1, LFA1)
  • the ITGB2 protein product is the integrin beta chain beta 2.
  • Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. A given chain may combine with multiple partners resulting in different integrins. For example, beta 2 combines with the alpha L chain to form the integrin LFA-1, and combines with the alpha M chain to form the integrin Mac-1. Integrins are known to participate in cell adhesion as well as cell-surface mediated signalling.
  • ADRB3 Adrenergic beta-3- receptor
  • the ADRB3 gene product, beta-3 -adrenergic receptor is located mainly in adipose tissue and is involved in the regulation of lipolysis and thermogenesis. Beta adrenergic receptors are involved in the epenephrine and norepinephrine-induced activation of adenylate cyclase through the action of G proteins.
  • CYP4F8 encodes a member of the cytochrome P450 superfamily of enzymes.
  • the cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and functions as a 19-hydroxylase of prostaglandins in seminal vesicles.
  • This gene is part of a cluster of cytochrome P450 genes on chromosome 19. Another member of this family, CYP4F3, is approximately 18 kb away.
  • Coagulation factor XIII is the last zymogen to become activated in the blood coagulation cascade.
  • Plasma factor XIII is a heterotetramer composed of 2 A subunits and 2 B subunits. The A subunits have catalytic function, and the B subunits do not have enzymatic activity and may serve as a plasma canier molecules. Platelet factor XIII is comprised only of 2 A subunits, which are identical to those of plasma origin.
  • the plasma factor XIII Upon activation by the cleavage ofthe activation peptide by thrombin and in the presence of calcium ion, the plasma factor XIII dissociates its B subunits and yields the same active enzyme, factor XHIa, as platelet factor XIII.
  • This enzyme acts as a transglutaminase to catalyze the formation of gamma-glutamyl-epsilon-lysine crosslinking between fibrin molecules, thus stabilizing the fibrin clot. It also crosslinks alpha-2-plasmin inhibitor, or fibronectin, to the alpha chains of fibrin.
  • Factor XIII deficiency is classified into two categories: type I deficiency, characterized by the lack of both the A and B subunits; and type II deficiency, characterized by the lack of the A subunit alone. These defects can result in a lifelong bleeding tendency, defective wound healing, and habitual abortion.
  • Beta adaptin The beta adaptin subunit is part of the clathrin coat assembly complex which links clathrin to receptors in coated pits and vesicles. These vesicles are involved in endocytosis and Golgi processing.
  • the beta 1 subunit is one ofthe assembly proteins which binds to clathrin and initiates coat formation.
  • Transforming growth factor-beta 1 regulates cell proliferation, differentiation, and apoptosis
  • Transforming growth factor-beta 3 Transforming growth factor-beta 3; transmits signals through transmembrane serine/- threonine kinases, may be required for normal development of the lung and palate; member of family of cytokines, very strongly similar to murine Tgfb3.
  • Flavin containing monooxygenase 1 Metabolic N-oxidation ofthe diet-derived amino-trimethylamine (TMA) is mediated by flavin-containing monooxygenase and is subject to an inherited FMO3 poly- mo ⁇ hism in man resulting in a small subpopulation with reduced TMA N-oxidation capacity resulting in fish odor syndrome Trimethylaminuria.
  • TMA diet-derived amino-trimethylamine
  • Three forms of the enzyme, FMO1 found in fetal liver, FMO2 found in adult liver, and FMO3 are encoded by genes clustered in the Iq23-q25 region.
  • Flavin-containing monooxy- genases are NADPH-dependent flavoenzymes that catalyzes the oxidation of soft nucleophilic heteroatom centers in drugs, pesticides, and xenobiotics.
  • This gene encodes vitamin K-dependent coagulation factor IX that circulates in the blood as an inactive zymogen. This factor is converted to an active form by factor
  • factor IXla which excises the activation peptide and thus generates a heavy chain and a light chain held together by one or more disulfide bonds.
  • the role of this activated factor IX in the blood coagulation cascade is to activate factor X to its active form through interactions with Ca+2 ions, membrane phospholipids, and factor VIII. Alterations of this gene, including point mutations, insertions and deletions, cause factor IX deficiency, which is a recessive X-linked disorder, also called hemophilia B or Christmas disease.
  • Apical protein, Xenopus laevis-like (APXL) May be an amiloride-sensitive sodium channel; similar to Xenopus laevis Apical
  • SNPs are linked to other SNPs in neighboring genes on a chromosome (Linkage Disequilibrium) those SNPs could also be used as marker SNPs.
  • Linkage Disequilibrium those SNPs could also be used as marker SNPs.
  • SNPs are linked over 100 kb in some cases more than
  • alleles refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.
  • allelic variant of a polymo ⁇ hic region of a gene refers to a region of a gene having one of several nucleotide sequences found in that region of the gene in other individuals.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for pu ⁇ oses of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function ofthe number of matching or homologous positions shared by the sequences. An "unrelated” or “non-homologous” sequence shares less than 40%) identity, though preferably less than 25% identity, with one of the sequences ofthe present invention.
  • a homologue of a nucleic acid refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof.
  • a homologue of a double stranded nucleic acid having SEQ ID NO. X is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with SEQ ID NO. X or with the complement thereof.
  • Prefened homologous of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.
  • interact as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a hybridization assay.
  • interact is also meant to include “binding" interactions between molecules.
  • Interactions may be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature.
  • intra sequence or "intronic nucleotide sequence” refers to the nucleo- tide sequence of an intron or portion thereof.
  • isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule.
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occuning as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • lipid shall refer to a fat or fat-like substance that is insoluble in polar solvents such as water.
  • lipid is intended to include true fats (e.g. esters of fatty acids and glycerol); lipids (phospholipids, cerebrosides, waxes); sterols (cholesterol, ergosterol) and lipoproteins (e.g. HDL, LDL and VLDL).
  • locus refers to a specific position in a chromosome.
  • a locus of a gene refers to the chromosomal position ofthe gene.
  • modulation refers to both up-regulation, (i.e., activation or stimulation), for example by agonizing, and down-regulation (i.e. inhibition or suppression), for example by antagonizing of a bioactivity (e.g. expression of a gene).
  • molecular structure of a gene or a portion thereof refers to the structure as defined by the nucleotide content (including deletions, substitutions, additions of one or more nucleotides), the nucleotide sequence, the state of methylation, and/or any other modification ofthe gene or portion thereof.
  • mutated gene refers to an allelic form of a gene, which is capable of altering the phenotype of a subject having the mutated gene relative to a subject which does not have the mutated gene. If a subject must be homozygous for this mutation to have an altered phenotype, the mutation is said to be recessive. If one copy of the mutated gene is sufficient to alter the genotype of the subject, the mutation is said to be dominant. If a subject has one copy of the mutated gene and has a phenotype that is intermediate between that of a homozygous and that of a heterozygous (for that gene) subject, the mutation is said to be co-dominant.
  • nucleic acid refers to polynucleotides such as deoxy- ribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxy- ribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, including peptide nucleic acids (PNA), mo ⁇ holino oligonucleotides (J. Summerton and D. Weller, Antisense and Nucleic Acid Drug Development 7:187 (1997)) and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • PNA peptide nucleic acids
  • PNA peptide nucleic acids
  • mo ⁇ holino oligonucleotides J. Summerton and D. Weller, Antisense and Nucleic Acid Drug Development 7:187
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine.
  • a nucleotide of a nucleic acid which can be DNA or an RNA
  • the term "adenosine”, “cytidine”, “guanosine”, and “fhymidine” are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.
  • nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO. x refers to the nucleotide sequence of the complementary strand of a nucleic acid strand having SEQ ID NO. x.
  • complementary strand is used herein interchangeably with the term “complement”.
  • the complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non- coding strand.
  • the complement of a nucleic acid having SEQ ID NO. x refers to the complementary strand of the strand having SEQ ID NO.
  • nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID NO. x or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID NO. x.
  • the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID NO. x.
  • the nucleotide sequences and complementary sequences thereof are always given in the 5' to 3' direction.
  • the term “complement” and "reverse comple- ment” are used interchangeably herein.
  • operably linked is intended to mean that the promoter is associated with the nucleic acid in such a manner as to facilitate transcription ofthe nucleic acid.
  • polymo ⁇ hism refers to the coexistence of more than one form of a gene or portion thereof.
  • a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is refened to as a "polymo ⁇ hic region of a gene".
  • a polymo ⁇ hic region can be a single nucleotide, the identity of which differs in different alleles.
  • a polymo ⁇ hic region can also be several nucleotides long.
  • a “polymo ⁇ hic gene” refers to a gene having at least one polymo ⁇ hic region.
  • an "ambiguity code” that stands for the possible variations of nucleotides in one site. The list of ambiguity codes is summarized in the following table:
  • a "R” in a nucleotide sequence means that either an "a” or a “g” could be at that position.
  • protein protein
  • polypeptide peptide
  • a “regulatory element”, also termed herein "regulatory sequence is intended to include elements which are capable of modulating transcription from a basic promoter and include elements such as enhancers and silencers.
  • the term “enhancer”, also refened to herein as “enhancer element”, is intended to include regulatory elements capable of increasing, stimulating, or enhancing transcription from a basic promoter.
  • the term “silencer”, also refened to herein as “silencer element” is intended to include regulatory elements capable of decreasing, inhibiting, or repressing transcription from a basic promoter. Regulatory elements are typically present in 5' flanking regions of genes. However, regulatory elements have also been shown to be present in other regions of a gene, in particular in introns.
  • genes have regulatory elements located in introns, exons, coding regions, and 3' flanking sequences.
  • Such regulatory elements are also intended to be encompassed by the present invention and can be identified by any of the assays that can be used to identify regulatory elements in 5' flanking regions of genes.
  • regulatory element further encompasses "tissue specific” regulatory elements, i.e., regulatory elements which effect expression of the selected DNA sequence preferentially in specific cells (e.g., cells of a specific tissue), gene expression occurs preferentially in a specific cell if expression in this cell type is significantly higher than expression in other cell types.
  • regulatory element also encompasses non-tissue specific regulatory elements, i.e., regulatory elements which are active in most cell types.
  • a regulatory element can be a constitutive regulatory element, i.e., a regulatory element which constitutively regulates transcription, as opposed to a regulatory element which is inducible, i.e., a regulatory element which is active primarily in response to a stimulus.
  • a stimulus can be, e.g., a molecule, such as a hormone, cytokine, heavy metal, phorbol ester, cyclic AMP (cAMP), or retinoic acid.
  • transcription factor is intended to include proteins or modified forms thereof, which interact preferentially with specific nucleic acid sequences, i.e., regulatory elements, and which in appropriate conditions stimulate or repress transcription.
  • transcription factors are active when they are in the form of a monomer.
  • transcription factors are active in the form of a dimer consisting of two identical proteins or different proteins (heterodimer).
  • Modified forms of transcription factors are intended to refer to transcription factors having a post- translational modification, such as the attachment of a phosphate group.
  • the activity of a transcription factor is frequently modulated by a post-translational modification.
  • certain transcription factors are active only if they are phosphorylated on specific residues.
  • transcription factors can be active in the absence of phosphorylated residues and become inactivated by phosphorylation.
  • a list of known transcription factors and their DNA binding site can be found, e.g., in public databases, e.g., TFMATRIX Transcription Factor Binding Site Profile database.
  • the term “specifically hybridizes” or “specifically detects” refers to the ability of a nucleic acid molecule of the invention to hybridize to at least approximately 6, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or 140 consecutive nucleotides of either strand of a gene.
  • wild-type allele refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
  • Candidate gene as used herein includes genes that can be assigned to either normal cardiovascular function or to metabolic pathways that are related to onset and/or progression of cardiovascular diseases. As the development of cardiovascular diseases is not completely understood, the term “candidate gene” may also comprise genes with presently unknown function.
  • CA SNP cardiac disease associated SNP
  • CA gene cardiac disease associated gene
  • CA gene polypeptide refers to a polypeptide encoded at least in part by a CA gene
  • Cardiovascular status refers to the physiological status of an individual's cardiovascular system as reflected in one or more markers or indicators.
  • Status markers include without limitation clinical measurements such as, e.g., blood pressure, electrocardiographic profile, and differentiated blood flow analysis as well as measurements of LDL- and HDL- Cholesterol levels, other lipids and other well established clinical parameters that are standard in the art.
  • Status markers according to the invention include diagnoses of one or more cardiovascular syndromes, such as, e.g., hypertension, acute myocardial infarction, silent myocardial infarction, stroke, and atherosclerosis.
  • a diagnosis of a cardiovascular syndrome made by a medical practitioner encompasses clinical measurements and medical judgement.
  • Status markers according to the invention are assessed using conventional methods well known in the art. Also included in the evaluation of cardiovascular status are quantitative or qualitative changes in status markers with time, such as would be used, e.g., in the determination of an individual's response to a particular therapeutic regimen.
  • the methods are ca ⁇ ied out by the steps of:
  • polymo ⁇ hic pattern of the individual is, preferably, highly similar and, most preferably, identical to the polymo ⁇ hic pattern of individuals who exhibit particular status markers, cardiovascular syndromes, and/or particular patterns of response to therapeutic interventions.
  • Polymo ⁇ hic patterns may also include polymo ⁇ hic positions in other genes which are shown, in combination with one or more polymo ⁇ hic positions in the genes listed in the Examples, to conelate with the presence of particular status markers.
  • the method involves comparing an individual's polymo ⁇ hic pattern with polymo ⁇ hic patterns of individuals who have been shown to respond positively or negatively to a particular therapeutic regimen.
  • Therapeutic regimen refers to treatments aimed at the elimination or amelioration of symptoms and events associated cardiovascular disease.
  • Such treatments include without limitation one or more of alteration in diet, lifestyle, and exercise regimen; invasive and noninvasive surgical techniques such as atherectomy, angioplasty, and coronary bypass surgery; and pharmaceutical interventions, such as administration of ACE inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antago- nists, cardiac glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor antagonists, calcium channel blockers, HMG-CoA reductase inhibitors, imidazoline receptor blockers, endothelin receptor blockers, organic nitrites, and modulators of protein function of genes listed in the Examples.
  • Interventions with pharmaceutical agents not yet known whose activity conelates with particular polymo ⁇ hic patterns associated with cardiovascular disease are also encompassed. It is contemplated, for example, that patients who are candidates for a particular therapeutic regimen will be screened for polymo ⁇ hic patterns that conelate with responsivity to that particular regimen.
  • the method involves comparing an individual's polymo ⁇ hic pattern with polymo ⁇ hic patterns of individuals who exhibit or have exhibited one or more markers of cardiovascular disease, such as, e.g., elevated LDL- Cholesterol levels, high blood pressure, abnormal electrocardiographic profile, myocardial infarction, stroke, or atherosclerosis.
  • an individual's polymo ⁇ hic pattern can be established by obtaining DNA from the individual and determining the sequence at predetermined polymo ⁇ hic positions in the genes such as those described in this file.
  • the DNA may be obtained from any cell source.
  • Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Cells may also be obtained from body fluids, including without limitation blood, saliva, sweat, urine, cerebrospinal fluid, feces, and tissue exudates at the site of infection or inflammation.
  • DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source.
  • the present invention provides methods for determining the molecular structure of at least one polymo ⁇ hic region of a gene, specific allelic variants of said polymo ⁇ hic region being associated with cardiovascular disease.
