WO2004033710A2 - Itgb3 gene haplotypes and atorvastatin dose effects on hdl cholesterol - Google Patents

Itgb3 gene haplotypes and atorvastatin dose effects on hdl cholesterol Download PDF

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WO2004033710A2
WO2004033710A2 PCT/US2003/032361 US0332361W WO2004033710A2 WO 2004033710 A2 WO2004033710 A2 WO 2004033710A2 US 0332361 W US0332361 W US 0332361W WO 2004033710 A2 WO2004033710 A2 WO 2004033710A2
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haplotypes
haplotype
itgb3
individual
psi
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PCT/US2003/032361
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French (fr)
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WO2004033710A3 (en
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Steven C. Bentivegna
Karen M. Bieglecki
Carlos D. Brain
Bradley J. Dain
Gina-Marie Cappola
Richard S. Judson
Michael Lachowicz
Helen H. Lee
Lynne Litvyn
Chad Messer
Nathan Petersen
Carol R. Reed
Eileen M. Rounds
David P. Russo
Andreas K. Windemuth
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2003284111A priority Critical patent/AU2003284111A1/en
Publication of WO2004033710A2 publication Critical patent/WO2004033710A2/en
Publication of WO2004033710A3 publication Critical patent/WO2004033710A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates to the field of genomics and pharmacogenetics. More specifically, this invention relates to genetic markers of the gene for integrin, beta 3 (ITGB3) and their use as predictors of response to treatment with statins.
  • Cardiovascular disease is a major health problem in the United States and worldwide (R. H. Knopp, jV. Engl. J. Med. 341:498-511, 1999).
  • the major cause of cardiovascular disease is atherosclerosis, which results from the fonnation of lipid-laden cellular lesions in one or more of the coronary arteries that supply the heart muscle with blood (Leff, T. and Gruber, P.J., "Cardiovascular Diseases” in: Meyers. R. Molecular Biology and Biotechnology (NCR Publishers 1995) pp. 149-153).
  • High levels of low-density lipoprotein cholesterol (“LDLC”) have long been associated with an increased risk of developing atherosclerosis (Leff and Gruber, supra).
  • LDL Apo B LDL apolipoprotein B
  • lipid- modifying drugs Patients with one or more of the above risk factors are frequently treated with one or more lipid- modifying drugs to achieve certain target levels of LDLC and HDLC that are recommended by the current National Cholesterol Education Program guidelines for treatment of hypercholesterolemia.
  • LIsual medical practice is to direct initial drug therapy toward elevated LDLC with treatment of low HDLC a secondary endpoint that is often managed by addition, after some weeks, of a second therapeutic agent.
  • statins which inhibit the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for cholesterol fonnation in the liver and other tissues.
  • HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A
  • increases in HDLC levels were observed, with a mean increase of 2% - 12%, depending upon the specific statin compound and the conditions under which it was studied.
  • statin therapy While most of the common side effects of statin therapy are mild, transient and reversible (e.g., dyspepsia, abdominal pain and flatulence), more severe, long-term adverse reactions to statins occur and include hepatitis, peripheral neuropathy, insomnia, difficulty in concentrating, and elevation of creatine phosphokinase, which is correlated with rhabdomyolysis (Knopp, supra, Lupattelli G et al., Nucl. Med. Commim. 22(5): 575-8, 2001; Moghadasian MH et al., Expert Opin. Pharmacother. 1(4): 683-95, 2000).
  • statins sold in the United States: lovastatin and simvastatin (sold by Merck as Mevacor ® and Zocor 8 , respectively); atorvastatin calcium (sold as Lipitor ® by the Parke Davis Division of Pfizer); fluvastatin sodium (sold as Lescol ® by Novartis); and pravastatm sodium (sold as Pravachol ® by Bristol-Myers Squibb) (Knopp, supra).
  • a sixth statin, cerivastatin sodium was previously sold as Baycol ® by Bayer, but was voluntarily removed from the market in 2001 because of safety concerns. Five of these drugs are metabolized by cytochrome P-450 enzyme systems, while the sixth, pravastatin sodium, is metabolized by sulfation and possibly other mechanisms (Knopp, supra).
  • the mean LDLC decreases and HDLC increases for pravastatm sodium administered once daily at bedtime were 22% and 7% at 10 mg/day, 32% and 2% at 20 mg/day, and 34% and 12% at 40 mg/day (Physicians' Desk Reference, 2000, p. 846).
  • statins differ in some of their clinical properties relevant to reducing the risks of atherosclerosis.
  • simvastatin resulted in larger increases in HDLC than atorvastatin (Illingworth DR et al. (2001) C r Med Res Opin 17(l):43-abstract only). Wierzbicki & Mikhailidis (2002 IntJ. Cardiol.
  • any particular individual treated with a statin may experience a 10% to 70% reduction in LDLC (Aguilar- Salinas SA et al.. Atherosclerosis 141:203-207, 1998).
  • physicians have observed that some patients treated with statins exhibit minimal or no increase in HDLC, which is not an optimal response for patients with low HDLC levels.
  • physicians currently are unable to identify patients who are at risk for reduced efficacy of statin therapy, which can be expensive and is not without risk.
  • physicians must currently rely on trial and error to determine which statin and dose combination will produce the best LDLC or HDLC response in any particular patient. Thus it would be useful to understand the biological basis for variability of response to statins.
  • ITGB3 insulin glycoprotein Ilia
  • GP Ilia platelet glycoprotein Ilia
  • ITGB3 insulin glycoprotein Ia
  • the ITGB3 protein is a member of the integrin family of receptors that bind cell adhesion molecules.
  • the integrin family consists of heterodimeric molecules composed of alpha and beta subunits.
  • ITGB3 is the beta subunit for at least tliree integrin receptors having different alpha subunits: the platelet complex, ITGA2B/ITGB3 (GP Ilb/DIa) receptor; the fibronectin receptor and the vitronectin receptor, hi view of its currently understood biological functions, it is an attractive target for new therapeutics to treat cardiovascular disease.
  • the major integrins are found on platelets and participate in the platelet adhesion process. Platelet activation is initiated by the presence of thrombin as well as various mechanical and chemical stimuli. Activation of platelets is also associated with the activation of the ITGA2B/ITGB3 (GP Ilb/IIIa) receptor complex (Lefkovits et al., N. Engl. J. Med. 1995; 332(23):1553-1559). ITGA2B and ITGB3 conespond to the alpha and beta subunits, respectively, of the platelet glycoprotein receptor.
  • ITGA2B and ITGB3 conespond to the alpha and beta subunits, respectively, of the platelet glycoprotein receptor.
  • Activation of platelets results in changes in platelet shape causing a shift of the ITGA2B/ITGB3 complex from a ligand-unreceptive state to a ligand-receptive state.
  • the ITGA2B/ITGB3 complex is able to bind fibrinogen, which forms a bridge between adjacent platelet molecules facilitating platelet aggregation (Lefkovits et al., supra).
  • ITGA2B ITGB3 Diminished levels of the ITGA2B ITGB3 are observed in Glanzman's thrombocytopenia, a disease characterized by a lack of platelet aggregation and recuirent mucocutaneous bleeding. The disease is associated with deficient platelet aggregation in response to all physiological stimuli, and is considered a paradigm for inliibition of ITGA2B ITGB3.
  • ITGA2B/ITGB3 receptor inhibitors are a class of drags with the potential to prevent events associated with coronary artery disease.
  • Examples of these inhibitors include abciximab (ReoPro®; Eli Lilly and Company), a chimeric immunoglobulin, as well as natural products such as trigramin, isolated from the venom of the viper Trimeresurus gramineus.
  • abciximab ReoPro®; Eli Lilly and Company
  • a chimeric immunoglobulin as well as natural products such as trigramin, isolated from the venom of the viper Trimeresurus gramineus.
  • RGD recognition sequence Arg-Gly-Asp
  • Examples include G4120 (Genentech, South San Francisco, CA), MK-852 (Merck, West Point, PA), and integrelin (COR Therapeutics, South San Francisco, CA).
  • ITGA2B/ITGB3 inhibitors to control platelet function has been demonstrated in clinical studies.
  • abciximab Patients undergoing angioplasty or atherectomy who were administered abciximab had 35% fewer ischemic events compared to individuals given a placebo. The principal effect of abciximab was to reduce the incidence of myocardial infarction (Lefkovitz et al., 1995; J Am Coll Cardiol (special issue):81A). In patients with unstable angina, the ITGA2B/ITGB3 inhibitor lamifiban has been shown to reduce the incidence of myocardial infarction and death (Theroux et al., 1994; Circulation:90:I-232).
  • the integrin, beta 3 (platelet glycoprotein Ilia, antigen CD61) gene is located on chromosome 17q21.32 and contains 15 exons that encode a 788 amino acid protein.
  • a reference sequence for the ITGB3 gene comprises the contiguous lines of Figure 1, which is a composite genomic sequence comprising Genaissance Reference Nos. 8070284, 8070315, 8070331, 8070343, 8070353 and 8070359 (SEQ ID NO:l).
  • Reference sequences for the coding sequence (GenBank Accession No. NM_000212.1) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.
  • Such infonnation would assist the treating physician in developing the most appropriate therapy regimen for patients with cardiovascular disease.
  • the ability to identify populations of patients who are at risk for minimal or no increase in HDLC upon treatment with a statin would be useful. Identification of these patients would enable physicians to select the most appropriate therapy regimen for those individuals at the time of initiation of treatment for hypercholesterolemia, thus saving the patient weeks of possibly inadequate therapy and potentially improving compliance.
  • Such infonnation would assist the treating physician in developing the most appropriate therapy regimen for patients at risk for or diagnosed with cardiovascular disease.
  • the inventors have identified correlations between haplotypes in the ITGB3 gene and differential HDLC response to treatment with atorvastatin calcium in a cohort of individuals participating in a a randomized, 16-week, open-label investigation of drug response in relationship to gene variants in adult subjects with primary hypercholesterolemia.
  • ITGB3 haplotypes affect the change in HDLC level resulting after treatment with atoi ⁇ astatin calcium at the highest permitted dose compared to the change in HDLC resulting after the lowest pennitted dose.
  • the ITGB3 haplotypes shown to have association with dose effects on HDLC changes in response to treatment with atorvastatin calcium are shown in Tables 1 A, IB and IC below.
  • statin response marker II zero copy of any of haplotypes 101 to 159 in Table 1A or haplotypes 201 to 463 in Table IB or two copies of any of haplotypes 160-194 in Table 1A or haplotypes 501-515 in Table 1 C are defined herein as a statin response marker II and are correlated with a more favorable change in HDLC (generally a percent increase) upon increasing atoi ⁇ astatin dose (See Tables 10 and 11).
  • statin response markers I and statin response markers II are summarized in Tables 1A, IB and IC below.
  • statin response marker I experienced a larger mean percent decrease in HDLC in response to increasing atoi ⁇ astatin calcium dose than did the group of mdividuals having a statin response marker II. Presence or absence of statin response marker 1 did not significantly affect the mean percent change in HDLC observed in the groups of individuals treated with increasing doses of simvastatin or pravastatin sodium. Thus, testing for the presence of any of these statin response markers I or II in patients will provide valuable information that can be used by the treating physician to devise the most effective statin treatment regimen.
  • statin response markers I and II may readily be identified based on linkage disequilibrium between the above ITGB3 haplotypes or their component polymorphisms and other haplotypes or polymorphisms, respectively, that are located in the ITGB3 gene or other genes.
  • statin response markers of the invention include those comprising haplotypes that are in linkage disequilibrium with any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC, hereinafter referred to as "linked haplotypes", as well as '"substitute haplotypes" for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC in which one or more of the polymo ⁇ hic sites in the original haplotype is substituted with another polymoiphic site, wherein the allele at the substituting polymoiphic site is in linkage disequilibrium with the allele at the replaced or substituted polymoiphic site.
  • statin response markers The correlations between the different types of statin response markers and varying changes in HDLC in response to treatment with increasing doses of statins suggest that testing for the presence of a statin response marker I or a statin response marker II in patients would provide valuable infonnation that can be used by the treating physician to choose the most effective statin or combination therapy for achieving a desired effect on LDLC and HDLC levels.
  • statin response marker I or statin response marker II in patients would provide valuable infonnation that can be used by the treating physician to choose the most effective statin or combination therapy for achieving a desired effect on LDLC and HDLC levels.
  • any clinical trial of a statin should include in its design or analysis a consideration of the potential effect of statin response markers on the efficacy of statin response.
  • the invention provides methods and kits for determining whether an individual has a statin response marker I or a statin response marker II. These methods and kits are useful for predicting the expected therapeutic response of an individual to treatment with statins, selecting an optimal statin for an individual or choosing appropriate therapy for an individual.
  • a method for determining whether an individual has a statin response marker I or a statin response marker II comprises determining the copy number present in the individual of a particular haplotype.
  • the haplotype is any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC.
  • the individual has a statin response marker I if the individual has at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • the individual has a statin response marker II if the individual has zero copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or if the individual has two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501- 515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501- 515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • the haplotype comprises one of haplotypes 201, 205, 209, 214, 225, and 288 in Table 1; preferably the haplotype comprises one ofhapiotypes 201, 205, and 209.
  • a method for assigning an individual to a first or second statin response marker group comprises determining the copy number present in the individual of a particular haplotype and assigning the individual to a statin response marker group based on the copy number of that haplotype.
  • the individual is assigned to the first statin response marker group if the individual has at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • the individual is assigned to the second stat response marker group if the mdividual has zero copy of any of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or if the mdividual has two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160- 194 in Table 1A and haplotypes 501-515 in Table IC.
  • the haplotype comprises one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB; preferably the haplotype comprises one of haplotypes 201, 205. and 209 in Table IB.
  • kits for determining whether an individual has a statin response marker I or a statin response marker II comprises a set of oligonucleotides designed for identifying at least one of the alleles present at each polymo ⁇ hic site (PS) in a set of polymo ⁇ hic sites.
  • the set of polymoiphic sites comprises the set of PSs comprising any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; the set of PSs comprising a linked haplotype to any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; or the set of PSs for a substitute haplotype comprising any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC, hi a preferred embodiment, the set of PSs comprises PS3, PS12 and PS42; PSI, PS12 and PS42; PS3 and PS42; PSI and PS42; PSI, PS3, PS12 and PS42; or PS39.
  • the set of PSs comprises PS3, PS 12 and PS42.
  • the kit comprises a manual with instructions for performing one or more reactions on a human nucleic acid sample to identify the allele(s) present in the individual at each polymo ⁇ hic site in the set of polymoiphic sites and determining if the individual has a statin response marker I or a statin response marker II based on the identified allele(s).
  • Another aspect of the invention is a method of selecting a statin therapy to provide an optimal HDLC response in an individual.
  • the method comprises determining whether the individual has a statin response marker I or a statin response marker ⁇ and selecting a statin therapy based on the results of the determining step. If the individual has a statin response marker ⁇ , then the selected statin therapy comprises any dose of atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid.
  • the selected statin therapy comprises a low dose of atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid, a higher dose of atorvastatin or a phannaceutically acceptable salt of atorvastatin acid in conjunction with an HDLC-modulating therapy, or another statin.
  • the invention provides a method for predicting an individual's HDLC response to treatment with a statin.
  • the statin may preferably be atoi ⁇ astatin or a phannaceutically acceptable salt of atorvastatin acid.
  • the method comprises determining whether the individual has a statin response marker I or a statin response marker II and making a response prediction based on the results of the determining step, hi some embodiments, if the individual is determined to have a statin response marker I, then the response prediction is that the individual will experience an unfavorable HDLC response if treated with atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid at a dose comparable to a dose of atorvastatin calcium greater than about 10 mg/day.
  • the response prediction is that the individual will likely experience a favorable HDLC response if treated with atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid at any dose comparable to a dose of aton'astatin calcium ranging from about 10 to about 80 mg/day.
  • the invention provides: (i) a method of seeking regulator ' approval for marketing a pharmaceutical formulation comprising a statin as at least one active ingredient for treating a disease or condition in a population partially or wholly defined by having a statin response marker, (ii) an article of manufacture comprising the pharmaceutical formulation that is marketed for treating the defined population, (iii) a method of manufacturing a drug product comprising the pha ⁇ naceutical formulation, and (iv) a method of marketing the drug product for treating the defined population, hi preferred embodiments, the disease or condition is a cardiovascular or coronary artery disorder, e.g., hypercholesterolemia.
  • the method of seeking regulatory approval comprises conducting at least one clinical trial which comprises administering the pha ⁇ naceutical fomiulation to a fust treatment group of patients and atorvastatin or a pharmaceutically acceptable salt of atorvastatin to a second treatment group of patients.
  • Each patient has the disease or condition and also has a statin response marker I.
  • the method further comprises demonstrating that the second treatment group exhibits a mean per cent change in High Density Lipoprotein Cholesterol (HDLC) that is worse than the mean per cent change in HDLC exhibited by the fust treatment group at any given dose of the phannaceutical fo ⁇ nulation and of atoi ⁇ astatin or a pharmaceutically acceptable salt of aton'astatin that achieves a comparable reduction in Low Density Lipoprotein Cholesterol.
  • HDLC High Density Lipoprotein Cholesterol
  • the statin is preferably simvastatin, a pharmaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid.
  • the method further comprises filing with a regulatory agency an application for marketing approval of the pharmaceutical formulation with a label stating that the pharmaceutical fo ⁇ nulation is indicated for patients having the statin response marker I.
  • the regulatory agency is the United States Food and Drug Administration (FDA) or the European Agency for the Evaluation of Medicinal Products (EMEA), or a future equivalent of these agencies.
  • the article of manufacture comprises the pharmaceutical formulation and at least one indicium identifying a population for whom the pharmaceutical formulation is indicated. The identified population is partially or wholly defined by having a statin response marker I.
  • an article of manufacture according to the invention comprises a pharmaceutical fomiulation comprising aton'astatin or a pharmaceutically acceptable salt of atoivastatin with an HDLC modulating agent and the defining statin response marker is a statin response marker I.
  • Another embodiment of the article of manufacture comprises packaging material and the pharmaceutical fo ⁇ nulation contained within the packaging material, wherein the packaging material comprises a label approved by a regulatory agency for the pharmaceutical fo ⁇ nulation, wherein the label states that the pha ⁇ naceutical formulation is indicated for a population partially or wholly defined by having a statin response marker I.
  • the method for manufacturing the drug product comprises combining in a package a pha ⁇ naceutical fo ⁇ nulation comprising a statin as at least one active ingredient and a label which states that the drug product is indicated for freating a population defined wholly or partially by having a statin response marker I.
  • the method for marketing the drug product comprises promoting to a target audience the use of the drug product for treating individuals who belong to the defined population.
  • each ITGB3 haplotype constitutes a code, or genetic marker, that defines a variant fomi of the gene that exists in the human population.
  • each ITGB3 haplotype represents a naturally occurring isofonn (also refened to herein as an "isogene") of the ITGB3 gene.
  • the inventors have dete ⁇ nined what pairs of these ITGB3 haplotypes are present in a patient cohort of individuals with primary hypercholesterolemia who were freated with atorvastatin calcium, pravastatin sodium, or simvastatin, as well as in a human reference population representing four major population groups: African descent, Asian, Caucasian and Hispanic/Latino.
  • the locations of the polymo ⁇ hic sites (PS) that comprise the ITGB3 haplotypes and haplotype pairs described herein and the identities of the polymo ⁇ hisms at each of these sites are shown in Table 3, which is located after the Examples below.
  • the composition of the different haplotypes and haplotype pairs for this set of polymo ⁇ hic sites that were found in the patient cohort and reference population are shown in Tables 5 and 4, respectively, which are located after the Examples below.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymo ⁇ hic variant of a reference sequence for the ITGB3 gene or a fragment thereof.
  • the reference sequence comprises the contiguous sequences shown in Figure 1 and the polymoiphic variant comprises at least one polymo ⁇ hism selected from the group consisting of the novel polymoiphisms shown in Table 3, presented following the Examples.
  • a particularly preferred polymoiphic variant is an isogene of the ITGB3 gene.
  • An ITGB3 isogene of the invention is defined by one of the haplotypes shown in Table 5 below, presented following the Examples.
  • the invention also provides a collection of at least two ITGB3 isogenes, refeixed to herem as an ITGB3 genome anthology.
  • the invention provides a polynucleotide comprising a polymo ⁇ hic variant of a reference sequence for a ITGB3 cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises at least one polymo ⁇ hism selected from the group consisting of adenine at a position corresponding to nucleotide 40, thymine at a position corresponding to nucleotide 57, thymine at a position conesponding to nucleotide 58, cytosine at a position con-esponding to nucleotide 176, guanine at a position co ⁇ -esponding to nucleotide 197, cytosine at a position conesponding to nucleotide 342, cytosine at a position corresponding to nucleotide 882, cytosine at a position corresponding to nucleotide 1143, adenine at a position corresponding to nucleotide 1333, guanine at a position corresponding to nucleotide 1533, adenine at a position corresponding to nucleo
  • polynucleotides complementary to these ITGB3 genomic and cDNA variants are also provided by the invention. It is believed that polymoiphic variants of the ITGB3 gene will be useful in studying the expression and function of ITGB3, and in expressing ITGB3 protein variants for use in screening for candidate drugs to treat diseases related to ITGB3 activity.
  • the invention provides a recombinant expression vector comprising one of the polymorphic genomic and cDNA variants operably linked to expression regulator ⁇ ' elements as well as a recombinant host cell fransformed or fransfected with the expression vector. The recombinant vector and host cell may be used to express ITGB3 for protein structure analysis and drug binding studies.
  • the invention provides a polypeptide comprising a polymo ⁇ hic variant of a reference amino acid sequence for the ITGB3 protein.
  • the reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymoiphic variant comprises at least one variant amino acid selected from the group consisting of methionine at a position conesponding to amino acid position 14, proline at a position co ⁇ esponding to amino acid position 59, arginine at a position corresponding to amino acid position 66, methionine at a position corresponding to amino acid position 445, and glutamine at a position con-esponding to amino acid position 515.
  • a polymo ⁇ hic variant of ITGB3 is useful in studying the effect of the variation on the biological activity of ITGB3 as well as on the binding affinity of candidate drugs targeting ITGB3 for the freatment of coronary heart disease or other disorders of cholesterol metabolism.
  • the present invention also provides antibodies that recognize and bind to the above polymo ⁇ hic ITGB3 protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.
  • the present invention also provides nonhuman transgenic animals comprising one or more of the ITGB3 polymo ⁇ hic genomic variants described herein and methods for producing such animals. The transgenic animals are useful for studying expression of the ITGB3 isogenes in vivo, for in vivo screening and testing of drugs targeted against ITGB3 protein, and for testing the efficacy of therapeutic agents and compounds for coronary heart disease or other disorders of cholesterol metabolism in a biological system.
  • the invention provides a method, composition and kit for genotyping and haplotyping the ITGB3 gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more ITGB3 polymo ⁇ hic sites selected from the novel polymo ⁇ hic sites shown in Table 3 below in both copies of the individual's ITGB3 gene.
  • the genotyping method may also comprise identifying the nucleotide pah that is present at one or more polymo ⁇ hic sites selected from the group consisting of the previously reported ITGB3 polymoiphic sites shown in Table 3 below
  • a genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region contaming, or adjacent to, one of these novel ITGB3 polymo ⁇ hic sites, hi one embodiment, a kit of the invention comprises a set of oligonucleotides designed to genotype one or more of the ITGB3 polymorphic sites disclosed herein.
  • the kit comprises a set of oligonucleotides designed to genotype each of PS1-PS44 shown in Table 3 below.
  • at least one PS in the set of two or more polymoiphic sites is selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7. PS8. PS9, PS10, PS11, PS12, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44. h another preferred embodiment of the kit.
  • the PSs are selected from the group consisting of: PSI, PS3, PS4, PSS, PS6, PS10. PS12, PS15, PS16. PS19. PS20. PS21, PS26, PS28, PS30, PS37, PS38, PS39, and PS42. Most preferably, the PSs are selected from the group consisting of: PSI, PS3, PS12, PS39, and PS42.
  • the genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below, thereby identifying the particular isogenes of ITGB3 present in the individual.
  • the invention also provides a method for haplotyping the ITGB3 gene in an individual.
  • the haplotyping method comprises detennining, for one copy of the individual's ITGB3 gene, the identity' of the nucleotide at one or more polymo ⁇ hic sites selected from the group of polymo ⁇ hic sites shown in Table 3 below.
  • the haplotyping method comprises determining whether one copy of the individual's ITGB3 gene is defined by one of the ITGB3 haplotypes shown in Table 5, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies of the individual's ITGB3 gene are defined by one of the ITGB3 haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. Establishing the ITGB3 haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with ITGB3 activity, e.g., coronary heart disease and other disorders of cholesterol metabolism.
  • diseases associated with ITGB3 activity e.g., coronary heart disease and other disorders of cholesterol metabolism.
  • the haplotyping method can be used by the pha ⁇ naceutical research scientist to validate ITGB3 as a candidate target for treating a specific condition or disease predicted to be associated with ITGB3 activity.
  • Detenninmg for a particular population the frequency of one or more of the individual ITGB3 haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue ITGB3 as a target for treating the specific disease of interest.
  • variable ITGB3 activity is associated with the disease, then one or more ITGB3 haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable ITGB3 activity has little, if any, involvement with that disease, h either case, the pha ⁇ naceutical research scientist can, without a priori knowledge as to the phenotypic effect of any ITGB3 haplotype or haplotype pair, apply the information derived from detecting ITGB3 haplotypes in an individual to decide whether modulating ITGB3 activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting ITGB3 to treat a specific condition or disease predicted to be associated with ITGB3 activity. For example, detecting which of the ITGB3 haplotypes or haplotype pairs disclosed herem are present in individual members of a population with the specific disease of interest enables the pha ⁇ naceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the ITGB3 isoforms present in the disease population, or for only the most frequent ITGB3 isoforms present in the disease population.
  • the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
  • Haplotyping the ITGB3 gene in an mdividual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with ITGB3 activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the ITGB3 haplotype(s) disclosed herein aie present in individual patients enables the phannaceutical scientist to distribute
  • ITGB3 haplotypes and/or haplotype pairs evenly to freatment and control groups, thereby reducing the potential for bias hi the results that could be introduced by a larger frequency of an ITGB3 haplotype or haplotype pair that is associated with response to the drug being studied in the frial, even if this association was previously unknown.
  • the scientist can more confidently rely on the information learned from the trial, without first detenmining the phenotypic effect of any ITGB3 haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and an ITGB3 genotype, haplotype, or haplotype pair for one or more of the novel polymo ⁇ hic sites described herein.
  • the method comprises comparing the frequency of the ITGB3 genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the ITGB3 genotype or haplotype in a reference population.
  • a different frequency of the ITGB3 genotype, haplotype, or haplotype pa - in the trait population than in the reference population indicates the trait is associated with the ITGB3 genotype, haplotype, or haplotype pair.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the ITGB3 haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic freatments for coronary heart disease or other disorders of cholesterol metabolism, as well as for other disorders in which receptors comprising ITGB3 are implicated.
  • Figure 1 illustrates a reference sequence for the ITGB3 gene (contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • SEQ ID NO:226 is a modified version of SEQ ID NO:l that shows the context sequence of each polymo ⁇ hic site enumerated in Table 5 in a uniform fonnat to facilitate electronic searching.
  • SEQ ID NO:226 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymo ⁇ hic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein.
  • Figure 2 illustrates a reference sequence for the ITGB3 coding sequence (contiguous lines; SEQ
  • Figure 3 illustrates a reference sequence for the ITGB3 protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymo ⁇ hism(s) of Figure 2 positioned below the polymo ⁇ hic site in the sequence.
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence, or one of the alternative polymoiphisms found at a polymoiphic site.
  • Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at a set of one or more polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in an individual.
  • Genotyping A process for dete ⁇ nining a genotype of an individual.
  • Haplotype - A 5' to 3' sequence of nucleotides found at a set of one or more polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5' to 3' sequence of nucleotides seen at a subset of one or more polymoiphic sites in a locus on a single chromosome from a single mdividual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for dete ⁇ nining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • Haplotype data Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in an individual or in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
  • Isoform - A particular fonn of a gene, mRNA, cDNA, coding sequence or the protein encoded thereby, distinguished from other fonns by its particular sequence and or structure.
  • Isogene - One of the isofonns (e.g., alleles) of a gene found in a population.
  • An isogene (or allele) contains all of the polymorphisms present in the particular isofonn of the gene.
  • Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention. Locus - A location on a chromosome or DNA molecule conesponding to a gene or a physical or phenotypic feature, where physical features include polymoiphic sites.
  • Nucleotide pair The nucleotides found at a polymorphic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pairs for two or more polymoiphic sites in a locus, phased means the combination of nucleotides present at those polymo ⁇ hic sites on a shigle copy of the locus is known.
  • Polymorphism The sequence variation observed in an individual at a polymo ⁇ hic site.
  • Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
  • Population Group A group of individuals sharing a common ethnogeographic origin.
  • Reference Population A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
  • Set of Polymorphic Sites one or more polymo ⁇ hic sites.
  • Single Nucleotide Polymorphism (SNP) Typically, the specific pah- of nucleotides observed at a single polymo ⁇ hic site. In rare cases, three or four nucleotides may be found.
  • Statin Response Marker I at least one copy of any of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • Statin Response Marker H zero copy of any of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be detennined.
  • Treatment A stimulus administered internally or externally to a subject.
  • Unphased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymoiphic sites on a single copy of the locus is not known. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the inventors herein have discovered novel variants of the ITGB3 gene and identified conelations between these variants and change in HDLC in response to increasing dose of atorvastatin calcium.
  • the inventors herein discovered 98 isogenes of the ITGB3 gene by characterizing the ITGB3 gene found in genomic DNAs isolated from immortalized cell lines from 854 humans.
  • This experimental population comprised an Index Repository that contains 93 human individuals and a patient cohort of 679 individuals participating in a randomized, 16-week, open-label investigation of drug response in relationship to gene variants in adult subjects with primary hypercholesterolemia. The experimental population is described in more detail in Examples 1 and 2.
  • the ITGB3 isogenes present in the human experimental population are defined by haplotypes for 44 polymoiphic sites in the ITGB3 gene.
  • the ITGB3 polymoiphic sites identified by the inventors are referred to as PS1-PS44 to designate the order in which they are located in the gene (see Table 3, presented following the Examples).
  • the human genotypes and haplotypes found in the experimental population for the ITGB3 gene include those shown in Tables 4 and 5, respectively (both presented at the end of the Examples).
  • Each statin response marker of the invention is a combination of a particular haplotype, or genetic marker, and the copy number for that haplotype, or genetic marker.
  • the genetic marker component of the statin response marker is one of the ITGB3 haplotypes shown in Tables 1A, IB or IC.
  • the ITGB3 polymo ⁇ hic sites in these ITGB3 haplotypes are referred to herein as PSI, PS3, PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42 and are located in the ITGB3 gene at positions in Figure 1 (SEQ ID NO:l) con-esponding to those identified in Table 3.
  • Figure 1 SEQ ID NO:l
  • nucleic acid molecules containing a particular gene may be complementary double stranded molecules and thus reference to a particular site or haplotype on the sense strand refers as well to the corresponding site or haplotype on the complementary antisense strand. Further, reference may be made to detecting a genetic marker or haplotype for one strand and it will be understood by the skilled artisan that this includes detection of the complementary haplotype on the other strand.
  • the location of a polymo ⁇ hic site in an individual's ITGB3 gene or fragment may be identified by aligning the sequence of the gene or fragment against the corresponding region of SEQ ED NO: 1. Alignment of a polymo ⁇ hism in SEQ ID NO: 1 against an alternative ITGB3 sequence to determine the corresponding position of the polymo ⁇ hic site in that alternative ITGB3 sequence should use a context sequence from SEQ ED NO:l ranging from about 25 to about 500 nucleotides with the polymo ⁇ hism in any position of the context sequence. The alignment should require a degree of homology appropriate for the length of the context sequence in establishing alignment between the two sequences.
  • the context sequence from SEQ ED NO: 1 is about 50 to about 300 nucleotides, with the polymo ⁇ hism positioned in the approximate center of the context sequence.
  • statin response markers of the invention are based on the discover ⁇ ' by the inventors of correlations between certain haplotypes in the ITGB3 gene and variation in change in HDLC levels in response to atorvastatin treatment in a cohort of individuals participating in a randomized, 16-week, open-label investigation of drug response in lelationship to gene variants in adult subjects with primary hypercholesterolemia.
  • the inventors herein discovered that copy number of ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB and
  • the group of patients with one or two copies of any of haplotypes 101 to 159 in Table 1A and haplotypes 201 -463 in Table 1 B or with zero or one copy of any of haplotypes 160- 194 in Table 1 A and haplotypes 501 to 515 in Table IC are conelated with a mean percent reduction in HDLC after a high dose atorvastatin treatment (See Tables 10A, 10B, 11 A and 1 IB) while the patient group having zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB or two copies of any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC are conelated with a negligible percent change or small percent increase in HDLC after a high dose atorvastatin freatment. No significant variation in differential change in HDLC in response to increasing dose of simvastatin or pravastatin sodium was observed for different copy numbers of any ITGB3 haplotypes
  • haplotypes in combination with then haplotype copy number, can be used to differentiate the HDLC response that would be predicted to occur in an individual or a trial population after treatment with a high dose of atoiYastatin. Consequently, one or two copies of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB or zero or one copy of any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC are referred to herein as a statin response marker I, while zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB or two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC are referred to herein as a statin response marker II.
  • a "favorable”, “better” or “best” HDLC response after treatment denotes that relative to the initial baseline HDLC measured that the change in HDLC measured after statin freatment shows a larger or largest increase in measured value. For example, no change or an increase in HDLC after treatment relative to the baseline measurement is a better response than a decrease in HDLC after treatment relative to the baseline measurement.
  • an "unfavorable”, “worse” or “worst” HDLC response after treatment denotes herem that the change in HDLC relative to the initial baseline HDLC value that is measured after statin treatment shows a smaller increase or a decrease in measured value.
  • no change or a decrease in HDLC after treatment relative to the baseline measurement is a worse response than an increase in HDLC after treatment relative to the baseline measurement.
  • the comparison of two or more values for a change in HDLC relative to baseline after a treatment may be for a single individual before and after two different therapy regimens, e.g., a low dose vs. a high dose regimen.
  • the comparison of two or more values for a change in HDLC relative to baseline after a treatment may be between two or more single individuals or two or more population groups before and after two different therapy regimens.
  • AtoiYastatin herein may refer to any chemical forms of atorvastatin, atorvastatin derivatives or phannaceutically acceptable salts of atoiYastatin acid.
  • atorvastatin atorvastatin derivatives or phannaceutically acceptable salts of atoiYastatin acid.
  • the skilled artisan would expect that there might be additional polymo ⁇ hisms in the
  • ⁇ 2 measures how well an allele X at a first polymorphic site predicts the occunence of an allele Y at a second polymo ⁇ hic site on the same chromosome. The measure only reaches 1.0 when the prediction is perfect (e.g., X if and only if Y).
  • the skilled artisan would expect that all of the embodiments of the invention described herein may frequently be practiced by substituting the allele at any (or all) of the specifically identified ITGB3 polymo ⁇ hic sites in ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB or 501-515 in Table IC with an allele at another polymo ⁇ hic site that is in high LD with the allele at the specifically identified polymo ⁇ hic site.
  • This "substitute polymo ⁇ hic site” may be one that is currently known or subsequently discovered and may be present at a polymo ⁇ hic site in the ITGB3 gene or elsewhere on chromosome 17.
  • the substitute polymoiphic site is present in the ITGB3 gene or in a genomic region of about 100 kilobases spanning the ITGB3 gene.
  • any ITGB3 statin response marker comprising an ITGB3 haplotype with a guanine at PS3 may be substituted with an alternative haplotype replacing guanine at PS3 with cytosine at PS9 and be nearly as well predictive of an individual's atorvastatin dose response as the ITGB3 statin response marker.
  • the inventors herein identified other alleles that are in high LD with one or more of the alleles found in the list of ITGB3 haplotypes shown in Table 1 and believe that ITGB3 haplotypes comprising one or more of these alleles substituted for the original allele can also function to predict the obsen'ed results.
  • the LD relationships identified by the inventors herein are shown in Table 2 below, which lists the values for ⁇ 2 in the total experimental population and for each of the 4 ethnic population groups within that population.
  • CA, AF, HL, and AS stand for Caucasian, African-American, Hispanic-Latino, and Asian, respectively.
  • the linked haplotype is present in the ITGB3 gene or in a genomic region of about 100 kilobases spanning the ITGB3 gene.
  • the linkage disequilibrium between any one of ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB or 501-515 in Table IC and a linked haplotype can also be measured using ⁇ 2 .
  • the linkage disequilibrium between the allele at a polymo ⁇ hic site in any of the ITGB3 haplotypes in Table 1 and the allele at a substitute polymo ⁇ hic site to replace it, or between any of the ITGB3 haplotypes in Table 1 and a linked haplotype has a ⁇ " value, as measured in a suitable reference population, of at least 0.75, more preferably at least 0.80, even more preferably at least 0.85 or at least 0.90, yet more preferably at least 0.95, and most preferably 1.0.
  • a suitable reference population for this ⁇ 2 measurement is preferably selected from a population with the distribution of the ethnic background of its members reflecting the population of patients to be treated with statins, which may be the general population, a population using statins, a population with coronary heart disease (CHD), cardiovascular disease (CVD) or CHD (or CVD) risk factors, and the like.
  • statins which may be the general population, a population using statins, a population with coronary heart disease (CHD), cardiovascular disease (CVD) or CHD (or CVD) risk factors, and the like.
  • LD patterns in genomic regions are readily determined empirically in appropriately chosen samples using various techniques known in the art for detennining whether any two alleles (at two different polymoiphic sites or two haplotypes) are in linkage disequilibrium (Weir B.S. 1996 Genetic Data Analysis II, Sinauer Associates, Inc. Publishers, Sunderland, MA). The skilled artisan may readily select which method of detennining LD will be best suited for a particular sample size and genomic region.
  • statin response marker I is deemed to include at least one copy of haplotypes that (A) either (1) have a polymoiphism sequence that is similar to that of any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, but in which the allele at one or more of the specifically identified ITGB3 polymoiphic sites in that haplotype has been substituted with the allele at a polymoiphic site in high LD with the allele at the specifically identified polymo ⁇ hic site (a "substitute haplotype"); or (2) are in high linkage disequilibrium with any one of haplotype
  • statin response marker I is deemed to include zero or one copy of haplotypes that (A) either (1) are a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, or (2) are a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC; and (B) behave similarly to zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC in tenns of predicting an individual's HDLC response to increased atoiYastatin dose.
  • statin response marker II is deemed to include zero copy of haplotypes that (A) either (1) have a polymo ⁇ hism sequence that is similar to that of any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, but in which the allele at one or more of the specifically identified ITGB3 polymo ⁇ hic sites in that haplotype has been substituted with the allele at a polymoiphic site in high LD with the allele at the specifically identified polymo ⁇ hic site (a "substitute haplotype”); or (2) are in high linkage disequilibrium with any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB (a "linked haplotype”); and (B) behave similarly to zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB in terms of predicting an individual's HDLC response to increased atorvastat
  • statin response marker II is deemed to include two copies of haplotypes that (A) either (1) are a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, or (2) are a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC; and (B) behave similarly to two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC in tenns of predicting an individual's HDLC response to increased atoiYastatin dose.
  • statin response markers of the invention are associated with effects on mean percent change in HDLC in response to freatment with increased doses of atoiYastatin calcium.
  • the invention provides a method and kit for determining whether an individual has a statin response marker I or a statin response marker H
  • the invention provides a method for detennining whether an individual has a statin response marker I or II.
  • the method comprises dete ⁇ nining the copy number present in the individual of a haplotype selected from the group consisting of haplotypes 101 to 194 in Table 1A, 201- 463 hi Table IB and 501-515 in Table IC, a linked haplotype to any one of haplotypes 101 to 194 in Table 1A, 201-463 hi Table IB and 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101 to 194 in Table 1A, 201-463 in Table IB and 501-515 in Table IC.
  • the individual has a statin response marker I if the individual has at least one copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, a haplotype linked to any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, a haplotype linked to any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC, or a substitute haplotype for any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC.
  • the individual has a statin response marker II if the individual has zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, a haplotype linked to any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any of haplotypes 101 to 159 ' in Table 1A and haplotypes 201-463 in Table IB; or if the individual has two copies of any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC, a haplotype linked to any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, or a substitute haplotype for any of haplotypes 160- 194 in Table 1A and haplotypes 501 to 515 in Table IC.
  • the invention provides a method for assigning an individual to a first or second statin response marker group.
  • the method comprises determining the copy number present in the individual of a haplotype selected from the group consisting of ITGB3 haplotypes 101-194 in Table 1 A, haplotypes 201 -463 in Table 1 B and haplotypes 501 to 515 in Table 1 C; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and assigning the individual to a statin response marker group based on the copy number of that haplotype.
  • the individual is assigned to the first statin response marker group if the mdividual has at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160- 194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160- 194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • the individual is assigned to the second statin response marker group if the individual has zero copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB; or if the individual has two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
  • the haplotype comprises any one of 201, 205,
  • the selected haplotype is one of haplotypes 201, 205, and 209 in Table IB.
  • the mdividual is Caucasian and may be diagnosed with a coronary artery disease or a cardiovascular disease, such as Type Ila or Type lib hypercholesterolemia, may have risk factors associated with cardiovascular disease, or may be a candidate for treatment with a statin for an alternative reason.
  • the dete ⁇ nining step may comprise genotyping each polymo ⁇ hic site in the set of polymo ⁇ hic sites comprising the selected haplotype; and using the results of the genotyping step to identify the haplotype pair present in the individual.
  • the genotyping step may be performed by any methods known to the art, including but not limited to those methods described herein.
  • the detennining step may also comprise consulting a data repository, such as a medical data card or a medical record for the individual, that provides information on the copy number present in the individual for the selected haplotype.
  • Determining the copy number" of a haplotype may in some instances mean determining if zero, one or two copies is present in the individual, i.e. identifying the haplotype present on each chromosomal copy of the individual. In other instances, determining the copy number of a haplotype in an individual may mean detennining a lower or upper limit on the number of copies, such as determining that there is at least one copy or fewer than two copies present in the individual, hi these latter instances, the haplotype for only one chromosomal copy of the individual is identified.
  • haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC For some individuals and some haplotypes selected from haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC, this is an adequate amount of infonnation to detennine if the individual has a statin response marker I or II or belongs to the first or second statin response marker group.
  • the individual may still be identified as having a statin response marker I or be assigned to the first statin response marker group.
  • the individual could have either a statin response marker I or II or belong to either statin response marker group, hi such instances infonnation on the haplotype of the individual's second chromosomal copy would be needed to determine which marker is present in the individual or for assigning the individual to a statin response marker group.
  • statin response marker I or II may be determined by a variety of indirect or direct methods well known in the art for determining haplotypes or haplotype pairs for a set of polymo ⁇ hic sites in one or both copies of the individual's genome, including those discussed below.
  • the genotype for a polymoiphic site in an individual may be detennined by methods known in the art or as described below.
  • Tables 1A, IB and IC present in an individual is by prediction based on the individual's genotype detennined at one or more of the polymo ⁇ hic sites in the set of polymo ⁇ hic sites comprising die haplotype and using the detennined genotype at each site to determine the ITGB3 haplotypes present in the individual.
  • the presence of zero, one or two copies of an ITGB3 haplotype of interest can be detennined by visual inspection of the alleles at the polymo ⁇ hic sites that comprise the haplotype.
  • the ITGB3 haplotype pair is assigned by comparing the individual's genotype at each polymoiphic site in the set of polymoiphic sites with the genotypes at the same set of polymorphic sites coiresponding to the haplotype pairs known to exist in the general population or in a specific population group or to the haplotype pairs that are theoretically possible based on the alternative alleles possible at each polymorphic site, and determining which haplotype pair is most likely to exist in the individual.
  • this haplotype pah prediction method comprises identifying a genotype for the individual at the set of polymo ⁇ hic sites comprising the selected haplotype, accessing data containing haplotype pairs identified in a reference population for a set of polymorphic sites comprising the polymo ⁇ hic sites of the selected haplotype, and assigning to the individual a haplotype pair that is consistent with the individual's genotype.
  • the haplotype pair can be assigned by comparing the individual's genotype with the genotypes conesponding to the haplotype pans known to exist in the general population or in a specific population group, and dete ⁇ nining which haplotype pair is consistent with the genotype of the individual. In some embodiments, the comparing step may be perfonned by visual inspection. When the genotype of the individual is consistent with more than one haplotype pair, frequency data may be used to detemiine which of these haplotype pairs is most likely to be present in the individual.
  • haplotype pair frequency data used in this determination is preferably for a reference population comprising the same ethnogeographic group as the individual.
  • the detemiination of the haplotype pair of the individual may also be perfonned in some embodiments by visual inspection. In other embodiments, the comparison may be made by a computer- implemented algorithm with the genotype of the individual and the reference haplotype data stored in computer-readable formats.
  • one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing ITGB3 haplotype pairs frequency data detennined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the individual.
  • the reference population is composed of randomly-selected individuals representing one or more of the major ethnogeographic groups of the world.
  • a prefened reference population for use in the methods of the present invention consists of Caucasian individuals, the number of which is chosen based on how rare a haplotype is that one wants to be guaranteed to see.
  • a prefened reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty.
  • a particularly prefe ⁇ ed reference population includes a 3-generation Caucasian family to serve as a control for checking quality of haplotyping procedures. If the reference population comprises more than one ethnogeographic group, the frequency data for each group is examined to detennine whether it is consistent with Hardy- Weinberg equilibrium.
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or enors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are obsened in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
  • CLASPER System TM technology U.S. Patent No. 5,866,404
  • single molecule dilution single
  • the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pan- is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the mdividual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • the haplotype pair in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al. 1990, Mol Bio Evol 7: 111- 22 or WO 01/80156) or through a commercial haplotyping service such as offered by Genaissance Pha ⁇ naceuticals, Inc. (New Haven, CT).
  • a commercial haplotyping service such as offered by Genaissance Pha ⁇ naceuticals, Inc. (New Haven, CT).
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra).
  • an individual with the C/G, T/C genotype could possess either of the following haplotype pahs: CT/GC or GT/CC, and thus could have either 1 copy of ITGB3 haplotype 214 which is a statin response marker I, or 0 copy of ITGB3 haplotype 214 which is a statin response marker II.
  • frequency information may be used to determine the most probable haplotype pair and therefore the most likely number of copies of ITGB3 haplotype 214 in the individual.
  • haplotype pair with the highest frequency is the most likely to be present in the individual.
  • the copy number of the haplotype of interest in this haplotype pair may then be determined by visual inspection of the alleles at the polymo ⁇ hic sites that comprise the response marker for each haplotype in the pair.
  • the individual's genotype for the desired set of PSs may be detennined using a variety of methods well-known in the art. Such methods typically include isolating from the individual a genomic DNA sample comprising both copies of the gene or locus of interest, amplifying from the sample one or more target regions containing the polymo ⁇ hic sites to be genotyped, and detecting the nucleotide pair present at each PS of interest in the amplified target region(s). It is not necessary to use the same procedure to determine the genotype for each PS of interest.
  • the identity of the allele(s) present at any of the novel polymorphic sites described herein may be indirectly detennined by haplotyping or genotyping another polymoiphic site that is in linkage disequilibrium with the polymoiphic site that is of interest.
  • the linkage disequilibrium data in Table 2 shows that ⁇ 2 for LD between PS3 and PS9 is close to 1 in the experimental population, therefore detennining the allele at PS 9 in an individual might be used to determine the allele at PS 3 in that individual indirectly.
  • Polymoiphic sites in linkage disequilibrium with the presently disclosed polymoiphic sites may be located hi regions of the gene or in other genomic regions not examined herein.
  • Detection of the allele(s) present at a polymo ⁇ hic site in linkage disequilibrium with the novel polymoiphic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymo ⁇ hic site.
  • the presence in an individual of a haplotype or haplotype pair for a set of PSs comprising a statin response marker may be detemiined by directly haplotyping at least one of the copies of the individual's genomic region of interest, or suitable fragment thereof, using methods known in the art.
  • Such direct haplotyping methods typically involve treating a genomic nucleic acid sample isolated from the individual in a manner that produces a hemizygous DNA sample that only has one of the two "copies" of the individual's genomic region which, as readily understood by the skilled artisan, may be the same allele or different alleles, amplifying from the sample one or more target regions containing the polymo ⁇ hic sites to be genotyped, and detecting the nucleotide present at each PS of interest h the amplified target region(s).
  • the nucleic acid sample may be obtained using a variety of methods known in the art for preparing hemizygous DNA samples, which include: targeted in vivo cloning (TEVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614; generating hemizygous DNA targets using an allele specific oligonucleotide in combination with primer extension and exonuclease degradation as described in U.S. Patent No. 5,972,614; shigle molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruai ⁇ o et al., 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).
  • TEVC targeted in vivo cloning
  • any individual clone will typically only provide haplotype infonnation on one of the two genomic copies present in an individual. If haplotype information is desired for the individual's other copy, additional clones will usually need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the genomic locus in an individual, hi some cases, however, once the haplotype for one genomic allele is directly detennined, the haplotype for the other allele may be inferred if the individual has a known genotype for the polymo ⁇ hic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
  • direct haplotyping of both copies of the gene is preferably perfo ⁇ ned with each copy of the gene being placed in separate containers, it is also envisioned that direct haplotyping could be performed in the same container if the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable.
  • first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymoiphic site(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second gene copy.
  • the nucleic acid sample used in the above indirect and direct haplotyping methods is typically isolated from a biological sample taken from the individual, such as a blood sample or tissue sample. Suitable tissue samples include whole blood, saliva, tears, urine, skin and hair.
  • the target region(s) containing the PS of interest may be amplified using any oligonucleotide- directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965, 188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88: 189- 193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241 : 1077-1080, 1988).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • Other known nucleic acid amplification procedures may be used to amplify the target region(s) including transcription-based amplification systems (U.S. Patent No.
  • the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site(s) in the amplified target region may be detemiined by sequencing the amplified region(s) using conventional methods. If both copies of the gene are represented in the amplified target, it will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, refened to as negative-type identification.
  • a site may be positively detennined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively detennined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • a polymoiphic site in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art.
  • allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pah- showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele-specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymo ⁇ hic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be perfo ⁇ ned with both entities in solution, or such hybridization may be perfo ⁇ ned when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, sfreptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
  • Detecting the nucleotide or nucleotide pair at a PS of interest may also be detennined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Whiter et al., Proc. Natl. Acad. Sci.
  • variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et ah, Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).
  • SSCP single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method ( ⁇ V092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524.
  • Related methods are disclosed in WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283.
  • Extended primers contaming the complement of the polymo ⁇ hism may be detected by mass specfrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruai ⁇ o et al., Nucl. Acids Res. 17:8392, 1989; Ruai ⁇ o et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635- 1641, 1995).
  • multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).
  • the genotype or haplotype for the ITGB3 gene of an individual may also be detennined by hybridization of a nucleic acid sample containing one or both copies of the gene, mRNA, cDNA or fragment(s) thereof, to nucleic acid ai ⁇ ays and subanays such as described in WO 95/11995.
  • the anays would contain a battery of allele-specific oligonucleotides representing each of the polymo ⁇ hic sites to be included in the genotype or haplotype.
  • the invention also provides a kit for determining whether an individual has a statin response marker I or a statin response marker II.
  • the kit comprises a set of oligonucleotides designed for determining the allele(s) present at a set of polymoiphic sites (PSs).
  • the set of polymo ⁇ hic sites comprises a set of polymoiphic sites selected from the group consisting of (1) PSI and PS42; (2) PS19 and PS42; (3) PS3, PS12, and PS42; (4) PSI, PS12, and PS42; (5) PS3, PS19, and PS42; (6) PSI, PS4, and PS42; (7) PS3, PS19, and PS39; (8) PSI, PS37, and PS42; (9) PS5, PS19, and PS42; (10) PSI, PS3, PS12, and PS42; (11) PS3, PS4, PS12, and PS42; (12) PSI, PS12, PS37 and PS42; (13) PS3, PS12, PS37 and PS42; (14) PSI, PS4, PS12, and PS42; (15) PS3, PS12, PS19 and PS42; (16) PS3, PS12, PS16 and PS42; (17) PSI, PS12, PS26 and PS42; (18) PSI, PS3, PS19, and PS42; (19) PS3, PS12, PS26
  • Preferred sets of polymo ⁇ hic sites comprise the sets of PSs for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB. More preferably the set of polymo ⁇ hic sites comprises the set of PSs for any one of haplotypes 201, 205, or 209 in Table IB.
  • the set of polymo ⁇ hic sites comprises PS3, PS12 and PS42; PSI, PS12 and PS42; PS3 and PS42; PSI and PS42; PSI, PS3, PS12 and PS42; or PS39. More preferably, the set of PSs comprises PS3, PS12 and PS42.
  • the kit comprises a set of oligonucleotides designed for identifying at least one of the alleles at each polymo ⁇ hic site (PS) hi a set of two or more polymo ⁇ hic sites (PSs) selected from the group consisting of PSI to PS44.
  • At least one PS in the set of two or more polymoiphic sites is selected from the group consisting of PSI, PS2, PS3, PS4, PSS, PS6, PS7, PS8, PS9, PS10, PS11, PSI 2, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44.
  • the PSs are selected from the group consisting of:
  • the kit comprises oligonucleotides for detecting at least one allele for each polymo ⁇ hic site in the set of polymo ⁇ hic sites, while in other embodiments the kit comprises oligonucleotides for detecting both alleles at each member of the set of polymo ⁇ hic sites.
  • Each oligonucleotide provided in the kit may be placed in the same or separate receptacles and may be provided together in a package.
  • a oligonucleotide for genotyping a polymo ⁇ hic site is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, a polymoiphic site of interest such as one of the polymo ⁇ hic sites comprising a statin response marker described herein.
  • the temi "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a prefened oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length.
  • oligonucleotide The exact length of the oligonucleotide will depend on the nature of the genomic region containing the PS of interest as well as the genotyping assay to be performed and can readily be detemiined by the skilled artisan.
  • the oligonucleotides used to practice the invention may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R.
  • Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • Oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a polynucleotide containing a desired locus.
  • specific hybridization means the oligonucleotide fonns an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to fonn such a structure when incubated with another region in the polynucleotide or with a polynucleotide lacking the desired locus under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • oligonucleotide probes and primers suitable for detecting polymoiphisms in the ITGB3 gene or adjacent regions of chromosome 17 in linkage disequilibrium with one of the selected ITGB3 haplotypes using the polymo ⁇ hism infonnation provided herein in conjunction with the known sequence infonnation for the ITGB3 gene, and adjacent regions of chromosome 17, and routine techniques.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if ever ⁇ ' nucleotide of one of the molecules is complementary to the nucleotide at the conesponding position of the other molecule.
  • a nucleic acid molecule is "substantially complementary” to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J.
  • an oligonucleotide primer may have a non-complementary fragment at its 5' end, with the remainder of the primer being complementaty to the target region.
  • non-complementary nucleotides may be interspersed into the probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Prefened oligonucleotides of the invention useful in dete ⁇ nining if an individual has a statin response marker I or II, are allele-specific oligonucleotides.
  • the te ⁇ n allele-specific oligonucleotide means an oligonucleotide that is able, under sufficiently stringent conditions, to hybridize specifically to one allele of a gene, or other locus, at a target region containing a polymo ⁇ hic site while not hybridizing to the conesponding region in another allele(s).
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps. Examples of hybridization and washing conditions typically used for ASO probes are found in Kogan et al., "Genetic Prediction of Hemophilia A” in PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990 and Ruano et al., 87 Proc. Natl. Acad. Sci. USA 6296-6300, 1990. Typically, an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele. Allele-specific oligonucleotides of the invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • An ASO primer of the invention has a 3' terminal nucleotide, or preferably a 3' penultimate nucleotide, that is complementary to only one of the nucleotide alleles of a particular polymoiphic site, thereby acting as a primer for polymerase-mediated extension only if that nucleotide allele is present at the PS in the sample being genotyped.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a prefened ASO probe for detecting ITGB3 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of: PSI GGGAAGAYCCAGGGA (SEQ ID NO 4) and its complement,
  • PS2 AAAATAGRTAAAGTC SEQ ID NO 5
  • PS23 GGAAAGASTCTCCCC (SEQ ID NO 26 and its complement
  • a preferred ASO primer for detecting ITGB3 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:
  • PSI AGTCTGGGGAAGAYC SEQ ID NO 43
  • TTTGAGTCCCTGGRT SEQ ID NO: 49
  • PS2 TTGAGGAAAATAGRT (SEQ ID NO 50) • CTTTGGGACTTTAYC (SEQ ID NO: 51) ;
  • PS3 ATCTAGAGAAGCCSG (SEQ ID NO 52) • GCTTCCTCCCCTCSG (SEQ ID NO: 53) ;
  • PS4 CCGCGGGCGGCGGYG (SEQ ID NO: 54) , CCCCACAGTGGGCRC (SEQ ID NO: 55) ;
  • PS5 CGCCGCGGGAGGCSG SEQ ID NO 56
  • CTCGCATCTCGTCSG SEQ ID NO: 57
  • PS6 GCTCTGGGCGACTRT SEQ ID NO 58
  • CCCAGCGCCAGCAYA SEQ ID NO: 59
  • PS7 TGGCGCTGGGGGCKC SEQ ID NO 60 • CAACGCCCGCCAGMG (SEQ IDNO:61) ;
  • PS8 GGCGCTGGGGGCGYT (3EQ ID NO 62) • CCAACGCCCGCCARC (SEQ ID NO: 63) ;
  • PS10 GCAAACGCGGAGGRC (SEQ ID NO 66) ; CGCGCGGGACCAGYC (SEQ ID NO: 67) ;
  • PS11 GCGCTGGGAATGCRC SEQ ID NO 68
  • CiSCCAGGACACGYG SEQ ID NO: 69
  • PS12 CGGGAGCTGGGGAYC SEQ 70
  • CCGGGCCAGGAAGRT SEQ ID NO:71
  • PS22 AGTTCTAAGCTTTRG (SEQ 90) ; CATCTCTAGGGTCYA (SEQ ID NO: 91) ;
  • PS36 AATGCAGCCCCCGRG (SEQ :118) CGGGCTGACCCTCYC (SEQ ID (-3:119) ;
  • PS37 TTGCCTTAATCACYG (SEQ :120) GGAGAGAGGACACRG (SEQ ID NO 121);
  • PS41 TCATTGGCCTTGCYG (SEQ :128) AGATGAGCAGGGCRG (SEQ ID NO 129) ;
  • PS42 ACACAGTAAGAGAYG (SEQ :130) AACGCCCAGCCCCRT (SEQ ID NO 131);
  • PS43 TGAGACTCTTAAGKG (SEQ :132) ATCTGCTGCTTCCMC (SEQ ID NO 133) ;
  • oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymoiphic sites identified herein. Such oligonucleotides are useful in poly erase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisiiis described herein and therefore such oligonucleotides are refei ⁇ ed to herein as, "primer- extension oligonucleotides".