  • determining the molecular structure of a polymo ⁇ hic region of a gene comprises determining the identity of the allelic variant.
  • a polymo ⁇ hic region of a gene, of which specific alleles are associated with cardiovascular disease can be located in an exon, an intron, at an intron/exon border, or in the promoter ofthe gene.
  • the invention provides methods for determining whether a subject has, or is at risk, of developing a cardiovascular disease.
  • Such disorders can be associated with an abenant gene activity, e.g., abnormal binding to a form of a lipid, or an abenant gene protein level.
  • An abenant gene protein level can result from an abenant transcription or post-transcriptional regulation.
  • allelic differences in specific regions of a gene can result in differences of gene protein due to differences in regulation of expression.
  • some of the identified polymo ⁇ hisms in the human gene may be associated with differences in the level of transcription, RNA maturation, splicing, or translation ofthe gene or transcription product.
  • the methods of the invention can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a specific allelic variant of one or more polymo ⁇ hic regions of a gene.
  • allelic differences can be: (i) a difference in the identity of at least one nucleotide or (ii) a difference in the number of nucleotides, which difference can be a single nucleotide or several nucleotides.
  • a prefened detection method is allele specific hybridization using probes overlapping the polymo ⁇ hic site and having about 5, 10, 20, 25, or 30 nucleotides around the polymo ⁇ hic region.
  • probes for detecting specific allelic variants of the polymo ⁇ hic region located in intron X are probes comprising a nucleotide sequence set forth in any of SEQ ID NO. X.
  • several probes capable of hybridizing specifically to allelic variants are attached to a solid phase support, e.g., a "chip".
  • Oligonucleotides can be bound to a solid support by a variety of processes, including lithography.
  • a chip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix). Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe anays" is described e.g., in Cronin et al. (1996) Human Mutation 7:244 and in Kozal et al. (1996) Nature Medicine 2:753.
  • a chip comprises all the allelic variants of at least one polymo ⁇ hic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.
  • allelic variant of the nucleotide polymo ⁇ hism of nucleotide T or G at position 140 of Seq ID 1 (baySNP10948) and that of other possible polymo ⁇ hic regions can be determined in a single hybridization experiment.
  • Amplification can be performed, e.g., by PCR and/or LCR, according to methods known in the art.
  • genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA.
  • the primers are located between 40 and 350 base pairs apart. Prefened primers for amplifying gene fragments of genes of this file are listed in Table 2 in the Examples.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of a gene and detect allelic variants, e.g., mutations, by comparing the sequence of the sample sequence with the conesponding wild-type (control) sequence.
  • Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463).
  • a specific allele of a gene in DNA from a subject can be shown by restriction enzyme analysis.
  • a specific nucleotide polymo ⁇ hism can result in a nucleotide sequence comprising a restriction site which is absent from the nucleotide sequence of another allelic variant.
  • alterations in eiectrophoretic mobility is used to identify the type of gene allelic variant.
  • single strand conformation polymo ⁇ hism SSCP
  • SSCP single strand conformation polymo ⁇ hism
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in eiectrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in eiectrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the identity of an allelic variant of a polymo ⁇ hic region is obtained by analyzing the movement of a nucleic acid comprising the polymo ⁇ hic region in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).
  • oligonucleotide probes may be prepared in which the known polymo ⁇ hic nucleotide is placed centrally (allele-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543).
  • oligonucleotide hybridization techniques may be used for the simultaneous detection of several nucleotide changes in different polymo ⁇ hic regions of gene. For example, oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid.
  • Oligonucleotides used as primers for specific amplification may carry the allelic variant of interest in the center ofthe molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension
  • identification of the allelic variant is canied out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science 241:1077-1080 (1988).
  • OLA oligonucleotide ligation assay
  • the OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target.
  • One of the oligonucleotides is linked to a separation marker, e.g,. biotinylated, and the other is detectably labeled.
  • oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand.
  • Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
  • U.S. Pat. No. 5,593,826 discloses an OLA using an oligonucleotide having 3'-amino group and a 5 '-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage.
  • OLA OLA combined with PCR permits typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e.
  • each LA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase.
  • This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.
  • the invention further provides methods for detecting single nucleotide polymo ⁇ hisms in a gene. Because single nucleotide polymo ⁇ hisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation and it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymo ⁇ hisms.
  • the single base polymo ⁇ hism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127).
  • a primer complementary to the allelic sequence immediately 3' to the polymo ⁇ hic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymo ⁇ hic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be inco ⁇ orated onto the end of the hybridized primer.
  • a solution-based method is used for deter- mining the identity of the nucleotide of a polymo ⁇ hic site.
  • Patent 2,650,840; PCT Appln. No. WO91/02087) As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3' to a polymo ⁇ hic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymo ⁇ hic site will become inco ⁇ orated onto the terminus ofthe primer.
  • GBA TM Genetic Bit Analysis
  • Goelet, P. et al. PCT Appln. No. 92/157112.
  • the method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymo ⁇ hic site.
  • the labeled terminator that is inco ⁇ orated is thus determined by, and complementary to, the nucleotide present in the polymo ⁇ hic site ofthe target molecule being evaluated.
  • the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
  • allelic variants of a polymo ⁇ hic region located in the coding region of a gene yet other methods than those described above can be used. For example, identification of an allelic variant which encodes a mutated gene protein can be performed by using an antibody specifically recognizing the mutant protein in, e.g, immunohistochemistry or immunoprecipitation. Antibodies to wild- type gene protein are described, e.g, in Acton et al. (1999) Science 271 :518 (anti- mouse gene antibody cross-reactive with human gene). Other antibodies to wild-type gene or mutated forms of gene proteins can be prepared according to methods known in the art.
  • Binding assays are known in the art and involve, e.g, obtaining cells from a subject, and performing binding experiments with a labeled lipid, to determine whether binding to the mutated form of the receptor differs from binding to the wild-type ofthe receptor.
  • the identity of the allelic variant can be determined by determining the molecular structure ofthe mRNA, pre-mRNA, or cDNA.
  • the molecular structure can be determined using any of the above described methods for determining the molecular structure ofthe genomic DNA, e.g, sequencing and SSCP.
  • sample nucleic acid for using in the above-described diagnostic and prognostic methods can be obtained from any cell type or tissue of a subject.
  • a subject's bodily fluid e.g. blood
  • venipuncture e.g. venipuncture
  • human tissues like heart e.g
  • Fetal nucleic acid samples for prenatal diagnostics can be obtained from maternal blood as described in International Patent Application No. WO91/07660 to Bianchi. Alternatively, amniocytes or chorionic villi may be obtained for performing prenatal testing.
  • Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
  • Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J, 1992, PCR in situ hybridization: protocols and applications, Raven Press, New York).
  • profiles may also be assessed in such detection schemes.
  • Finge ⁇ rint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
  • the distribution of polymo ⁇ hic patterns in a large number of individuals exhibiting particular markers of cardiovascular status is determined by any of the methods described above, and compared with the distribution of polymo ⁇ hic patterns in patients that have been matched for age, ethnic origin, and/or any other statistically or medically relevant parameters, who exhibit quantitatively or qualitatively different status markers.
  • Conelations are achieved using any method known in the art, including nominal logistic regression, chi square tests or standard least squares regression analysis. In this manner, it is possible to establish statistically significant conelations between particular polymo ⁇ hic patterns and particular cardiovascular statuses (given in p values).
  • the present invention provides isolated nucleic acids comprising the polymo ⁇ hic positions described herein for human genes; vectors comprising the nucleic acids; and transformed host cells comprising the vectors.
  • the invention also provides probes which are useful for detecting these polymo ⁇ hisms.
  • nucleic acids typically DNAs
  • the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini ofthe DNAs and/or vector by digesting back single-stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerase.
  • any site desired may be produced, e.g, by ligating nucleotide sequences (linkers) onto the termini. Such linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated by the use of the polymerase chain reaction (PCR). See, e.g, Saiki et al,
  • the cleaved vector and the DNA fragments may also be modified if required by homopolymeric tailing.
  • the nucleic acids may be isolated directly from cells or may be chemically synthesized using known methods. Alternatively, the polymerase chain reaction
  • PCR PCR method
  • Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate inco ⁇ oration into a given vector for recombinant expression.
  • the nucleic acids of the present invention may be flanked by native gene sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like.
  • the 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 occuning nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, mo ⁇ holines etc.) and with charged linkages (e.g, phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, mo ⁇ holines etc.
  • 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. Further- more, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • the invention also provides nucleic acid vectors comprising the gene sequences or derivatives or fragments thereof of genes described in the Examles.
  • 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.
  • suitable vectors include without limitation pUC plasmids, pET plasmids
  • vector/host is not critical to the practice ofthe invention.
  • Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl 2 mediated DNA uptake, fungal or viral infection, microinjection, microprojectile, or other established methods.
  • Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells.
  • a large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced peptides and polypeptides encoded by genes of the Examples.
  • Nucleic acids encoding peptides or polypeptides from gene sequences of the Examples may also be introduced into cells by recombination events.
  • such a sequence can be introduced into a cell and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene.
  • Other recombination-based methods such as non- homologous recombinations or deletion of endogenous genes by homologous recombination may also be used.
  • both or all subunits have to be expressed in one system or cell.
  • nucleic acids of the present invention find use as probes for the detection of genetic polymo ⁇ hisms and as templates for the recombinant production of normal or variant peptides or polypeptides encoded by genes listed in the Examples.
  • Probes in accordance with the present invention comprise without limitation isolated nucleic acids of about 10-100 bp, preferably 15-75 bp and most preferably 17-25 bp in length, which hybridize at high stringency to one or more of the polymo ⁇ hic sequences disclosed herein or to a sequence immediately adjacent to a polymo ⁇ hic position.
  • a full-length gene sequence may be used as a probe.
  • the probes span the polymo ⁇ hic positions in genes disclosed herein.
  • the probes conespond to sequences immediately adjacent to the polymo ⁇ hic positions.
  • the present invention encompasses isolated peptides and polypeptides encoded by genes listed in the Examples comprising polymo ⁇ hic positions disclosed herein.
  • the peptides and polypeptides are useful screening targets to identify cardiovascular drugs.
  • the peptides and polypeptides are capable of eliciting antibodies in a suitable host animal that react specifically with a polypeptide comprising the polymo ⁇ hic position and distinguish it from other polypeptides having a different 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 poly- peptides are described below. Many conventional techniques in protein biochemistry and immunology are used. Such techniques are well known and are explained in Immunochemical Methods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press, London); Scopes, 1987, Protein Purification: Principles and Practice, Second Edition (Springer- Verlag, N.Y.) and Handbook of Experimental
  • Nucleic acids comprising protein-coding sequences can be used to direct the ITT recombinant expression of polypeptides encoded by genes disclosed herein in intact cells or in cell-free translation systems.
  • the known genetic code tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences.
  • the polypeptides may be isolated from human cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an appropriate protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins.
  • Peptides and polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
  • polypeptides are preferably prepared by solid phase peptide synthesis as described by Menifield, 1963, J. Am. Chem. Soc. 85:2149.
  • polypeptide purification is well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed- phase HPLC, gel filtration, ion exchange and partition chromatography, and counter- cunent distribution.
  • the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence.
  • the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies produced against peptides encoded by genes disclosed herein can be used as purification reagents. Other purification methods are possible.
  • nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e, function-conservative variants.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids.
  • the isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.
  • the present invention also encompasses antibodies that specifically recognize the polymo ⁇ hic positions of the invention and distinguish a peptide or polypeptide containing a particular polymo ⁇ hism from one that contains a different sequence at that position.
  • Such polymo ⁇ hic position-specific antibodies according to the present invention include polyclonal and monoclonal antibodies.
  • the antibodies may be elicited in an animal host by immunization with peptides encoded by genes disclosed herein or may be formed by in vitro immunization of immune cells.
  • the immuno- genie components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems.
  • the antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA.
  • the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains.
  • the antibodies include hybrid antibodies (i.e, containing two sets of heavy chain/light chain combinations, each of which recognizes a different antigen), chimeric antibodies (i.e, in which either the heavy chains, light chains, or both, are fusion proteins), and univalent antibodies (i.e, comprised of a heavy chain/light chain complex bound to the constant region of a second heavy chain).
  • Fab fragments including Fab' and F(ab).sub.2 fragments of antibodies.
  • Panels of monoclonal antibodies produced against peptides encoded by genes disclosed herein 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 countercunent distribution. Purification methods for antibodies are disclosed, e.g, in The Art of Antibody Purification, 1989, Amicon Division, W. R. Grace & Co. General protein purification methods are described in Protein Purification: Principles and Practice, R. K. Scopes, Ed, 1987, Springer-Verlag, New York, N.Y.
  • antibodies elicited in response to a peptide comprising a particular polymo ⁇ hic sequence can be tested for their ability to specifically recognize that polymo ⁇ hic sequence, i.e, to bind differentially to a peptide or polypeptide comprising the polymo ⁇ hic sequence and thus distinguish it from a similar peptide or polypeptide containing a different sequence at the same position.
  • the invention provides diagnostic methods, e.g, for determining the identity of the allelic variants of polymo ⁇ hic regions present in the gene loci of genes disclosed herein, wherein specific allelic variants of the polymo ⁇ hic region are associated with cardiovascular diseases.
  • the diagnostic kit can be used to detennine whether a subject is at risk of developing a cardiovascular disease. This information could then be used, e.g, to optimize treatment of such individuals.
  • the kit comprises a probe or primer which is capable of hybridizing to a gene and thereby identifying whether the gene contains an allelic variant of a polymo ⁇ hic region which is associated with a risk for cardiovascular disease.
  • the kit preferably further comprises instructions for use in diagnosing a subject as having, or having a predisposition, towards developing a cardiovascular disease.
  • the probe or primers of the kit can be any of the probes or primers described in this file.
  • kits for amplifying a region of a gene comprising a polymo ⁇ hic region of interest comprise one, two or more primers.
  • the invention also provides antibody-based methods for detecting polymo ⁇ hic patterns in a biological sample.
  • the methods comprise the steps of: (i) contacting a sample with one or more antibody preparations, wherein each of the antibody preparations is specific for a particular polymo ⁇ hic form of the proteins encoded by genes disclosed herein, under conditions in which a stable antigen-antibody complex can form between the antibody and antigenic components in the sample; and (ii) detecting any antigen-antibody complex formed in step (i) using any suitable means known in the art, wherein the detection of a complex indicates the presence of the particular polymo ⁇ hic form in the sample.
  • immunoassays use either a labelled antibody or a labelled antigenic component (e.g, that competes with the antigen in the sample for binding to the antibody).
  • Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules.
  • Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labelled immunoassays, such as ELISA assays.
  • Diagnostic kits typically include one or more of the following compo- nents:
  • 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
  • the kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.
  • kits refened to above may include instructions for conducting the test. Furthermore, in prefened embodiments, the diagnostic kits are adaptable to high- throughput and/or automated operation. Drug Targets and Screening Methods
  • nucleotide sequences derived from genes disclosed herein and peptide sequences encoded by genes disclosed herein, particularly those that contain one or more polymo ⁇ hic sequences comprise useful targets to identify cardiovascular drugs, i.e, compounds that are effective in treating one or more clinical symptoms of cardiovascular disease. Furthermore, especially when a protein is a multimeric protein that are build of two or more subunits, is a combination of different polymo ⁇ hic subunits very useful.