  • the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complenientaiy to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
  • PS2 AGGAAAATAG SEQ ID NO 138
  • TGGGACTTTA SEQ ID NO 139
  • PS3 TAGAGAAGCC SEQ ID NO 140
  • TCCTCCCCTC SEQ ID NO 141
  • PS4 CGGGCGGCGG SEQ ID NO 142
  • CACAGTGGGC SEQ ID NO 143
  • PS6 CTGGGCGACT SEQ ID NO 146
  • AGCGCCAGCA SEQ ID NO 147
  • PS10 AACGCGGAGG (SEQ ID NO 154) GCGGGACCAG (SEQ ID NO 155); psii CTGGGAATGC (SEQ ID NO 156) CCAGGACACG (SEQ ID NO :157); PS12 GAGCTGGGGA SEQ ID NO 158) , GGCCAGGAAG ( SEQ ID NO 159)
  • PS40 CCCCAGGAGG SEQ ID NO 214)
  • TTCTCCCTCT SEQ ID NO :215)
  • Termination mixes are chosen to temiinate extension of the oligonucleotide at the polymo ⁇ hic site of interest, or one base thereafter, depending on the alternative nucleotides present at the polymoiphic site.
  • Prefened ASO probes or primers and preferred primer extension oligonucleotides for detecting the alleles at the polymorphic sites comprising the prefened embodiments of the statin response markers I and II comprise the nucleotide sequences for PSI, PS3. PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42.
  • Particularly prefened ASO probes and primers and primer extension oligonucleotides for genotyping PS3, PS12 and PS42 are SEQ ED NO:6, 15 and 45, and their complements; SEQ ID NOS:52-53, 70-71, 130-131, and SEQ ED NOS: 140-141, 158-159, and 218-219.
  • the genotyping oligonucleotides in a kit of the invention have different labels to allow probing of the identity of nucleotides or nucleotide pairs at two or more polymoiphic sites simultaneously. It is also contemplated that a kit of the invention may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site in a statin response marker.
  • the oligonucleotides comprising a kit of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019).
  • a solid surface such as a microchip, bead, or glass slide
  • Such immobilized oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized oligonucleotides useful in practicing the invention may comprise an ordered a ⁇ ay of oligonucleotides designed to rapidly screen a nucleic acid sample for polymo ⁇ hisms in multiple genes at the same time.
  • Kits of the invention may also contain other components such as hybridization buffer (e.g., where the oligonucleotides are to be used as allele-specific probes) or dideoxynucleotide triphosphates (ddNTPs; e.g., where the alleles at the polymoiphic sites are to be detected by primer extension).
  • the set of oligonucleotides consists of primer extension oligonucleotides.
  • the kit may also contain a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase.
  • kits may also include detection reagents, such as biotin- or fluorescent-tagged oligonucleotides or ddNTPs and/or an enzyme-labeled antibody and one or more subsfrates that generate a detectable signal when acted on by the enzyme.
  • detection reagents such as biotin- or fluorescent-tagged oligonucleotides or ddNTPs and/or an enzyme-labeled antibody and one or more subsfrates that generate a detectable signal when acted on by the enzyme.
  • each of the oligonucleotides and all other reagents in the kit have been quality tested for optimal performance in an assay for detennining the alleles at a set of polymo ⁇ hic sites comprising a statin response marker I or statin response marker II.
  • the kit comprises a manual with instructions for performing genotyping assays on a nucleic acid sample from an individual and detennining if the individual has a statin response marker I or a statin response marker II based on the results of the assay.
  • the instructions may also contain infonnation to help a physician determine whether or how to use particular statins, alone or in combination with other therapies affecting HDLC levels, to freat an mdividual with the determined statin response marker.
  • the methods and kits of the invention are useful for helping physicians make decisions about how to treat an individual. They can be used to predict the change in HDLC of an individual in response to freatment with a statin or in selecting a statin therapy for an individual.
  • the invention provides a method for predicting the HDLC response of an individual to treatment with a statin.
  • the method comprises determining whether the individual has a statin response marker I or a statin response marker II and making a response prediction based on the results of the detennining step.
  • the statin is atorvastatin or a pharmaceutically acceptable salt of atoiYastatin acid.
  • the statin is atoiYastatin calcium.
  • the response prediction is that the individual will experience an unfavorable HDLC response if treated with atoiYastatin calcium at a dose greater than about 10 mg/day.
  • the determining step comprises consulting a data repository that states whether the individual has a statin response marker I or a statin response marker II.
  • the data repositoiy may be the individual ' s medical records or a medical data card.
  • the determining step comprises detennining the copy number of a haplotype selected from the group consisting of: haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC.
  • the selected haplotype is one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB
  • a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB then the individual has a statin response marker I if the individual has at least one copy of the selected marker and a statin response marker II if the individual has zero copy of the selected marker.
  • the selected haplotype is one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC
  • a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC then the individual has a statin response marker I if the individual has zero or one copy of the selected haplotype and a statin response marker II if the individual has two copies of the selected haplotype.
  • Prefened haplotypes are haplotypes 201, 205, 209, 214, 225, or 288 in Table IB.
  • Values for the linkage disequilibrium, ⁇ 2 , for linked haplotypes or substituting polymoiphic sites in substitute haplotypes are at least 0.75, preferably at least 0.80, more preferably at least 0.85, yet more preferably at least 0.90, and most preferably at least 0.95 or 1.0.
  • the determination of the statin response marker present in an individual can be made using one of the direct or indirect methods described herein or known in the art.
  • the detennining step comprises identifying for one or both copies of the genomic locus present in the individual the identity of the nucleotide or nucleotide pair at each member of the set of polymo ⁇ hic sites comprising the selected haplotype.
  • the individual is Caucasian.
  • the invention also provides a method of selecting a statin therapy to provide an optimal HDLC response in an individual.
  • the method comprises detennining whether the individual has a statin response marker I or a statm response marker II and selecting a statin therapy based on the results of the detennining step.
  • the selected statin therapy comprises any dose of atoiYastatin or a pharmaceutically acceptable salt of atoiYastatin acid and if the individual has a statin response marker I, then the selected statin therapy comprises a low dose of atorvastatin, a low dose of a pharmaceutically acceptable salt of atorvastatin acid, a higher dose of atoiYastatin in conjunction with an HDLC-modulating therapy, or another statin.
  • the preferred phannaceutically acceptable salt of atorvastatin acid is atorvastatin calcium.
  • a low dose means a dose that is at the lower end of the range of doses permitted by a regulatory agency, while a high dose or higher dose means a dose that is at the higher end of the range of permitted doses.
  • a low dose comprises about 10 mg day, while a higher dose of atorvastatin calcium conesponds to doses greater than about 10 mg/day, preferably greater than about 40 mg/day, most preferably about 80 mg/day.
  • the determination of the statin response marker present in an individual can be made using one of the direct or indirect methods described herein or known in the art.
  • One method to determine whether a statin response marker I or II is present in the individual comprises determining the copy number of any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; a linked haplotype to any one of haplotypes 101-194 in Table 1 A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC.
  • Preferred haplotypes are haplotypes 201, 205, 209, 214, 225, or 288 in Table IB. Values for the linkage disequilibrium, ⁇ 2 , for linked haplotypes or substituting polymorphic sites in substitute haplotypes are at least 0.75, preferably at least 0.80, more preferably at least 0.85, yet more preferably at least 0.90, and most preferably at least 0.95, if not 1.0.
  • the detennining step may comprise consulting a data repositoiy that states the individual's copy number for one or more haplotypes comprising a statin response marker I or II.
  • the data repository may be the individual's medical records or a medical data card. In prefened embodiments, the individual is Caucasian.
  • an article of manufacture comprises a pha ⁇ naceutical fomiulation and at least one indicium identifying a population for which the phaniiaceutical fomiulation is indicated.
  • the phaniiaceutical fomiulation comprises a statin as at least one active ingredient.
  • the statin may be simvastatin, a phannaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a phannaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid.
  • the pharmaceutical formulation may be regulated and the indicium may comprise the approved label for the phaniiaceutical fo ⁇ nulation.
  • the identified population is partially or wholly defined by having a statin response marker I.
  • the identified population preferably may be further defined as Caucasian.
  • a population wholly defined by having a statin response marker is one for which there are no other factors which should be considered in identifying the population for which the pharmaceutical formulation is indicated, hi contrast, a population that is partially defined by having a statin response marker is one for which other factors may be pertinent to identification of the population for which the pharmaceutical fomiulation is indicated. Examples of other such factors are age, weight, gender, disease state, possession of other genetic markers or biomarkers, or the like.
  • the statin response marker I comprises a copy number of a specific haplotype.
  • the statin response marker I is at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 hi Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501- 515 in Table IC.
  • the phamiaceutical fonnulation is fonnulated, in any way known in the art, as a sustained release fo ⁇ nulation, but most preferably as a transdermal patch,
  • the pharmaceutical formulation is a tablet or capsule and the article may further comprise an additional indicium comprising the color or shape of the table or capsule, h further embodiments, the article may further comprise an additional indicium comprising a symbol stamped on the tablet or capsule, or a S ⁇ mbol or logo printed on the approved label
  • a frial population having the statin response marker I exhibits a better HDLC response to the phamiaceutical formulation than to treatment with atoiYastatin or a pharmaceutically acceptable salt of atoiYastatin acid.
  • the approved label may state that the phamiaceutical formulation provides a better HDLC response in a population having the statin response marker I.
  • the approved label may further state that the phamiaceutical fomiulation is indicated for individuals identified as having the statin response marker I on a specified test, preferably a specified genetic test, hi some or all of these embodiments, the label may describe the mean change in HDLC expected for the identified population.
  • the pha ⁇ naceutical fomiulation comprises as separate active ingredients a statin, which may be atoiYastatin or a phannaceutically acceptable salt of atorvastatin acid, and an HDL cholesterol (HDLC) modulating agent, hi prefened embodiments, the statin is atorvastatin calcium.
  • the identified population is partially or wholly defined by having a statin response marker I.
  • a trial population having the statin response marker I may exhibit a worse mean HDLC response to treatment with only atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid than to treatment with the pharmaceutical formulation.
  • a trial population havmg the statin response marker I may exhibit a worse mean HDLC response to treatment with only atorvastatin or a pharmaceutically acceptable salt of aton'astatin acid than a trial population lacking the statin response marker I.
  • the statin is present hi the pha ⁇ naceutical formulation at an amount effective to reduce LDL cholesterol levels.
  • the effective amount ranges from about 10 to about 80 mg.
  • An additional embodiment of the article of manufacture provided by the invention comprises packaging material and a pharmaceutical fomiulation contained within said packaging material.
  • the pharmaceutical fomiulation comprises a statin as at least one active ingredient.
  • the statin may be simvastatin, a pharmaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceuticaUy acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a phannaceutically acceptable salt of rosuvastatin acid, atorvastatin, a phannaceutically acceptable salt of atoiYastatin acid, pravastatin, or a phannaceutically acceptable salt of pravastatin acid.
  • the pharmaceutical fo ⁇ nulation may further comprise an HDLC-modulating agent, such as niaciii.
  • the packaging material may comprise a label that may state that the pharmaceutical fo ⁇ nulation is indicated for a population partly or wholly defined by having a statin response marker I.
  • the label may further state the mean percent change in HDLC expected for the identified population.
  • a population having a statin response marker I exhibits a better HDLC response to the pharmaceutical fomiulation than to treatment with atoiYastatin or a phannaceutically acceptable salt of atorvastatin acid.
  • the indicated population in any of the above articles may preferably be further defined as Caucasian.
  • the label may further state that a specified test can be used to identify members of the indicated population.
  • the specified test is a genetic test.
  • statin may be simvastatin, a phannaceutically acceptable salt of simvastatin acid, lovastatin. a phannaceutically acceptable salt of lovastatin, fluvastatin, a phannaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, aton'astatin, a phannaceutically acceptable salt of atorvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid.
  • the method comprises combining in a package a phamiaceutical fo ⁇ nulation comprising the statin and a label.
  • the label states that the phamiaceutical formulation is indicated for freating a population partially or wholly defined by having a statin response marker I.
  • the indicated population having the defining statin response marker preferably may be further defined as Caucasian.
  • the indicated and/or contraindicated populations may be identified on the pharmaceutical fo ⁇ nulation, on the label or on the package by at least one indicium, such as a symbol or logo, color, or the like. Detecting the presence of a statin response marker I or II in an individual is also useful in a method of seeking regulatory approval for marketing a phamiaceutical fo ⁇ nulation for treating a disease or condition in a population defined by the statin response marker.
  • the method comprises conducting at least one clinical trial with first and second treatment groups of patients having the disease or condition, wherein each patient has a statin response marker I.
  • Each patient in the first freatment group is treated with the phamiaceutical fomiulation and each patient in the second treatment group is treated with atorvastatin, a pharmaceutically acceptable salt of aton'astatin, or a derivative thereof.
  • the method also comprises demonstrating that the first treatment group exhibits a mean percent change in HDLC that is better than the mean percent change exhibited by the second treatment group; and filing with a regulatory agency an application for marketing approval of the pha ⁇ naceutical formulation with a label stating that the phamiaceutical formulation is indicated for patients having the statin response marker I.
  • the pharmaceutical fonnulation may comprise any statin known in the art other than atorvastatin or pharmaceutically acceptable salts of atoiYastatin acid.
  • the statin may be simvastatin, a phannaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid.
  • the pharmaceutical formulation comprises a statin and an HDLC modulating agent as two separate active ingredients
  • the statin may be atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid. More preferably in these latter embodiments, the pharmaceutical fonnulation comprises atoiYastatin calcium.
  • the clinical trial may be conducted by recruiting patients with the disease or condition, determining whether they have a statm response marker I or II and assigning the patients to the first and second treatment groups based on the results of the detennining step.
  • the disease or condition may include any for which statin therapy is indicated, e.g., hyperlipidemia, hypercholesterolemia, cardiovascular disease (CVD), presence of CVD risk factors, coronary artery disease, and the like.
  • the patients in each treatment group are preferably administered the same dose of the pharmaceutical fomiulation, which includes a statin compound as at least one active ingredient.
  • the pha ⁇ naceutical formulation may contain other active ingredients, for example another compound known or believed to have therapeutic activity hi treating the disease or condition examined in the study or a compound that sen'es to reduce or block one or more side effects caused by the statin compound.
  • the regulatory agency may be any person or group authorized by the government of a country anywhere in the world to control the marketing or distribution of drugs in that country.
  • the regulatory agency is authorized by the government of a major industrialized country, such as Australia, Canada, China, a member of the European Union. Japan, and the like.
  • Most preferably the regulatory agency is authorized by the government of the United States and the type of application for approval that is filed will depend on the legal requirements set forth in the last enacted version of the Food, Drug and Cosmetic Act that are applicable for the pha ⁇ naceutical fonnulation and may also include other considerations such as the cost of making the regulatory filing and the marketing strategy for the composition.
  • the application might be a paper NDA, a supplemental NDA or an abbreviated NDA, but the application would be a full NDA if the pharmaceutical formulation has never been approved before; with these terms havmg the meanings applied to them by those skilled in the phamiaceutical arts or as defined in the Drug Price Competition and Patent Term Restoration Act of 1984.
  • the individual's ITGB3 haplotype content or statin response marker may be determined by consulting a data repository such as the individual's patient records, a medical data card, a file (e.g. a flat ASCII file) accessible by a computer or other electronic or non-electronic media on which infonnation about the individual's ITGB3 haplotype content or statin response marker can be stored.
  • a data repository such as the individual's patient records, a medical data card, a file (e.g. a flat ASCII file) accessible by a computer or other electronic or non-electronic media on which infonnation about the individual's ITGB3 haplotype content or statin response marker can be stored.
  • a medical data card is a portable storage device such as a magnetic data card, a smart card, which has an on-board processing unit and which is sold by vendors such as Siemens of Kunststoff Germany, or a flash-memory card.
  • the medical data card may be, but does not have to be, credit-card sized so that it easily fits into pocketbooks, wallets and other such objects ca ⁇ ied by the individual.
  • the medical data card may be swiped through a device designed to access infonnation stored on the data card.
  • portable data storage devices other than data cards can be used.
  • a touch- memory device such as the "i-button” produced by Dallas Semiconductor of Dallas, Texas can store information about an individual's ITGB3 haplotype content or statin response marker, and this device can be incorporated into objects such as jewelry.
  • the data storage device may be implemented so that it can wirelessly communicate with routing/intelligence devices through EEEE 802.11 wireless networking technology or through other methods well known to the skilled artisan.
  • infonnation about an individual's ITGB3 haplotype content or statin response marker can also be stored in a file accessible by a computer; such files may be located on various media, including: a server, a client, a hard disk, a CD, a DVD, a personal digital assistant such as a Palm Pilot, a tape, a zip disk, the computer's internal ROM (read-only-memory) or the intemet or worldwide web.
  • Other media for the storage of files accessible by a computer will be obvious to one skilled in the art.
  • any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer.
  • the computer may execute a program that assigns ITGB3 haplotype pairs and/or a statin response marker I or II to individuals based on genotype data inputted by a laboratoiy technician or treating physician.
  • the computer may output the predicted change in one or more lipoprotein levels in response to a statin following input of the individual's ITGB3 haplotype content or statin response marker, which was either detennined by the computer program or input by the technician or physician.
  • Data on which statin response markers were detected in an individual may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files) containing other clinical and/or haplotype data for the individual.
  • a relational database e.g., an instance of an Oracle database or a set of ASCII flat files
  • These data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the ITGB3 gene or a fragment of the gene which contains at least one of the novel polymoiphic sites described herein.
  • nucleotide sequence of a variant ITGB3 gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymo ⁇ hic sites PSI, PS2, PS3, PS4, PS5, PS6, PS7, PSS, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44.
  • nucleotide sequence of a variant fragment of the ITGB3 gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the ITGB3 gene, which is defined by haplotype 39, (or other reported ITGB3 sequences) or to portions of the reference sequence (or other reported ITGB3 sequences), except for the haplotyping and genotyping oligonucleotides described above.
  • the location of a polymo ⁇ hism in a variant ITGB3 gene or fragment is preferably identified by aligning its sequence against SEQ ED NO:l.
  • the polymo ⁇ hism is selected from the group consisting of thymine at PSI, guanine at PS2, cytosine at PS3, thymine at PS4, cytosine at PS5, adenine at PS6, thymine at PS7, thymine at PSS, guanine at PS9, adenine at PS10, adenine at PSI 1, thymine at PS12, adenine at PS13, thymine at PS14, cytosine at PS15, guanine at PS16, cytosine at PS17, adenine at
  • the polymo ⁇ hic variant comprises a naturally-occurring isogene of the ITGB3 gene which is defined by any one of haplotypes 1- 38
  • Polymoiphic variants of the invention may be prepared by isolating a clone containing the ITGB3 gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymoiphic sites described herein.
  • Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by perforating in vitro mutagenesis using procedures well-known in the art.
  • Any particular ITGB3 variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
  • ITGB3 isogenes, or fragments thereof may be isolated using any method that allows separation of the two "copies" of the ITGB3 gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TEVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • TEVC targeted in vivo cloning
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • the invention also provides ITGB3 genome anthologies, which are collections of at least two ITGB3 isogenes found in a given population.
  • the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same gender population.
  • An ITGB3 genome anthology may comprise individual ITGB3 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the ITGB3 isogenes in the anthology may be stored in separate containers.
  • a prefened ITGB3 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements hi a recombinant expression vector capable of being propagated and expressing the encoded ITGB3 protein in a prokaryotic or a eukaiyotic host cell.
  • expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence hi a given host cell.
  • the conect combinations of expression regulatory elements will depend on the host system used.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
  • Host cells which may be used to express the variant ITGB3 sequences of the invention include, but are not limited to, eukaiyotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known hi the art.
  • the recombinant expression vector may be infroduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning.
  • eukaryotic expression vectors that function in eukaiyotic cells, and preferably mammalian cells, are used.
  • Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
  • Preferred eukaiyotic cell lines include COS cells, CHO cells, HeLa cells, NEH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282:1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • ITGB3 mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules. These mRNAs can be used for the preparation of an
  • ITGB3 cDNA comprising a nucleotide sequence which is a polymoiphic variant of the ITGB3 reference coding sequence shown in Figure 2.
  • the invention also provides ITGB3 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ED NO:2 (Fig.
  • a particularly preferred polymoiphic cDNA variant is selected from the group consisting of A, B, C, D, E, F, G, H, I, K, J, L, M, N, O, P and Q represented in Table 8. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain one or more of the novel polymoiphisms described herein.
  • the invention specifically excludes polynucleotides identical to previously identified ITGB3 mRNAs or cDNAs, and previously described fragments thereof.
  • Polynucleotides comprising a variant ITGB3 RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • a polymo ⁇ hic variant of an ITGB3 gene fragment, mRNA fragment or cDNA fragment comprises at least one novel polymo ⁇ hism identified herein and has a length of at least 10 nucleotides and may range up to the full length of the gene.
  • such fragments are between 100 and 3000 nucleotides in length, and more preferably between 100 and 2000 nucleotides in length, and most preferably between 100 and 500 nucleotides in length.
  • nucleic acid molecules containing the ITGB3 gene or cDNA may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand.
  • reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymorphic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the ITGB3 genomic, mRNA and cDNA variants described herein.
  • Polynucleotides comprising a polymoiphic gene variant or fragment of the invention may be useful for therapeutic memeposes.
  • an expression vector encoding the isofonn may be administered to the patient.
  • the patient may be one who lacks the ITGB3 isogene encoding that isofonn or may already have at least one copy of that isogene.
  • ITGB3 isogene In other situations, it may be desirable to decrease or block expression of a particular ITGB3 isogene.
  • Expression of an ITGB3 isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA or antisense RNA for the isogene or fragment thereof.
  • oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3 ' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are prefened.
  • inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense oligonucleotides may also be designed to block translation of ITGB3 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of ITGB3 mRNA transcribed from a particular isogene.
  • the unfranslated mRNA, antisense RNA or antisense oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo. Alternatively, such molecules may be fo ⁇ mulated as a phamiaceutical composition for administration to the patient. Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
  • Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
  • the invention also provides an isolated polypeptide comprising a polymoiphic variant of (a) the reference ITGB3 amino acid sequence shown in Figure 3 or (b) a fragment of this reference sequence.
  • the location of a variant amino acid in an ITGB3 polypeptide or fragment of the invention is preferably identified by aligning its sequence against SEQ ID NO:3 (Fig. 3).
  • AN ITGB3 protein variant (or isofomi) of the invention comprises an amino acid sequence identical to SEQ ED NO:3 for those regions of SEQ ID NO: 3 that are encoded by examined portions of the ITGB3 gene (as described in the Examples below), except for having one or more variant amino acids selected from the group consisting of methionine at a position conesponding to amino acid position 14, proline at a position conesponding to amino acid position 59, arginine at a position conesponding to amino acid position 66, methionine at a position conesponding to amino acid position 445, and glutamine at a position conesponding to amino acid position 515.
  • an ITGB3 protein fragment of the invention is any fragment of an ITGB3 protein variant that contains one or more of the novel amino acid variations described herein.
  • the invention specifically excludes amino acid sequences identical to those previously identified for ITGB3, including SEQ ID NO:3, and previously described fragments thereof.
  • ITGB3 protein variants included within the invention comprise all amino acid sequences based on SEQ ED NO:3 and having any of the novel combination of amino acid variations described herein.
  • an ITGB3 protein variant is selected from the group consisting of A, B, C, D, E, F, and G represented in Table 9.
  • An ITGB3 peptide variant of the invention is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long.
  • Such ITGB3 peptide variants may be useful as antigens to generate antibodies specific for one of the above ITGB3 isofo ⁇ ns.
  • the ITGB3 peptide variants may be useful in drag screening assays.
  • An ITGB3 variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing an appropriate variant ITGB3 genomic or cDNA sequence described above.
  • the ITGB3 protein variant may be isolated from a biological sample of an individual having an ITGB3 isogene which encodes the variant protein.
  • a particular ITGB3 isofonn of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular ITGB3 isofonn but does not bind to the other ITGB3 isofonn.
  • ITGB3 protein or peptide variant may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isofonn of the ITGB3 protein or peptide as discussed further below.
  • ITGB3 variant proteins and peptides can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et.
  • a polymorphic variant ITGB3 gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric ITGB3 protein.
  • the non-ITGB3 portion of the chimeric protein may be recognized by a commercially available antibody.
  • the chimeric protein may also be engineered to contain a cleavage site located between the ITGB3 and non-ITGB3 portions so that the 1TGB3 protein may be cleaved and purified away from the non-ITGB3 portion.
  • An additional embodiment of the invention relates to using a novel ITGB3 protein isofomi, or a fragment thereof, in any of a variety of drug screening assays.
  • Such screening assays may be perfomied to identify agents that bind specifically to all known ITGB3 protein isoforms or to only a subset of one or more of these isoforms.
  • the agents may be from chemical compound libraries, peptide libraries and the like.
  • the ITGB3 protein or peptide variant may be free in solution or affixed to a solid support.
  • high throughput screening of compounds for binding to an ITGB3 variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the ITGB3 protein(s) of interest and then washed. Bound ITGB3 protein(s) are then defected using methods well-known in the art.
  • a novel ITGB3 protein isofonn may be used in assays to measure the binding affinities of one or more candidate drugs targeting the ITGB3 protein.
  • the invention provides antibodies specific for and immunoreactive with one or more of the novel ITGB3 protein or peptide variants described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the ITGB3 protein or peptide variant used to generate the antibodies may be from natural or recombinant sources (in vitro or in vivo) or produced by chemical synthesis or semi-synthetic synthesis using synthesis techniques known in the art. If the ITGB3 protein or peptide variant is of insufficient size to be antigenic, it may be concatenated or conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide.
  • carrier molecules include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Ten, Appleton and Lange, Nonvalk Connecticut, San Mateo, California).
  • an antibody specifically immunoreactive with one of the novel protein or peptide variants described herein is administered to an individual to neutralize activity of the ITGB3 isofonn expressed by that individual.
  • the antibody may be fonnulated as a phamiaceutical composition which includes a phannaceutically acceptable carrier.
  • Antibodies specific for and immunoreactive with one of the novel protein isofo ⁇ ns described herein may be used to immunoprecipitate the ITGB3 protein variant from solution as well as react with ITGB3 protein isofomis on Western or immunoblots of polyaciylamide gels on membrane supports or substrates.
  • the antibodies will detect ITGB3 protein isofomis in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques.
  • an antibody specifically immunoreactive with one of the novel ITGB3 protein variants described herein is used in immunoassays to detect this variant in biological samples, hi this method, an antibody of the present invention is contacted with a biological sample and the formation of a complex between the ITGB3 protein variant and the antibody is detected.
  • suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
  • Neoman Stockton Press, New York, New York; Cunent Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York). Standard techniques known hi the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984. Methods in Immunology, W.A. Benjamin, Inc.). Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
  • Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Cun-ent Protocols hi Molecular Biology, supra.
  • Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site.
  • Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam).
  • the antibodies or antigen binding fragments thereof may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication numbers WO 9014443 and WO 9014424, and in Huse et al., 1989, Science, 246: 1275-1281.
  • the antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci. USA 86; 10029). Effect(s) of the polymorphisms identified herein on expression of ITGB3 may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts of the ITGB3 gene, cDNA or fragment thereof, or by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the ITGB3 gene.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA(s) into ITGB3 protein(s) (including effects of polymo ⁇ hisms on codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the desired ITGB3 isogene, cDNA or coding sequence may be infroduced into the cell in a vector such that the isogene, cDNA or coding sequence remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the ITGB3 isogene, cDNA or coding sequence is infroduced into a cell in such a way that it recombines with the endogenous ITGB3 gene present in the cell. Such recombination requires the occunence of a double recombination event, thereby resulting in the desired ITGB3 gene polymoiphism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector constmct may be used in the invention.
  • Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral fransduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
  • Examples of cells into which the ITGB3 isogene, cDNA or coding sequence may be introduced include, but are not limited to, continuous culture cells, such as COS, CHO, NIH 3T3, and primary or culture cells of the relevant tissue type, i.e., they express the ITGB3 isogene, cDNA or coding sequence.
  • Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant ITGB3 gene, cDNA or coding sequence are prepared using standard procedures known in the art.
  • a construct comprising the variant gene, cDNA or coding sequence is introduced into a nonhuman animal or an ancestor of the animal at an embiyonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
  • Transgenic animals carrying the constracts of the invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes (or cDNA or coding sequence) of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053.
  • Another method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells.
  • mice examples include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice” and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272).
  • Transgenic animals stably expressing a human ITGB3 isogene, cDNA or coding sequence and producing the encoded human ITGB3 protein can be used as biological models for studying diseases related to abnormal ITGB3 expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel ITGB3 isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel ITGB3 isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one of the novel ITGB3 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel ITGB3 isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel ITGB3 isogene is reduced and/or eliminated.
  • the composition also comprises a phannaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered salhie, dextrose, and water.
  • a phannaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered salhie, dextrose, and water.
  • Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the phamiaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
  • Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, mtrameduUary, infrathecal, infraventricular, infradermal, transdermal, subcutaneous, infraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Phamiaceutical Sciences (Maack Publishing Co., Easton, PA). For any composition, determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art. For example, the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage will be detennined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of adminisfration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • the invention also provides a method for determining the frequency of an ITGB3 genotype, haplotype, or haplotype pah in a population.
  • the method comprises, for each member of the population, detennining the genotype, haplotype or the haplotype pair for the novel ITGB3 polymorphic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
  • the population may be e.g., a reference population, a family population, a same gender population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic freatment).