  • Drug targets include without limitation (i) isolated nucleic acids derived from the genes disclosed herein, and (ii) isolated peptides and polypeptides encoded by genes disclosed herein, each of which comprises one or more polymo ⁇ hic positions.
  • an isolated nucleic acid comprising one or more polymo ⁇ hic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner.
  • the methods comprise:
  • an isolated peptide or polypeptide comprising one or more polymo ⁇ hic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner.
  • the screening methods involve:
  • high-throughput screening protocols are used to survey a large number of test compounds for their ability to bind the genes or peptides disclosed above in a sequence-specific manner.
  • Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Menimack, N.H.), and Micro- source (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories
  • Intact cells or whole animals expressing polymo ⁇ hic variants of genes disclosed herein can be used in screening methods to identify candidate cardiovascular drugs.
  • a permanent cell line is established from an individual exhibiting a particular polymo ⁇ hic pattern.
  • cells including without limitation mammalian, insect, yeast, or bacterial cells
  • Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to particular polymo ⁇ hic variants of proteins encoded by genes disclosed herein; (ii) assays that measure the ability of a test compound to modify
  • transgenic animals are created in which (i) one or more human genes disclosed herein, having different sequences at particular polymo ⁇ hic positions are stably inserted into the genome of the transgenic animal; and/or (ii) the endogenous genes disclosed herein are inactivated and replaced with human genes disclosed herein, having different sequences at particular polymo ⁇ hic positions.
  • Such animals can be treated with candidate compounds and monitored for one or more clinical markers of cardiovascular status . The following are intended as non-limiting examples ofthe invention.
  • a PCR is set up to amplify the flanking regions around a SNP.
  • 2 ng of genomic DNA patient sample
  • a primerset (20 - 40 pmol) producing a 75 to 320 bp PCR fragment with 0,3 to 1 U Qiagens Hot Star Taq PolymeraseTM in a total volume of 20 ⁇ L.
  • One primer is biotinylated depending on the direction of the sequencing primer. To force the biotinylated primer to be inco ⁇ orated it is used 0,8 fold.
  • PCR setup is performed by a BioRobot 3000 TM from Qiagen. PCR takes place in TI or Tgradient Thermocyclers TM from Biometra.
  • T A *V A prepared using the Sample Prep Tool and SNP Reagent Kit from Pyrosequencing according to their instructions.
  • Tool as follows: Place the cover on the desk, retract the 4 attachment rods by separating the handle from the magnetic rod holder, fit the magnetic rods into the holes of the cover plate, push the handle downward until a click is heard.
  • the PSQ 96 Sample Prep Tool is now ready for use. 2. To transfer beads from one plate to another, place the covered tool into the PSQ 96 Plate containing the samples and lower the magnetic rods by separating the handle from the magnetic rod holder. Move the tool up and down a few times then wait for 30-60 seconds. Transfer the beads into a new PSQ 96 plate containing the solution of choice.
  • Biotinylated PCR products are immobilized on streptavidin-coated DynabeadsTM M-
  • Qiagen Genomics formerly Rapigene
  • Rapigene is a service contractor for genotyping SNPs in patient samples. Their method is based on a primer extension method where two complementary primers are designed for each genotype that are labeled with different tags. Depending on the genotype only one primer will be elongated together with a certain tag. This tag can be detected with mass spectrometry and is a measure for the respective genotype. The method is described in the following patent: "Detection and identification of nucleic acid molecules - using tags which may be detected by non- fluorescent spectrometry or potentiometry" (WO 9727325).
  • Samples were collected anonymous and labeled with a patient number.
  • the baySNP number refers to an internal numbering of the CA SNPs. Primer sequences are listed for preamplification of the genomic fragments (primers EF and EF).
  • the baySNP number refers to an internal numbering of the CA SNPs. Primer sequences are listed for preamplification of the genomic fragments and for sequencing ofthe SNP using the pyrosequencing method.
  • the baySNP number refers to an internal numbering of the CA SNPs. Listed are the different polymo ⁇ hisms found in our association study. Also accession numbers and descriptions of those gene loci are given that are most homologous to the CA genes as listed in the sequences section (see below). Homologous genes and their accession numbers could be found by those skilled in the art in the Genbank database.
  • CVD MAL CASE/CTRL Individuals with diagnosis of cardiovascular disease and healthy controls (male)
  • the baySNP number refers to an internal numbering of the CA SNPs.
  • GTYPE P and A PVAL provide the p values obtained through chi square tests when comparing COHORTS A and B.
  • GTYPE P p value of genotypes
  • the number of patients in cohort A carrying genotypes 11, 12 or 22 were compared with the respective patients in cohort B (FQ11 B, FQ 12 B, FQ 22 B) resulting in a chi square test with a 3x2 matrix.
  • a PVAL p value of alleles
  • SIZE A and B Number of patients in cohorts A and B, respectively. See table 4 for definition of COHORTS A and B.
  • case and control populations represent any case-control-group pair, or bad(case)-good(control)-group pair, respectively.
  • a value RR1>1, RR2>1, and RR3>1 indicates an increased risk for individuals carrying genotype 1, genotype 2, and genotype 3, respectively.
  • the baySNP number refers to an internal numbering ofthe CA S ⁇ Ps and can be found in the sequence listing.

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Abstract

The present invention relates to isolated polynucleotides encoding a cardiovascular associated (CA) gene polypeptide useful in methods to identify therapeutic agents and useful for preparation of a medicament to treat cardiovascular disease, the polynucleotide is selected from the group comprising:SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 with allelic variation as indicated in the sequences section contained in a functional surrounding like full length cDNA for CA gene polypeptide and with or without the CA gene promoter sequence.The invention also provides diagnostic methods and kits including antibodies determining whether a human subject is at risk for a cardiovascular disease. The invention provides further polymorphic sequences and other genes.

Description

Single Nucleotide Polymorphisms predicting Cardiovascular Disease and Medication Efficacy
Technical Field
This invention relates to genetic polymorphisms useful for assessing cardiovascular risks in humans, including, but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, arterial inflammation, myocardial infarction, and stroke. Specifically, the present invention identifies and describes gene variations which are individually present in humans with cardiovascular disease states, relative to humans with normal, or non-cardiovascular disease states, and/or in response to medications relevant to cardiovascular disease. Further, the present invention provides methods for the identification and therapeutic use of compounds as treatments of cardiovascular disease. Moreover, the present invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease, and for monitoring the efficacy of compounds in clinical trials. Still further, the present invention provides methods to use gene variations to predict personal medication schemes omitting adverse drug reactions. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various cardiovascular diseases, and for the identification of subjects exhibiting a predisposition to such conditions.
Background ofthe Invention
Cardiovascular disease is a major health risk throughout the industrialized world.
Cardiovascular diseases include but are not limited by the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, atherosclerosis, ischemic diseases ofthe heart, coronary heart disease, all kinds of atrial and ventricular aπhythmias, hypertensive vascular diseases and peripheral vascular diseases. Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent ofthe underlying cause.
Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously nanowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included as well as the acute treatment of MI and the prevention of complications.
Ischemic diseases are conditions in which the coronary flow is restricted resulting in an perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases include stable angina, unstable angina and asymptomatic ischemia.
Arrhythmias include all forms of atrial and ventricular tachyanhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation) as well as bradycardic forms of arrhythmias.
Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others).
Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders. Atherosclerosis, the most prevalent of vascular diseases, is the principal cause of heart attack, stroke, and gangrene of the extremities, and thereby the principal cause of death. Atherosclerosis is a complex disease involving many cell types and molecular factors (for a detailed review, see Ross, 1993, Nature 362: 801-809 and Lusis, A. J., Nature 407, 233-241 (2000)). The process, in normal circumstances a protective response to insults to the endothelium and smooth muscle cells (SMCs) of the wall of the artery, consists of the formation of fibrofatty and fibrous lesions or plaques, preceded and accompanied by inflammation. The advanced lesions of atherosclerosis may occlude the artery concerned, and result from an excessive inflammatory-fibroproliferative response to numerous different forms of insult. For example, shear stresses are thought to be responsible for the frequent occunence of atherosclerotic plaques in regions of the circulatory system where turbulent blood flow occurs, such as branch points and inegular structures.
The first observable event in the formation of an atherosclerotic plaque occurs when blood-borne monocytes adhere to the vascular endothelial layer and transmigrate through to the sub-endothelial space. Adjacent endothelial cells at the same time produce oxidized low density lipoprotein (LDL). These oxidized LDLs are then taken up in large amounts by the monocytes through scavenger receptors expressed on their surfaces. In contrast to the regulated pathway by which native LDL (nLDL) is taken up by nLDL specific receptors, the scavenger pathway of uptake is not regulated by the monocytes.
These lipid-filled monocytes are called foam cells, and are the major constituent of the fatty streak. Interactions between foam cells and the endothelial and SMCs which sunound them lead to a state of chronic local inflammation which can eventually lead to smooth muscle cell proliferation and migration, and the formation of a fibrous plaque. Such plaques occlude the blood vessel concerned and thus restrict the flow of blood, resulting in ischemia. Ischemia is a condition characterized by a lack of oxygen supply in tissues of organs due to inadequate perfusion. Such inadequate perfusion can have number of natural causes, including atherosclerotic or restenotic lesions, anemia, or stroke, to name a few. Many medical interventions, such as the interruption of the flow of blood during bypass surgery, for example, also lead to ischemia. In addition to sometimes being caused by diseased cardiovascular tissue, ischemia may sometimes affect cardiovascular tissue, such as in ischemic heart disease. Ischemia may occur in any organ, however, that is suffering a lack of oxygen supply.
The most common cause of ischemia in the heart is atherosclerotic disease of epicardial coronary arteries. By reducing the lumen of these vessels, atherosclerosis causes an absolute decrease in myocardial perfusion in the basal state or limits appropriate increases in perfusion when the demand for flow is augmented. Coronary blood flow can also be limited by arterial thrombi, spasm, and, rarely, coronary emboli, as well as by ostial nanowing due to luetic aortitis. Congenital abnormalities, such as anomalous origin of the left anterior descending coronary artery from the pulmonary artery, may cause myocardial ischemia and infarction in infancy, but this cause is very rare in adults. Myocardial ischemia can also occur if myocardial oxygen demands are abnormally increased, as in severe ventricular hypertrophy due to hypertension or aortic stenosis. The latter can be present with angina that is indistinguishable from that caused by coronary atherosclerosis. A reduction in the oxygen-carrying capacity of the blood, as in extremely severe anemia or in the presence of carboxy-hemoglobin, is a rare cause of myocardial ischemia. Not infrequently, two or more causes of ischemia will coexist, such as an increase in oxygen demand due to left ventricular hypertrophy and a reduction in oxygen supply secondary to coronary atherosclerosis.
The foregoing studies are aimed at defining the role of particular gene variations presumed to be involved in the misleading of normal cellular function leading to cardiovascular disease. However, such approaches cannot identify the full panoply of gene variations that are involved in the disease process. At present, the only available treatments for cardiovascular disorders are pharmaceutical based medications that are not targeted to an individual's actual defect; examples include angiotensin converting enzyme (ACE) inhibitors and diuretics for hypertension, insulin supplementation for non-insulin dependent diabetes mellitus
(NIDDM), cholesterol reduction strategies for dyslipidaemia, anticoagulants, β blockers for cardiovascular disorders and weight reduction strategies for obesity. If targeted treatment strategies were available it might be possible to predict the response to a particular regime of therapy and could markedly increase the effective- ness of such treatment. Although targeted therapy requires accurate diagnostic tests for disease susceptibility, once these tests are developed the opportunity to utilize targeted therapy will become widespread. Such diagnostic tests could initially serve to identify individuals at most risk of hypertension and could allow them to make changes in lifestyle or diet that would serve as preventative measures. The benefits associated by coupling the diagnostic tests with a system of targeted therapy could include the reduction in dosage of administered drugs and thus the amount of unpleasant side effects suffered by an individual. In more severe cases a diagnostic test may suggest that earlier surgical intervention would be useful in preventing a further deterioration in condition.
It is an object of the invention to provide genetic diagnosis of predisposition or susceptibility for cardiovascular diseases. Another related object is to provide treatment to reduce or prevent or delay the onset of disease in those predisposed or susceptible to this disease. A further object is to provide means for carrying out this diagnosis.
Accordingly, a first aspect of the invention provides a method of diagnosis of disease in an individual, said method comprising determining one, various or all genotypes in said individual ofthe genes listed in the Examples.
In another aspect, the invention provides a method of identifying an individual predisposed or susceptible to a disease, said method comprising determining one, various or all genotypes in said individual ofthe genes listed in the Examples.
The invention is of advantage in that it enables diagnosis of a disease or of certain disease states via genetic analysis which can yield useable results before onset of disease symptoms, or before onset of severe symptoms. The invention is further of advantage in that it enables diagnosis of predisposition or susceptibility to a disease or of certain disease states via genetic analysis.
The invention may also be of use in confirming or conoborating the results of other diagnostic methods. The diagnosis of the invention may thus suitably be used either as an isolated technique or in combination with other methods and apparatus for diagnosis, in which latter case the invention provides a further test on which a diagnosis may be assessed.
The present invention stems from using allelic association as a method for genotyping individuals; allowing the investigation of the molecular genetic basis for cardiovascular diseases. In a specific embodiment the invention tests for the polymorphisms in the sequences of the listed genes in the Examples. The invention demonstrates a link between this polymorphisms and predispositions to cardiovascular diseases by showing that allele frequencies significantly differ when individuals with "bad" serum lipids are compared to individuals with "good" serum levels. The meaning of "good and bad" serum lipid levels is defined in Table 1.
Certain disease states would benefit, that is to say the suffering of the patient may be reduced or prevented or delayed, by administration of treatment or therapy in advance of disease appearance; this can be more reliably canied out if advance diagnosis of predisposition or susceptibility to disease can be diagnosed. Detailed Description ofthe Invention
The present invention is based at least in part on the discovery that a specific allele of a polymorphic region of a so called "candidate gene" is associated with CVD.
"Candidate gene" as used herein includes genes that can be assigned to either normal cardiovascular function or to metabolic pathways that are related to onset and/or progression of cardiovascular diseases. As the development of cardiovascular diseases is not completely understood, the term "candidate gene" may also comprise genes with presently unknown function.
For the present invention the following candidate genes were analyzed: -Genes found to be expressed in cardiac tissue (Hwang et al., Circulation 1997, 96:4146-4203). -Genes from the following metabolic pathways and their regulatory elements:
Lipid metabolism
Numerous studies have shown a connection between serum lipid levels and cardiovascular diseases. Candidate genes falling into this group include but are not limited by genes of the cholesterol pathway, apolipoproteins and their modifiying factors.
Coagulation
Ischemic diseases of the heart and in particular myocardial infarction may be caused by a thrombotic occlusion. Genes falling into this group include all genes of the coagulation cascade and their regulatory elements.
Inflammation
Complications of atherosclerosis are the most common causes of death in Western societies. In broad outline atherosclerosis can be considered to be a form of chronic inflammation resulting from interaction modified lipoproteins, monocyte-derived macrophages,T cells, and the normal cellular elements of the arterial wall. This inflammatory process can ultimately lead to the development of complex lesions, or plaques, that protrude into the arterial lumen. Finally plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction and stroke (Glass et al., Cell 2001, 104:503-516).
It follows that all genes related to inflammatory processes, including but not limited by cytokines, cytokine receptors and cell adhesion molecules are candidate genes for CVD.
Glucose and energy metabolism
As glucose and energy metabolism is interdependent with the metabolism of lipids (see above) also the former pathways contain candidate genes. Energy metabolism in general also relates to obesity, which is an independent risk factor for CVD (Melanson et al., Cardiol Rev 2001 9:202-207). In addition high blood glucose levels are associated with many microvascular and macrovascular complications and may therefore affect an individuals disposition to CVD (Duckworth, Cun Atheroscler Rep 2001, 3:383-391).
Hypertension
As hypertension is an independent risk factor for CVD, also genes that are involved in the regulation of systolic and diastolic blood pressure affect an individuals risk for CVD (Safer, Cun Opin Cardiol 2000, 15:258-263). interestingly hypertension and diabetes (see above) appear to be interdependent, since hypertension is approximately twice as frequent in patients with diabetes compared with patients without the disease. Conversely, recent data suggest that hypertensive persons are more predisposed to the development of diabetes than are normotensive persons (Sowers et al., Hypertension 2001, 37:1053-1059). Unclassified genes
As stated above, the mechanisms that lead to cardiovascular diseases are not completely elucidated. Hence also candidate genes were analysed, which could not be assigned to the above listed categories. The present invention is based at least in part on the discovery of polymoφhisms, that lie in genomic regions of unknown physiological function.
Results
After conducting an association study, we suφrisingly found polymoφhic sites in a number of candidate genes which show a strong conelation between healthy and CVD prone individuals. "Healthy" as used herein refers to individuals that neither suffer from existing CVD, nor exihibit an increased risk for CVD through their serum lipid level profile. "CVD prone" as used herein refers to individuals with existing CVD and/or a serum lipid profile that confers a high risk to get CVD (see
Table 1 for definitions of healthy and CVD prone serum lipid levels). Polymoφhic sites in candidate genes that were found to be significantly associated with "healthy" or "CVD prone" phenotypes will be refered to as "CVD associated SNPs" (CA SNPs). The respective genomic loci that harbour CA SNPs will be refered to as "CVD associated genes" (CA genes), inespective of the actual function of this gene locus.
In particular we suφrisingly found CA SNPs in the following genes:
Apolipoprotein E
Chylomicron remnants and very low density lipoprotein (VLDL) remnants are rapidly removed from the circulation by receptor-mediated endocytosis in the liver. Apolipoprotein E, a main apoprotein of the chylomicron, binds to a specific receptor on liver cells and peripheral cells. ApoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents. The APOE gene is mapped to chromosome 19 in a cluster with APOC1 and APOC2. Defects in apolipoprotein E result in familial dysbetalipoproteinemia, or type III hyperlipoproteinemia (HLP III), in which increased plasma cholesterol and triglycerides are the consequence of impaired clearance of chylomicron and VLDL remnants.
Apolipoprotein e receptor 2 (low density lipoprotein receptor-related protein 8,
LPR8)
This gene encodes an apolipoprotein E receptor, a member of the low density lipoprotein receptor (LDLR) family. Apolipoprotein E is a small lipophilic plasma protein and a component of lipoproteins such as chylomicron remnants, very low density lipoprotein (VLDL), and high density lipoprotein (HDL). The apolipoprotein E receptor is involved in cellular recognition and internalization of these lipoproteins. Alternative splicing generates three transcript variants for this gene; additional variants have been described, but their full length nature has not been determined.
Diazepam binding inhibitor, Endozepine
Diazepam binding inhibitor (acyl-CoA-binding protein); binds and induces medium- chain acyl-CoA ester synthesis
Nonmuscle type myosin heavy chain 9
Non-muscle myosin heavy chain 9; motor protein that provides force for muscle contraction, cytokinesis and phagocytosis; contains an ATPase head domain and a rod-like tail domain
Apolipoprotein A-I and C-III genomic locus
APOA1 promotes cholesterol efflux from tissues to the liver for excretion. Apolipoprotein A-I is the major protein component of high density lipoprotein (HDL) in the plasma. Synthesized in the liver and small intestine, it consists of two identical chains of 77 amino acids; an 18-amino acid signal peptide is removed co- translationally and a 6-amino acid propeptide is cleaved post-translationally. Variation in the latter step, in addition to modifications leading to so-called isoforms, is responsible for some of the polymoφhism observed. APOA1 is a cofactor for lecithin cholesterolacyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters. The APOA1, APOC3 and APOA4 genes are closely linked in both rat and human genomes. The A-I and A-IV genes are transcribed from the same strand, while the C-III gene is transcribed convergently in relation to A-I.
Defects in the apolipoprotein A-l gene are associated with HDL deficiency and Tangier disease.
Apolipoprotein C-III is a very low density lipoprotein (VLDL) protein. APOC3 inhibits lipoprotein lipase and hepatic lipase; it is thought to delay catabolism of triglyceride-rich particles. The APOA1, APOC3 and APOA4 genes are closely linked in both rat and human genomes. The A-I and A-IV genes are transcribed from the same strand, while the A-l and C-III genes are convergently transcribed. An increase in apoC-III levels induces the development of hypertriglyceridemia.
Apolipoprotein B
Apolipoprotein B (ApoB) is the main apolipoprotein of chylomicrons and low density lipoproteins (LDL). The protein occurs in the plasma in 2 main isoforms, apoB-48 and apoB-100. The first is synthesized exclusively by the gut, the second by the liver. The intestinal (B-48) and hepatic (B-100) forms of apoB are coded by a single gene and by a single mRNA transcript larger than 16 kb. The 2 proteins share a common amino terminal sequence. In the ApoB- 100 isoform the precursor has 4,563 amino acids, and the mature apoB-100 has 4,536 amino acid residues. Mature, circulating B-48 is homologous over its entire length (estimated to be between 2,130 and 2,144 amino acid residues) with the amino-terminal portion of B-100 and contains no sequence from the carboxyl end of B-100. From structural studies, it is thought that apoB-48 represents the amino-terminal 47% of apoB-100 and that the carboxyl terminus of apoB-48 is in the vicinity of residue 2151 of apoB-100. Apolipoprotein B-48 may be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C-to-U change in the codon CAA encoding
Gln(2153) in apoB-100 mRNA. Since only the sequence that codes B-100 is present in genomic DNA, this presents the possibility of an organ-specific introduction of a stop codon to an mRNA and the change from CAA to UAA of codon 2153 of the message as a unique RNA editing process.
LIM domain kinase 1
There are approximately 40 known eukaryotic LIM proteins, so named for the LIM domains they contain. LIM domains are highly conserved cysteine-rich structures containing 2 zinc fingers. Although zinc fingers usually function by binding to DNA or RNA, the LIM motif probably mediates protein-protein interactions. LIM kinase- 1 and LIM kinase-2 belong to a small subfamily with a unique combination of 2 N- terminal LIM motifs and a C-terminal protein kinase domain. LIMKl is likely to be a component of an intracellular signaling pathway and may be involved in brain development. LIMKl hemizygosity is implicated in the impaired visuospatial constructive cognition of Williams syndrome. Two splice variant have been identified.
Thermostable phenol sulfotransferase (STP2)
Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurofransmitters, and steroid hormones. The phenol sulfotransferase STP2 maps to chromosome 16 (Dooley and Huang, 1996) and preferentially catalyzes the sulfonation of 'simple' planar phenols.
Low Density Lipoprotein Receptor
The low density lipoprotein receptor (LDLR) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. Low density lipoprotein (LDL) is normally bound at the cell membrane and taken into the cell ending up in lysosomes where the protein is degraded and the cholesterol is made available for repression of microsomal enzyme 3-hydroxy-3-methylglutaryl coen- zyme A (HMG CoA) reductase, the rate-limiting step in cholesterol synthesis. At the same time, a reciprocal stimulation of cholesterol ester synthesis takes place. Mutations in the LDL receptor (LDLR) gene cause the autosomal dominant disorder, familial hypercholesterolemia.
ATP-binding cassette, sub-family B (MDR/TAP), member 1 The membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and infra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance. The protein encoded by this gene is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs. This protein also functions as a transporter in the blood-brain barrier.
Annexin VI (Calphobindin II)
Annexin VI belongs to a family of calcium-dependent membrane and phospholipid binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. The annexin VI gene is approximately 60 kbp long and contains 26 exons. It encodes a protein of about 68 kDa that consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to human annexins I and II sequences, each of which contain four such repeats. Exon 21 of annexin VI is alternatively spliced, giving rise to two isoforms that differ by a 6-amino acid insertion at the start of the seventh repeat. Annexin VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. Protein C inhibitor (PCI-B)
Marlar and Griffin (J. Clin. Invest. 1980, 66: 1186-1189) identified in normal plasma a protein inhibitor of activated protein C. They showed, furthermore, that this inhibitor is deficient in combined factor V and VIII deficiency. Activated protein C is a potent anticoagulant.
Protein C inhibitor (plasminogen activator inhibitor III); may be a serine protease inhibitor; member ofthe seφin family of serine protease inhibitors.
NADH dehydrogenase 5
Subunit of NADH-ubiquinone oxidoreductase (complex I); transports electrons from NADH to ubiquinone.
Lysosomal alpha-glucosidase (acid maltase) This gene encodes acid alpha-glucosidase, which is essential for the degradation of glycogen to glucose in lysosomes. Different forms of acid alpha-glucosidase are obtained by proteolytic processing. Defects in this gene are the cause of glycogen storage disease π, also known as Pompe's disease, which is an autosomal recessive disorder with a broad clinical spectrum.
In classic cases of Pompe disease, affected children are prostrate and markedly hypotonic with large hearts. The tongue may be enlarged. The liver is rarely enlarged (except as a result of heart failure), and hypoglycemia and acidosis do not occur as they do in type I glycogen storage disease. Death usually occurs in the first year of life in the classic form ofthe disorder and cardiac involvement is striking.
Interleukin 6
Interleukin 6 (interferon-beta 2); induces the maturation of B cells into immunoglobulin-secreting cells. Fibrinogen gamma chain/fibrinogen alpha chain genes
The protein encoded by these genes is fibrinogen, a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots. In addition, various cleavage products of fibrinogen and fibrin regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types. Mutations in this gene lead to several disorders, including dysfibrinogenemia, hypofibrinogenemia and thrombo- philia. Alternative splicing results in two isoforms, varying in the carboxy-terminus.
Paraoxonase 2 (PON2)
Paraoxonase/arylesterase 2; possibly functions in protecting low density lipoprotein against oxidative modification; member of a family that hydrolyzes toxic organo- phosphates.
Defensin, alpha 6
Defensins are a family of microbicidal and cytotoxic peptides thought to be involved in host defense. They are abundant in the granules of neutrophils and also found in the epithelia of mucosal surfaces such as those of the intestine, respiratory tract, urinary tract, and vagina. Members of the defensin family are highly similar in protein sequence and distinguished by a conserved cysteine motif. Several alpha defensin genes appear to be clustered on chromosome 8. The protein encoded by this gene, defensin, alpha 6, is highly expressed in the secretory granules of Paneth cells ofthe small intestine, and likely plays a role in host defense of human bowel.
Tetracycline transporter-like protein
Cardiac beta myosin heavy chain, myosin, heavy polypeptide 7
MYH7 encodes the cardiac muscle beta (or slow) isoform of myosin. Changes in the relative abundance of MYH7 and MYH6 (the alpha, or fast, isoform of cardiac myosin heavy chain) conelate with the contractile velocity of cardiac muscle. Mutations in MYH7 are associated with familial hypertrophic cardiomyopathy.
Endothelial leukocyte adhesion molecule 1 (ELAM-1) The endothelial leukocyte adhesion molecule- 1 is expressed by cytokine-stimulated endothelial cells. It is thought to be responsible for the accumulation of blood leukocytes at sites of inflammation by mediating the adhesion of cells to the vascular lining. It exhibits structural features such as the presence of lectin- and EGF-like domains followed by short consensus repeat (SCR) domains that contain 6 conserved cysteine residues. These proteins are part of the selectin family of cell adhesion molecules. This gene is present in single copy in the human genome and contains 14 exons spanning about 13 kb of DNA. Adhesion molecules participate in the interaction between leukocytes and the endothelium and appear to be involved in the pathogenesis of atherosclerosis.
Lamin B2 (LAMB2)
Lamin B2 is a member of a family of structural nuclear envelope proteins.
Estrogen receptor 2 (ER beta) Estrogen receptor beta 2 is a transcriptional activator involved in regulation of reproduction; exists in five isoforms.
Tissue plasminogen activator
This gene encodes tissue-type plasminogen activator, a secreted serine protease which converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme.
Tissue-type plasminogen activator is synthesized as a single chain which is cleaved by plasmin to a two chain disulfide linked protein. This enzyme plays a role in cell migration and tissue remodeling. Increased enzymatic activity causes hyper- fibrinolysis, which manifests as excessive bleeding; decreased activity leads to hypofibrinolysis which can result in thrombosis or embolism. Alternative splicing of this gene produces three transcripts. Laminin receptor 1
Laminins, a family of extracellular matrix glycoproteins, are the major non- collagenous constituent of basement membranes. They have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. Many of the effects of laminin are mediated through interactions with cell surface receptors. These receptors include members of the integrin family, as well as non-integrin laminin-binding proteins. This gene encodes a high-affinity, non-integrin family, laminin receptor 1. This receptor has been variously called 67 kD laminin receptor, 37 kD laminin receptor precursor (37LRP) and p40 ribosome-associated protein. The amino acid sequence of laminin receptor 1 is highly conserved through evolution, suggesting a key biological function. It has been observed that the level of the laminin receptor transcript is higher in colon carcinoma tissue and lung cancer cell line than their normal counteφarts. Also, there is a conelation between the upregulation of this polypeptide in cancer cells and their invasive and metastatic phenotype. Multiple copies of this gene exist, however, most of them are pseudogenes thought to have arisen from retropositional events.
Integrin, beta 3 (platelet glycoprotein Ilia, antigen CD61)
The ITGB3 protein product is the integrin beta chain beta 3. Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. A given chain may combine with multiple partners resulting in different integrins. Integrin beta 3 is found along with the alpha lib chain in platelets. Integrins are known to participate in cell adhesion as well as cell-surface mediated signalling.
Myeloid cell differentiation protein (MCL1)
Rinkenberger et al. (Genes Dev. 2000, 14: 23-27) disrupted the Mcll locus in murine
ES cells to determine the developmental roles of this Bcl2 family member. Deletion of Mcll resulted in periimplantation embryonic lethality. Homozygous Mcll- deficient embryos did not implant in utero, but could be recovered at E3.5 to E4.0. Null blastocysts failed to hatch or attach in vitro, indicating a frophectoderm defect, although the inner cell mass could grow in culture. Of note, homozygous Mcll- deficient blastocysts showed no evidence of increased apoptosis, but exhibited a delay in maturation beyond the precompaction stage. This model indicates that Mcll is essential for preimplantation development and implantation, and suggests that it has a function beyond regulating apoptosis.
Tumor necrosis factor type 1 receptor associated protein (TRAP1)
TRAP is a highly conserved member of a class of molecular chaperones with multiple functions. It is involved in the maturation of a subset of proteins involved in signal transduction.