  • ITGB3 haplotype frequencies in a trait population having a medical condition and a control population lacking the medical condition are used in a method of validating the ITGB3 protein as a candidate target for freating a medical condition predicted to be associated with ITGB3 activity.
  • the method comprises comparing the frequency of each ITGB3 haplotype shown in Table 5 in the trait population and hi a control population and making a decision whether to pursue ITGB3 as a target.
  • the composition of the control population will be dependent upon the specific study and may be a reference population or it may be an appropriately matched population with regards to age, gender, and clinical symptoms for example.
  • At least one ITGB3 haplotype is present at a frequency i the trait population that is different from the frequency in the confrol population at a statistically significant level, a decision to pursue the ITGB3 protein as a target should be made.
  • the frequencies of each of the ITGB3 haplotypes are not statistically significantly different between the trait and control populations, a decision not to pursue the ITGB3 protein as a target is made.
  • the statistically significant level of difference in the frequency may be defined by the skilled artisan practicing the method using any conventional or operationally convenient means known to one skilled in the art, taking into consideration that this level should help the artisan to make a rational decision about pursuing ITGB3 protein as a target.
  • each of the trait and controls populations may be comprised of different ethnogeographic origins, including but not limited to Caucasian, Hispanic Latino, African American, and Asian, while in other embodiments, the trait and reference population may be comprised of just one ethnogeographic origin.
  • frequency data for ITGB3 haplotypes are determined h a population having a condition or disease predicted to be associated with ITGB3 activity and used in a method for screening for compounds targeting the ITGB3 protein to treat such condition or disease.
  • frequency data are determined in the population of interest for the ITGB3 haplotypes shown in Table 5.
  • the frequency data for this population may be obtained by genotyping or haplotyping each individual in the population using one or more of the methods described above.
  • the haplotypes for this population may be detennined directly or, alternatively, by a predictive genotype to haplotype approach as described above.
  • the frequency data for this population are obtained by accessing previously determined frequency data, which may be in written or electronic fonn.
  • the frequency data may be present in a database that is accessible by a computer.
  • the ITGB3 isoforms conesponding to ITGB3 haplotypes occuning at a frequency greater than or equal to a desired frequency in this population are then used in screening for a compound, or compounds, that displays a desired agonist (enhancer) or antagonist (inhibitor) activity for each ITGB3 isofonn.
  • the desired frequency for the haplotypes might be chosen to be the frequency of the most frequent haplotype, greater than some cut-off value, such as 10% in the population, or the desired frequency might be detennined by ranking the haplotypes by frequency and then choosing the frquency of the third most frequent haplotype as the cut-off value.
  • the desired level of agonist or antagonist level displayed in the screening process could be chosen to be greater than or equal to a cut-off value, such as activity levels in the top 10% of values detennined.
  • Embodiments may employ cell-free or cell-based screening assays known in the art.
  • the compounds used in the screening assays may be from chemical compound libraries, peptide libraries and the like.
  • the ITGB3 isofo ⁇ ns used in the screening assays may be free in solution, affixed to a solid support, or expressed in an appropriate cell line.
  • the condition or disease associated with ITGB3 activity is coronary heart disease or other disorders of cholesterol metabolism.
  • frequency data for ITGB3 genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and a ITGB3 genotype, haplotype, or haplotype pah.
  • the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as hi a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one or more of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic fonn.
  • the frequency data may be present in a database that is accessible by a computer.
  • the frequencies of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared, hi a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If the frequency of a particular ITGB3 genotype, haplotype, or haplotype pair is different in the trait population than in the reference population to a statistically significant degree, then the trait is predicted to be associated with that ITGB3 genotype, haplotype or haplotype pair.
  • the ITGB3 genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the genotypes and haplotypes shown in Tables 4 and 5, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
  • clinical population In order to deduce a conelation between clinical response to a treatment and an ITGB3 genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been ran and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • the term "clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.
  • the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetennined criteria. It is contemplated that hi many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the ITGB3 gene for each individual hi the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • Correlations may be produced in several ways. In one method, individuals are grouped by their ITGB3 genotype or haplotype (or haplotype pair) (also refened to as a polymoiphism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
  • the correlation is found using a genetic algorithm approach as described in WO 01/01218. Correlations may also be analyzed using analysis of variation (ANOVA) techniques to detemiine how much of the variation in the clinical data is explained by different subsets of the polymoiphic sites in the ITGB3 gene. As described in WO 01/01218, ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10).
  • ANOVA analysis of variation
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of ITGB3 genotype or haplotype content.
  • the model is validated in one or more follow-up clinical trials designed to test the model.
  • the identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the ITGB3 gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the freatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug.
  • the diagnostic method will detect the presence in an individual of the genotype, haplotype or haplotype pair that is associated with the clinical response and may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the ITGB3 gene), a serological test, or a physical exam measurement.
  • this diagnostic method uses the predictive haplotyping method described above.
  • Another embodiment of the invention comprises a method for reducing the potential for bias in a clinical trial of a candidate drug for treating a disease or condition predicted to be associated with ITGB3 activity.
  • Haplotyping one or both copies of the ITGB3 gene in those individuals participating in the trial will allow the pharmaceutical scientist conducting the clinical trial to assign each individual fiom the tiial one of the ITGB3 haplotypes or haplotype pairs shown in Tables 5 and 4, respectively, or a ITGB3 sub-haplotype or sub-haplotype pair thereof.
  • the haplotypes may be determined directly, or alternatively, by a predictive genotype to haplotype approach as decribed above.
  • this can be accomplished by haplotyping individuals participating in a clinical trial by identifying, for example, in one or both copies of the individual's ITGB3 gene, the phased sequence of nucleotides present at each of PS 1 -PS44. Determining the ITGB3 haplotype or haplotype pair present in individuals participating in the clinical frial enables the pharmaceutical scientist to assign individuals possessing a specific haplotype or haplotype pair evenly to treatment and control groups. Typical clinical trials conducted may include, but are not limited to, Phase I, II, and III clinical trials.
  • each individual in the trial may produce a specific response to the candidate drug based upon the individual's haplotype or haplotype pair.
  • each treatment and control group are assigned an even distribution (or equal numbers) of individuals having a particular ITGB3 haplotype or haplotype pair.
  • the phamiaceutical scientist requires no a priori knowledge of any effect a ITGB3 haplotype or haplotype pair may have on the results of the trial.
  • Diseases or conditions predicted to be associated with ITGB3 activity include, e.g., Glanzman's thrombocytopenia and cardiovascular disease. It is also contemplated that the above described methods and compositions of the invention may be utilized in combination with identifying genotype(s) and/or haplotype(s) for other genomic regions.
  • Example 1 This example illustrates the clinical and biochemical characterization of 679 patients in the patient cohort.
  • a multicenter, 17-week, (16 weeks controlled), open-label, clinical discovery trial was designed to assess the relationship between genetic haplotype markers and treatment response associated with 4 different commercially available medications, all of which act as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (cerivastatin sodium [BaycolTM], aton'astatin calcium [Lipitor®], simvastatin [Zocor®], and pravastatin sodium [Pravachol®]) in adult subjects with primaiy hypercholesterolemia. Study medications were packaged by their respective manufacturers and dispensed in a non-blinded fashion by a commercial pha ⁇ nacist. The cerivastatin sodium aim of the study was discontinued at the time of the withdrawal of the drug from the market by the manufacturer and therefore data from the partially completed arm are excluded from this analysis.
  • HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors
  • Subject inclusion criteria were based upon medical history assessments and laboratory determinations of cholesterol levels as described by the National Cholesterol Education Program (NCEP)-recommended goal for LDL-cholesterol (> 160 mg/dL for subjects with 0 to 1 coronary heart disease [CHD] risk factor, > 130 mg/dL for those with 2 or more CHD risk factors, or > 100 mg/dL for those with documented CHD or peripheral vascular disease) and had triglyceride levels ⁇ 400 mg/dL prior to randomization.
  • Eligible subjects had an LDL-cholesterol level ⁇ 240 mg/dL at screening and baseline. Subjects had to demonstrate dietary compliance with the AHA Step I or Step II diet as measured by a food diary at baseline to be eligible for randomization.
  • the entire patient cohort comprised 679 patients. Subjects were randomly assigned to 1 of 4 treatment groups: 0.4 mg/day cerivastatin sodium, 10 mg/day atorvastatin calcium, 20 mg/day simvastatin, or 10 mg/day pravastatin sodium at baseline. All medication was taken once daily hi the evening.
  • the doses for the treatment groups were as follows: 0.8 mg/day cerivastatin sodium, 80 mgday atoiYastatin calcium, 80 mg/day simvastatin, and 40 mg/day pravastatin sodium. All medication was taken once daily in the evening.
  • the primary phenotypic endpoint used in the association of treatment response to genetic variability was the percent change from baseline in LDL-cholesterol values after 8 weeks and after 16 weeks of treatment, separately.
  • the final Week 8 value was defined as the mean of the last 2 measurements (Weeks 6 and 8) during the first 8 weeks (low dose) of therapy.
  • the final Week 16 value was defined as the mean of the last 2 measurements (Weeks 14 and 16) during the final 8 weeks (high dose) of therapy.
  • Baseline was defined as the mean of the measurements taken at screening and baseline.
  • the patient cohorts for the diree completed statin arms were characterized with respect to statin taken in treatment as shown below.
  • Demographics, baseline characteristics, and lipid changes for the low-dose compliant population are Demographics, baseline characteristics, and lipid changes for the low-dose compliant population.
  • HDL-C* BL (mg/dL) 50.6 ⁇ 13.7 47.3 ⁇ 11.2 48.9 ⁇ 12.4 48.9 ⁇ 12.5
  • TGJ BL (mg/dL) 164(70,361) 173(60,384) 166(54,370) 167(54,384)
  • This example illustrates examination of various regions of the ITGB3 gene in the 854 individuals of the experimental population, including the Index Repository and the patient cohort described in Example 1, for polymoiphic sites.
  • the human individuals in the Index Repository included a reference population of 82 unrelated individuals, of which 79 were self-identified as belonging to one of four major population groups: Caucasian (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic Latino (18 individuals).
  • the Index Repositoiy contains three unrelated indigenous American Indians (one from each of North, Cenfral and South America), one three- generation Caucasian family (from the CEPH LJtah cohort) and one two-generation African-American family AMPLIFICATION OF TARGET REGIONS
  • the following target regions of the ITGB3 gene were amplified using 'tailed' PCR primers, each of which includes a universal sequence fonning a noncomplemeiitary 'tail' attached to the 5' end of each unique sequence in the PCR primer pairs.
  • the universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 ' (SEQ ED NO:224) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5'-AGGAAACAGCTATGACCAT-3 r (SEQ ED NO:225).
  • the nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and conespond to positions in SEQ ED NO:l ( Figure 1).
  • Fragment 16 24385-24406 complement of 24930-24909 546 nt
  • Amplification profile 97°C - 2 min. 1 cycle
  • PCR PRODUCTS The PCR products were purified using a Whatman/Polyfiltronics 100 ⁇ l 384 well unifilter plate essentially according to the manufacturers protocol. The purified DNA was eluted in 50 ⁇ l of distilled water. Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol. The purified PCR products were sequenced in both directions using the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
  • This example illustrates analysis of the ITGB3 polymo ⁇ hisms identified in the Index Repositoiy and the patient cohort for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in unrelated members of the reference population are shown in Table 4 below, following the examples, with the haplotype pair indicating the combination of haplotypes detemiined for the individual using the haplotype derivation protocol described below.
  • homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides.
  • the haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented algorithm for assigning haplotypes to unrelated individuals in a population sample, as described in WO 01/80156.
  • each of the ITGB3 haplotypes comprises a 5 ' - 3 ' ordered sequence of 44 polymoiphisms whose positions in SEQ ED NO:l and alleles are set forth in Table 5.
  • Table 5 the column labeled "Region Examined” provides the nucleotide positions in SEQ ID NO: 1 conesponding to sequenced regions of the gene.
  • PS No. and PS Position provide the polymo ⁇ hic site number designation (see Table 3) and the coiresponding nucleotide position of this polymoiphic site within SEQ ED NO: 1 or SEQ ID NO:226.
  • the columns beneath the "Haplotype Number” heading are labeled to provide a unique number designation for each ITGB3 haplotype.
  • Table 6 shows the number of chromosomes characterized by a given ITGB3 haplotype for all unrelated individuals in the Index Repositoiy for which haplotype data was obtained.
  • the number of these unrelated individuals who have a given ITGB3 haplotype pair is shown in Table 7 below, following the Examples.
  • the "Total" column shows this frequency data for all of these unrelated individuals, while the other columns show the frequency data for these unrelated individuals categorized according to their self-identified ethnogeographic origin.
  • the size and composition of the Index Repositoiy were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population.
  • this repository contains approximately equal sample sizes of African-descent, Asian-American, European-American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background.
  • the number of unrelated mdividuals in the Index Repositoiy provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population.
  • a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group.
  • the size and composition of the Index Repositoiy means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the ITGB3 gene are likely to be similar to the relative frequencies of these ITGB3 haplotypes and haplotype pairs hi the general U.S. population and in the four population groups represented in the Index Repository.
  • the genetic diversity obseiYed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.
  • Each ITGB3 haplotype shown in Table 5 defines an ITGB3 isogene.
  • the ITGB3 isogene defined by a given ITGB3 haplotype comprises the examined regions of Figure 1 (SEQ ED NO: 1) indicated in Table 5, with the corresponding ordered sequence of nucleotides occurring at each polymorphic site within the ITGB3 gene shown in Table 5 for that defining haplotype.
  • Each ITGB3 isogene defined by one of the haplotypes shown in Table 5 will further correspond to a particular ITGB3 coding sequence variant.
  • Each of these ITGB3 coding sequence variants comprises the regions of Figure 2 (SEQ ED NO:2) examined and is defined by the 5 ' - 3 ' ordered sequence of nucleotides occurring at each polymorphic site within the coding sequence of the ITGB3 gene, as shown in Table 8, following the Examples.
  • the column labeled 'Region Examined' provides the nucleotide positions in Figure 2 (SEQ ID NO:2) corresponding to sequenced regions of the gene; the columns labeled 'PS No.' and 'PS Position' provide the polymoiphic site number designation (see Table 3) and the corresponding nucleotide position of this polymoiphic site within Figure 2 (SEQ ID NO:2).
  • the columns beneath the 'Coding Sequence Number' heading are numbered to conespond to the haplotype number defining the ITGB3 isogene from which the coding sequence variant is derived.
  • ITGB3 coding sequence variants that differ from the reference ITGB3 coding sequence are denoted in Table 8 by a letter (A, B. etc) identifying each unique novel coding sequence.
  • the same letter at the top of more than one column denotes that a given novel coding sequence is present in multiple novel ITGB3 isogenes.
  • each ITGB3 coding sequence represented in Table 8 encodes an ITGB3 protein variant.
  • Each of the ITGB3 protein variants encoded by the 216 ITGB3 isogenes described herein comprises the regions of Figure 3 (SEQ ID NO:3) examined by sequencing and is defined by the N- ten ⁇ inus to C-tenninus sequence of amino acids resulting from the observed polymo ⁇ hisms at the polymo ⁇ hic sites within the coding sequence of the ITGB3 gene, as presented in Table 9, following the Examples.
  • the column labeled 'Region Examined' provides amino acid positions in Figure 3 (SEQ ID NO:3) conesponding to sequenced regions of the gene.
  • EXAMPLE 4 This example illustrates analysis of the ITGB3 haplotypes in Table 4 for association with individuals' response to Lipitor®.
  • Low-dose percent change in HDLC (Low-dose follow-up HDLC - Baseline HDLC)/(Baseline HDLC)* 100.
  • Baseline HDLC the average of the screening and baseline visits' HDLC values, unless the screening and baseline LDL values were more than 15% apart from one another, in which case a second baseline sample of all lipids was collected, and baseline HDLC was the average of the two baseline HDLC values.
  • High-dose follow-up HDLC the average of the 14-week and 16-week values if both were available; otherwise the last single value from among the 12-week, 14-week, 16-week and early termination (if applicable) values was used.
  • a haplotype marker is defined as a prescription for the genotype at all the polymo ⁇ hic sites h a single copy of a particular gene.
  • the prescription allows for a site to be either inelevant ('*'), or requires the genotype at the site to be one of the possible alleles (e.g., A or G).
  • a particular marker can be expressed as a string of characters, one for each site. If a gene A has 5 known polymo ⁇ hic sites, for example, we can define a particular marker to be A:*A*CG. In order to have a copy of the example marker A:*A*CG, a person has to have a copy of the gene with A, C and G at polymo ⁇ hic sites 2, 4 and 5, respectively.
  • the column labeled Sites indicates the number of polymo ⁇ hic sites present in the marker.
  • the Marker column represents the sites by the polymoiphic site number (as per Table 3) and the nucleotide present at that site.
  • the marker IC, 42C indicates that the marker is composed of a cytosine at PSI and a cytosine at PS42; the identities of the nucleotides present at the other ITGB3 polymo ⁇ hic sites presented herein are irrelevant to the statistical significance of the association.
  • the model column indicates whether the In group is defined by havmg 1 or 2 copies of the Marker (Dominant) or 2 copies of the Marker (Recessive).
  • the In Mean and Out Mean columns present for the hi and Out groups, respectively, the mean difference in per cent change i HDLC relative to baseline after the 16 week high dose regimen vs. after the 8 week low dose regimen .
  • hi Count and Out Count present the number of members of the Lipitor® cohort belonging to the hi and Out groups, respectively.
  • the column labeled Delta presents the difference between the In mean and Out mean to illustrate the magnitude in the difference in response of members of the two groups.
  • Raw P presents the p value calculated for the marker-pheiiotype association using the t-test, while Adjusted P presents the p value for the association detennined by the permutation test described above.
  • the In group with 1 or 2 copies of that haplotype showed a mean difference in per cent change in HDLC from baseline aftei the week 16 determination at high dose of atorvastatin relative to per cent change in HDLC from baseline after the week 8 determination at low dose of atorvastatin that was worse, fiom a clinical perspective, than the mean difference in per cent change in HDLC from baseline ,at high dose lelative to low dose of atorvastatin hkel ⁇ to be observed m an individual having no copy of that haplotype.
  • the HDLC levels of patients having at least one copy (i.e., the "In Group") of an ITGB3 haplotype shown in Table 10A were found to be more sensitive to atoiYastatin dose than patients having zero copy of these haplotypes (the Out group for these haplotypes).
  • These patients in the hi group for the ITGB3 haplotypes shown in Table 10A responded to treatment with the high dose of atoiYastatin with a reduction in their HDLC relative to the effect on HDLC they experienced in response to treatment with the low dose of atorvastatin.
  • HDLC levels for patients belonging to the Out group were found to be little changed or to increase upon freatment with the highest dose of atoiYastatin, compared to freatment with the lowest dose.
  • the Out group comprising those with only 0 or 1 copies of that haplotype, showed a mean difference in per cent change in HDLC from baseline after the week 16 determination at high dose of atoiYastatin relative to per cent change in HDLC from baseline after the week 8 determination at low dose of aton'astatin that was worse, from a clinical perspective, than the mean difference in per cent change in HDLC from baseline at high dose relative to low dose of atorvastatin likely to be observed in individuals having two copies of that haplotype (In group).
  • the group of patients (In Group) with two copies of an ITGB3 haplotype shown in Table 10B experienced a better adjusted mean per cent change in HDLC at high dose relative to low dose of atorvastatin than the group of patients having 0 or 1 copy of the marker (Out group).
  • the mean difference obsen'ed for the In group for the identified haplotypes in Table 10B ranged from +0.26% to +1.26%, while the mean difference observed for the Out group for these haplotypes ranged from —4.75% to -5.40%.
  • the HDLC levels of patients having two copies of any ITGB3 haplotype shown in Table 10B were found to be more likely to show at least a small increase in change in HDLC with increased atoiYastatin dose than patients having only zero or one copy of any of these ITGB3 haplotypes.
  • These patients in the In group responded to treatment with the high dose of atorvastatin with an increase in their HDLC relative to the effect on HDLC they experienced in response to treatment with the low dose of atorvastatin.
  • HDLC levels for patients belonging to the Out group were found to decrease upon treatment with the highest dose of atorvastatin, compared to the HDLC level detennined after freatment with the lowest dose.
  • any of these genetic markers or haplotypes in Tables 10A and 10 B are useful for identifying patients who may respond to higher doses of atorvastatin with a reduction in their HDLC levels rather than a small increase. Consequently, the haplotypes in Table 5, comprising all 44 polymo ⁇ hic sites identified by Applicants, that comprise any of these genetic markers are likewise useful for identifying these patients.
  • a reduced set of 13 polymo ⁇ hisms resulting in 91% of the haplotype diversity provided by the set of 44 polymo ⁇ hisms was also analyzed for correlations in HDLC response to dose of atorvastatin. All combinations of haplotypes including up to four polymoiphism were considered hi this analysis. Each unique haplotype with a frequency of >1% was tested for association with the clinical response. Each mdividual in the analysis cohort was classified as having 0, 1, or 2 copies of the haplotype. Only dominant (i.e., 1 or 2 copies of the marker vs. 0 copy of the marker) and recessive (i.e., 0 or 1 copy of the marker vs 2 copies) models were considered in this analysis.
  • ANCOVA covariance
  • Polyld is a unique identifier assigned to each PS by Genaissance Pha ⁇ naceuticals, Inc.
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions sequenced;
  • PS polymo ⁇ hic site;
  • Alleles for ITGB3 haplotypes are presented 5' to 3 ' in each column. Table 5(Part 2). Haplotypes of the ITGB3 gene.
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions sequenced;
  • PS polymo ⁇ hic site;
  • Alleles for ITGB3 haplotypes are presented 5' to 3 ' in each column. Table 5(Part 3). Haplotypes of the ITGB3 gene.
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions ; sequenced;
  • PS polymo ⁇ hic site;
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions t sequenced;
  • PS polymo ⁇ hic site;
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions : sequenced;
  • PS polymo ⁇ hic site;
  • Alleles for ITGB3 haplotypes are presentee I 5 ' to : 5 ' in each column. Table 5(Part 7). Haplotypes of the ITGB3 gene.
  • Region examhied represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions : sequenced;
  • PS polymoiphic site;
  • Alleles for ITGB3 haplotypes are presentee 1 5 ' to 2 V in ⁇ 2ach column. Table 5(Patt 8). Haplotypes of the ITGB3 gene.

Abstract

Haplotypes of the ITGB3 gene that are associated with atorvastatin dose effects on changes in HDL cholesterol are disclosed. Compositions and methods for detecting and using these ITGB3 haplotypes in a variety of clinical applications, as well as in pharmaceutical research and development contexts, are disclosed. Such applications include articles of manufacture comprising a statin composition approved for treating patients having one of these ITGB3 haplotypes, methods and kits for predicting the response of an individual to atorvastatin treatment based on their haplotype profile, and methods for treating individuals with hyperlipidemia based on their haplotype profile.

Description

ITGB3 GENE HAPLOTYPES AND ATORVASTATIN DOSE EFFECTS ON HDL
CHOLESTEROL
RELATED APPLICATIONS This application claims the benefit of US Provisional Application 60/417,743 filed October 9,
2002.
FIELD OF THE INVENTION
This invention relates to the field of genomics and pharmacogenetics. More specifically, this invention relates to genetic markers of the gene for integrin, beta 3 (ITGB3) and their use as predictors of response to treatment with statins.
BACKGROUND OF THE INVENTION
Cardiovascular disease is a major health problem in the United States and worldwide (R. H. Knopp, jV. Engl. J. Med. 341:498-511, 1999). The major cause of cardiovascular disease is atherosclerosis, which results from the fonnation of lipid-laden cellular lesions in one or more of the coronary arteries that supply the heart muscle with blood (Leff, T. and Gruber, P.J., "Cardiovascular Diseases" in: Meyers. R. Molecular Biology and Biotechnology (NCR Publishers 1995) pp. 149-153). High levels of low-density lipoprotein cholesterol ("LDLC") have long been associated with an increased risk of developing atherosclerosis (Leff and Gruber, supra). However, it is now widely accepted that high levels of plasma tnglycerides ("TG") and low levels of high-density lipoprotein cholesterol ("HDLC") are associated with coronary artery disease as well (Gotto AM, American Journal of Cardiology, 87 (5 Suppl.) 13-18, 2001. Another risk factor for cardiovascular disease is high levels of LDL apolipoprotein B ("LDL Apo B"), which is the major lipoprotein associated with LDLC particles (American Heart Association National Center, New Release NR 96-4430 (Circ/apo B), August 1, 1996).
Patients with one or more of the above risk factors are frequently treated with one or more lipid- modifying drugs to achieve certain target levels of LDLC and HDLC that are recommended by the current National Cholesterol Education Program guidelines for treatment of hypercholesterolemia. LIsual medical practice is to direct initial drug therapy toward elevated LDLC with treatment of low HDLC a secondary endpoint that is often managed by addition, after some weeks, of a second therapeutic agent.
One class of lipid-modifying drugs that are particularly useful for reducing elevated LDLC levels are statins, which inhibit the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for cholesterol fonnation in the liver and other tissues (Vaughan et al., J. Amer. College Cardiol. 35:1-10, 2000; Knopp, supra). In addition, in clinical trials of various statin compounds, increases in HDLC levels were observed, with a mean increase of 2% - 12%, depending upon the specific statin compound and the conditions under which it was studied. While most of the common side effects of statin therapy are mild, transient and reversible (e.g., dyspepsia, abdominal pain and flatulence), more severe, long-term adverse reactions to statins occur and include hepatitis, peripheral neuropathy, insomnia, difficulty in concentrating, and elevation of creatine phosphokinase, which is correlated with rhabdomyolysis (Knopp, supra, Lupattelli G et al., Nucl. Med. Commim. 22(5): 575-8, 2001; Moghadasian MH et al., Expert Opin. Pharmacother. 1(4): 683-95, 2000). Currently, there are five statins sold in the United States: lovastatin and simvastatin (sold by Merck as Mevacor® and Zocor8, respectively); atorvastatin calcium (sold as Lipitor® by the Parke Davis Division of Pfizer); fluvastatin sodium (sold as Lescol® by Novartis); and pravastatm sodium (sold as Pravachol® by Bristol-Myers Squibb) (Knopp, supra). A sixth statin, cerivastatin sodium, was previously sold as Baycol® by Bayer, but was voluntarily removed from the market in 2001 because of safety concerns. Five of these drugs are metabolized by cytochrome P-450 enzyme systems, while the sixth, pravastatin sodium, is metabolized by sulfation and possibly other mechanisms (Knopp, supra).
Extensive studies have been perfoπned with cerivastatin sodium, atorvastatin calcium, simvastatin, and pravastatin sodium to determine efficacy in the treatment of hypercholesterolemia. In three multicenter, placebo-controlled, dose-response studies of cerivastatin sodium, subjects with primary hypercholesterolemia experienced significantly reduced levels of total-cholesterol, LDLC, apolipoprotein B (apo B), tnglycerides, total-cholesterol/HDLC ratio, and LDLC HDLC ratios following treatment with cerivastatin sodium for an 8-week period. The mean decreases in LDLC and mean increases in HDLC for cerivastatin sodium administered once daily in the evening were 25% and 9% at 0.2 mg/day, 31% and 8% at 0.3 mg/day, and 34% and 7% at 0.4 mg/day (Physicians' Desk Reference, 2000, p. 675). Similarly, in two multicenter, placebo-controlled, dose-response studies, atorvastatin calcium given as a single dose over a six-week period significantly reduced total- cholesterol, LDLC, Apo B, and triglycerides. Atorvastatin calcium at 10, 20. 40, and 80 mg/day, resulted in mean LDLC decreases/HDLC increases of 39%/6%, 43%/9%, 50%/6%, and 60%/5%, respectively (Physicians' Desk Reference, 2000, p. 2255). Also, a multicenter, double-blind, placebo- controlled, dose-response study of simvastatin showed a significant decrease in total-cholesterol, LDLC, total cholesterol HDLC ratio, and LDLC /HDLC ratios in subjects with familial or non-familial hypercholesterolemia. (Physicians' Desk Reference, 2000, p. 1917). In comparative studies of simvastatin at a low daily dose versus a high daily dose, the mean percent decreases in LDLC and mean percent increases in HDLC observed were 26% and 10% for 5 mg, 30% and 12% for 10 mg, 41% and 9% for 40 mg, and 47% and 8% for 80 mg. Finally, in multicenter, placebo-controlled studies of pravastatin sodium given in daily doses from 10-40 mg, subjects with primary hypercholesterolemia showed consistent and significant decreases in total-cholesterol, LDLC, triglycerides, total- cholesterol/HDLC ratio, and LDLC HDLC ratios. The mean LDLC decreases and HDLC increases for pravastatm sodium administered once daily at bedtime were 22% and 7% at 10 mg/day, 32% and 2% at 20 mg/day, and 34% and 12% at 40 mg/day (Physicians' Desk Reference, 2000, p. 846).
Other comparative studies have suggested that statins differ in some of their clinical properties relevant to reducing the risks of atherosclerosis. A recent double-blind, randomized, parallel, 36-week dose escalation study with 826 hypercholesterolemic patients compared simvastatin and atorvastatin at 40 or 80 mg/day. As dose increased, simvastatin resulted in larger increases in HDLC than atorvastatin (Illingworth DR et al. (2001) C r Med Res Opin 17(l):43-abstract only). Wierzbicki & Mikhailidis (2002 IntJ. Cardiol. 84(1): 53 -57) reviewed five studies comparing the dose-response effects of atorvastatin and simvastatin on HDLC in hypercholesterolemic patients to compare daily doses for both drugs ranging from 10 to 80 mg. HDLC was significantly and consistently increased by all doses of simvastatin. However while atorvastatin showed increases in HDLC at low dose, the pooled data from all five studies suggest a negative dose-response effect with smaller increases in HDLC with increasing atorvastatin concentration. The studies described above report population means for changes in LDLC and HDLC that disguise substantial evidence of significant interindividual variation in response to statins. Indeed, any particular individual treated with a statin may experience a 10% to 70% reduction in LDLC (Aguilar- Salinas SA et al.. Atherosclerosis 141:203-207, 1998). In addition, physicians have observed that some patients treated with statins exhibit minimal or no increase in HDLC, which is not an optimal response for patients with low HDLC levels. However, physicians currently are unable to identify patients who are at risk for reduced efficacy of statin therapy, which can be expensive and is not without risk. Also, physicians must currently rely on trial and error to determine which statin and dose combination will produce the best LDLC or HDLC response in any particular patient. Thus it would be useful to understand the biological basis for variability of response to statins. Part of this biological basis may be genetic variation in proteins involved in lipid metabolism and/or atherogenecity (Kuivenhoven et al., supra). One protein demonstrated to be involved in atherogenicity is the integrin, beta 3 (ITGB3) protein, also known as platelet glycoprotein Ilia (GP Ilia) or antigen CD61. Therefore, one pharmaceutically-important gene for the treatment of cardiovascular disease and Glanzman's thrombocytopenia is the integrin, beta 3 (ITGB3) gene or its encoded product. The ITGB3 protein is a member of the integrin family of receptors that bind cell adhesion molecules. The integrin family consists of heterodimeric molecules composed of alpha and beta subunits. ITGB3 is the beta subunit for at least tliree integrin receptors having different alpha subunits: the platelet complex, ITGA2B/ITGB3 (GP Ilb/DIa) receptor; the fibronectin receptor and the vitronectin receptor, hi view of its currently understood biological functions, it is an attractive target for new therapeutics to treat cardiovascular disease.
The major integrins are found on platelets and participate in the platelet adhesion process. Platelet activation is initiated by the presence of thrombin as well as various mechanical and chemical stimuli. Activation of platelets is also associated with the activation of the ITGA2B/ITGB3 (GP Ilb/IIIa) receptor complex (Lefkovits et al., N. Engl. J. Med. 1995; 332(23):1553-1559). ITGA2B and ITGB3 conespond to the alpha and beta subunits, respectively, of the platelet glycoprotein receptor. Activation of platelets results in changes in platelet shape causing a shift of the ITGA2B/ITGB3 complex from a ligand-unreceptive state to a ligand-receptive state. In the latter configuration, the ITGA2B/ITGB3 complex is able to bind fibrinogen, which forms a bridge between adjacent platelet molecules facilitating platelet aggregation (Lefkovits et al., supra).
Diminished levels of the ITGA2B ITGB3 are observed in Glanzman's thrombocytopenia, a disease characterized by a lack of platelet aggregation and recuirent mucocutaneous bleeding. The disease is associated with deficient platelet aggregation in response to all physiological stimuli, and is considered a paradigm for inliibition of ITGA2B ITGB3. ITGA2B/ITGB3 receptor inhibitors are a class of drags with the potential to prevent events associated with coronary artery disease. Examples of these inhibitors include abciximab (ReoPro®; Eli Lilly and Company), a chimeric immunoglobulin, as well as natural products such as trigramin, isolated from the venom of the viper Trimeresurus gramineus. In addition, a number of synthetic peptides based on the recognition sequence Arg-Gly-Asp (RGD) present in ITGA2B ITGB3 receptors have been developed. Examples include G4120 (Genentech, South San Francisco, CA), MK-852 (Merck, West Point, PA), and integrelin (COR Therapeutics, South San Francisco, CA). The ability of ITGA2B/ITGB3 inhibitors to control platelet function has been demonstrated in clinical studies. Patients undergoing angioplasty or atherectomy who were administered abciximab had 35% fewer ischemic events compared to individuals given a placebo. The principal effect of abciximab was to reduce the incidence of myocardial infarction (Lefkovitz et al., 1995; J Am Coll Cardiol (special issue):81A). In patients with unstable angina, the ITGA2B/ITGB3 inhibitor lamifiban has been shown to reduce the incidence of myocardial infarction and death (Theroux et al., 1994; Circulation:90:I-232). Similar results for unstable angina have been shown in pilot studies of abciximab and integrelin (Simoons et al, 1994; Circulation:90:I-232; Schulman et al, 1993; Circulation 88:1-608). These data demonstrate that inhibition of platelet aggregation is a useful strategy in combating cardiovascular disease.
The integrin, beta 3 (platelet glycoprotein Ilia, antigen CD61) gene is located on chromosome 17q21.32 and contains 15 exons that encode a 788 amino acid protein. A reference sequence for the ITGB3 gene comprises the contiguous lines of Figure 1, which is a composite genomic sequence comprising Genaissance Reference Nos. 8070284, 8070315, 8070331, 8070343, 8070353 and 8070359 (SEQ ID NO:l). Reference sequences for the coding sequence (GenBank Accession No. NM_000212.1) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.
Because of the demonstrated potential for variation in the ITGB3 gene to affect the expression and function of the encoded protein, as well as HDLC levels, it would be useful to know whether additional polymorphisms exist in the ITGB3 gene, as well as how such polymorphisms are combined in different copies of the gene. Such infonnation could be applied for studying the biological function of ITGB3 as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function. None of the studies cited above assessed the clinical relevance of haplotypes of multiple ITGB3 polymorphisms in affecting response to atorvastatin therapy. Thus, it would also be useful to determine whether any ITGB3 haplotypes are associated with response to treatment with atorvastatin. Such infonnation would assist the treating physician in developing the most appropriate therapy regimen for patients with cardiovascular disease. In particular, as noted above, the ability to identify populations of patients who are at risk for minimal or no increase in HDLC upon treatment with a statin would be useful. Identification of these patients would enable physicians to select the most appropriate therapy regimen for those individuals at the time of initiation of treatment for hypercholesterolemia, thus saving the patient weeks of possibly inadequate therapy and potentially improving compliance. Such infonnation would assist the treating physician in developing the most appropriate therapy regimen for patients at risk for or diagnosed with cardiovascular disease.
SUMMARY OF THE INVENTION Accordingly, the inventors have identified correlations between haplotypes in the ITGB3 gene and differential HDLC response to treatment with atorvastatin calcium in a cohort of individuals participating in a a randomized, 16-week, open-label investigation of drug response in relationship to gene variants in adult subjects with primary hypercholesterolemia.
The inventors have discovered that the copy number of these ITGB3 haplotypes affect the change in HDLC level resulting after treatment with atoiΥastatin calcium at the highest permitted dose compared to the change in HDLC resulting after the lowest pennitted dose. The ITGB3 haplotypes shown to have association with dose effects on HDLC changes in response to treatment with atorvastatin calcium are shown in Tables 1 A, IB and IC below. One or two copies of any of haplotypes 101 to 159 in Table 1A or haplotypes 201 to 463 in Table IB or zero or one copy of any of haplotypes 160-194 in Table 1A or haplotypes 501-515 in Table IC aie defined herein as a statin response marker I and are conelated with a mean percent decrease in HDLC upon increasing the atorvastatin dose (See Tables 10 and 11). Correspondingly, zero copy of any of haplotypes 101 to 159 in Table 1A or haplotypes 201 to 463 in Table IB or two copies of any of haplotypes 160-194 in Table 1A or haplotypes 501-515 in Table 1 C are defined herein as a statin response marker II and are correlated with a more favorable change in HDLC (generally a percent increase) upon increasing atoiΥastatin dose (See Tables 10 and 11). The
ITGB3 haplotypes and copy number that comprise statin response markers I and statin response markers II are summarized in Tables 1A, IB and IC below.
Figure imgf000006_0001
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
a An asterck in icates the polymorphic ste is not part of t e aplotype.
Figure imgf000015_0001
hi the patient cohort, the group of individuals having a statin response marker I experienced a larger mean percent decrease in HDLC in response to increasing atoiΥastatin calcium dose than did the group of mdividuals having a statin response marker II. Presence or absence of statin response marker 1 did not significantly affect the mean percent change in HDLC observed in the groups of individuals treated with increasing doses of simvastatin or pravastatin sodium. Thus, testing for the presence of any of these statin response markers I or II in patients will provide valuable information that can be used by the treating physician to devise the most effective statin treatment regimen. In addition, as described in more detail below, the inventors believe that additional statin response markers I and II may readily be identified based on linkage disequilibrium between the above ITGB3 haplotypes or their component polymorphisms and other haplotypes or polymorphisms, respectively, that are located in the ITGB3 gene or other genes. In particular, statin response markers of the invention include those comprising haplotypes that are in linkage disequilibrium with any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC, hereinafter referred to as "linked haplotypes", as well as '"substitute haplotypes" for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC in which one or more of the polymoφhic sites in the original haplotype is substituted with another polymoiphic site, wherein the allele at the substituting polymoiphic site is in linkage disequilibrium with the allele at the replaced or substituted polymoiphic site.
The correlations between the different types of statin response markers and varying changes in HDLC in response to treatment with increasing doses of statins suggest that testing for the presence of a statin response marker I or a statin response marker II in patients would provide valuable infonnation that can be used by the treating physician to choose the most effective statin or combination therapy for achieving a desired effect on LDLC and HDLC levels. In addition, these correlations suggest that any clinical trial of a statin should include in its design or analysis a consideration of the potential effect of statin response markers on the efficacy of statin response. Accordingly, some aspects of the invention are based on the correlations of statin response markers I and II with a differential HDLC response to treatment with increasing doses of atorvastatin or pharmaceutically acceptable salts of atorvastatin acid. In one aspect, the invention provides methods and kits for determining whether an individual has a statin response marker I or a statin response marker II. These methods and kits are useful for predicting the expected therapeutic response of an individual to treatment with statins, selecting an optimal statin for an individual or choosing appropriate therapy for an individual. hi one embodiment, a method for determining whether an individual has a statin response marker I or a statin response marker II comprises determining the copy number present in the individual of a particular haplotype. The haplotype is any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC. The individual has a statin response marker I if the individual has at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC. The individual has a statin response marker II if the individual has zero copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or if the individual has two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501- 515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501- 515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC. In preferred embodiments of these methods, the haplotype comprises one of haplotypes 201, 205, 209, 214, 225, and 288 in Table 1; preferably the haplotype comprises one ofhapiotypes 201, 205, and 209. hi another embodiment of the invention, a method for assigning an individual to a first or second statin response marker group comprises determining the copy number present in the individual of a particular haplotype and assigning the individual to a statin response marker group based on the copy number of that haplotype. The individual is assigned to the first statin response marker group if the individual has at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC. The individual is assigned to the second stat response marker group if the mdividual has zero copy of any of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or if the mdividual has two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160- 194 in Table 1A and haplotypes 501-515 in Table IC. hi prefened embodiments of these methods, the haplotype comprises one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB; preferably the haplotype comprises one of haplotypes 201, 205. and 209 in Table IB.
One embodiment of a kit for determining whether an individual has a statin response marker I or a statin response marker II comprises a set of oligonucleotides designed for identifying at least one of the alleles present at each polymoφhic site (PS) in a set of polymoφhic sites. The set of polymoiphic sites (PSs) comprises the set of PSs comprising any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; the set of PSs comprising a linked haplotype to any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; or the set of PSs for a substitute haplotype comprising any one of ITGB3 haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC, hi a preferred embodiment, the set of PSs comprises PS3, PS12 and PS42; PSI, PS12 and PS42; PS3 and PS42; PSI and PS42; PSI, PS3, PS12 and PS42; or PS39. More preferably, the set of PSs comprises PS3, PS 12 and PS42. In a further embodiment, the kit comprises a manual with instructions for performing one or more reactions on a human nucleic acid sample to identify the allele(s) present in the individual at each polymoφhic site in the set of polymoiphic sites and determining if the individual has a statin response marker I or a statin response marker II based on the identified allele(s).
Another aspect of the invention is a method of selecting a statin therapy to provide an optimal HDLC response in an individual. The method comprises determining whether the individual has a statin response marker I or a statin response marker π and selecting a statin therapy based on the results of the determining step. If the individual has a statin response marker π, then the selected statin therapy comprises any dose of atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid. If the individual has a statin response marker I, then the selected statin therapy comprises a low dose of atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid, a higher dose of atorvastatin or a phannaceutically acceptable salt of atorvastatin acid in conjunction with an HDLC-modulating therapy, or another statin..
In yet another embodiment, the invention provides a method for predicting an individual's HDLC response to treatment with a statin. The statin may preferably be atoiΥastatin or a phannaceutically acceptable salt of atorvastatin acid. The method comprises determining whether the individual has a statin response marker I or a statin response marker II and making a response prediction based on the results of the determining step, hi some embodiments, if the individual is determined to have a statin response marker I, then the response prediction is that the individual will experience an unfavorable HDLC response if treated with atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid at a dose comparable to a dose of atorvastatin calcium greater than about 10 mg/day. If the individual is determined to have a statin response marker II, then the response prediction is that the individual will likely experience a favorable HDLC response if treated with atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid at any dose comparable to a dose of aton'astatin calcium ranging from about 10 to about 80 mg/day.
In other aspects, the invention provides: (i) a method of seeking regulator ' approval for marketing a pharmaceutical formulation comprising a statin as at least one active ingredient for treating a disease or condition in a population partially or wholly defined by having a statin response marker, (ii) an article of manufacture comprising the pharmaceutical formulation that is marketed for treating the defined population, (iii) a method of manufacturing a drug product comprising the phaπnaceutical formulation, and (iv) a method of marketing the drug product for treating the defined population, hi preferred embodiments, the disease or condition is a cardiovascular or coronary artery disorder, e.g., hypercholesterolemia. hi some embodiments, the method of seeking regulatory approval comprises conducting at least one clinical trial which comprises administering the phaπnaceutical fomiulation to a fust treatment group of patients and atorvastatin or a pharmaceutically acceptable salt of atorvastatin to a second treatment group of patients. Each patient has the disease or condition and also has a statin response marker I. The method further comprises demonstrating that the second treatment group exhibits a mean per cent change in High Density Lipoprotein Cholesterol (HDLC) that is worse than the mean per cent change in HDLC exhibited by the fust treatment group at any given dose of the phannaceutical foπnulation and of atoiΥastatin or a pharmaceutically acceptable salt of aton'astatin that achieves a comparable reduction in Low Density Lipoprotein Cholesterol. The statin is preferably simvastatin, a pharmaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid. hi some embodiments, the method further comprises filing with a regulatory agency an application for marketing approval of the pharmaceutical formulation with a label stating that the pharmaceutical foπnulation is indicated for patients having the statin response marker I. In preferred embodiments, the regulatory agency is the United States Food and Drug Administration (FDA) or the European Agency for the Evaluation of Medicinal Products (EMEA), or a future equivalent of these agencies. In one embodiment, the article of manufacture comprises the pharmaceutical formulation and at least one indicium identifying a population for whom the pharmaceutical formulation is indicated. The identified population is partially or wholly defined by having a statin response marker I. In these embodiments, a trial population having the statm response marker I exhibits a better HDLC response to the pharmaceutical fomiulation than to treatment with atorvastatin or a phannaceutically acceptable salt of atorvastatin acid, hi yet other embodiments, an article of manufacture according to the invention comprises a pharmaceutical fomiulation comprising aton'astatin or a pharmaceutically acceptable salt of atoivastatin with an HDLC modulating agent and the defining statin response marker is a statin response marker I. Another embodiment of the article of manufacture comprises packaging material and the pharmaceutical foπnulation contained within the packaging material, wherein the packaging material comprises a label approved by a regulatory agency for the pharmaceutical foπnulation, wherein the label states that the phaπnaceutical formulation is indicated for a population partially or wholly defined by having a statin response marker I.
The method for manufacturing the drug product comprises combining in a package a phaπnaceutical foπnulation comprising a statin as at least one active ingredient and a label which states that the drug product is indicated for freating a population defined wholly or partially by having a statin response marker I.
The method for marketing the drug product comprises promoting to a target audience the use of the drug product for treating individuals who belong to the defined population.
Additionally, the inventors herein have identified a set of 98 human haplotypes for a set of 44 ITGB3 polymoφhic sites. Each of these ITGB3 haplotypes constitutes a code, or genetic marker, that defines a variant fomi of the gene that exists in the human population. Thus, each ITGB3 haplotype represents a naturally occurring isofonn (also refened to herein as an "isogene") of the ITGB3 gene. In addition, the inventors have deteπnined what pairs of these ITGB3 haplotypes are present in a patient cohort of individuals with primary hypercholesterolemia who were freated with atorvastatin calcium, pravastatin sodium, or simvastatin, as well as in a human reference population representing four major population groups: African descent, Asian, Caucasian and Hispanic/Latino.
The locations of the polymoφhic sites (PS) that comprise the ITGB3 haplotypes and haplotype pairs described herein and the identities of the polymoφhisms at each of these sites are shown in Table 3, which is located after the Examples below. The composition of the different haplotypes and haplotype pairs for this set of polymoφhic sites that were found in the patient cohort and reference population are shown in Tables 5 and 4, respectively, which are located after the Examples below.
In one embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymoφhic variant of a reference sequence for the ITGB3 gene or a fragment thereof. The reference sequence comprises the contiguous sequences shown in Figure 1 and the polymoiphic variant comprises at least one polymoφhism selected from the group consisting of the novel polymoiphisms shown in Table 3, presented following the Examples.
A particularly preferred polymoiphic variant is an isogene of the ITGB3 gene. An ITGB3 isogene of the invention is defined by one of the haplotypes shown in Table 5 below, presented following the Examples. The invention also provides a collection of at least two ITGB3 isogenes, refeixed to herem as an ITGB3 genome anthology. hi another embodiment, the invention provides a polynucleotide comprising a polymoφhic variant of a reference sequence for a ITGB3 cDNA or a fragment thereof. The reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises at least one polymoφhism selected from the group consisting of adenine at a position corresponding to nucleotide 40, thymine at a position corresponding to nucleotide 57, thymine at a position conesponding to nucleotide 58, cytosine at a position con-esponding to nucleotide 176, guanine at a position coπ-esponding to nucleotide 197, cytosine at a position conesponding to nucleotide 342, cytosine at a position corresponding to nucleotide 882, cytosine at a position corresponding to nucleotide 1143, adenine at a position corresponding to nucleotide 1333, guanine at a position corresponding to nucleotide 1533, adenine at a position corresponding to nucleotide 1544, adenine at a position conesponding to nucleotide 1545, and thymine at a position corresponding to nucleotide 2208. A particularly preferred polymoiphic cDNA variant is selected from the group consisting of A, B, C, D, E, F, G, H, I, K, J, L, M, N, O, P and Q represented in Table 8.
Polynucleotides complementary to these ITGB3 genomic and cDNA variants are also provided by the invention. It is believed that polymoiphic variants of the ITGB3 gene will be useful in studying the expression and function of ITGB3, and in expressing ITGB3 protein variants for use in screening for candidate drugs to treat diseases related to ITGB3 activity. In other embodiments, the invention provides a recombinant expression vector comprising one of the polymorphic genomic and cDNA variants operably linked to expression regulator}' elements as well as a recombinant host cell fransformed or fransfected with the expression vector. The recombinant vector and host cell may be used to express ITGB3 for protein structure analysis and drug binding studies. In yet another embodiment, the invention provides a polypeptide comprising a polymoφhic variant of a reference amino acid sequence for the ITGB3 protein. The reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymoiphic variant comprises at least one variant amino acid selected from the group consisting of methionine at a position conesponding to amino acid position 14, proline at a position coιτesponding to amino acid position 59, arginine at a position corresponding to amino acid position 66, methionine at a position corresponding to amino acid position 445, and glutamine at a position con-esponding to amino acid position 515. A polymoφhic variant of ITGB3 is useful in studying the effect of the variation on the biological activity of ITGB3 as well as on the binding affinity of candidate drugs targeting ITGB3 for the freatment of coronary heart disease or other disorders of cholesterol metabolism.
The present invention also provides antibodies that recognize and bind to the above polymoφhic ITGB3 protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods. The present invention also provides nonhuman transgenic animals comprising one or more of the ITGB3 polymoφhic genomic variants described herein and methods for producing such animals. The transgenic animals are useful for studying expression of the ITGB3 isogenes in vivo, for in vivo screening and testing of drugs targeted against ITGB3 protein, and for testing the efficacy of therapeutic agents and compounds for coronary heart disease or other disorders of cholesterol metabolism in a biological system.
In other aspects, the invention provides a method, composition and kit for genotyping and haplotyping the ITGB3 gene in an individual. The genotyping method comprises identifying the nucleotide pair that is present at one or more ITGB3 polymoφhic sites selected from the novel polymoφhic sites shown in Table 3 below in both copies of the individual's ITGB3 gene. In some embodiments, the genotyping method may also comprise identifying the nucleotide pah that is present at one or more polymoφhic sites selected from the group consisting of the previously reported ITGB3 polymoiphic sites shown in Table 3 below A genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region contaming, or adjacent to, one of these novel ITGB3 polymoφhic sites, hi one embodiment, a kit of the invention comprises a set of oligonucleotides designed to genotype one or more of the ITGB3 polymorphic sites disclosed herein. In another embodiment, the kit comprises a set of oligonucleotides designed to genotype each of PS1-PS44 shown in Table 3 below. In one prefened embodiment of the kit, at least one PS in the set of two or more polymoiphic sites is selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7. PS8. PS9, PS10, PS11, PS12, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44. h another preferred embodiment of the kit. the PSs are selected from the group consisting of: PSI, PS3, PS4, PSS, PS6, PS10. PS12, PS15, PS16. PS19. PS20. PS21, PS26, PS28, PS30, PS37, PS38, PS39, and PS42. Most preferably, the PSs are selected from the group consisting of: PSI, PS3, PS12, PS39, and PS42. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below, thereby identifying the particular isogenes of ITGB3 present in the individual. Determining the haplotype or haplotype pair of the individual is also useful for determining whether the individual has a statin response marker I or a statin response marker II. The invention also provides a method for haplotyping the ITGB3 gene in an individual. In one embodiment, the haplotyping method comprises detennining, for one copy of the individual's ITGB3 gene, the identity' of the nucleotide at one or more polymoφhic sites selected from the group of polymoφhic sites shown in Table 3 below. In another embodiment, the haplotyping method comprises determining whether one copy of the individual's ITGB3 gene is defined by one of the ITGB3 haplotypes shown in Table 5, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies of the individual's ITGB3 gene are defined by one of the ITGB3 haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. Establishing the ITGB3 haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with ITGB3 activity, e.g., coronary heart disease and other disorders of cholesterol metabolism.
For example, the haplotyping method can be used by the phaπnaceutical research scientist to validate ITGB3 as a candidate target for treating a specific condition or disease predicted to be associated with ITGB3 activity. Detenninmg for a particular population the frequency of one or more of the individual ITGB3 haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue ITGB3 as a target for treating the specific disease of interest. In particular, if variable ITGB3 activity is associated with the disease, then one or more ITGB3 haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls. Conversely, if each of the observed ITGB3 haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable ITGB3 activity has little, if any, involvement with that disease, h either case, the phaπnaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any ITGB3 haplotype or haplotype pair, apply the information derived from detecting ITGB3 haplotypes in an individual to decide whether modulating ITGB3 activity would be useful in treating the disease.
The claimed invention is also useful in screening for compounds targeting ITGB3 to treat a specific condition or disease predicted to be associated with ITGB3 activity. For example, detecting which of the ITGB3 haplotypes or haplotype pairs disclosed herem are present in individual members of a population with the specific disease of interest enables the phaπnaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the ITGB3 isoforms present in the disease population, or for only the most frequent ITGB3 isoforms present in the disease population. Thus, without requiring any a priori knowledge of the phenotypic effect of any particular ITGB3 haplotype or haplotype pair, the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
Haplotyping the ITGB3 gene in an mdividual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with ITGB3 activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the ITGB3 haplotype(s) disclosed herein aie present in individual patients enables the phannaceutical scientist to distribute
ITGB3 haplotypes and/or haplotype pairs evenly to freatment and control groups, thereby reducing the potential for bias hi the results that could be introduced by a larger frequency of an ITGB3 haplotype or haplotype pair that is associated with response to the drug being studied in the frial, even if this association was previously unknown. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first detenmining the phenotypic effect of any ITGB3 haplotype or haplotype pair.
In another embodiment, the invention provides a method for identifying an association between a trait and an ITGB3 genotype, haplotype, or haplotype pair for one or more of the novel polymoφhic sites described herein. The method comprises comparing the frequency of the ITGB3 genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the ITGB3 genotype or haplotype in a reference population. A different frequency of the ITGB3 genotype, haplotype, or haplotype pa - in the trait population than in the reference population indicates the trait is associated with the ITGB3 genotype, haplotype, or haplotype pair. In preferred embodiments, the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug. In a particularly preferred embodiment, the ITGB3 haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic freatments for coronary heart disease or other disorders of cholesterol metabolism, as well as for other disorders in which receptors comprising ITGB3 are implicated.
BRIEF DESCRIPTION OF THE DRA WINGS
Figure 1 illustrates a reference sequence for the ITGB3 gene (contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymoφhic site(s) and polymoφhism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence. SEQ ID NO:l is equivalent to Figure 1, with the two alternative allelic variants of each polymoiphic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WIPO standard ST.25). SEQ ID NO:226 is a modified version of SEQ ID NO:l that shows the context sequence of each polymoφhic site enumerated in Table 5 in a uniform fonnat to facilitate electronic searching. For each polymorphic site, SEQ ID NO:226 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymoφhic site at the 30th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein. Figure 2 illustrates a reference sequence for the ITGB3 coding sequence (contiguous lines; SEQ
ID NO:2), with the polymoiphic site(s) and polymoφhism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymoφhic site in the sequence.
Figure 3 illustrates a reference sequence for the ITGB3 protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymoφhism(s) of Figure 2 positioned below the polymoφhic site in the sequence.
DEFINITIONS hi the context of this disclosure, the terms below shall be defined as follows unless otherwise indicated:
Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence, or one of the alternative polymoiphisms found at a polymoiphic site.
Gene - A segment of DNA that contains the coding sequence for a protein, wherein the segment may include promoters, exons, introns, and other untranslated regions that control expression.
Genotype - An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at a set of one or more polymoφhic sites in a locus on a pair of homologous chromosomes in an individual.
Genotyping - A process for deteπnining a genotype of an individual.
Haplotype - A 5' to 3' sequence of nucleotides found at a set of one or more polymoφhic sites in a locus on a single chromosome from a single individual.
Sub-haplotype - The 5' to 3' sequence of nucleotides seen at a subset of one or more polymoiphic sites in a locus on a single chromosome from a single mdividual.
Haplotype pair - The two haplotypes found for a locus in a single individual.
Haplotyping - A process for deteπnining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
Haplotype data - Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in an individual or in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait. Isoform - A particular fonn of a gene, mRNA, cDNA, coding sequence or the protein encoded thereby, distinguished from other fonns by its particular sequence and or structure.
Isogene - One of the isofonns (e.g., alleles) of a gene found in a population. An isogene (or allele) contains all of the polymorphisms present in the particular isofonn of the gene.
Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term "isolated" is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention. Locus - A location on a chromosome or DNA molecule conesponding to a gene or a physical or phenotypic feature, where physical features include polymoiphic sites.
Nucleotide pair - The nucleotides found at a polymorphic site on the two copies of a chromosome from an individual.
Phased - As applied to a sequence of nucleotide pairs for two or more polymoiphic sites in a locus, phased means the combination of nucleotides present at those polymoφhic sites on a shigle copy of the locus is known.
Polymorphic site (PS) - A position on a chromosome or DNA molecule at which at least two alternative sequences are found in a population. Polymorphic variant or variant - A gene, mRNA, cDNA, polypeptide, protein or peptide whose nucleotide or amino acid sequence varies from a reference sequence due to the presence of a polymoφhism in the gene.
Polymorphism - The sequence variation observed in an individual at a polymoφhic site. Polymoφhisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
Population Group - A group of individuals sharing a common ethnogeographic origin. Reference Population - A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%. Set of Polymorphic Sites — one or more polymoφhic sites. Single Nucleotide Polymorphism (SNP) - Typically, the specific pah- of nucleotides observed at a single polymoφhic site. In rare cases, three or four nucleotides may be found.
Statin Response Marker I — at least one copy of any of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
Statin Response Marker H — zero copy of any of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC.
Subject - A human individual whose genotypes or haplotypes or response to treatment or disease state are to be detennined.
Treatment - A stimulus administered internally or externally to a subject. Unphased - As applied to a sequence of nucleotide pairs for two or more polymoφhic sites in a locus, unphased means the combination of nucleotides present at those polymoiphic sites on a single copy of the locus is not known. DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the present invention, the inventors herein have discovered novel variants of the ITGB3 gene and identified conelations between these variants and change in HDLC in response to increasing dose of atorvastatin calcium. As described in more detail below, the inventors herein discovered 98 isogenes of the ITGB3 gene by characterizing the ITGB3 gene found in genomic DNAs isolated from immortalized cell lines from 854 humans. This experimental population comprised an Index Repository that contains 93 human individuals and a patient cohort of 679 individuals participating in a randomized, 16-week, open-label investigation of drug response in relationship to gene variants in adult subjects with primary hypercholesterolemia. The experimental population is described in more detail in Examples 1 and 2. The ITGB3 isogenes present in the human experimental population are defined by haplotypes for 44 polymoiphic sites in the ITGB3 gene. The ITGB3 polymoiphic sites identified by the inventors are referred to as PS1-PS44 to designate the order in which they are located in the gene (see Table 3, presented following the Examples). The human genotypes and haplotypes found in the experimental population for the ITGB3 gene include those shown in Tables 4 and 5, respectively (both presented at the end of the Examples).