Xanthine dehydrogenase/oxidase
Xanthine dehydrogenase belongs to the group of molybdenum-containing hydroxyl- ases involved in the oxidative metabolism of purines. The enzyme is a homodimer.
Xanthine dehydrogenase can be converted to xanthine oxidase by reversible sulfhydryl oxidation or by ineversible proteolytic modification. Defects in xanthine dehydrogenase cause xanthinuria, may contribute to adult respiratory stress syndrome, and may potentiate influenza infection through an oxygen metabolite- dependent mechanism.
Stromal cell-derived factor 1 (SDF 1)
Stromal cell-derived factor 1 is a lymphocyte chemoattractant that signals through the receptor CXCR4.
Diacylglycerol kinase delta
Diacylglycerol kinase delta phosphorylates the arachidonoyl type of diacylglycerol; contains a pleckstrin homology domain and an EPH domain. Smooth muscle myosin heavy chain, MYHll
The protein encoded by this gene is a smooth muscle myosin belonging to the myosin heavy chain family. The gene product is a subunit of a hexameric protein that consists of 2 heavy chain subunits and 2 pairs of non-identical light chain subunits. It functions as a major contractile protein, converting chemical energy into mechanical energy through the hydrolysis of ATP. The gene encoding a human ortholog of rat NUDEl is transcribed from the reverse strand of MYHl l gene, and its 3' end overlaps with that of the latter. The pericentric inversion of chromosome 16 [inv(16)(pl3q22)] produces a chimeric transcript consisting of the first 165 residues from the N terminus of core-binding factor beta in a fusion with the C-terminal portion of the smooth muscle myosin heavy chain. This chromosomal reaπangement is associated with acute myeloid leukemia of the M4Eo subtype. Alternative splicing generates isoforms that are differentially expressed, with ratios changing during muscle cell maturation. Additional splice variants have been described but their full- length nature has not been determined.
Caveolin 1
The scaffolding protein encoded by this gene is the main component of the caveolae plasma membranes found in most cell types. The protein links integrin subunits to the tyrosine kinase FYN, an initiating step in coupling integrins to the Ras-ERK pathway and promoting cell cycle progression. The gene is a tumor suppressor gene candidate and a negative regulator of the Ras-p42/44 MAP kinase cascade. CAV1 and CAV2 are located next to each other on chromosome 7 and express colocalizing proteins that form a stable hetero-oligomeric complex. By using alternative initiation codons in the same reading frame, two isoforms (alpha and beta) are encoded by a single transcript from this gene.
AP-2 beta
Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. It was shown that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl- X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism it was shown that transfection of AP-2 into cell lines in vitro strongly suppresses c- myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6pl2-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymoφhic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene (Moser et al., Genes Dev 1997, 11:1938-1948).
Transforming growth factor beta 2 (TGFB2)
Transforming growth factor-beta 2 (glioblastoma-derived T cell suppressor factor); suppresses IL2 - dependent growth of T cells and is a member of a family of cytokines that transmits signals through transmembrane serine/threonine kinases.
Tumor necrosis factor, alpha-induced protein 2 (TNFAIP2, B94)
Secreted by vascular endothelium, expression is induced by tumor necrosis factor alpha, interleukin-1 beta, and lipopolysaccharide.
Protein phosphatase IA (formerly 2C), magnesium-dependent, alpha isoform Magnesium- or manganese-dependent alpha protein phosphatase IA; regulates cell stress responses.
ATPase, Ca++ transporting, plasma membrane 3 (PMCA3)
Osteonidogen (nidogen 2) ATP-binding cassette, sub-family A (ABC1), member 1
The membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20,
White). This protein is a member of the ABC1 subfamily. Members of the ABC1 subfamily comprise the only major ABC subfamily found exclusively in multicellular eukaryotes. With cholesterol as its substrate, this protein functions as a cholesteral efflux pump in the cellular lipid removal pathway. Mutations in this gene have been associated with Tangier's disease and familial high-density lipoprotein deficiency.
Bromodomain-containing 3 (BRD3)
This gene was identified based on its homology to the gene encoding the RING3 protein, a serine/threonine kinase. The gene localizes to 9q34, a region which contains several major histocompatibility complex (MHC) genes. The function ofthe encoded protein is not known.
Peroxisome proliferator activated receptor-gamma (PPA gamma)
Peroxisome proliferator activated receptor-gamma (PPAR gamma) regulates adipocyte and macrophage gene expression and differentiation.
Tumor necrosis factor beta (Lymphotoxin alpha, LTA)
Lymphotoxin alpha, a member of the tumor necrosis factor family, is a cytokine produced by lymphocytes. LTA is highly inducible, secreted, and exists as homo- trimeric molecule. LTA forms heterotrimers with lymphotoxin-beta which anchors lymphotoxin-alpha to the cell surface. LTA mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. LTA is also involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis. DEK oncogene
Site-specific DNA binding protein; involved in transcriptional regulation and signal transduction.
S-adenosyl methionine transferase
S-adenosyl methionine transferase catalyzes the formation of S-adenosylmethionine from methionine and ATP. Both the beta and alpha isoforms may be encoded by one gene. Methionine adenosyltransferase deficiency is known to be caused by recessive as well as dominant mutations, the latter identified in autosomal dominant persistant hypermethioninemia.
Coagulation factor III (thromboplastin, tissue factor)
This gene encodes coagulation factor III which is a cell surface glycoprotein. This factor enables cells to initiate the blood coagulation cascades, and it functions as the high-affinity receptor for the coagulation factor VII. The resulting complex provides a catalytic event that is responsible for initiation ofthe coagulation protease cascades by specific limited proteolysis. Unlike the other cofactors of these protease cascades, which circulate as nonfunctional precursors, this factor is a potent initiator that is fully functional when expressed on cell surfaces. There are 3 distinct domains of this factor: extracellular, transmembrane, and cytoplasmic. This protein is the only one in the coagulation pathway for which a congenital deficiency has not been described.
Membrane protein palmitoylated 2 (MPP2)
Palmitoylated membrane protein 2 is a member of a family of membrane-associated proteins termed MAGUKs (membrane-associated guanylate kinase homologs).
MAGUKs interact with the cytoskeleton and regulate cell proliferation, signaling pathways, and intracellular junctions. Palmitoylated membrane protein 2 contains a conserved sequence, called the SH3 (src homology 3) motif, found in several other proteins that associate with the cytoskeleton and are suspected to play important roles in signal transduction. Granulocyte-macrophage colony stimulating factor 2 receptor, beta chain (CSF2RB)
CSF2RB is a common beta chain of the high affinity receptor for IL-3, IL-5 and CSF. Defective CSF2RB has been reported to be associated with protein alveolar proteinosis
Coagulation factor II (Prothrombin, F2)
Coagulation factor II is proteolytically cleaved to form thrombin in the first step of the coagulation cascade which ultimately results in the stemming of blood loss. F2 also plays a role in maintaining vascular integrity during development and postnatal life. Mutations in F2 leads to various forms of thrombosis and dysprothrombinemia.
Lipoprotein lipase (LPL)
LPL encodes lipoprotein lipase, which is expressed in heart, muscle, and adipose tissue. LPL functions as a homodimer, and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Severe mutations that cause LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.
activin beta-C chain (Inhibin beta C)
This gene encodes the beta C chain of inhibin, a member of the TGF-beta super- family. This subunit forms heterodimers with beta A and beta B subunits. Inhibins and activins, also members of the TGF-beta superfamily, are hormones with opposing actions and are involved in hypothalamic, pituitary, and gonadal hormone secretion, as well as growth and differentiation of various cell types.
Methylenetetrahydrofolate dehydrogenase, NADP+ dependent (MTHFD)
This gene encodes a protein that possesses three distinct enzymatic activities, 5,10- methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclo- hydrolase and 10-formyltetrahydrofolate synthetase. Each of these activities catalyzes one of three sequential reactions in the interconversion of 1 -carbon derivatives of tetrahydrofolate, which are substrates for methionine, thymidylate, and de novo purine syntheses. The trifunctional enzymatic activities are confened by two major domains, an aminoterminal portion containing the dehydrogenase and cyclohydrolase activities and a larger synthetase domain.
PACAP gene for pituitary adenylate cyclase activating polypeptide This gene encodes adenylate cyclase activating polypeptide 1. Mediated by adenylate cyclase activating polypeptide 1 receptors, this polypeptide stimulates adenylate cyclase and subsequently increases the cAMP level in target cells. Adenylate cyclase activating polypeptide 1 is not only a hypophysiotropic hormone, but also functions as a neurotransmitter and neuromodulator. In addition, it plays a role in paracrine and autocrine regulation of certain types of cells. This gene is composed of five exons. Exons 1 and 2 encode the 5' UTR and signal peptide, respectively; exon 4 encodes an adenylate cyclase activating polypeptide 1 -related peptide; and exon 5 encodes the mature peptide and 3' UTR. This gene encodes three different mature peptides, including two isotypes: a shorter form and a longer form.
Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase), polypeptide 8
This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and its expression is induced by phenobarbital. The enzyme is known to metabolize many xenobiotics, including the anticonvulsive drug mephenytoin, benzo(a)pyrene, 7-ethyoxycoumarin, and the anti- cancer drug taxol. Two transcript variants for this gene have been described; it is thought that the longer form does not encode an active cytochrome P450 since its protein product lacks the heme binding site. This gene is located within a cluster of cytochrome P450 genes on chromosome 10q24. Leucocyte adhesion receptor, L-selectin (SELL)
SELL is a cell surface component that is a member of a family of adhesion/homing receptors which play important roles in leukocyte-endothelial cell interactions. The molecule is composed of multiple domains: one homologous to lectins, one to epidermal growth factor, and two to the consensus repeat units found in C3/C4 binding proteins.
Mitochondrial ATP synthase c subunit (PI form)
Isoform 1 (PI) of subunit c, H+-translocating subunit of FO ATP synthase; catalyzes the synthesis of ATP during oxidative phosphorylation.
Calmodulin
Calmodulin binds Ca2+, regulates proteins and enzymes in a Ca2+-dependent manner
WNT1 inducible signaling pathway protein 1 (WISPl) gene
WISP1 is a member ofthe connective tissue growth factor family
Ribophorin I Ribophorin I; subunit of oligosaccharyltransferase that binds ribosomes.
Nonsyndromic hearing impairment protein (DFNA5)
Hearing impairment is a heterogeneous condition with over 40 loci described. The protein encoded by this gene is expressed in fetal cochlea, however, its function is not known. Nonsyndromic hearing impairment is associated with a mutation in this gene.
Cytochrome P450 2E1 (CYP2E1)
This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and is induced by ethanol, the diabetic state, and starvation. The enzyme metabolizes both endogenous substrates, such as ethanol, acetone, and acetal, as well as exogenous substrates including benzene, carbon tetrachloride, ethylene glycol, and nitrosamines which are premutagens found in cigarette smoke. Due to its many substrates, this enzyme may be involved in such varied processes as gluconeogenesis, hepatic cinhosis, diabetes, and cancer.
Vacuolar H+ ATPase E subunit (ATP6E) Subunit E of the vacuolar H+- ATPase proton pump; regulates ATP binding and hydrolysis by the A and B subunits
Retinoid X receptor, alpha
Retinoid X receptors (RXRs) and retinoic acid receptors (RARs), are nuclear receptors that mediate the biological effects of retinoids by their involvement in retinoic acid-mediated gene activation. These receptors exert their action by binding, as homodimers or heterodimers, to specific sequences in the promoters of target genes and regulating their transcription. The protein encoded by this gene is a member of the steroid and thyroid hormone receptor superfamily of transcriptional regulators.
Peroxisome proliferative activated receptor, delta (PPARD)
Peroxisome proliferator-activated receptor delta is a member of the steroid hormone receptor superfamily
Ataxin (SCA1)
The autosomal dominant cerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders characterized by progressive degeneration of the cerebellum, brain stem and spinal cord. Clinically, ADCA has been divided into three groups: ADCA types I-III. ADCAI is genetically heterogeneous, with five genetic loci, designated spinocerebellar ataxia (SCA) 1, 2, 3, 4 and 6, being assigned to five different chromosomes. ADC All, which always presents with retinal degeneration (SCA7), and ADCAIII often refened to as the pure' cerebellar syndrome (SCA5), are most likely homogeneous disorders. Several SCA genes have been cloned and shown to contain CAG repeats in their coding regions. ADCA is caused by the expansion of the CAG repeats, producing an elongated polyglutamine tract in the conesponding protein. The expanded repeats are variable in size and unstable, usually increasing in size when transmitted to successive generations. The function of the ataxins is not known. The SCA1 locus has been mapped to chromosome 6, and it has been determined that the diseased allele contains 41-81 CAG repeats, compared to 6-39 in the normal allele. Several transcript variants of
SCA1 in the 5' UTR have been described; however, their full-length nature is not known.
Adducin 1 (alpha) Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). Alpha- and gamma-adducins are ubiquitously expressed. In contrast, beta- adducin is expressed at high levels in brain and hematopoietic tissues. Adducin is a heterodimeric protein that consists of related subunits, alpha and beta, which are produced from distinct genes but share a similar structure. Alpha- and beta-adducin include a protease-resistant N-terminal region and a protease-sensitive, hydrophilic
C-terminal region. Adducin binds with high affinity to Ca(2+)/calmodulin and is a substrate for protein kinases A and C.
Zinc finger protein 202 (ZNF202) Zinc-finger protein 202; may repress genes involved in lipid metabolism; contains zinc fingers
Mevalonate pyrophosphate decarboxylase
The enzyme mevalonate pyrophosphate decarboxylase catalyzes the conversion of mevalonate pyrophosphate into isopentenyl pyrophosphate in one ofthe early steps in cholesterol biosynthesis. It decarboxylates and dehydrates its substrate while hydrolyzing ATP.
Integrin, beta 2 (ITGB2, lymphocyte function-associated antigen 1, LFA1) The ITGB2 protein product is the integrin beta chain beta 2. Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. A given chain may combine with multiple partners resulting in different integrins. For example, beta 2 combines with the alpha L chain to form the integrin LFA-1, and combines with the alpha M chain to form the integrin Mac-1. Integrins are known to participate in cell adhesion as well as cell-surface mediated signalling.
Adrenergic beta-3- receptor (ADRB3)
The ADRB3 gene product, beta-3 -adrenergic receptor, is located mainly in adipose tissue and is involved in the regulation of lipolysis and thermogenesis. Beta adrenergic receptors are involved in the epenephrine and norepinephrine-induced activation of adenylate cyclase through the action of G proteins.
Cytochrome P450, subfamily IVF, polypeptide 8
This gene-, CYP4F8, encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and functions as a 19-hydroxylase of prostaglandins in seminal vesicles. This gene is part of a cluster of cytochrome P450 genes on chromosome 19. Another member of this family, CYP4F3, is approximately 18 kb away.