The inventors have discovered haplotypes in the ITGB3 gene that are associated with variation in change in HDLC in response to freatment with increasing dose of aton'astatin calcium. The inventors have also discovered that the copy number of these ITGB3 haplotypes affects the HDLC changes in response to aton'astatin. Each statin response marker of the invention is a combination of a particular haplotype, or genetic marker, and the copy number for that haplotype, or genetic marker. Preferably the genetic marker component of the statin response marker is one of the ITGB3 haplotypes shown in Tables 1A, IB or IC. The ITGB3 polymoφhic sites in these ITGB3 haplotypes are referred to herein as PSI, PS3, PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42 and are located in the ITGB3 gene at positions in Figure 1 (SEQ ID NO:l) con-esponding to those identified in Table 3. In describing the polymoiphic sites in the statin response markers of the invention, reference is made to the sense strand of a gene for convenience. However, as recognized by the skilled artisan, nucleic acid molecules containing a particular gene may be complementary double stranded molecules and thus reference to a particular site or haplotype on the sense strand refers as well to the corresponding site or haplotype on the complementary antisense strand. Further, reference may be made to detecting a genetic marker or haplotype for one strand and it will be understood by the skilled artisan that this includes detection of the complementary haplotype on the other strand.
The location of a polymoφhic site in an individual's ITGB3 gene or fragment may be identified by aligning the sequence of the gene or fragment against the corresponding region of SEQ ED NO: 1. Alignment of a polymoφhism in SEQ ID NO: 1 against an alternative ITGB3 sequence to determine the corresponding position of the polymoφhic site in that alternative ITGB3 sequence should use a context sequence from SEQ ED NO:l ranging from about 25 to about 500 nucleotides with the polymoφhism in any position of the context sequence. The alignment should require a degree of homology appropriate for the length of the context sequence in establishing alignment between the two sequences. Determining the degree of homology or the permissible number of mismatches between the two sequences is well within the skills of the routine practitioner of sequence alignment algorithms. Preferably, the context sequence from SEQ ED NO: 1 is about 50 to about 300 nucleotides, with the polymoφhism positioned in the approximate center of the context sequence. The number of nucleotides in SEQ ED NO: 1 separating any two polymoφhic sites in a haplotype represented in Table 5, including nucleotide positions in SEQ ED NO: 1 not sequenced by Applicants, may not describe the number of nucleotides separating those polymoiphic sites in a different ITGB3 reference sequence. The skilled practitioner would recognize the potential for variation in the relative spacing of the polymoφhic sites depending on chosen reference sequence and would be able to use the context sequences for each polymoφhic site to determine the presence of any particular combination of polymoφhisms comprising a haplotype within the different ITGB3 reference sequence. The skilled practitioner would recognize that similar aligning procedures against a suitable reference sequence could be performed to identify the location of a polymoiphic site in a coding sequence for ITGB3 or in an ITGB3 polypeptide sequence. As described in more detail in Examples 1-4, the statin response markers of the invention are based on the discover}' by the inventors of correlations between certain haplotypes in the ITGB3 gene and variation in change in HDLC levels in response to atorvastatin treatment in a cohort of individuals participating in a randomized, 16-week, open-label investigation of drug response in lelationship to gene variants in adult subjects with primary hypercholesterolemia. In particular, the inventors herein discovered that copy number of ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB and
501-515 in Table IC significantly affected the change in HDLC levels obsen'ed in patients participating in the study following treatment at high dose with Lipitor® (atoiYastatin calcium) compared to the change in HDLC those patients experienced following treatment at the lowest dose of Lipitor®. The group of patients with one or two copies of any of haplotypes 101 to 159 in Table 1A and haplotypes 201 -463 in Table 1 B or with zero or one copy of any of haplotypes 160- 194 in Table 1 A and haplotypes 501 to 515 in Table IC are conelated with a mean percent reduction in HDLC after a high dose atorvastatin treatment (See Tables 10A, 10B, 11 A and 1 IB) while the patient group having zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB or two copies of any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC are conelated with a negligible percent change or small percent increase in HDLC after a high dose atorvastatin freatment. No significant variation in differential change in HDLC in response to increasing dose of simvastatin or pravastatin sodium was observed for different copy numbers of any ITGB3 haplotypes.
Therefore these haplotypes, in combination with then haplotype copy number, can be used to differentiate the HDLC response that would be predicted to occur in an individual or a trial population after treatment with a high dose of atoiYastatin. Consequently, one or two copies of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB or zero or one copy of any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC are referred to herein as a statin response marker I, while zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB or two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC are referred to herein as a statin response marker II.
The NCEP ATPIII report mdicates that low HDLC is a risk factor for CHD. Therefore, herein, a "favorable", "better" or "best" HDLC response after treatment denotes that relative to the initial baseline HDLC measured that the change in HDLC measured after statin freatment shows a larger or largest increase in measured value. For example, no change or an increase in HDLC after treatment relative to the baseline measurement is a better response than a decrease in HDLC after treatment relative to the baseline measurement. Conversely, an "unfavorable", "worse" or "worst" HDLC response after treatment denotes herem that the change in HDLC relative to the initial baseline HDLC value that is measured after statin treatment shows a smaller increase or a decrease in measured value. For example, no change or a decrease in HDLC after treatment relative to the baseline measurement is a worse response than an increase in HDLC after treatment relative to the baseline measurement. The comparison of two or more values for a change in HDLC relative to baseline after a treatment may be for a single individual before and after two different therapy regimens, e.g., a low dose vs. a high dose regimen. The comparison of two or more values for a change in HDLC relative to baseline after a treatment may be between two or more single individuals or two or more population groups before and after two different therapy regimens.
AtoiYastatin herein may refer to any chemical forms of atorvastatin, atorvastatin derivatives or phannaceutically acceptable salts of atoiYastatin acid. In addition, the skilled artisan would expect that there might be additional polymoφhisms in the
ITGB3 gene or elsewhere on cluOmosome 17 that are in high LD with one or more of the polymoφhisms in the haplotypes comprising a statin response marker I or a statin response marker II. Two particular nucleotide alleles at different polymoφhic sites are said to be in LD if the presence of one of the alleles at one of the sites tends to predict the presence of the other allele at the other site on the same chromosome (Stevens, JC, Mol. Diag. 4: 309-17, 1999). One of the most frequently used measures of linkage disequilibrium is Δ2, which is calculated using the fonnula described in Devlin, B. and Risch, N. (1995, Genomics, 29(2):311-22). Basically, Δ2 measures how well an allele X at a first polymorphic site predicts the occunence of an allele Y at a second polymoφhic site on the same chromosome. The measure only reaches 1.0 when the prediction is perfect (e.g., X if and only if Y). Thus, the skilled artisan would expect that all of the embodiments of the invention described herein may frequently be practiced by substituting the allele at any (or all) of the specifically identified ITGB3 polymoφhic sites in ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB or 501-515 in Table IC with an allele at another polymoφhic site that is in high LD with the allele at the specifically identified polymoφhic site. This "substitute polymoφhic site" may be one that is currently known or subsequently discovered and may be present at a polymoφhic site in the ITGB3 gene or elsewhere on chromosome 17. Preferably, the substitute polymoiphic site is present in the ITGB3 gene or in a genomic region of about 100 kilobases spanning the ITGB3 gene.
For example, as may be seen in the table below essentially perfect LD exists between PS3 and PS9 (Δ2 = 0.97654 for the total experimental population examined herein). Thus, it would be expected by the skilled artisan that any ITGB3 statin response marker comprising an ITGB3 haplotype with a guanine at PS3 may be substituted with an alternative haplotype replacing guanine at PS3 with cytosine at PS9 and be nearly as well predictive of an individual's atorvastatin dose response as the ITGB3 statin response marker. The inventors herein identified other alleles that are in high LD with one or more of the alleles found in the list of ITGB3 haplotypes shown in Table 1 and believe that ITGB3 haplotypes comprising one or more of these alleles substituted for the original allele can also function to predict the obsen'ed results. The LD relationships identified by the inventors herein are shown in Table 2 below, which lists the values for Δ2 in the total experimental population and for each of the 4 ethnic population groups within that population.
Figure imgf000029_0001
CA, AF, HL, and AS stand for Caucasian, African-American, Hispanic-Latino, and Asian, respectively.
Further, the inventors contemplate that there will be other haplotypes in the ITGB3 gene or elsewhere on chromosome 17 that are in high LD with any one of ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB or 501-515 in Table IC and that these highly linked haplotypes would therefore also be predictive of the HDLC response to atoiYastatin dose. Preferably, the linked haplotype is present in the ITGB3 gene or in a genomic region of about 100 kilobases spanning the ITGB3 gene. The linkage disequilibrium between any one of ITGB3 haplotypes 101 to 194 in Table 1A, 201-463 in Table IB or 501-515 in Table IC and a linked haplotype can also be measured using Δ2. hi preferred embodiments, the linkage disequilibrium between the allele at a polymoφhic site in any of the ITGB3 haplotypes in Table 1 and the allele at a substitute polymoφhic site to replace it, or between any of the ITGB3 haplotypes in Table 1 and a linked haplotype, has a Δ" value, as measured in a suitable reference population, of at least 0.75, more preferably at least 0.80, even more preferably at least 0.85 or at least 0.90, yet more preferably at least 0.95, and most preferably 1.0. A suitable reference population for this Δ2 measurement is preferably selected from a population with the distribution of the ethnic background of its members reflecting the population of patients to be treated with statins, which may be the general population, a population using statins, a population with coronary heart disease (CHD), cardiovascular disease (CVD) or CHD (or CVD) risk factors, and the like.
LD patterns in genomic regions are readily determined empirically in appropriately chosen samples using various techniques known in the art for detennining whether any two alleles (at two different polymoiphic sites or two haplotypes) are in linkage disequilibrium (Weir B.S. 1996 Genetic Data Analysis II, Sinauer Associates, Inc. Publishers, Sunderland, MA). The skilled artisan may readily select which method of detennining LD will be best suited for a particular sample size and genomic region. Similarly, the ability of substitute haplotypes, that contain an allele at one or more substituting polymoiphic sites, or of linked haplotypes, that are in high LD with one or more of the ITGB3 haplotypes in Table 1, to predict the LDLC response to one of the statins studied herein may also be readily tested by the skilled artisan Thus, reference herein to a statin response marker I is deemed to include at least one copy of haplotypes that (A) either (1) have a polymoiphism sequence that is similar to that of any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, but in which the allele at one or more of the specifically identified ITGB3 polymoiphic sites in that haplotype has been substituted with the allele at a polymoiphic site in high LD with the allele at the specifically identified polymoφhic site (a "substitute haplotype"); or (2) are in high linkage disequilibrium with any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB (a "linked haplotype"); and (B) behave similarly to at least one copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB in terms of predicting an individual's HDLC response to increased atorvastatin dose. Additionally, reference herein to a statin response marker I is deemed to include zero or one copy of haplotypes that (A) either (1) are a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, or (2) are a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC; and (B) behave similarly to zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC in tenns of predicting an individual's HDLC response to increased atoiYastatin dose. Similarly, reference herein to a statin response marker II is deemed to include zero copy of haplotypes that (A) either (1) have a polymoφhism sequence that is similar to that of any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, but in which the allele at one or more of the specifically identified ITGB3 polymoφhic sites in that haplotype has been substituted with the allele at a polymoiphic site in high LD with the allele at the specifically identified polymoφhic site (a "substitute haplotype"); or (2) are in high linkage disequilibrium with any one of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB (a "linked haplotype"); and (B) behave similarly to zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB in terms of predicting an individual's HDLC response to increased atorvastatin dose. Additionally, reference herein to a statin response marker II is deemed to include two copies of haplotypes that (A) either (1) are a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, or (2) are a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC; and (B) behave similarly to two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC in tenns of predicting an individual's HDLC response to increased atoiYastatin dose.
As described above and in the Examples below, the statin response markers of the invention are associated with effects on mean percent change in HDLC in response to freatment with increased doses of atoiYastatin calcium. Thus, the invention provides a method and kit for determining whether an individual has a statin response marker I or a statin response marker H
In one embodiment, the invention provides a method for detennining whether an individual has a statin response marker I or II. The method comprises deteπnining the copy number present in the individual of a haplotype selected from the group consisting of haplotypes 101 to 194 in Table 1A, 201- 463 hi Table IB and 501-515 in Table IC, a linked haplotype to any one of haplotypes 101 to 194 in Table 1A, 201-463 hi Table IB and 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101 to 194 in Table 1A, 201-463 in Table IB and 501-515 in Table IC. The individual has a statin response marker I if the individual has at least one copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, a haplotype linked to any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, a haplotype linked to any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC, or a substitute haplotype for any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC. The individual has a statin response marker II if the individual has zero copy of any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, a haplotype linked to any of haplotypes 101 to 159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any of haplotypes 101 to 159' in Table 1A and haplotypes 201-463 in Table IB; or if the individual has two copies of any of haplotypes 160-194 in Table 1 A and haplotypes 501 to 515 in Table IC, a haplotype linked to any of haplotypes 160-194 in Table 1A and haplotypes 501 to 515 in Table IC, or a substitute haplotype for any of haplotypes 160- 194 in Table 1A and haplotypes 501 to 515 in Table IC.
In another embodiment, the invention provides a method for assigning an individual to a first or second statin response marker group. The method comprises determining the copy number present in the individual of a haplotype selected from the group consisting of ITGB3 haplotypes 101-194 in Table 1 A, haplotypes 201 -463 in Table 1 B and haplotypes 501 to 515 in Table 1 C; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and assigning the individual to a statin response marker group based on the copy number of that haplotype. The individual is assigned to the first statin response marker group if the mdividual has at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or if the individual has zero or one copy of any of haplotypes 160- 194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160- 194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC. The individual is assigned to the second statin response marker group if the individual has zero copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB; or if the individual has two copies of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC. hi preferred embodiments of the above methods, the haplotype comprises any one of 201, 205,
209, 214, 225, and 288 in Table IB. More preferably, the selected haplotype is one of haplotypes 201, 205, and 209 in Table IB. In some embodiments, the mdividual is Caucasian and may be diagnosed with a coronary artery disease or a cardiovascular disease, such as Type Ila or Type lib hypercholesterolemia, may have risk factors associated with cardiovascular disease, or may be a candidate for treatment with a statin for an alternative reason.
In each of the above methods, the deteπnining step may comprise genotyping each polymoφhic site in the set of polymoφhic sites comprising the selected haplotype; and using the results of the genotyping step to identify the haplotype pair present in the individual. The genotyping step may be performed by any methods known to the art, including but not limited to those methods described herein. The detennining step may also comprise consulting a data repository, such as a medical data card or a medical record for the individual, that provides information on the copy number present in the individual for the selected haplotype.
"Determining the copy number" of a haplotype may in some instances mean determining if zero, one or two copies is present in the individual, i.e. identifying the haplotype present on each chromosomal copy of the individual. In other instances, determining the copy number of a haplotype in an individual may mean detennining a lower or upper limit on the number of copies, such as determining that there is at least one copy or fewer than two copies present in the individual, hi these latter instances, the haplotype for only one chromosomal copy of the individual is identified. For some individuals and some haplotypes selected from haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC, this is an adequate amount of infonnation to detennine if the individual has a statin response marker I or II or belongs to the first or second statin response marker group. For example, if it is determined that an individual has one copy of haplotype 201, but the haplotype of the second chromosomal copy is not detennined, the individual may still be identified as having a statin response marker I or be assigned to the first statin response marker group. On the other hand, if an individual is detemiined to have one copy of haplotype 501 and the haplotype of the individual's second chromosomal copy is not identified, the individual could have either a statin response marker I or II or belong to either statin response marker group, hi such instances infonnation on the haplotype of the individual's second chromosomal copy would be needed to determine which marker is present in the individual or for assigning the individual to a statin response marker group. The presence in an individual of a statin response marker I or II may be determined by a variety of indirect or direct methods well known in the art for determining haplotypes or haplotype pairs for a set of polymoφhic sites in one or both copies of the individual's genome, including those discussed below. The genotype for a polymoiphic site in an individual may be detennined by methods known in the art or as described below. One indirect method for determining the copy number of any one of the ITGB3 haplotypes in
Tables 1A, IB and IC present in an individual is by prediction based on the individual's genotype detennined at one or more of the polymoφhic sites in the set of polymoφhic sites comprising die haplotype and using the detennined genotype at each site to determine the ITGB3 haplotypes present in the individual. The presence of zero, one or two copies of an ITGB3 haplotype of interest can be detennined by visual inspection of the alleles at the polymoφhic sites that comprise the haplotype. The ITGB3 haplotype pair is assigned by comparing the individual's genotype at each polymoiphic site in the set of polymoiphic sites with the genotypes at the same set of polymorphic sites coiresponding to the haplotype pairs known to exist in the general population or in a specific population group or to the haplotype pairs that are theoretically possible based on the alternative alleles possible at each polymorphic site, and determining which haplotype pair is most likely to exist in the individual.
In a related indirect haplotyping method, the copy number present in an individual of an ITGB3 haplotype disclosed herein is predicted from the individual's genotype for a set of polymoφhic sites comprising the selected haplotype using infonnation on haplotype pairs blown to exist in a reference population, hi one embodiment, this haplotype pah prediction method comprises identifying a genotype for the individual at the set of polymoφhic sites comprising the selected haplotype, accessing data containing haplotype pairs identified in a reference population for a set of polymorphic sites comprising the polymoφhic sites of the selected haplotype, and assigning to the individual a haplotype pair that is consistent with the individual's genotype. Whether the individual has a statin response marker I or a statin response marker II can be subsequently determined based on the assigned haplotype pair. The haplotype pair can be assigned by comparing the individual's genotype with the genotypes conesponding to the haplotype pans known to exist in the general population or in a specific population group, and deteπnining which haplotype pair is consistent with the genotype of the individual. In some embodiments, the comparing step may be perfonned by visual inspection. When the genotype of the individual is consistent with more than one haplotype pair, frequency data may be used to detemiine which of these haplotype pairs is most likely to be present in the individual. If a particular haplotype pair consistent with the genotype of the individual is more frequent in the reference population than other haplotype pairs consistent with the genotype, then that haplotype pa - with the highest frequency is the most likely to be present hi the individual. The haplotype pair frequency data used in this determination is preferably for a reference population comprising the same ethnogeographic group as the individual. The detemiination of the haplotype pair of the individual may also be perfonned in some embodiments by visual inspection. In other embodiments, the comparison may be made by a computer- implemented algorithm with the genotype of the individual and the reference haplotype data stored in computer-readable formats. For example, as described in WO 01/80156, one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing ITGB3 haplotype pairs frequency data detennined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the individual. Typically, the reference population is composed of randomly-selected individuals representing one or more of the major ethnogeographic groups of the world. A prefened reference population for use in the methods of the present invention consists of Caucasian individuals, the number of which is chosen based on how rare a haplotype is that one wants to be guaranteed to see. For example, if one wants to have a q% chance of not missing a haplotype that exists in the population at a p% frequency of occuning in the reference population, the number of individuals (n) who must be sampled is given by 2n=log(l-q)/log(l-p) where p and q are expressed as fractions. A prefened reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty. A particularly prefeιτed reference population includes a 3-generation Caucasian family to serve as a control for checking quality of haplotyping procedures. If the reference population comprises more than one ethnogeographic group, the frequency data for each group is examined to detennine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L. Hard et al., Principles of Population Genomics, Sinauer Associates (Sunderland, MA), 3rd Ed., 1997) postulates that the frequency of finding the haplotype pair H, / H2 is equal to pH_w(H H ) = 2p(Hx)p(H2) if H, ≠ H2 and pH_w (H, I Hf) = p(H, )p(H2) if H, = H2 . A statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or enors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are obsened in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System™ technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
In one embodiment of this method for predicting a haplotype pair for an individual, the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pan- is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the mdividual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair. Alternatively, the haplotype pair in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al. 1990, Mol Bio Evol 7: 111- 22 or WO 01/80156) or through a commercial haplotyping service such as offered by Genaissance Phaπnaceuticals, Inc. (New Haven, CT). hi rare cases, either no haplotypes in the reference population are consistent with the possible haplotype pahs, or alternatively, multiple reference haplotype pairs are consistent with the possible haplotype pairs, hi such cases, the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System™ technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra).
Determination of the number of haplotypes present in the individual from the genotypes is illustrated here for ITGB3 haplotype 214 in Table IB, comprising guanine at PS3 and cytosine at PS42.
Possible copy numbers of ITGB3 Haplotype 214 based on the enot es at PS3 and PS42
Figure imgf000035_0001
There are tliree genotypes that may possibly occur at each polymoφhic site; thus there are 9 genotypes that could be detected at PS3 and PS42, using both chromosomal copies from an individual. Eight of the nine possible genotypes for the two sites allow unambiguous determination of the number of copies of the ITGB3 haplotype 214 present in the individual and therefore would allow unambiguous determination of whether the individual has a statin response marker I or II. However, an individual with the C/G, T/C genotype could possess either of the following haplotype pahs: CT/GC or GT/CC, and thus could have either 1 copy of ITGB3 haplotype 214 which is a statin response marker I, or 0 copy of ITGB3 haplotype 214 which is a statin response marker II. For this instance where there is ambiguity in the haplotype pah underlying the detemiined genotype C/G, T/C, frequency information may be used to determine the most probable haplotype pair and therefore the most likely number of copies of ITGB3 haplotype 214 in the individual. If a particular ITGB3 haplotype pair consistent with the genotype of the individual is more frequent in a reference population than other haplotype pairs consistent with the genotype, then that haplotype pair with the highest frequency is the most likely to be present in the individual. The copy number of the haplotype of interest in this haplotype pair may then be determined by visual inspection of the alleles at the polymoφhic sites that comprise the response marker for each haplotype in the pair.
The individual's genotype for the desired set of PSs may be detennined using a variety of methods well-known in the art. Such methods typically include isolating from the individual a genomic DNA sample comprising both copies of the gene or locus of interest, amplifying from the sample one or more target regions containing the polymoφhic sites to be genotyped, and detecting the nucleotide pair present at each PS of interest in the amplified target region(s). It is not necessary to use the same procedure to determine the genotype for each PS of interest.
In addition, the identity of the allele(s) present at any of the novel polymorphic sites described herein may be indirectly detennined by haplotyping or genotyping another polymoiphic site that is in linkage disequilibrium with the polymoiphic site that is of interest. For example, the linkage disequilibrium data in Table 2 shows that Δ2 for LD between PS3 and PS9 is close to 1 in the experimental population, therefore detennining the allele at PS 9 in an individual might be used to determine the allele at PS 3 in that individual indirectly. Polymoiphic sites in linkage disequilibrium with the presently disclosed polymoiphic sites may be located hi regions of the gene or in other genomic regions not examined herein. Detection of the allele(s) present at a polymoφhic site in linkage disequilibrium with the novel polymoiphic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymoφhic site. Alternatively, the presence in an individual of a haplotype or haplotype pair for a set of PSs comprising a statin response marker may be detemiined by directly haplotyping at least one of the copies of the individual's genomic region of interest, or suitable fragment thereof, using methods known in the art. Such direct haplotyping methods typically involve treating a genomic nucleic acid sample isolated from the individual in a manner that produces a hemizygous DNA sample that only has one of the two "copies" of the individual's genomic region which, as readily understood by the skilled artisan, may be the same allele or different alleles, amplifying from the sample one or more target regions containing the polymoφhic sites to be genotyped, and detecting the nucleotide present at each PS of interest h the amplified target region(s). The nucleic acid sample may be obtained using a variety of methods known in the art for preparing hemizygous DNA samples, which include: targeted in vivo cloning (TEVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614; generating hemizygous DNA targets using an allele specific oligonucleotide in combination with primer extension and exonuclease degradation as described in U.S. Patent No. 5,972,614; shigle molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruaiϊo et al., 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).
As will be readily appreciated by those skilled in the art, any individual clone will typically only provide haplotype infonnation on one of the two genomic copies present in an individual. If haplotype information is desired for the individual's other copy, additional clones will usually need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the genomic locus in an individual, hi some cases, however, once the haplotype for one genomic allele is directly detennined, the haplotype for the other allele may be inferred if the individual has a known genotype for the polymoφhic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
While direct haplotyping of both copies of the gene is preferably perfoπned with each copy of the gene being placed in separate containers, it is also envisioned that direct haplotyping could be performed in the same container if the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable. For example, if first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymoiphic site(s), then detecting a combination of the first and third dyes would identify the polymoφhism in the first gene copy while detecting a combination of the second and third dyes would identify the polymoφhism in the second gene copy. The nucleic acid sample used in the above indirect and direct haplotyping methods is typically isolated from a biological sample taken from the individual, such as a blood sample or tissue sample. Suitable tissue samples include whole blood, saliva, tears, urine, skin and hair.
The target region(s) containing the PS of interest may be amplified using any oligonucleotide- directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965, 188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88: 189- 193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241 : 1077-1080, 1988). Other known nucleic acid amplification procedures may be used to amplify the target region(s) including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992).
In both the direct and indirect haplotyping methods, the identity of a nucleotide (or nucleotide pair) at a polymoφhic site(s) in the amplified target region may be detemiined by sequencing the amplified region(s) using conventional methods. If both copies of the gene are represented in the amplified target, it will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymoφhic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site. The polymoφhism may be identified directly, known as positive-type identification, or by inference, refened to as negative-type identification. For example, where a polymoφhism is known to be guanine and cytosine in a reference population, a site may be positively detennined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site. Alternatively, the site may be negatively detennined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine). A polymoiphic site in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art. Typically, allele-specific oligonucleotides are utilized in performing such methods. The allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pah- showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant. In some embodiments, more than one polymoφhic site may be detected at once using a set of allele-specific oligonucleotides or oligonucleotide pairs. Preferably, the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymoφhic sites being detected.
Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be perfoπned with both entities in solution, or such hybridization may be perfoπned when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, sfreptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid. Detecting the nucleotide or nucleotide pair at a PS of interest may also be detennined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Whiter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variant alleles can be identified by single strand conformation polymoφhism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et ah, Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).
A polymerase-mediated primer extension method may also be used to identify the polymoφhism(s). Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis" method (ΛV092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524. Related methods are disclosed in WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283. Extended primers contaming the complement of the polymoφhism may be detected by mass specfrometry as described in U.S. Patent No. 5,605,798. Another primer extension method is allele-specific PCR (Ruaiϊo et al., Nucl. Acids Res. 17:8392, 1989; Ruaiϊo et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635- 1641, 1995). Ei addition, multiple polymoφhic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).
The genotype or haplotype for the ITGB3 gene of an individual may also be detennined by hybridization of a nucleic acid sample containing one or both copies of the gene, mRNA, cDNA or fragment(s) thereof, to nucleic acid aiτays and subanays such as described in WO 95/11995. The anays would contain a battery of allele-specific oligonucleotides representing each of the polymoφhic sites to be included in the genotype or haplotype.
The invention also provides a kit for determining whether an individual has a statin response marker I or a statin response marker II. The kit comprises a set of oligonucleotides designed for determining the allele(s) present at a set of polymoiphic sites (PSs). In some embodiments, the set of polymoφhic sites comprises a set of polymoiphic sites selected from the group consisting of (1) PSI and PS42; (2) PS19 and PS42; (3) PS3, PS12, and PS42; (4) PSI, PS12, and PS42; (5) PS3, PS19, and PS42; (6) PSI, PS4, and PS42; (7) PS3, PS19, and PS39; (8) PSI, PS37, and PS42; (9) PS5, PS19, and PS42; (10) PSI, PS3, PS12, and PS42; (11) PS3, PS4, PS12, and PS42; (12) PSI, PS12, PS37 and PS42; (13) PS3, PS12, PS37 and PS42; (14) PSI, PS4, PS12, and PS42; (15) PS3, PS12, PS19 and PS42; (16) PS3, PS12, PS16 and PS42; (17) PSI, PS12, PS26 and PS42; (18) PSI, PS3, PS19, and PS42; (19) PS3, PS12, PS26 and PS42; (20) PS3, PS4, PS19, and PS42; (21) PSI, PS10, PS12 and PS42; (22) PS3, PS10, PS12, and PS42; (23) PSI, PS12, PS16 and PS42; (24) PSI, PS12, PS21 and PS42; (25) PS3, PS12, PS21 and PS42; (26) PSI, PS12, PS20 and PS42; (27) PS3, PS12, PS20 and PS42; (28) PS3, PS16, PS19 and PS42; (29) PS3, PS19, PS37 and PS42; (30) PSI, PS3, PS19 and PS42; (31) PSI, PS3, PS4, PS12, and PS42; (32) PSI, PS12, PS21, PS37, and PS42; (33) PS3, PS12, PS21, PS37, and PS42; (34) PSI, PS3, PS12, PS37, and PS42; (35) PSI, PS4, PS12, PS37, and PS42; (36) PS3, PS4, PS12, PS37, and PS42; (37) PSI, PS12, PS20, PS37, and PS42; (38) PSI, PS3, PS12, PS19 and PS42; (39) PS3/PS12, PS20, PS37, and PS42; (40) PS3, PS4, PS12, PS19 and PS42; (41) PS3, PS12, PS19, PS37, and PS42; (42) PSI, PS3, PS12, PS16 and PS42; (43) PS3, PS4, PS12, PS16 and PS42; (44) PSI, PS4, PS12, PS19 and PS42; (45) PSI, PS3, PS12, PS26 and PS42; (46) PSI, PS4, PS12, PS26 and PS42; (47) PS3, PS4, PS12, PS26, and PS42; (48) PSI, PS12, PS16, PS37, and PS42; (49) PSI, PS3, PS4, PS19 and PS42; (50) PSI, PS12, PS19, PS37, and PS42; (51) PS3, PS12, PS16, PS37 and PS42; (52) PSI, PS3, PS10, PS12 and PS42; (53) PSI, PS4,PS10, PS12 and PS42; (54) PS3, PS4, PS10, PS12, and PS42; (55) PSI, PS3, PS12, PS21 and PS42; (56) PSI, PS4, PS12, PS12 and PS42; (57) PSI, PS4, PS12, PS16 and PS42; (58) PS3, PS4, PS12, PS21, and PS42; (59) PS3, PS12, PS16, PS19 and PS42;' (60) PSI, PS3, PS12, PS20 and PS42; (61) PSI, PS4, PS12, PS20 and PS42; (62) PS3, PS4, PS12, ' ' PS20, and PS42; (63) PS3, PS12, PS19, PS26 and PS42; (64) PS3, PS10, PS12, PS19, and PS42; (65) PSI, PS12, PS16, PS19, and PS42; (66) PS21 and PS39; (67) PS10 and PS39; (68) PS26 and PS39; (69) PS20 and PS39; (70) PS15, PS21 and PS39; (71) PS10, PS15 and PS39; (72) PS15, PS26 and PS39: (73) PS15, PS20 and PS39; (74) PS21, PS30 and PS39; (75) PS10, PS30 and PS39; (76) PS5, PS21 and PS39; (77) PS26, PS30 and PS39; (78) PS20, PS30 and PS39: (79) PS21, PS37 and PS39; (80) PS4, PS15 and PS39; (81) PS5, PS10 and PS39; (82) PS15, PS37 and PS39; (83) PS5, PS26 and PS39; (84) PS15, PS21 and PS39; (85) PS10, PS15 and PS39; (86) PS5, PS15, PS21 and PS39; (87) PS15, PS26, PS30 and PS39; (88) PS15, PS20, PS30 and PS39; (89) PS15, PS21, PS37 and PS39; (90) PS5, PS21, PS30 and PS39; (91) PS5, PS10, PS15 and PS39; (92) PS5, PS15, PS20 and PS39; (93) PS5, PSI 5, PS26 and PS39; (94) PS15, PS20, PS37 and PS39; (95) PS5, PS10, PS30 and PS39; (96) PS5, PS15, PS21 and PS39; (97) PS5, PS10, PS15, PS30 and PS39; (98) PS5, PS15, PS20, PS30 and PS39; (99) PS5, PS15, PS26, PS30 and PS39; (100) PS15, PS21, PS30, PS37 and PS39; (101) PS3 and PS42; (102) PSI, PS3 and PS42; (103) PSI, PS28 and PS42; (104) PS12, PS16 and PS42; (105) PS30 and PS39; (106) PS3 and PS39; (107) PS39; (108) PS28, PS30 and PS39; (109) PSI and PS39; (110) PS28, PS30 and PS42; (111) PS28 and PS39; (112) PS12, PS28, PS30 and PS42; (113) PS3, PS16 and PS42; (114) PSI, PS38 and PS42; (115) PS28 and PS42; (116) PSI, PS16 and PS42; (117) PS12, PS28 and PS42; (118) PS12, PS28, PS38 and PS42; (119) PS3, PS38 and PS42; (120) PS16, PS30 and PS39; (121) PS3, PS16 and PS39; (122) PS16 and PS39; (123) PS16, PS28, PS30 and PS39; (124) PSI, PS16 and PS39; (125) PS16, PS28, and PS39; (126) PS28, PS38 and PS42; (127) PSI, PS16, PS38 and PS42; (128) PS 16, PS28 and PS42; (129) a set of polymoφhic sites comprising a linked haplotype to any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; or (130) a set of polymoiphic sites comprising a substitute haplotype for any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC in which the allele at one or more of the polymorphic sites in the original haplotype is replaced with an allele at a substitute polymoiphic site in linkage disequilibrium with the allele at the replaced or substituted polymorphic site. Preferred sets of polymoφhic sites comprise the sets of PSs for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB. More preferably the set of polymoφhic sites comprises the set of PSs for any one of haplotypes 201, 205, or 209 in Table IB. Thus in some preferred embodiments, the set of polymoφhic sites comprises PS3, PS12 and PS42; PSI, PS12 and PS42; PS3 and PS42; PSI and PS42; PSI, PS3, PS12 and PS42; or PS39. More preferably, the set of PSs comprises PS3, PS12 and PS42. In other embodiments of a kit of the invention, the kit comprises a set of oligonucleotides designed for identifying at least one of the alleles at each polymoφhic site (PS) hi a set of two or more polymoφhic sites (PSs) selected from the group consisting of PSI to PS44. In some of these embodiments, at least one PS in the set of two or more polymoiphic sites is selected from the group consisting of PSI, PS2, PS3, PS4, PSS, PS6, PS7, PS8, PS9, PS10, PS11, PSI 2, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44. In another embodiment of the kit, the PSs are selected from the group consisting of:
PSI, PS3, PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS37, PS38, PS39, and PS42; most preferably, the PSs are selected from the group consisting of: PSI, PS3, PS 12, PS39, and PS42. In one embodiment, the kit comprises oligonucleotides for detecting at least one allele for each polymoφhic site in the set of polymoφhic sites, while in other embodiments the kit comprises oligonucleotides for detecting both alleles at each member of the set of polymoφhic sites. Each oligonucleotide provided in the kit may be placed in the same or separate receptacles and may be provided together in a package.
As used herein, a oligonucleotide for genotyping a polymoφhic site is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, a polymoiphic site of interest such as one of the polymoφhic sites comprising a statin response marker described herein. The temi "oligonucleotide" refers to a polynucleotide molecule having less than about 100 nucleotides. A prefened oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length. The exact length of the oligonucleotide will depend on the nature of the genomic region containing the PS of interest as well as the genotyping assay to be performed and can readily be detemiined by the skilled artisan. The oligonucleotides used to practice the invention may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives. Alternatively, oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc. (1995), pages 617-620). Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion. The oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like. Oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a polynucleotide containing a desired locus. As used herein, specific hybridization means the oligonucleotide fonns an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to fonn such a structure when incubated with another region in the polynucleotide or with a polynucleotide lacking the desired locus under the same hybridizing conditions. Preferably, the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions. The skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymoiphisms in the ITGB3 gene or adjacent regions of chromosome 17 in linkage disequilibrium with one of the selected ITGB3 haplotypes, using the polymoφhism infonnation provided herein in conjunction with the known sequence infonnation for the ITGB3 gene, and adjacent regions of chromosome 17, and routine techniques.
A nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect" or "complete" complement of another nucleic acid molecule if ever}' nucleotide of one of the molecules is complementary to the nucleotide at the conesponding position of the other molecule. A nucleic acid molecule is "substantially complementary" to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1 89) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, ERL Press,
Washington, D.C. (1985). While perfectly complementary oligonucleotides are preferred for detecting polymoφhisms, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region. For example, an oligonucleotide primer may have a non-complementary fragment at its 5' end, with the remainder of the primer being complementaty to the target region. Alternatively, non-complementary nucleotides may be interspersed into the probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
Prefened oligonucleotides of the invention, useful in deteπnining if an individual has a statin response marker I or II, are allele-specific oligonucleotides. As used herein, the teπn allele-specific oligonucleotide (ASO) means an oligonucleotide that is able, under sufficiently stringent conditions, to hybridize specifically to one allele of a gene, or other locus, at a target region containing a polymoφhic site while not hybridizing to the conesponding region in another allele(s). As understood by the skilled artisan, allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps. Examples of hybridization and washing conditions typically used for ASO probes are found in Kogan et al., "Genetic Prediction of Hemophilia A" in PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990 and Ruano et al., 87 Proc. Natl. Acad. Sci. USA 6296-6300, 1990. Typically, an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele. Allele-specific oligonucleotides of the invention include ASO probes and ASO primers. ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymoφhic site in the target region (e.g., approximately the 7th or 8th position in a 15mer, the 8th or 9th position in a 16mer, and the 10th or 11th position in a 20mer). An ASO primer of the invention has a 3' terminal nucleotide, or preferably a 3' penultimate nucleotide, that is complementary to only one of the nucleotide alleles of a particular polymoiphic site, thereby acting as a primer for polymerase-mediated extension only if that nucleotide allele is present at the PS in the sample being genotyped. ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention. ASO probes and primers listed below use the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WIPO standard ST.25) at the position of the polymoφhic site to represent that the ASO contains either of the two alternative allelic variants observed at that polymoiphic site.
A prefened ASO probe for detecting ITGB3 gene polymoφhisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of: PSI GGGAAGAYCCAGGGA (SEQ ID NO 4) and its complement,
PS2 AAAATAGRTAAAGTC (SEQ ID NO 5) and its complement,
PS3 AGAAGCCSGAGGGGA (SEQ ID NO- 6) and its complement,
PS4 GCGGCGGYGCCCACT (SEQ ID NO- 7) and its complement,
PSS GGGAGGCSGACGAGA (SEQ ID NO 8) and its complement,
PS6 GGCGACTRTGCTGGC (SEQ ID NO 9) and its complement,
PS7 TGGGGGCKCTGGCGG (SEQ ID NO 10) and its complement,
PS8 GGGGGCGYTGGCGGG (SEQ ID NO 11 and its complement,
PS9 CTGCGCCSCGGTCAA (SEQ ID NO 12 and its complement,
PS10 GCGGAGGRCTGGTCC (SEQ ID NO 13 and its complement,
PS11 GGAATGCRCGTGTCC (SEQ ID NO 14 and its complement,
PS12 CTGGGGAYCTTCCTG (SEQ ID NO 15 and its complement,
PS13 AATGTACRGGGTAAA (SEQ ID NO 16 and its complement,
PS14 GCCATAGYTCTGATT (SEQ ID NO 17 and its complement,
PS15 CTGCCTCYGGGCTCA (SEQ ID NO 18 and its complement,
PSI 6 TGTGACCKGAAGGAG (SEQ ID NO 19 and its complement,
PS17 AGAGGATYGCACTCC (SEQ ID NO 20 and its complement,
PS18 AAGΔCAARGATGAGG (SEQ ID NO 21 and its complement,
PSI 9 TATATTTKTCCCCTC (SEQ ID NO 22 and its complement,
PS20 TCCAGCCYAATGACG (SEQ ID NO 23 and its complement,
PS21 ACCACCTRTGGTTTC (SEQ ID NO 24 and its complement,
PS22 AAGCTTTRGACCCTA (SEQ ID NO 25 and its complement,
PS23 GGAAAGASTCTCCCC (SEQ ID NO 26 and its complement,
PS24 ACCAGCTYCCTTTGG (SEQ ID NO 27 and its complement,
PS25 ATAGTCCYGCGGAGA (SEQ ID NO 28 and its complement,
PS26 TAGTCCCRCGGAGAG (SEQ ID NO 29 and its complement,
PS27 GTCCCGCRGAGAGTC (SEQ ID NO 30 and its complement,
PS28 GAGTCCAYCTCATTT (SEQ ID NO 31 and its complement,
PS29 TCTACCAYACGGTCT (SEQ ID NO 32 and its complement,
PS30 TTGGGATRGAACTGG (SEQ ID NO 33 and its complement,
PS31 CTAAAGTMGAGCTGG (SEQ ID NO 34 and its complement,
PS32 TTCCTTTKGTAGTGA (SEQ ID NO 35 and its complement,
PS33 AAAGCCCRTGGGCTT (SEQ ID NO 36 and its complement,
PS34 AGGACGARTGCAGCC (SEQ ID NO 37 and its complement,
PS35 AGCCCCCRGGAGGGT (SEQ ID NO 38 and its complement,
PS36 GCCCCCGRGAGGGTC (SEQ ID NO 39 and its complement,
PS37 TAATCACYGTGTCCT (SEQ ID NO 40 and its complement,
PS38 ATGGAAGYGTGACTT (SEQ ID NO 41 and its complement,
PS39 ACTTCCCYGGAAGTC (SEQ ID NO 42 and its complement,
PS40 CAGGAGGRAGAGGGA (SEQ ID NO 43 and its complement,
PS41 GCCTTGCYGCCCTGC (SEQ ID NO 44 and its complement,
PS42 TAAGAGAYGGGGCTG (SEQ ID NO :45 ) and its complement,
PS43 TCTTAAGKGGAAGCA (SEQ ID NO 46 and its complement, and
PS44 CTCTGTTSTGGAAAC (SEQ ID NO 47 and its complement. A preferred ASO primer for detecting ITGB3 gene polymoφhisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
PSI AGTCTGGGGAAGAYC (SEQ ID NO 43) • TTTGAGTCCCTGGRT (SEQ ID NO: 49) ;
PS2 TTGAGGAAAATAGRT (SEQ ID NO 50) • CTTTGGGACTTTAYC (SEQ ID NO: 51) ;
PS3 ATCTAGAGAAGCCSG (SEQ ID NO 52) • GCTTCCTCCCCTCSG (SEQ ID NO: 53) ;
PS4 CCGCGGGCGGCGGYG (SEQ ID NO: 54) , CCCCACAGTGGGCRC (SEQ ID NO: 55) ;
PS5 CGCCGCGGGAGGCSG (SEQ ID NO 56) • CTCGCATCTCGTCSG (SEQ ID NO: 57) ;
PS6 GCTCTGGGCGACTRT (SEQ ID NO 58) • CCCAGCGCCAGCAYA (SEQ ID NO: 59) ;
PS7 TGGCGCTGGGGGCKC (SEQ ID NO 60) • CAACGCCCGCCAGMG (SEQ IDNO:61) ;
PS8 GGCGCTGGGGGCGYT (3EQ ID NO 62) • CCAACGCCCGCCARC (SEQ ID NO: 63) ;
PS9 CAGGATCTGCGCCSC (SEQ ID NO 64) CGCAACTTGACCGSG (SEQ ID NO: 65) ;
PS10 GCAAACGCGGAGGRC (SEQ ID NO 66) ; CGCGCGGGACCAGYC (SEQ ID NO: 67) ;
PS11 GCGCTGGGAATGCRC (SEQ ID NO 68) ; CiSCCAGGACACGYG (SEQ ID NO: 69) ; PS12 CGGGAGCTGGGGAYC SEQ 70) ; CCGGGCCAGGAAGRT (SEQ ID NO:71)
PS13 TGCTCCAATGTACRG SEQ 72); TAAGAGTTTACCCYG (SEQ ID NO:73)
PS14 AGAGTCGCCATAGYT SEQ 74); TCCAGCAATCAGARC (SEQ ID NO:75)
PS15 CAGGCCCTGCCTCYG ( SEQ 76); GCGAGGTGAGCCCRG (SEQ ID NO: 77)
PS16 CCTCGCTGTGACCKG SEQ 78); CAGATTCTCCTTCMG (SEQ ID NO:79)
PS17 GTCCCCAGAGGATYG SEQ 80); GGAGCCGGAGTGCRA (SEQ ID NO:81)
PS18 GGTGGGAAGACAARG SEQ 82); CTCCCCCCTCATCYT (SEQ IDNO:83) ;
PS19 ACTATCTATATTTKT SEQ 84); GAAAGAGAGGGGAMA (SEQ ID NO: 85)
PS20 GCATTGTCCAGCCYA (SEQ 86); ACTGCCCGTCATTRG (SEQ ID NO: 87)
PS21 TCTGGGACCACCTRT (SEQ 88); TGAATAGAAACCAYA (SEQ ID NO:89)
PS22 AGTTCTAAGCTTTRG (SEQ 90) ; CATCTCTAGGGTCYA (SEQ ID NO: 91) ;
PS23 CAATCAGGAAAGAST (SEQ 92); GGATTAGGGGAGAST (SEQ ID NO: 93)
PS24 GAGACCACCAGCTYC (SEQ 94); AGCTTACCAAAGGRA (SEQ ID NO: 95)
PS25 CTGGGAATAGTCCYG (SEQ 96); GTGGACTCTCCGCRG (SEQ ID NO: 97)
PS26 TGGGAATAGTCCCRC SEQ 98); GGTGGACTCTCCGYG (SEQ ID NO:99
PS27 GGAATAGTCCCGCRG SEQ 100) GAGGTGGACTCTCYG (SEQ ID NO: 101
PS28 CGCGGAGAGTCCAYC SEQ 102) TAAGCCAAATGAGRT (SEQ ID NO: 103
PS29 TGCCACTCTACCAYA SEQ 104) GCAGTAAGACCGTRT S?EQ ID NO:105);
PS30 TTTGGCTTGGGATRG SEQ 106) CAAGAGCCAGTTCYA (SEQ ID NO: 107)
PS31 TCCGTTCTAAAGTMG (SEQ 108) GCACTTCCAGCTCKA (SEQ ID NO 109) ;
PS32 CCT.TTCTTCCTTTKG (SEQ 110) TTTTAGTCACTACMA (SEQ ID NO ill);
PS33 TACCATAAAGCCCRT (SEQ 112); TCCTTGAAGCCCAYG (SEQ ID NO: 113);
PS34 CCCAGCAGGACGART (SEQ NO :114) CCCGGGGGCTGCAYT (SEQ ID NO 115);
PS35 GAATGCAGCCCCCRG (SEQ NO :116) GGGCTGACCCTCCYG (SEQ ID NO 117);
PS36 AATGCAGCCCCCGRG (SEQ :118) CGGGCTGACCCTCYC (SEQ ID (-3:119) ;
PS37 TTGCCTTAATCACYG (SEQ :120) GGAGAGAGGACACRG (SEQ ID NO 121);
PS38 TGTAAAATGGAAGYG (SEQ :122) AGGTAGAAGTCACRC (SEQ ID NO 123);
PS39 TCACATACTTCCCYG (SEQ :124) TCACAGGACTTCCRG (SEQ ID NO 125);
PS40 CTGCCCCAGGAGGRA (SEQ :126) TGGTTCTCCCTCTYC (SEQ ID NO: 127) ;
PS41 TCATTGGCCTTGCYG (SEQ :128) AGATGAGCAGGGCRG (SEQ ID NO 129) ;
PS42 ACACAGTAAGAGAYG (SEQ :130) AACGCCCAGCCCCRT (SEQ ID NO 131);
PS43 TGAGACTCTTAAGKG (SEQ :132) ATCTGCTGCTTCCMC (SEQ ID NO 133) ;
PS44 ATTATCCTCTGTTST (SEQ :134) and CTCCCAGTTTCCASA (SEQ ID NO: 135)
Other oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymoiphic sites identified herein. Such oligonucleotides are useful in poly erase-mediated primer extension methods for detecting one of the novel polymoφhisiiis described herein and therefore such oligonucleotides are refeiτed to herein as, "primer- extension oligonucleotides". In a prefened embodiment, the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complenientaiy to the nucleotide located immediately adjacent to the polymoφhic site.
A particularly preferred oligonucleotide primer for detecting ITGB3 gene polymoφhisms by primer extension tenninates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
PSI CTGGGGAAGA (SEQ ID NO 136) GAGTCCCTGG (SEQ ID NO 137);
PS2 AGGAAAATAG (SEQ ID NO 138) TGGGACTTTA (SEQ ID NO 139);
PS3 TAGAGAAGCC (SEQ ID NO 140) • TCCTCCCCTC (SEQ ID NO 141) ;
PS4 CGGGCGGCGG (SEQ ID NO 142) CACAGTGGGC (SEQ ID NO 143);
PS5 CGCGGGAGGC (SEQ ID NO 144) GCATCTCGTC (SEQ ID NO 145);
PS6 CTGGGCGACT (SEQ ID NO 146) AGCGCCAGCA (SEQ ID NO 147);
PS7 CGCTGGGGGC (SEQ ID NO 148) CGCCCGCCAG (SEQ ID NO :149) ;
PS8 GCTGGGGGCG (SEQ ID NO 150) ACGCCCGCCA (SEQ ID NO 151);
PS9 GATCTGCGCC (SEQ ID MO 152) AACTTGACCG (SEQ ID NO 153);
PS10 AACGCGGAGG (SEQ ID NO 154) GCGGGACCAG (SEQ ID NO 155); psii CTGGGAATGC (SEQ ID NO 156) CCAGGACACG (SEQ ID NO :157); PS12 GAGCTGGGGA SEQ ID NO 158) , GGCCAGGAAG ( SEQ ID NO 159)
PS13 TCCAATGTAC SEQ ID NO 160) , GAGTTTACCC ( SEQ ID NO 161)
PS14 GTCGCCATAG SEQ ID NO 162) , AGCAATCAGA ( SEQ ID NO 163)
PS15 GCCCTGCCTC SEQ ID NO 164) , AGGTGAGCCC ( SEQ ID NO :165)
PS16 CGCTGTGACC SEQ ID NO 166) , ATTCTCCTTC ( SEQ ID NO 167)
PS17 CCCAGAGGAT SEQ ID NO 168) , GCCGGAGTGC SEQ ID NO 169)
PS18 GGGAAGACAA SEQ ID NO 170) , CCCCCTCATC SEQ ID NO 171)
PS19 ATCTATATTT SEQ ID NO 172) , AGAGAGGGGA SEQ ID NO 173)
PS20 TTGTCCAGCC SEQ ID NO 174) , GCCCGTCATT SEQ ID NO 175)
PS21 GGCACCACCT SEQ ID NO 176) , ATAGAAACCA SEQ ID NO 177)
PS22 TCTAAGCTTT SEQ ID NO 178) , CTCTAGGGTC SEQ ID NO 179)
PS23 TCAGGAAAGA SEQ ID NO 180) , TTAGGGGAGA SEQ ID NO 181)
PS24 ACCACCAGCT SEQ ID NO 182) , TTACCAAAGG SEQ ID NO 183)
PS25 GGAATAGTCC SEQ ID NO 184) , GACTCTCCGC SEQ ID NO 185)
PS26 GAATAGTCCC SEQ ID NO 186) GGACTCTCCG SEQ ID NO 187)
PS27 ATAGTCCCGC SEQ ID NO 188) GTGGACTCTC SEQ ID NO 189)
PS28 GGAGAGTCCA SEQ ID NO 190) GCCAAATGAG SEQ ID NO 191)
PS29 CAGTCTACCA SEQ ID NO 192) GTAAGACCGT SEQ ID NO 193)
PS30 GGCTTGGGAT SEQ ID NO 194) GAGCCAGTTC SEQ ID NO 195)
PS31 GTTCTAAAGT SEQ ID NO 196) CTTCCAGCTC SEQ ID NO 197)
PS32 TTCTTCCTTT SEQ ID NO 198) • TAGTCACTAC SEQ ID NO 199)
PS33 CATAAAGCCC SEQ ID NO 200) TTGAAGCCCA SEQ ID NO 201)
PS34 AGCAGGACGA SEQ ID NO 202) GGGGGCTGCA SEQ ID NO 203)
PS35 TGCAGCCCCC SEQ ID NO 204) CTGACCCTCC SEQ ID NO 205)
PS36 GCAGCCCCCG SEQ ID NO 206) ; GCTGACCCTC (SEQ ID NO 207)
PS37 CCTTAATCAC SEQ ID NO 208) GAGAGGACAC SEQ ID NO 209)
PS38 AAAATGGAAG SEQ ID NO 210) TAGAAGTCAC SEQ ID NO 211)
PS39 CATACTTCCC SEQ ID NO 212) CAGGACTTCC SEQ ID NO 213)
PS40 CCCCAGGAGG (SEQ ID NO 214) TTCTCCCTCT (SEQ ID NO :215)
PS41 TTGGCCTTGC SEQ ID NO 216) TGAGCAGGGC SEQ ID NO 217)
PS42 CAGTAAGAGA (SEQ ID NO 218) GCCCAGCCCC SEQ ID NO 219)
PS43 GACTCTTAAG (SEQ ID NO 220) TGCTGCTTCC SEQ ID NO 221)
PS44 ATCCTCTGTT (SEQ ID NO 222) and CCAGTTTCC; ^ (SEQ ID NO:2 1223)
Termination mixes are chosen to temiinate extension of the oligonucleotide at the polymoφhic site of interest, or one base thereafter, depending on the alternative nucleotides present at the polymoiphic site. Prefened ASO probes or primers and preferred primer extension oligonucleotides for detecting the alleles at the polymorphic sites comprising the prefened embodiments of the statin response markers I and II comprise the nucleotide sequences for PSI, PS3. PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42. Particularly prefened ASO probes and primers and primer extension oligonucleotides for genotyping PS3, PS12 and PS42 are SEQ ED NO:6, 15 and 45, and their complements; SEQ ID NOS:52-53, 70-71, 130-131, and SEQ ED NOS: 140-141, 158-159, and 218-219.
In some embodiments, the genotyping oligonucleotides in a kit of the invention have different labels to allow probing of the identity of nucleotides or nucleotide pairs at two or more polymoiphic sites simultaneously. It is also contemplated that a kit of the invention may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymoφhic site in a statin response marker.
The oligonucleotides comprising a kit of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized oligonucleotides may be used in a variety of polymoφhism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized oligonucleotides useful in practicing the invention may comprise an ordered aιτay of oligonucleotides designed to rapidly screen a nucleic acid sample for polymoφhisms in multiple genes at the same time. Kits of the invention may also contain other components such as hybridization buffer (e.g., where the oligonucleotides are to be used as allele-specific probes) or dideoxynucleotide triphosphates (ddNTPs; e.g., where the alleles at the polymoiphic sites are to be detected by primer extension). In a preferred embodiment, the set of oligonucleotides consists of primer extension oligonucleotides. The kit may also contain a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase. Preferred kits may also include detection reagents, such as biotin- or fluorescent-tagged oligonucleotides or ddNTPs and/or an enzyme-labeled antibody and one or more subsfrates that generate a detectable signal when acted on by the enzyme. It will be understood by the skilled artisan that the set of oligonucleotides and reagents for perfonning the genotyping or haplotyping assay will be provided in separate receptacles placed in the container if appropriate to preserve biological or chemical activity and enable proper use in the assay.
In a particularly prefened embodiment, each of the oligonucleotides and all other reagents in the kit have been quality tested for optimal performance in an assay for detennining the alleles at a set of polymoφhic sites comprising a statin response marker I or statin response marker II. In a further embodiment, the kit comprises a manual with instructions for performing genotyping assays on a nucleic acid sample from an individual and detennining if the individual has a statin response marker I or a statin response marker II based on the results of the assay. The instructions may also contain infonnation to help a physician determine whether or how to use particular statins, alone or in combination with other therapies affecting HDLC levels, to freat an mdividual with the determined statin response marker.
The methods and kits of the invention are useful for helping physicians make decisions about how to treat an individual. They can be used to predict the change in HDLC of an individual in response to freatment with a statin or in selecting a statin therapy for an individual.
Thus, the invention provides a method for predicting the HDLC response of an individual to treatment with a statin. The method comprises determining whether the individual has a statin response marker I or a statin response marker II and making a response prediction based on the results of the detennining step. The statin is atorvastatin or a pharmaceutically acceptable salt of atoiYastatin acid. Preferably, the statin is atoiYastatin calcium. In some embodiments, if the individual is detennined to have a statin response marker I, then the response prediction is that the individual will experience an unfavorable HDLC response if treated with atoiYastatin calcium at a dose greater than about 10 mg/day. Also, if the individual is detennined to have a statin response marker II, then the response prediction is that the individual will likely experience a favorable HDLC response if freated with aton'astatin calcium at any dose between about 10 to about 80 mg/day. hi some embodiments, the determining step comprises consulting a data repository that states whether the individual has a statin response marker I or a statin response marker II. The data repositoiy may be the individual's medical records or a medical data card. In other embodiments, the determining step comprises detennining the copy number of a haplotype selected from the group consisting of: haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC; a linked haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 Table IC; and a substitute haplotype for any of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501 to 515 in Table IC. If the selected haplotype is one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, then the individual has a statin response marker I if the individual has at least one copy of the selected marker and a statin response marker II if the individual has zero copy of the selected marker. If the selected haplotype is one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, then the individual has a statin response marker I if the individual has zero or one copy of the selected haplotype and a statin response marker II if the individual has two copies of the selected haplotype. Prefened haplotypes are haplotypes 201, 205, 209, 214, 225, or 288 in Table IB. Values for the linkage disequilibrium, Δ2, for linked haplotypes or substituting polymoiphic sites in substitute haplotypes are at least 0.75, preferably at least 0.80, more preferably at least 0.85, yet more preferably at least 0.90, and most preferably at least 0.95 or 1.0. The determination of the statin response marker present in an individual can be made using one of the direct or indirect methods described herein or known in the art. In some prefened embodiments, the detennining step comprises identifying for one or both copies of the genomic locus present in the individual the identity of the nucleotide or nucleotide pair at each member of the set of polymoφhic sites comprising the selected haplotype. In prefened embodiments, the individual is Caucasian.
The invention also provides a method of selecting a statin therapy to provide an optimal HDLC response in an individual. The method comprises detennining whether the individual has a statin response marker I or a statm response marker II and selecting a statin therapy based on the results of the detennining step. hi some embodiments, if the individual has a statin response marker II, then the selected statin therapy comprises any dose of atoiYastatin or a pharmaceutically acceptable salt of atoiYastatin acid and if the individual has a statin response marker I, then the selected statin therapy comprises a low dose of atorvastatin, a low dose of a pharmaceutically acceptable salt of atorvastatin acid, a higher dose of atoiYastatin in conjunction with an HDLC-modulating therapy, or another statin. The preferred phannaceutically acceptable salt of atorvastatin acid is atorvastatin calcium.