Coagulation factor XIII, A subunit
This gene encodes the coagulation factor XIII A subunit. Coagulation factor XIII is the last zymogen to become activated in the blood coagulation cascade. Plasma factor XIII is a heterotetramer composed of 2 A subunits and 2 B subunits. The A subunits have catalytic function, and the B subunits do not have enzymatic activity and may serve as a plasma canier molecules. Platelet factor XIII is comprised only of 2 A subunits, which are identical to those of plasma origin. Upon activation by the cleavage ofthe activation peptide by thrombin and in the presence of calcium ion, the plasma factor XIII dissociates its B subunits and yields the same active enzyme, factor XHIa, as platelet factor XIII. This enzyme acts as a transglutaminase to catalyze the formation of gamma-glutamyl-epsilon-lysine crosslinking between fibrin molecules, thus stabilizing the fibrin clot. It also crosslinks alpha-2-plasmin inhibitor, or fibronectin, to the alpha chains of fibrin. Factor XIII deficiency is classified into two categories: type I deficiency, characterized by the lack of both the A and B subunits; and type II deficiency, characterized by the lack of the A subunit alone. These defects can result in a lifelong bleeding tendency, defective wound healing, and habitual abortion.
Beta adaptin The beta adaptin subunit is part of the clathrin coat assembly complex which links clathrin to receptors in coated pits and vesicles. These vesicles are involved in endocytosis and Golgi processing. The beta 1 subunit is one ofthe assembly proteins which binds to clathrin and initiates coat formation.
Transforming growth factor-beta 1 (TGFB1)
Transforming growth factor-beta 1; regulates cell proliferation, differentiation, and apoptosis
Transforming growth factor-beta 3 (TGFB3) Transforming growth factor-beta 3; transmits signals through transmembrane serine/- threonine kinases, may be required for normal development of the lung and palate; member of family of cytokines, very strongly similar to murine Tgfb3.
Flavin containing monooxygenase 1 (FMO1) Metabolic N-oxidation ofthe diet-derived amino-trimethylamine (TMA) is mediated by flavin-containing monooxygenase and is subject to an inherited FMO3 poly- moφhism in man resulting in a small subpopulation with reduced TMA N-oxidation capacity resulting in fish odor syndrome Trimethylaminuria. Three forms of the enzyme, FMO1 found in fetal liver, FMO2 found in adult liver, and FMO3 are encoded by genes clustered in the Iq23-q25 region. Flavin-containing monooxy- genases are NADPH-dependent flavoenzymes that catalyzes the oxidation of soft nucleophilic heteroatom centers in drugs, pesticides, and xenobiotics.
Coagulation factor IX (F9)
This gene encodes vitamin K-dependent coagulation factor IX that circulates in the blood as an inactive zymogen. This factor is converted to an active form by factor
Xla, which excises the activation peptide and thus generates a heavy chain and a light chain held together by one or more disulfide bonds. The role of this activated factor IX in the blood coagulation cascade is to activate factor X to its active form through interactions with Ca+2 ions, membrane phospholipids, and factor VIII. Alterations of this gene, including point mutations, insertions and deletions, cause factor IX deficiency, which is a recessive X-linked disorder, also called hemophilia B or Christmas disease.
Apical protein, Xenopus laevis-like (APXL) May be an amiloride-sensitive sodium channel; similar to Xenopus laevis Apical
Protein
As SNPs are linked to other SNPs in neighboring genes on a chromosome (Linkage Disequilibrium) those SNPs could also be used as marker SNPs. In a recent publication it was shown that SNPs are linked over 100 kb in some cases more than
150 kb (Reich D.E. et al. Nature 411, 199-204, 2001). Hence SNPs lying in regions neighbouring CA SNPs could be linked to the latter and by this being a CVD marker. These associations could be performed as described for the gene polymoφhism in methods. Definitions
For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. Moreover, the definitions by itself are intended to explain a further background ofthe invention.
The term "allele", which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.
The term "allelic variant of a polymoφhic region of a gene" refers to a region of a gene having one of several nucleotide sequences found in that region of the gene in other individuals.
"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for puφoses of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function ofthe number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40%) identity, though preferably less than 25% identity, with one of the sequences ofthe present invention.
The term "a homologue of a nucleic acid" refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof. A homologue of a double stranded nucleic acid having SEQ ID NO. X is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with SEQ ID NO. X or with the complement thereof. Prefened homologous of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.
The term "interact" as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a hybridization assay.
The term interact is also meant to include "binding" interactions between molecules.
Interactions may be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature.
The term "intronic sequence" or "intronic nucleotide sequence" refers to the nucleo- tide sequence of an intron or portion thereof.
The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occuning as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
The term "lipid" shall refer to a fat or fat-like substance that is insoluble in polar solvents such as water. The term "lipid" is intended to include true fats (e.g. esters of fatty acids and glycerol); lipids (phospholipids, cerebrosides, waxes); sterols (cholesterol, ergosterol) and lipoproteins (e.g. HDL, LDL and VLDL).
The term "locus" refers to a specific position in a chromosome. For example, a locus of a gene refers to the chromosomal position ofthe gene.
The term "modulation" as used herein refers to both up-regulation, (i.e., activation or stimulation), for example by agonizing, and down-regulation (i.e. inhibition or suppression), for example by antagonizing of a bioactivity (e.g. expression of a gene).
The term "molecular structure" of a gene or a portion thereof refers to the structure as defined by the nucleotide content (including deletions, substitutions, additions of one or more nucleotides), the nucleotide sequence, the state of methylation, and/or any other modification ofthe gene or portion thereof.
The term "mutated gene" refers to an allelic form of a gene, which is capable of altering the phenotype of a subject having the mutated gene relative to a subject which does not have the mutated gene. If a subject must be homozygous for this mutation to have an altered phenotype, the mutation is said to be recessive. If one copy of the mutated gene is sufficient to alter the genotype of the subject, the mutation is said to be dominant. If a subject has one copy of the mutated gene and has a phenotype that is intermediate between that of a homozygous and that of a heterozygous (for that gene) subject, the mutation is said to be co-dominant.
As used herein, the term "nucleic acid" refers to polynucleotides such as deoxy- ribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, including peptide nucleic acids (PNA), moφholino oligonucleotides (J. Summerton and D. Weller, Antisense and Nucleic Acid Drug Development 7:187 (1997)) and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For puφoses of clarity, when refening herein to a nucleotide of a nucleic acid, which can be DNA or an RNA, the term "adenosine", "cytidine", "guanosine", and "fhymidine" are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.
The term "nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO. x" refers to the nucleotide sequence of the complementary strand of a nucleic acid strand having SEQ ID NO. x. The term "complementary strand" is used herein interchangeably with the term "complement". The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non- coding strand. When refening to double stranded nucleic acids, the complement of a nucleic acid having SEQ ID NO. x refers to the complementary strand of the strand having SEQ ID NO. x or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID NO. x. When refening to a single stranded nucleic acid having the nucleotide sequence SEQ ID NO. x, the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID NO. x. The nucleotide sequences and complementary sequences thereof are always given in the 5' to 3' direction. The term "complement" and "reverse comple- ment" are used interchangeably herein.
The term "operably linked" is intended to mean that the promoter is associated with the nucleic acid in such a manner as to facilitate transcription ofthe nucleic acid.
The term "polymoφhism" refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is refened to as a "polymoφhic region of a gene". A polymoφhic region can be a single nucleotide, the identity of which differs in different alleles. A polymoφhic region can also be several nucleotides long.
A "polymoφhic gene" refers to a gene having at least one polymoφhic region. To describe a "polymoφhic site" in a nucleotide sequence often there is used an "ambiguity code" that stands for the possible variations of nucleotides in one site. The list of ambiguity codes is summarized in the following table:
So, for example, a "R" in a nucleotide sequence means that either an "a" or a "g" could be at that position.
The terms "protein", "polypeptide" and "peptide" are used interchangeably herein when refening to a gene product.
A "regulatory element", also termed herein "regulatory sequence is intended to include elements which are capable of modulating transcription from a basic promoter and include elements such as enhancers and silencers. The term "enhancer", also refened to herein as "enhancer element", is intended to include regulatory elements capable of increasing, stimulating, or enhancing transcription from a basic promoter. The term "silencer", also refened to herein as "silencer element" is intended to include regulatory elements capable of decreasing, inhibiting, or repressing transcription from a basic promoter. Regulatory elements are typically present in 5' flanking regions of genes. However, regulatory elements have also been shown to be present in other regions of a gene, in particular in introns. Thus, it is possible that genes have regulatory elements located in introns, exons, coding regions, and 3' flanking sequences. Such regulatory elements are also intended to be encompassed by the present invention and can be identified by any of the assays that can be used to identify regulatory elements in 5' flanking regions of genes.
The term "regulatory element" further encompasses "tissue specific" regulatory elements, i.e., regulatory elements which effect expression of the selected DNA sequence preferentially in specific cells (e.g., cells of a specific tissue), gene expression occurs preferentially in a specific cell if expression in this cell type is significantly higher than expression in other cell types. The term "regulatory element" also encompasses non-tissue specific regulatory elements, i.e., regulatory elements which are active in most cell types. Furthermore, a regulatory element can be a constitutive regulatory element, i.e., a regulatory element which constitutively regulates transcription, as opposed to a regulatory element which is inducible, i.e., a regulatory element which is active primarily in response to a stimulus. A stimulus can be, e.g., a molecule, such as a hormone, cytokine, heavy metal, phorbol ester, cyclic AMP (cAMP), or retinoic acid.
Regulatory elements are typically bound by proteins, e.g., transcription factors. The term "transcription factor" is intended to include proteins or modified forms thereof, which interact preferentially with specific nucleic acid sequences, i.e., regulatory elements, and which in appropriate conditions stimulate or repress transcription.
Some transcription factors are active when they are in the form of a monomer.
Alternatively, other transcription factors are active in the form of a dimer consisting of two identical proteins or different proteins (heterodimer). Modified forms of transcription factors are intended to refer to transcription factors having a post- translational modification, such as the attachment of a phosphate group. The activity of a transcription factor is frequently modulated by a post-translational modification. For example, certain transcription factors are active only if they are phosphorylated on specific residues. Alternatively, transcription factors can be active in the absence of phosphorylated residues and become inactivated by phosphorylation. A list of known transcription factors and their DNA binding site can be found, e.g., in public databases, e.g., TFMATRIX Transcription Factor Binding Site Profile database.
As used herein, the term "specifically hybridizes" or "specifically detects" refers to the ability of a nucleic acid molecule of the invention to hybridize to at least approximately 6, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or 140 consecutive nucleotides of either strand of a gene.
The term "wild-type allele" refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
"Candidate gene" as used herein includes genes that can be assigned to either normal cardiovascular function or to metabolic pathways that are related to onset and/or progression of cardiovascular diseases. As the development of cardiovascular diseases is not completely understood, the term "candidate gene" may also comprise genes with presently unknown function.
"CA SNP" (cardiovascular disease associated SNP) refers to a polymoφhic site which shows a significant association with the risk to develop cardiovascular diseases.
"CA gene" (cardiovascular disease associated gene) refers to a genomic locus harbouring a CA SNP, inespective ofthe actual function of this gene locus. CA gene polypeptide refers to a polypeptide encoded at least in part by a CA gene
Methods for Assessing Cardiovascular Status
The present invention provides diagnostic methods for assessing cardiovascular status in a human individual. Cardiovascular status as used herein refers to the physiological status of an individual's cardiovascular system as reflected in one or more markers or indicators. Status markers include without limitation clinical measurements such as, e.g., blood pressure, electrocardiographic profile, and differentiated blood flow analysis as well as measurements of LDL- and HDL- Cholesterol levels, other lipids and other well established clinical parameters that are standard in the art. Status markers according to the invention include diagnoses of one or more cardiovascular syndromes, such as, e.g., hypertension, acute myocardial infarction, silent myocardial infarction, stroke, and atherosclerosis. It will be understood that a diagnosis of a cardiovascular syndrome made by a medical practitioner encompasses clinical measurements and medical judgement. Status markers according to the invention are assessed using conventional methods well known in the art. Also included in the evaluation of cardiovascular status are quantitative or qualitative changes in status markers with time, such as would be used, e.g., in the determination of an individual's response to a particular therapeutic regimen.
The methods are caπied out by the steps of:
(i) determining the sequence of one or more polymoφhic positions within one, sseveral or all of the genes listed in Examples or other genes mentioned in this file in the individual to establish a polymoφhic pattern for the individual; and (ii) comparing the polymoφhic pattern established in (i) with the polymoφhic patterns of humans exhibiting different markers of cardiovascular status. The polymoφhic pattern of the individual is, preferably, highly similar and, most preferably, identical to the polymoφhic pattern of individuals who exhibit particular status markers, cardiovascular syndromes, and/or particular patterns of response to therapeutic interventions. Polymoφhic patterns may also include polymoφhic positions in other genes which are shown, in combination with one or more polymoφhic positions in the genes listed in the Examples, to conelate with the presence of particular status markers. In one embodiment, the method involves comparing an individual's polymoφhic pattern with polymoφhic patterns of individuals who have been shown to respond positively or negatively to a particular therapeutic regimen. Therapeutic regimen as used herein refers to treatments aimed at the elimination or amelioration of symptoms and events associated cardiovascular disease. Such treatments include without limitation one or more of alteration in diet, lifestyle, and exercise regimen; invasive and noninvasive surgical techniques such as atherectomy, angioplasty, and coronary bypass surgery; and pharmaceutical interventions, such as administration of ACE inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antago- nists, cardiac glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor antagonists, calcium channel blockers, HMG-CoA reductase inhibitors, imidazoline receptor blockers, endothelin receptor blockers, organic nitrites, and modulators of protein function of genes listed in the Examples. Interventions with pharmaceutical agents not yet known whose activity conelates with particular polymoφhic patterns associated with cardiovascular disease are also encompassed. It is contemplated, for example, that patients who are candidates for a particular therapeutic regimen will be screened for polymoφhic patterns that conelate with responsivity to that particular regimen. In a prefened embodiment, the method involves comparing an individual's polymoφhic pattern with polymoφhic patterns of individuals who exhibit or have exhibited one or more markers of cardiovascular disease, such as, e.g., elevated LDL- Cholesterol levels, high blood pressure, abnormal electrocardiographic profile, myocardial infarction, stroke, or atherosclerosis.
In practicing the methods ofthe invention, an individual's polymoφhic pattern can be established by obtaining DNA from the individual and determining the sequence at predetermined polymoφhic positions in the genes such as those described in this file.
The DNA may be obtained from any cell source. Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Cells may also be obtained from body fluids, including without limitation blood, saliva, sweat, urine, cerebrospinal fluid, feces, and tissue exudates at the site of infection or inflammation. DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source.
Diagnostic and Prognostic Assays
The present invention provides methods for determining the molecular structure of at least one polymoφhic region of a gene, specific allelic variants of said polymoφhic region being associated with cardiovascular disease. In one embodiment, determining the molecular structure of a polymoφhic region of a gene comprises determining the identity of the allelic variant. A polymoφhic region of a gene, of which specific alleles are associated with cardiovascular disease can be located in an exon, an intron, at an intron/exon border, or in the promoter ofthe gene. The invention provides methods for determining whether a subject has, or is at risk, of developing a cardiovascular disease. Such disorders can be associated with an abenant gene activity, e.g., abnormal binding to a form of a lipid, or an abenant gene protein level. An abenant gene protein level can result from an abenant transcription or post-transcriptional regulation. Thus, allelic differences in specific regions of a gene can result in differences of gene protein due to differences in regulation of expression. In particular, some of the identified polymoφhisms in the human gene may be associated with differences in the level of transcription, RNA maturation, splicing, or translation ofthe gene or transcription product.
In prefened embodiments, the methods of the invention can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a specific allelic variant of one or more polymoφhic regions of a gene. The allelic differences can be: (i) a difference in the identity of at least one nucleotide or (ii) a difference in the number of nucleotides, which difference can be a single nucleotide or several nucleotides.
A prefened detection method is allele specific hybridization using probes overlapping the polymoφhic site and having about 5, 10, 20, 25, or 30 nucleotides around the polymoφhic region. Examples of probes for detecting specific allelic variants of the polymoφhic region located in intron X are probes comprising a nucleotide sequence set forth in any of SEQ ID NO. X. In a prefened embodiment of the invention, several probes capable of hybridizing specifically to allelic variants are attached to a solid phase support, e.g., a "chip". Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. For example a chip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix). Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe anays" is described e.g., in Cronin et al. (1996) Human Mutation 7:244 and in Kozal et al. (1996) Nature Medicine 2:753. In one embodiment, a chip comprises all the allelic variants of at least one polymoφhic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment. For example, the identity of the allelic variant of the nucleotide polymoφhism of nucleotide T or G at position 140 of Seq ID 1 (baySNP10948) and that of other possible polymoφhic regions can be determined in a single hybridization experiment.
In other detection methods, it is necessary to first amplify at least a portion of a gene prior to identifying the allelic variant. Amplification can be performed, e.g., by PCR and/or LCR, according to methods known in the art. In one embodiment, genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA. In prefened embodiments, the primers are located between 40 and 350 base pairs apart. Prefened primers for amplifying gene fragments of genes of this file are listed in Table 2 in the Examples.
Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In one embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of a gene and detect allelic variants, e.g., mutations, by comparing the sequence of the sample sequence with the conesponding wild-type (control) sequence. Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example, U.S. Pat. No. 5,547,835 and international patent application Publication Number WO 94/16101, entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S. Pat. No. 5,547,835 and international patent application Publication Number WO 94/21822 entitled "DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation" by H. Koster), and U.S. Pat. No. 5,605,798 and International Patent Application No. PCT/US96/03651 entitled DNA Diagnostics Based on Mass Spectrometry by H. Koster; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident to one skilled in the art that, for certain embodiments, the occunence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleotide is detected, can be canied out.
Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No. 5,580,732 entitled
"Method of DNA sequencing employing a mixed DNA-polymer chain probe" and U.S. Pat. No. 5,571,676 entitled "Method for mismatch-directed in vitro DNA sequencing".
In some cases, the presence of a specific allele of a gene in DNA from a subject can be shown by restriction enzyme analysis. For example, a specific nucleotide polymoφhism can result in a nucleotide sequence comprising a restriction site which is absent from the nucleotide sequence of another allelic variant.
In other embodiments, alterations in eiectrophoretic mobility is used to identify the type of gene allelic variant. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in eiectrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in eiectrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In another prefened embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in eiectrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
In yet another embodiment, the identity of an allelic variant of a polymoφhic region is obtained by analyzing the movement of a nucleic acid comprising the polymoφhic region in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately
40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).
Examples of techniques for detecting differences of at least one nucleotide between 2 nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes may be prepared in which the known polymoφhic nucleotide is placed centrally (allele-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such allele specific oligonucleotide hybridization techniques may be used for the simultaneous detection of several nucleotide changes in different polymoφhic regions of gene. For example, oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid.
Alternatively, allele specific amplification technology which depends on selective
PCR amplification may be used. Oligonucleotides used as primers for specific amplification may carry the allelic variant of interest in the center ofthe molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension
(Prossner (1993) Tibtech 11 :238; Newton et al. (1989) Nucl. Acids Res. 17:2503). This technique is also termed "PROBE" for Probe Oligo Base Extension. In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al (1992) Mol. Cell Probes 6:1).
In another embodiment, identification of the allelic variant is canied out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science 241:1077-1080 (1988). The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is linked to a separation marker, e.g,. biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
Several techniques based on this OLA method have been developed and can be used to detect specific allelic variants of a polymoφhic region of a gene. For example, U.S. Pat. No. 5,593,826 discloses an OLA using an oligonucleotide having 3'-amino group and a 5 '-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage. In another variation of OLA described in Tobe et al. ((1996)Nucleic Acids Res 24: 3728), OLA combined with PCR permits typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each LA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.
The invention further provides methods for detecting single nucleotide polymoφhisms in a gene. Because single nucleotide polymoφhisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation and it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymoφhisms.
In one embodiment, the single base polymoφhism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, a primer complementary to the allelic sequence immediately 3' to the polymoφhic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymoφhic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incoφorated onto the end of the hybridized primer. Such incoφoration renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymoφhic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.
In another embodiment of the invention, a solution-based method is used for deter- mining the identity of the nucleotide of a polymoφhic site. Cohen, D. et al. (French
Patent 2,650,840; PCT Appln. No. WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3' to a polymoφhic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymoφhic site will become incoφorated onto the terminus ofthe primer.
An alternative method, known as Genetic Bit Analysis or GBA TM is described by Goelet, P. et al. (PCT Appln. No. 92/15712). The method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymoφhic site. The labeled terminator that is incoφorated is thus determined by, and complementary to, the nucleotide present in the polymoφhic site ofthe target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
Recently, several primer-guided nucleotide incoφoration procedures for assaying polymoφhic sites in DNA have been described (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990);
Syvanen, A. -C, et al., Genomics 8:684-692 (1990), Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al. Hum. Mutat. 1 :159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al. Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA TM in that they all rely on the incoφoration of labeled deoxynucleotides to discriminate between bases at a polymoφhic site. In such a format, since the signal is proportional to the number of deoxynucleotides incoφorated, polymoφhisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A.-C, et al, Amer. J. Hum. Genet. 52:46-59 (1993)).
For determining the identity of the allelic variant of a polymoφhic region located in the coding region of a gene, yet other methods than those described above can be used. For example, identification of an allelic variant which encodes a mutated gene protein can be performed by using an antibody specifically recognizing the mutant protein in, e.g, immunohistochemistry or immunoprecipitation. Antibodies to wild- type gene protein are described, e.g, in Acton et al. (1999) Science 271 :518 (anti- mouse gene antibody cross-reactive with human gene). Other antibodies to wild-type gene or mutated forms of gene proteins can be prepared according to methods known in the art. Alternatively, one can also measure an activity of an gene protein, such as binding to a lipid or lipoprotein. Binding assays are known in the art and involve, e.g, obtaining cells from a subject, and performing binding experiments with a labeled lipid, to determine whether binding to the mutated form of the receptor differs from binding to the wild-type ofthe receptor.
If a polymoφhic region is located in an exon, either in a coding or non-coding region of the gene, the identity of the allelic variant can be determined by determining the molecular structure ofthe mRNA, pre-mRNA, or cDNA. The molecular structure can be determined using any of the above described methods for determining the molecular structure ofthe genomic DNA, e.g, sequencing and SSCP.
The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits, such as those described above, comprising at least one probe or primer nucleic acid described herein, which may be conveniently used, e.g, to determine whether a subject has or is at risk of developing a disease associated with a specific gene allelic variant. Sample nucleic acid for using in the above-described diagnostic and prognostic methods can be obtained from any cell type or tissue of a subject. For example, a subject's bodily fluid (e.g. blood) can be obtained by known techniques (e.g. venipuncture) or from human tissues like heart (biopsies, transplanted organs). Alternatively, nucleic acid tests can be performed on dry samples (e.g. hair or skin).
Fetal nucleic acid samples for prenatal diagnostics can be obtained from maternal blood as described in International Patent Application No. WO91/07660 to Bianchi. Alternatively, amniocytes or chorionic villi may be obtained for performing prenatal testing.
Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J, 1992, PCR in situ hybridization: protocols and applications, Raven Press, New York).
In addition to methods which focus primarily on the detection of one nucleic acid sequence, profiles may also be assessed in such detection schemes. Fingeφrint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
In practicing the present invention, the distribution of polymoφhic patterns in a large number of individuals exhibiting particular markers of cardiovascular status is determined by any of the methods described above, and compared with the distribution of polymoφhic patterns in patients that have been matched for age, ethnic origin, and/or any other statistically or medically relevant parameters, who exhibit quantitatively or qualitatively different status markers. Conelations are achieved using any method known in the art, including nominal logistic regression, chi square tests or standard least squares regression analysis. In this manner, it is possible to establish statistically significant conelations between particular polymoφhic patterns and particular cardiovascular statuses (given in p values). It is further possible to establish statistically significant conelations between particular polymoφhic patterns and changes in cardiovascular status such as, would result, e.g, from particular treatment regimens. In this manner, it is possible to conelate polymoφhic patterns with responsivity to particular treatments.
Isolated Polymoφhic Nucleic Acids. Probes, and Vectors
The present invention provides isolated nucleic acids comprising the polymoφhic positions described herein for human genes; vectors comprising the nucleic acids; and transformed host cells comprising the vectors. The invention also provides probes which are useful for detecting these polymoφhisms.
In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA, are used. Such techniques are well known and are explained fully in, for example, Sambrook et al, 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L.Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Ausubel et al, Cunent Protocols in Molecular Biology, 1997, (John Wiley and Sons); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds, respectively).
Insertion of nucleic acids (typically DNAs) comprising the sequences in a functional surrounding like full length cDNA of the present invention into a vector is easily accomplished when the termini of both the DNAs and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini ofthe DNAs and/or vector by digesting back single-stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerase. Alternatively, any site desired may be produced, e.g, by ligating nucleotide sequences (linkers) onto the termini. Such linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated by the use of the polymerase chain reaction (PCR). See, e.g, Saiki et al,
1988, Science 239:48. The cleaved vector and the DNA fragments may also be modified if required by homopolymeric tailing.
The nucleic acids may be isolated directly from cells or may be chemically synthesized using known methods. Alternatively, the polymerase chain reaction
(PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates. Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate incoφoration into a given vector for recombinant expression.
The nucleic acids of the present invention may be flanked by native gene sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like. The 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 occuning nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, moφholines 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. Further- more, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
The invention also provides nucleic acid vectors comprising the gene sequences or derivatives or fragments thereof of genes described in the Examles. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple cloning or protein expression. Non-limiting examples of suitable vectors include without limitation pUC plasmids, pET plasmids
(Novagen, Inc, Madison, Wis.), or pRSET or pREP (Invitrogen, San Diego, Calif), 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 ofthe invention.
Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl2 mediated DNA uptake, fungal or viral infection, microinjection, microprojectile, or other established methods. Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced peptides and polypeptides encoded by genes of the Examples. Nucleic acids encoding peptides or polypeptides from gene sequences of the Examples may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene. Other recombination-based methods such as non- homologous recombinations or deletion of endogenous genes by homologous recombination may also be used.
In case of proteins that form heterodimers or other multimers, both or all subunits have to be expressed in one system or cell.
The nucleic acids of the present invention find use as probes for the detection of genetic polymoφhisms and as templates for the recombinant production of normal or variant peptides or polypeptides encoded by genes listed in the Examples.
Probes in accordance with the present invention comprise without limitation isolated nucleic acids of about 10-100 bp, preferably 15-75 bp and most preferably 17-25 bp in length, which hybridize at high stringency to one or more of the polymoφhic sequences disclosed herein or to a sequence immediately adjacent to a polymoφhic position. Furthermore, in some embodiments a full-length gene sequence may be used as a probe. In one series of embodiments, the probes span the polymoφhic positions in genes disclosed herein. In another series of embodiments, the probes conespond to sequences immediately adjacent to the polymoφhic positions.
Polymorphic Polypeptides and Polymorphism-Specific Antibodies
The present invention encompasses isolated peptides and polypeptides encoded by genes listed in the Examples comprising polymoφhic positions disclosed herein. In one prefened embodiment, the peptides and polypeptides are useful screening targets to identify cardiovascular drugs. In another prefened embodiments, the peptides and polypeptides are capable of eliciting antibodies in a suitable host animal that react specifically with a polypeptide comprising the polymoφhic position and distinguish it from other polypeptides having a different 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 poly- peptides are described below. Many conventional techniques in protein biochemistry and immunology are used. Such techniques are well known and are explained in Immunochemical Methods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press, London); Scopes, 1987, Protein Purification: Principles and Practice, Second Edition (Springer- Verlag, N.Y.) and Handbook of Experimental
Immunology, 1986, Volumes I-IV (Weir and Blackwell eds.).
Nucleic acids comprising protein-coding sequences can be used to direct the ITT recombinant expression of polypeptides encoded by genes disclosed herein in intact cells or in cell-free translation systems. The known genetic code, tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The polypeptides may be isolated from human cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an appropriate protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins.
Peptides and polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
The polypeptides are preferably prepared by solid phase peptide synthesis as described by Menifield, 1963, J. Am. Chem. Soc. 85:2149.
Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed- phase HPLC, gel filtration, ion exchange and partition chromatography, and counter- cunent distribution. For some puφoses, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against peptides encoded by genes disclosed herein, can be used as purification reagents. Other purification methods are possible.
The present invention also encompasses derivatives and homologues of the poly- peptides. For some puφoses, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e, function-conservative variants. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids.
The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.
The present invention also encompasses antibodies that specifically recognize the polymoφhic positions of the invention and distinguish a peptide or polypeptide containing a particular polymoφhism from one that contains a different sequence at that position. Such polymoφhic position-specific antibodies according to the present invention include polyclonal and monoclonal antibodies. The antibodies may be elicited in an animal host by immunization with peptides encoded by genes disclosed herein or may be formed by in vitro immunization of immune cells. The immuno- genie components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems. The antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA. Alternatively, the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains. The antibodies include hybrid antibodies (i.e, containing two sets of heavy chain/light chain combinations, each of which recognizes a different antigen), chimeric antibodies (i.e, in which either the heavy chains, light chains, or both, are fusion proteins), and univalent antibodies (i.e, comprised of a heavy chain/light chain complex bound to the constant region of a second heavy chain). Also included are Fab fragments, including Fab' and F(ab).sub.2 fragments of antibodies. Methods for the production of all of the above types of antibodies and derivatives are well-known in the art and are discussed in more detail below. For example, techniques for producing and processing polyclonal antisera are disclosed in Mayer and Walker, 1987, Immunochemical Methods in Cell and Molecular Biology, (Academic Press, London). The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Ban vims. See, e.g, Schreier et al, 1980, Hybridoma Techniques; U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against peptides encoded by genes disclosed herein 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 countercunent distribution. Purification methods for antibodies are disclosed, e.g, in The Art of Antibody Purification, 1989, Amicon Division, W. R. Grace & Co. General protein purification methods are described in Protein Purification: Principles and Practice, R. K. Scopes, Ed, 1987, Springer-Verlag, New York, N.Y.
Methods for determining the immunogenic capability of the disclosed sequences and the characteristics of the resulting sequence-specific antibodies and immune cells are well-known in the art. For example, antibodies elicited in response to a peptide comprising a particular polymoφhic sequence can be tested for their ability to specifically recognize that polymoφhic sequence, i.e, to bind differentially to a peptide or polypeptide comprising the polymoφhic sequence and thus distinguish it from a similar peptide or polypeptide containing a different sequence at the same position.
Kits
As set forth herein, the invention provides diagnostic methods, e.g, for determining the identity of the allelic variants of polymoφhic regions present in the gene loci of genes disclosed herein, wherein specific allelic variants of the polymoφhic region are associated with cardiovascular diseases. In a prefened embodiment, the diagnostic kit can be used to detennine whether a subject is at risk of developing a cardiovascular disease. This information could then be used, e.g, to optimize treatment of such individuals.
In prefened embodiments, the kit comprises a probe or primer which is capable of hybridizing to a gene and thereby identifying whether the gene contains an allelic variant of a polymoφhic region which is associated with a risk for cardiovascular disease. The kit preferably further comprises instructions for use in diagnosing a subject as having, or having a predisposition, towards developing a cardiovascular disease. The probe or primers of the kit can be any of the probes or primers described in this file.
Prefened kits for amplifying a region of a gene comprising a polymoφhic region of interest comprise one, two or more primers.
Antibody-based diagnostic methods and kits
The invention also provides antibody-based methods for detecting polymoφhic patterns in a biological sample. The methods comprise the steps of: (i) contacting a sample with one or more antibody preparations, wherein each of the antibody preparations is specific for a particular polymoφhic form of the proteins encoded by genes disclosed herein, under conditions in which a stable antigen-antibody complex can form between the antibody and antigenic components in the sample; and (ii) detecting any antigen-antibody complex formed in step (i) using any suitable means known in the art, wherein the detection of a complex indicates the presence of the particular polymoφhic form in the sample.
Typically, immunoassays use either a labelled antibody or a labelled antigenic component (e.g, that competes with the antigen in the sample for binding to the antibody). Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labelled immunoassays, such as ELISA assays.
The present invention also provides kits suitable for antibody-based diagnostic applications. Diagnostic kits typically include one or more of the following compo- nents:
(i) Polymoφhism-specific antibodies. The antibodies may be pre-labelled; alternatively, the antibody may be unlabelled and the ingredients for labelling may be included in the kit in separate containers, or a secondary, labelled antibody is provided; and
(ii) Reaction components: The kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.
The kits refened to above may include instructions for conducting the test. Furthermore, in prefened embodiments, the diagnostic kits are adaptable to high- throughput and/or automated operation. Drug Targets and Screening Methods
According to the present invention, nucleotide sequences derived from genes disclosed herein and peptide sequences encoded by genes disclosed herein, particularly those that contain one or more polymoφhic sequences, comprise useful targets to identify cardiovascular drugs, i.e, compounds that are effective in treating one or more clinical symptoms of cardiovascular disease. Furthermore, especially when a protein is a multimeric protein that are build of two or more subunits, is a combination of different polymoφhic subunits very useful.
Drug targets include without limitation (i) isolated nucleic acids derived from the genes disclosed herein, and (ii) isolated peptides and polypeptides encoded by genes disclosed herein, each of which comprises one or more polymoφhic positions.
In vitro screening methods
In one series of embodiments, an isolated nucleic acid comprising one or more polymoφhic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner. The methods comprise:
(i) providing a first nucleic acid containing a particular sequence at a polymoφhic position and a second nucleic acid whose sequence is identical to that of the first nucleic acid except for a different sequence at the same polymoφhic position;
(ii) contacting the nucleic acids with a multiplicity of test compounds under conditions appropriate for binding; and
(iii) identifying those compounds that bind selectively to either the first or second nucleic acid sequence. Selective binding as used herein refers to any measurable difference in any parameter of binding, such as, e.g, binding affinity, binding capacity, etc.
In another series of embodiments, an isolated peptide or polypeptide comprising one or more polymoφhic positions is tested in vitro for its ability to bind test compounds in a sequence-specific manner. The screening methods involve:
(i) providing a first peptide or polypeptide containing a particular sequence at a polymoφhic position and a second peptide or polypeptide whose sequence is identical to the first peptide or polypeptide except for a different sequence at the same polymoφhic position;
(ii) contacting the polypeptides with a multiplicity of test compounds under conditions appropriate for binding; and
(iii) identifying those compounds that bind selectively to one of the nucleic acid sequences.
In prefened embodiments, high-throughput screening protocols are used to survey a large number of test compounds for their ability to bind the genes or peptides disclosed above in a sequence-specific manner.
Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Menimack, N.H.), and Micro- source (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories
(Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
In vivo screening methods
Intact cells or whole animals expressing polymoφhic variants of genes disclosed herein can be used in screening methods to identify candidate cardiovascular drugs.
In one series of embodiments, a permanent cell line is established from an individual exhibiting a particular polymoφhic pattern. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are programmed to express a gene comprising one or more polymoφhic sequences by introduction of appropriate
DNA. Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to particular polymoφhic variants of proteins encoded by genes disclosed herein; (ii) assays that measure the ability of a test compound to modify
(i.e, inhibit or enhance) a measurable activity or function of proteins encoded by genes disclosed herein; 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 of genes disclosed herein.
In another series of embodiments, transgenic animals are created in which (i) one or more human genes disclosed herein, having different sequences at particular polymoφhic positions are stably inserted into the genome of the transgenic animal; and/or (ii) the endogenous genes disclosed herein are inactivated and replaced with human genes disclosed herein, having different sequences at particular polymoφhic positions. See, e.g, Coffman, Semin. Nephrol. 17:404, 1997; Esther et al. Lab. Invest. 74:953, 1996; Murakami et al. Blood Press. Suppl. 2:36, 1996. Such animals can be treated with candidate compounds and monitored for one or more clinical markers of cardiovascular status . The following are intended as non-limiting examples ofthe invention.
Material and Methods
Genotyping of patient DNA with the Pyrosequencing™ Method as described in the patent application WO 9813523:
First a PCR is set up to amplify the flanking regions around a SNP. Therefor 2 ng of genomic DNA (patient sample) are mixed with a primerset (20 - 40 pmol) producing a 75 to 320 bp PCR fragment with 0,3 to 1 U Qiagens Hot Star Taq Polymerase™ in a total volume of 20 μL. One primer is biotinylated depending on the direction of the sequencing primer. To force the biotinylated primer to be incoφorated it is used 0,8 fold.
For primer design, programms like Oligo 6™ (Molecular Biology Insights) or Primer
Select™ (DNAStar) are used. PCR setup is performed by a BioRobot 3000 ™ from Qiagen. PCR takes place in TI or Tgradient Thermocyclers ™ from Biometra.
The whole PCR reaction is transfened into a PSQ plate ™ (Pyrosequencing) and
T A *V A prepared using the Sample Prep Tool and SNP Reagent Kit from Pyrosequencing according to their instructions.
Preparation of template for Pyrosequencing™:
Sample preparation using PSQ 96 Sample Prep Tool
1. Mount the PSQ 96 Sample Prep Tool Cover onto the PSQ 96 Sample Prep
Tool as follows: Place the cover on the desk, retract the 4 attachment rods by separating the handle from the magnetic rod holder, fit the magnetic rods into the holes of the cover plate, push the handle downward until a click is heard.
The PSQ 96 Sample Prep Tool is now ready for use. 2. To transfer beads from one plate to another, place the covered tool into the PSQ 96 Plate containing the samples and lower the magnetic rods by separating the handle from the magnetic rod holder. Move the tool up and down a few times then wait for 30-60 seconds. Transfer the beads into a new PSQ 96 plate containing the solution of choice.
3. Release the beads by lifting the magnetic rod holder, bringing it together with the handle. Move the tool up and down a few times to make sure that the beads are released.
All steps are performed at room temperature unless otherwise stated.
Immobilization of PCR product:
Biotinylated PCR products are immobilized on streptavidin-coated Dynabeads™ M-
280 Streptavidin. Parallel immobilization of several samples are performed in the PSQ 96 Plate.
1. Mix PCR product, 20 μl of a well optimized PCR, with 25 μl 2X BW-buffer II. Add 60-150 μg Dynabeads. It is also possible to add a mix of Dynabeads and 2X BW-buffer II to the PCR product yielding a final BW-buffer II concentration of approximately lx.
2. Incubate at 65°C for 15 min agitation constantly to keep the beads dispersed. For optimal immobilization of fragments longer than 300 bp use 30 min incubation time.
Strand separation
4. For strand separation, use the PSQ 96 Sample Prep Tool to transfer the beads with the immobilized sample to a PSQ 96 Plate containing 50 μl 0.50 M NaOH per well. Release the beads. 5. After approximately 1 min, transfer the beads with the immobilized strand to a PSQ 96 Plate containing 99 μl lx Annealing buffer per well and mix thoroughly.
6. Transfer the beads to a PSQ 96 Plate containing 45 μl of a mix of lx Annealing buffer and 3-15 pmoles sequencing primer per well.
7. Heat at 80°C for 2 minutes in the PSQ 96 Sample Prep Thermoplate and move to room temperature.
8. After reaching room temperature, continue with the sequencing reaction.
Sequencing reaction
1. Choose the method to be used ("SNP Method") and enter relevant information in the PSQ 96 Instrument Control software.
2. Place the cartridge and PSQ 96 Plate in the PSQ 96 Instrument.
3. Start the run.
Genotyping with a service contractor
Qiagen Genomics, formerly Rapigene, is a service contractor for genotyping SNPs in patient samples. Their method is based on a primer extension method where two complementary primers are designed for each genotype that are labeled with different tags. Depending on the genotype only one primer will be elongated together with a certain tag. This tag can be detected with mass spectrometry and is a measure for the respective genotype. The method is described in the following patent: "Detection and identification of nucleic acid molecules - using tags which may be detected by non- fluorescent spectrometry or potentiometry" (WO 9727325).
Examples
Table 1: Definition of "good" and "bad" serum lipid levels
An informed consent was signed by the patients and control people. Blood was taken by a physician according to medical standard procedures.
Samples were collected anonymous and labeled with a patient number.
DNA was extracted using kits from Qiagen.
Table 2a: Oligonucleotide primers used for genotyping using mass spectrometry
The baySNP number refers to an internal numbering of the CA SNPs. Primer sequences are listed for preamplification of the genomic fragments (primers EF and
ER) and for subsequent allele specific PCR ofthe SNP.
Table 2b: Oligonucleotide primers used for genotyping using Pyrosequencing
The baySNP number refers to an internal numbering of the CA SNPs. Primer sequences are listed for preamplification of the genomic fragments and for sequencing ofthe SNP using the pyrosequencing method.
Table 3: CA SNPs and putative CA genes
The baySNP number refers to an internal numbering of the CA SNPs. Listed are the different polymoφhisms found in our association study. Also accession numbers and descriptions of those gene loci are given that are most homologous to the CA genes as listed in the sequences section (see below). Homologous genes and their accession numbers could be found by those skilled in the art in the Genbank database.
Table 4: Cohorts
Given are names (as used in table 5) and formations ofthe various cohorts that were used for genotyping
COHORT Definition
HELD ALL GOOD/BAD Healthy elderly individuals of both genders with good or bad serum lipid profiles (as defined in table 1)
HELD FEM GOOD/BAD Healthy elderly individuals (female) with good or bad serum lipid profiles (as defined in table 1)
HELD MAL GOOD/BAD Healthy elderly individuals (male) with good or bad serum lipid profiles (as defined in table 1)
CVD ALL CASE/CTRL Individuals with diagnosis of cardiovascular disease and healthy controls (both genders)
CVD FEM CASE/CTRL individuals with diagnosis of cardiovascular disease and healthy controls (female)
CVD MAL CASE/CTRL Individuals with diagnosis of cardiovascular disease and healthy controls (male)
Table 5: Cohort sizes and p-values of CA SNPs
The baySNP number refers to an internal numbering of the CA SNPs. GTYPE P and A PVAL provide the p values obtained through chi square tests when comparing COHORTS A and B. For GTYPE P (p value of genotypes) the number of patients in cohort A carrying genotypes 11, 12 or 22 (FQ11 A, FQ 12 A, FQ 22 A) were compared with the respective patients in cohort B (FQ11 B, FQ 12 B, FQ 22 B) resulting in a chi square test with a 3x2 matrix. For A PVAL (p value of alleles) we compared the allele count of alleles 1 and 2 in cohorts A and B, respectively (chi square test with a 2x2 matrix). SIZE A and B: Number of patients in cohorts A and B, respectively. See table 4 for definition of COHORTS A and B.
we calculate
/ N11 + N12
RRl = Nl
N23/ N21 + N22
Here, the case and control populations represent any case-control-group pair, or bad(case)-good(control)-group pair, respectively. A value RR1>1, RR2>1, and RR3>1 indicates an increased risk for individuals carrying genotype 1, genotype 2, and genotype 3, respectively. For example, RR1=3 indicates a 3-fold risk of an individual carrying genotype 1 as compared to individuals carrying genotype 2 or 3 (a detailed description of relative risk calculation and statistics can be found in (Biostatistics, L. D. Fisher and G. van Belle, Wiley Interscience 1993)). The baySNP number refers to an internal numbering ofthe CA SΝPs and can be found in the sequence listing.

Claims

Claims
1. An isolated polynucleotide encoded by a cardiovascular associated (CA) gene; the polynucleotide is selected from the group comprising
SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 with allelic variation as indicated in the sequences section contained in a functional sunounding like full length cDNA for CA gene polypeptide and with or without the CA gene promoter sequence.
2. An expression vector containing one or more of the polynucleotides of claim
1.
3. A host cell containing the expression vector of claim 2.
4. A substantially purified CA gene polypeptide encoded by a polynucleotide of claim 1.
5. A method for producing a CA gene polypeptide, wherein the method comprises the following steps:
a) culturing the host cell of claim 3 under conditions suitable for the expression ofthe CA gene polypeptide; and
b) recovering the CA gene polypeptide from the host cell culture.
6. A method for the detection of a polynucleotide of claim 1 or a CA gene polypeptide of claim 4 comprising the steps of:
contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the CA gene polypeptide.
7. A method of screening for agents which regulate the activity of a CA gene comprising the steps of:
contacting a test compound with a CA gene polypeptide encoded by any polynucleotide of claim 1 ; and detecting CA gene activity of the polypeptide, wherein a test compound which increases the CA gene polypeptide activity is identified as a potential therapeutic agent for increasing the activity of the CA gene polypeptide and wherein a test compound which decreases the CA activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity ofthe CA gene polypeptide.
8. A reagent that modulates the activity of a CA polypeptide or a polynucleotide wherein said reagent is identified by the method ofthe claim 7.
9. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 8 and a pharmaceutically acceptable canier.
10. Use of the reagent according to claim 8 for the preparation of a medicament.
11. A method for determining whether a human subject has, or is at risk of developing a cardiovascular disease, comprising determining the identity of nucleotide variations as indicated in the sequences section of SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 of the CA gene locus of the subject; whereas a "risk" genotype has a risk ratio of greater than 1 as can be seen from table 6.
12. A kit for assessing cardiovascular status, said kit comprising
a) sequence determination primers and
b) sequence determination reagents,
wherein said primers are selected from the group comprising primers that hybridize to polymorphic positions in human CA genes according to claim 1 ; and primers that hybridize immediately adjacent to polymorphic positions in human CA genes according to claim 1.
13. A kit as defined in claims 12 detecting a combination of two or more, up to all, polymorphic sites selected from the groups of sequences as defined in claim 1.
14. A kit for assessing cardiovascular status, said kit comprising one or more antibodies specific for a polymorphic position defined in claim 1 within the human CA gene polypeptides and combinations of any ofthe foregoing.
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