A low dose means a dose that is at the lower end of the range of doses permitted by a regulatory agency, while a high dose or higher dose means a dose that is at the higher end of the range of permitted doses. For example, for atoiYastatin calcium, a low dose comprises about 10 mg day, while a higher dose of atorvastatin calcium conesponds to doses greater than about 10 mg/day, preferably greater than about 40 mg/day, most preferably about 80 mg/day. The determination of the statin response marker present in an individual can be made using one of the direct or indirect methods described herein or known in the art. One method to determine whether a statin response marker I or II is present in the individual comprises determining the copy number of any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; a linked haplotype to any one of haplotypes 101-194 in Table 1 A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC. Preferred haplotypes are haplotypes 201, 205, 209, 214, 225, or 288 in Table IB. Values for the linkage disequilibrium, Δ2, for linked haplotypes or substituting polymorphic sites in substitute haplotypes are at least 0.75, preferably at least 0.80, more preferably at least 0.85, yet more preferably at least 0.90, and most preferably at least 0.95, if not 1.0. Alternatively, the detennining step may comprise consulting a data repositoiy that states the individual's copy number for one or more haplotypes comprising a statin response marker I or II. The data repository may be the individual's medical records or a medical data card. In prefened embodiments, the individual is Caucasian.
In other aspects, the invention provides an article of manufacture. In one embodiment, an article of manufacture comprises a phaπnaceutical fomiulation and at least one indicium identifying a population for which the phaniiaceutical fomiulation is indicated. The phaniiaceutical fomiulation comprises a statin as at least one active ingredient. The statin may be simvastatin, a phannaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a phannaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid. Additionally, the pharmaceutical formulation may be regulated and the indicium may comprise the approved label for the phaniiaceutical foπnulation. The identified population is partially or wholly defined by having a statin response marker I. The identified population preferably may be further defined as Caucasian. A population wholly defined by having a statin response marker is one for which there are no other factors which should be considered in identifying the population for which the pharmaceutical formulation is indicated, hi contrast, a population that is partially defined by having a statin response marker is one for which other factors may be pertinent to identification of the population for which the pharmaceutical fomiulation is indicated. Examples of other such factors are age, weight, gender, disease state, possession of other genetic markers or biomarkers, or the like.
The statin response marker I comprises a copy number of a specific haplotype. The statin response marker I is at least one copy of any of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 hi Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB; or zero or one copy of any of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501- 515 in Table IC.
In some embodiments, the phamiaceutical fonnulation is fonnulated, in any way known in the art, as a sustained release foπnulation, but most preferably as a transdermal patch, hi other embodiments, the pharmaceutical formulation is a tablet or capsule and the article may further comprise an additional indicium comprising the color or shape of the table or capsule, h further embodiments, the article may further comprise an additional indicium comprising a symbol stamped on the tablet or capsule, or a S}mbol or logo printed on the approved label
In some embodiments of this article, a frial population having the statin response marker I exhibits a better HDLC response to the phamiaceutical formulation than to treatment with atoiYastatin or a pharmaceutically acceptable salt of atoiYastatin acid. The approved label may state that the phamiaceutical formulation provides a better HDLC response in a population having the statin response marker I. In these embodiments, the approved label may further state that the phamiaceutical fomiulation is indicated for individuals identified as having the statin response marker I on a specified test, preferably a specified genetic test, hi some or all of these embodiments, the label may describe the mean change in HDLC expected for the identified population. In yet another embodiment of this article of manufacture comprising a phamiaceutical fomiulation and at least one indicium identifying a population for which the phamiaceutical foπnulation is indicated, the phaπnaceutical fomiulation comprises as separate active ingredients a statin, which may be atoiYastatin or a phannaceutically acceptable salt of atorvastatin acid, and an HDL cholesterol (HDLC) modulating agent, hi prefened embodiments, the statin is atorvastatin calcium. The identified population is partially or wholly defined by having a statin response marker I. A trial population having the statin response marker I may exhibit a worse mean HDLC response to treatment with only atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid than to treatment with the pharmaceutical formulation. Alternatively, a trial population havmg the statin response marker I may exhibit a worse mean HDLC response to treatment with only atorvastatin or a pharmaceutically acceptable salt of aton'astatin acid than a trial population lacking the statin response marker I.
Additionally, in some or all of these embodiments, the statin is present hi the phaπnaceutical formulation at an amount effective to reduce LDL cholesterol levels. In embodiments in which the statin is atorvastatin calcium, the effective amount ranges from about 10 to about 80 mg.
An additional embodiment of the article of manufacture provided by the invention comprises packaging material and a pharmaceutical fomiulation contained within said packaging material. The pharmaceutical fomiulation comprises a statin as at least one active ingredient. The statin may be simvastatin, a pharmaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceuticaUy acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a phannaceutically acceptable salt of rosuvastatin acid, atorvastatin, a phannaceutically acceptable salt of atoiYastatin acid, pravastatin, or a phannaceutically acceptable salt of pravastatin acid. The pharmaceutical foπnulation may further comprise an HDLC-modulating agent, such as niaciii. In some embodiments, the packaging material may comprise a label that may state that the pharmaceutical foπnulation is indicated for a population partly or wholly defined by having a statin response marker I. The label may further state the mean percent change in HDLC expected for the identified population. A population having a statin response marker I exhibits a better HDLC response to the pharmaceutical fomiulation than to treatment with atoiYastatin or a phannaceutically acceptable salt of atorvastatin acid. The indicated population in any of the above articles may preferably be further defined as Caucasian. The label may further state that a specified test can be used to identify members of the indicated population. Preferably the specified test is a genetic test.
Additionally, other aspects of the invention provide a method of manufacturing a drug product comprising a statin as at least one active ingredient. The statin may be simvastatin, a phannaceutically acceptable salt of simvastatin acid, lovastatin. a phannaceutically acceptable salt of lovastatin, fluvastatin, a phannaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, aton'astatin, a phannaceutically acceptable salt of atorvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid. The method comprises combining in a package a phamiaceutical foπnulation comprising the statin and a label. In some embodiments, the label states that the phamiaceutical formulation is indicated for freating a population partially or wholly defined by having a statin response marker I.
The indicated population having the defining statin response marker preferably may be further defined as Caucasian. The indicated and/or contraindicated populations may be identified on the pharmaceutical foπnulation, on the label or on the package by at least one indicium, such as a symbol or logo, color, or the like. Detecting the presence of a statin response marker I or II in an individual is also useful in a method of seeking regulatory approval for marketing a phamiaceutical foπnulation for treating a disease or condition in a population defined by the statin response marker. The method comprises conducting at least one clinical trial with first and second treatment groups of patients having the disease or condition, wherein each patient has a statin response marker I. Each patient in the first freatment group is treated with the phamiaceutical fomiulation and each patient in the second treatment group is treated with atorvastatin, a pharmaceutically acceptable salt of aton'astatin, or a derivative thereof. The method also comprises demonstrating that the first treatment group exhibits a mean percent change in HDLC that is better than the mean percent change exhibited by the second treatment group; and filing with a regulatory agency an application for marketing approval of the phaπnaceutical formulation with a label stating that the phamiaceutical formulation is indicated for patients having the statin response marker I. In some embodiments, the pharmaceutical fonnulation may comprise any statin known in the art other than atorvastatin or pharmaceutically acceptable salts of atoiYastatin acid. For example, the statin may be simvastatin, a phannaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a pharmaceutically acceptable salt of pravastatin acid. In other embodiments in which the pharmaceutical formulation comprises a statin and an HDLC modulating agent as two separate active ingredients, the statin may be atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid. More preferably in these latter embodiments, the pharmaceutical fonnulation comprises atoiYastatin calcium.
The clinical trial may be conducted by recruiting patients with the disease or condition, determining whether they have a statm response marker I or II and assigning the patients to the first and second treatment groups based on the results of the detennining step. The disease or condition may include any for which statin therapy is indicated, e.g., hyperlipidemia, hypercholesterolemia, cardiovascular disease (CVD), presence of CVD risk factors, coronary artery disease, and the like. The patients in each treatment group are preferably administered the same dose of the pharmaceutical fomiulation, which includes a statin compound as at least one active ingredient. The phaπnaceutical formulation may contain other active ingredients, for example another compound known or believed to have therapeutic activity hi treating the disease or condition examined in the study or a compound that sen'es to reduce or block one or more side effects caused by the statin compound.
The regulatory agency may be any person or group authorized by the government of a country anywhere in the world to control the marketing or distribution of drugs in that country. Preferably, the regulatory agency is authorized by the government of a major industrialized country, such as Australia, Canada, China, a member of the European Union. Japan, and the like. Most preferably the regulatory agency is authorized by the government of the United States and the type of application for approval that is filed will depend on the legal requirements set forth in the last enacted version of the Food, Drug and Cosmetic Act that are applicable for the phaπnaceutical fonnulation and may also include other considerations such as the cost of making the regulatory filing and the marketing strategy for the composition. For example, if the phaπnaceutical formulation has previously been approved for the same cognitive function, then the application might be a paper NDA, a supplemental NDA or an abbreviated NDA, but the application would be a full NDA if the pharmaceutical formulation has never been approved before; with these terms havmg the meanings applied to them by those skilled in the phamiaceutical arts or as defined in the Drug Price Competition and Patent Term Restoration Act of 1984.
Further, in perforating any of the methods described herein which require information on the haplotype content of the individual (i.e., the haplotypes and haplotype copy number present in the individual for the polymoiphic sites in haplotypes comprising a statin response marker I or II) or which require knowing if a statin response marker I or II is present in the individual, the individual's ITGB3 haplotype content or statin response marker may be determined by consulting a data repository such as the individual's patient records, a medical data card, a file (e.g. a flat ASCII file) accessible by a computer or other electronic or non-electronic media on which infonnation about the individual's ITGB3 haplotype content or statin response marker can be stored. As used herein, a medical data card is a portable storage device such as a magnetic data card, a smart card, which has an on-board processing unit and which is sold by vendors such as Siemens of Munich Germany, or a flash-memory card. The medical data card may be, but does not have to be, credit-card sized so that it easily fits into pocketbooks, wallets and other such objects caπied by the individual. The medical data card may be swiped through a device designed to access infonnation stored on the data card. En an alternative embodiment, portable data storage devices other than data cards can be used. For example, a touch- memory device, such as the "i-button" produced by Dallas Semiconductor of Dallas, Texas can store information about an individual's ITGB3 haplotype content or statin response marker, and this device can be incorporated into objects such as jewelry. The data storage device may be implemented so that it can wirelessly communicate with routing/intelligence devices through EEEE 802.11 wireless networking technology or through other methods well known to the skilled artisan. Further, as stated above, infonnation about an individual's ITGB3 haplotype content or statin response marker can also be stored in a file accessible by a computer; such files may be located on various media, including: a server, a client, a hard disk, a CD, a DVD, a personal digital assistant such as a Palm Pilot, a tape, a zip disk, the computer's internal ROM (read-only-memory) or the intemet or worldwide web. Other media for the storage of files accessible by a computer will be obvious to one skilled in the art.
Any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer. For example, the computer may execute a program that assigns ITGB3 haplotype pairs and/or a statin response marker I or II to individuals based on genotype data inputted by a laboratoiy technician or treating physician. In addition, the computer may output the predicted change in one or more lipoprotein levels in response to a statin following input of the individual's ITGB3 haplotype content or statin response marker, which was either detennined by the computer program or input by the technician or physician. Data on which statin response markers were detected in an individual may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files) containing other clinical and/or haplotype data for the individual. These data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network. hi another embodiment, the invention provides an isolated polynucleotide comprising a polymoφhic variant of the ITGB3 gene or a fragment of the gene which contains at least one of the novel polymoiphic sites described herein. The nucleotide sequence of a variant ITGB3 gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymoφhic sites PSI, PS2, PS3, PS4, PS5, PS6, PS7, PSS, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44. Similarly, the nucleotide sequence of a variant fragment of the ITGB3 gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymoφhic sites described herein. Thus, the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the ITGB3 gene, which is defined by haplotype 39, (or other reported ITGB3 sequences) or to portions of the reference sequence (or other reported ITGB3 sequences), except for the haplotyping and genotyping oligonucleotides described above.
The location of a polymoφhism in a variant ITGB3 gene or fragment is preferably identified by aligning its sequence against SEQ ED NO:l. The polymoφhism is selected from the group consisting of thymine at PSI, guanine at PS2, cytosine at PS3, thymine at PS4, cytosine at PS5, adenine at PS6, thymine at PS7, thymine at PSS, guanine at PS9, adenine at PS10, adenine at PSI 1, thymine at PS12, adenine at PS13, thymine at PS14, cytosine at PS15, guanine at PS16, cytosine at PS17, adenine at
PS18, thymine at PS19, cytosine at PS20, guanine at PS21, guanine at PS22, cytosine at PS23, cytosine at PS24, thymine at PS25, adenine at PS26, adenine at PS27, thymine at PS28, thymine at PS29, adenine at PS30, cytosine at PS31, guanine at PS32, adenine at PS33, guanine at PS34, adenine at PS35, adenine at PS36, cytosine at PS37, thymine at PS38, cytosine at PS39, adenine at PS40, thymine at PS41, thymine at PS42, guanine at PS43 and guanine at PS44. In a preferred embodiment, the polymoφhic variant comprises a naturally-occurring isogene of the ITGB3 gene which is defined by any one of haplotypes 1- 38 and 40 - 98 shown in Table 5 below.
Polymoiphic variants of the invention may be prepared by isolating a clone containing the ITGB3 gene from a human genomic library. The clone may be sequenced to determine the identity of the nucleotides at the novel polymoiphic sites described herein. Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by perforating in vitro mutagenesis using procedures well-known in the art. Any particular ITGB3 variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
ITGB3 isogenes, or fragments thereof, may be isolated using any method that allows separation of the two "copies" of the ITGB3 gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TEVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets. Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al., 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).
The invention also provides ITGB3 genome anthologies, which are collections of at least two ITGB3 isogenes found in a given population. The population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same gender population. An ITGB3 genome anthology may comprise individual ITGB3 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the ITGB3 isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of such isogenes in a genome anthology may be stored in any convenient and stable fonn, including but not limited to in buffered solutions, as DNA precipitates, freeze-dried preparations and the like. A prefened ITGB3 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.
An isolated polynucleotide containing a polymoφhic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements hi a recombinant expression vector capable of being propagated and expressing the encoded ITGB3 protein in a prokaryotic or a eukaiyotic host cell. Examples of expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40. Other regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence hi a given host cell. Of course, the conect combinations of expression regulatory elements will depend on the host system used. In addition, it is understood that the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers. Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York). Host cells which may be used to express the variant ITGB3 sequences of the invention include, but are not limited to, eukaiyotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known hi the art. The recombinant expression vector may be infroduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York), hi a prefened aspect, eukaryotic expression vectors that function in eukaiyotic cells, and preferably mammalian cells, are used. Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, heφes virus vectors, and baculovirus transfer vectors. Preferred eukaiyotic cell lines include COS cells, CHO cells, HeLa cells, NEH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282:1145-1147). Particularly preferred host cells are mammalian cells.
As will be readily recognized by the skilled artisan, expression of polymoiphic variants of the ITGB3 gene will produce ITGB3 mRNAs varying from each other at any polymoφhic site retained in the spliced and processed mRNA molecules. These mRNAs can be used for the preparation of an
ITGB3 cDNA comprising a nucleotide sequence which is a polymoiphic variant of the ITGB3 reference coding sequence shown in Figure 2. Thus, the invention also provides ITGB3 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ED NO:2 (Fig. 2) (or its conesponding RNA sequence) for those regions of SEQ ED NO:2 that conespond to the examined portions of the ITGB3 gene (as described in the Examples below), except for having one or more polymoφhisms selected from the group consisting of adenine at a position conesponding to nucleotide 40, thymine at a position corresponding to nucleotide 57, thymine at a position con-esponding to nucleotide 58, cytosine at a position corresponding to nucleotide 176, guanine at a position con'esponding to nucleotide 197, cytosine at a position con-esponding to nucleotide 342, cytosine at a position con-esponding to nucleotide 882, cytosine at a position corresponding to nucleotide 1143, adenine at a position corresponding to nucleotide 1333, guanine at a position conesponding to nucleotide 1533, adenine at a position corresponding to nucleotide 1544, adenine at a position corresponding to nucleotide 1545, and thymine at a position corresponding to nucleotide 2208. A particularly preferred polymoiphic cDNA variant is selected from the group consisting of A, B, C, D, E, F, G, H, I, K, J, L, M, N, O, P and Q represented in Table 8. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain one or more of the novel polymoiphisms described herein. The invention specifically excludes polynucleotides identical to previously identified ITGB3 mRNAs or cDNAs, and previously described fragments thereof.
Polynucleotides comprising a variant ITGB3 RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
As used herein, a polymoφhic variant of an ITGB3 gene fragment, mRNA fragment or cDNA fragment comprises at least one novel polymoφhism identified herein and has a length of at least 10 nucleotides and may range up to the full length of the gene. Preferably, such fragments are between 100 and 3000 nucleotides in length, and more preferably between 100 and 2000 nucleotides in length, and most preferably between 100 and 500 nucleotides in length.
In describing the ITGB3 polymoφhic sites identified heiein, reference is made to the sense strand of the gene for convenience. However, as recognized by the skilled artisan, nucleic acid molecules containing the ITGB3 gene or cDNA may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand. Thus, reference may be made to the same polymoφhic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymorphic site. Thus, the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the ITGB3 genomic, mRNA and cDNA variants described herein.
Polynucleotides comprising a polymoiphic gene variant or fragment of the invention may be useful for therapeutic puiposes. For example, where a patient could benefit from expression, or increased expression, of a particular ITGB3 protein isofonn, an expression vector encoding the isofonn may be administered to the patient. The patient may be one who lacks the ITGB3 isogene encoding that isofonn or may already have at least one copy of that isogene.
In other situations, it may be desirable to decrease or block expression of a particular ITGB3 isogene. Expression of an ITGB3 isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA or antisense RNA for the isogene or fragment thereof. Alternatively, oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3 ' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are prefened. Similarly, inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). Antisense oligonucleotides may also be designed to block translation of ITGB3 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of ITGB3 mRNA transcribed from a particular isogene.
The unfranslated mRNA, antisense RNA or antisense oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo. Alternatively, such molecules may be foπmulated as a phamiaceutical composition for administration to the patient. Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life. Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
The invention also provides an isolated polypeptide comprising a polymoiphic variant of (a) the reference ITGB3 amino acid sequence shown in Figure 3 or (b) a fragment of this reference sequence. The location of a variant amino acid in an ITGB3 polypeptide or fragment of the invention is preferably identified by aligning its sequence against SEQ ID NO:3 (Fig. 3). AN ITGB3 protein variant (or isofomi) of the invention comprises an amino acid sequence identical to SEQ ED NO:3 for those regions of SEQ ID NO: 3 that are encoded by examined portions of the ITGB3 gene (as described in the Examples below), except for having one or more variant amino acids selected from the group consisting of methionine at a position conesponding to amino acid position 14, proline at a position conesponding to amino acid position 59, arginine at a position conesponding to amino acid position 66, methionine at a position conesponding to amino acid position 445, and glutamine at a position conesponding to amino acid position 515. Thus, an ITGB3 protein fragment of the invention, also refened to herein as an ITGB3 peptide variant, is any fragment of an ITGB3 protein variant that contains one or more of the novel amino acid variations described herein. The invention specifically excludes amino acid sequences identical to those previously identified for ITGB3, including SEQ ID NO:3, and previously described fragments thereof. ITGB3 protein variants included within the invention comprise all amino acid sequences based on SEQ ED NO:3 and having any of the novel combination of amino acid variations described herein. In preferred embodiments, an ITGB3 protein variant is selected from the group consisting of A, B, C, D, E, F, and G represented in Table 9.
An ITGB3 peptide variant of the invention is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long. Such ITGB3 peptide variants may be useful as antigens to generate antibodies specific for one of the above ITGB3 isofoπns. En addition, the ITGB3 peptide variants may be useful in drag screening assays.
An ITGB3 variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing an appropriate variant ITGB3 genomic or cDNA sequence described above.
Alternatively, the ITGB3 protein variant may be isolated from a biological sample of an individual having an ITGB3 isogene which encodes the variant protein. Where the sample contains two different ITGB3 isofonns (i.e., the individual has different ITGB3 isogenes), a particular ITGB3 isofonn of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular ITGB3 isofonn but does not bind to the other ITGB3 isofonn.
The expressed or isolated ITGB3 protein or peptide variant may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isofonn of the ITGB3 protein or peptide as discussed further below. ITGB3 variant proteins and peptides can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York, New York), hi the case of immunoaffinity chromatography, antibodies specific for a particular polymoφhic variant may be used. A polymorphic variant ITGB3 gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric ITGB3 protein. The non-ITGB3 portion of the chimeric protein may be recognized by a commercially available antibody. In addition, the chimeric protein may also be engineered to contain a cleavage site located between the ITGB3 and non-ITGB3 portions so that the 1TGB3 protein may be cleaved and purified away from the non-ITGB3 portion. An additional embodiment of the invention relates to using a novel ITGB3 protein isofomi, or a fragment thereof, in any of a variety of drug screening assays. Such screening assays may be perfomied to identify agents that bind specifically to all known ITGB3 protein isoforms or to only a subset of one or more of these isoforms. The agents may be from chemical compound libraries, peptide libraries and the like. The ITGB3 protein or peptide variant may be free in solution or affixed to a solid support. In one embodiment, high throughput screening of compounds for binding to an ITGB3 variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the ITGB3 protein(s) of interest and then washed. Bound ITGB3 protein(s) are then defected using methods well-known in the art. hi another embodiment, a novel ITGB3 protein isofonn may be used in assays to measure the binding affinities of one or more candidate drugs targeting the ITGB3 protein.
In yet another embodiment, when a particular ITGB3 haplotype or group of ITGB3 haplotypes encodes an ITGB3 protein variant with an amino acid sequence distinct from that of ITGB3 protein isofoπns encoded by other ITGB3 haplotypes, then detection of that particular ITGB3 haplotype or group of ITGB3 haplotypes may be accomplished by detecting expression of the encoded ITGB3 protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan, hi another embodiment, the invention provides antibodies specific for and immunoreactive with one or more of the novel ITGB3 protein or peptide variants described herein. The antibodies may be either monoclonal or polyclonal in origin. The ITGB3 protein or peptide variant used to generate the antibodies may be from natural or recombinant sources (in vitro or in vivo) or produced by chemical synthesis or semi-synthetic synthesis using synthesis techniques known in the art. If the ITGB3 protein or peptide variant is of insufficient size to be antigenic, it may be concatenated or conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide. Examples of carrier molecules, include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Ten, Appleton and Lange, Nonvalk Connecticut, San Mateo, California).
In one embodiment, an antibody specifically immunoreactive with one of the novel protein or peptide variants described herein is administered to an individual to neutralize activity of the ITGB3 isofonn expressed by that individual. The antibody may be fonnulated as a phamiaceutical composition which includes a phannaceutically acceptable carrier.
Antibodies specific for and immunoreactive with one of the novel protein isofoπns described herein may be used to immunoprecipitate the ITGB3 protein variant from solution as well as react with ITGB3 protein isofomis on Western or immunoblots of polyaciylamide gels on membrane supports or substrates. In another prefened embodiment, the antibodies will detect ITGB3 protein isofomis in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques. In another embodiment, an antibody specifically immunoreactive with one of the novel ITGB3 protein variants described herein is used in immunoassays to detect this variant in biological samples, hi this method, an antibody of the present invention is contacted with a biological sample and the formation of a complex between the ITGB3 protein variant and the antibody is detected. As described, suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Press, New York, New York; Cunent Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York). Standard techniques known hi the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984. Methods in Immunology, W.A. Benjamin, Inc.). Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Pres, NY, NY; and Oellirich, M., 1984, J. Clin. Chem. Clin. Biochem., 22:895-904). Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Cun-ent Protocols hi Molecular Biology, supra. Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site. Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam). The antibodies or antigen binding fragments thereof may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication numbers WO 9014443 and WO 9014424, and in Huse et al., 1989, Science, 246: 1275-1281. The antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci. USA 86; 10029). Effect(s) of the polymorphisms identified herein on expression of ITGB3 may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts of the ITGB3 gene, cDNA or fragment thereof, or by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymoφhic variant of the ITGB3 gene. As used herein, "expression" includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA(s) into ITGB3 protein(s) (including effects of polymoφhisms on codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
To prepare a recombinant cell of the invention, the desired ITGB3 isogene, cDNA or coding sequence may be infroduced into the cell in a vector such that the isogene, cDNA or coding sequence remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. In a prefened embodiment, the ITGB3 isogene, cDNA or coding sequence is infroduced into a cell in such a way that it recombines with the endogenous ITGB3 gene present in the cell. Such recombination requires the occunence of a double recombination event, thereby resulting in the desired ITGB3 gene polymoiphism. Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector constmct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral fransduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner. Examples of cells into which the ITGB3 isogene, cDNA or coding sequence may be introduced include, but are not limited to, continuous culture cells, such as COS, CHO, NIH 3T3, and primary or culture cells of the relevant tissue type, i.e., they express the ITGB3 isogene, cDNA or coding sequence. Such recombinant cells can be used to compare the biological activities of the different protein variants. Recombinant nonhuman organisms, i.e., transgenic animals, expressing a variant ITGB3 gene, cDNA or coding sequence are prepared using standard procedures known in the art. Preferably, a construct comprising the variant gene, cDNA or coding sequence is introduced into a nonhuman animal or an ancestor of the animal at an embiyonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage. Transgenic animals carrying the constracts of the invention can be made by several methods known to those having skill in the art. One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes (or cDNA or coding sequence) of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053. Another method involves directly injecting a transgene into the embryo. A third method involves the use of embryonic stem cells. Examples of animals into which the ITGB3 isogene, cDNA or coding sequences may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272). Transgenic animals stably expressing a human ITGB3 isogene, cDNA or coding sequence and producing the encoded human ITGB3 protein can be used as biological models for studying diseases related to abnormal ITGB3 expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases. An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel ITGB3 isogene described herein. The pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel ITGB3 isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one of the novel ITGB3 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel ITGB3 isogene described herein. Preferably, the composition contains the active ingredient in a therapeutically effective amount. By therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel ITGB3 isogene is reduced and/or eliminated. The composition also comprises a phannaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered salhie, dextrose, and water. Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist. The phamiaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound. Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, mtrameduUary, infrathecal, infraventricular, infradermal, transdermal, subcutaneous, infraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Phamiaceutical Sciences (Maack Publishing Co., Easton, PA). For any composition, determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art. For example, the dose can be estimated initially either in cell culture assays or in animal models. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be detennined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of adminisfration, other drugs being taken by the patient, and tolerance/response to the treatment. The invention also provides a method for determining the frequency of an ITGB3 genotype, haplotype, or haplotype pah in a population. The method comprises, for each member of the population, detennining the genotype, haplotype or the haplotype pair for the novel ITGB3 polymorphic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population. The population may be e.g., a reference population, a family population, a same gender population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic freatment).
En one embodiment of the invention, ITGB3 haplotype frequencies in a trait population having a medical condition and a control population lacking the medical condition are used in a method of validating the ITGB3 protein as a candidate target for freating a medical condition predicted to be associated with ITGB3 activity. The method comprises comparing the frequency of each ITGB3 haplotype shown in Table 5 in the trait population and hi a control population and making a decision whether to pursue ITGB3 as a target. It will be understood by the skilled artisan that the composition of the control population will be dependent upon the specific study and may be a reference population or it may be an appropriately matched population with regards to age, gender, and clinical symptoms for example. If at least one ITGB3 haplotype is present at a frequency i the trait population that is different from the frequency in the confrol population at a statistically significant level, a decision to pursue the ITGB3 protein as a target should be made. However, if the frequencies of each of the ITGB3 haplotypes are not statistically significantly different between the trait and control populations, a decision not to pursue the ITGB3 protein as a target is made. The statistically significant level of difference in the frequency may be defined by the skilled artisan practicing the method using any conventional or operationally convenient means known to one skilled in the art, taking into consideration that this level should help the artisan to make a rational decision about pursuing ITGB3 protein as a target. Any ITGB3 haplotype not present in a population is considered to have a frequency of zero, hi some embodiments, each of the trait and controls populations may be comprised of different ethnogeographic origins, including but not limited to Caucasian, Hispanic Latino, African American, and Asian, while in other embodiments, the trait and reference population may be comprised of just one ethnogeographic origin.
In another embodiment of the invention, frequency data for ITGB3 haplotypes are determined h a population having a condition or disease predicted to be associated with ITGB3 activity and used in a method for screening for compounds targeting the ITGB3 protein to treat such condition or disease. In some embodiments, frequency data are determined in the population of interest for the ITGB3 haplotypes shown in Table 5. The frequency data for this population may be obtained by genotyping or haplotyping each individual in the population using one or more of the methods described above. The haplotypes for this population may be detennined directly or, alternatively, by a predictive genotype to haplotype approach as described above. In another embodiment, the frequency data for this population are obtained by accessing previously determined frequency data, which may be in written or electronic fonn. For example, the frequency data may be present in a database that is accessible by a computer. The ITGB3 isoforms conesponding to ITGB3 haplotypes occuning at a frequency greater than or equal to a desired frequency in this population are then used in screening for a compound, or compounds, that displays a desired agonist (enhancer) or antagonist (inhibitor) activity for each ITGB3 isofonn. The desired frequency for the haplotypes might be chosen to be the frequency of the most frequent haplotype, greater than some cut-off value, such as 10% in the population, or the desired frequency might be detennined by ranking the haplotypes by frequency and then choosing the frquency of the third most frequent haplotype as the cut-off value. Other methods for choosing a desired frequency are possible, such as choosing a frequency based on the desired market size for treatment with the compound. The desired level of agonist or antagonist level displayed in the screening process could be chosen to be greater than or equal to a cut-off value, such as activity levels in the top 10% of values detennined. Embodiments may employ cell-free or cell-based screening assays known in the art. The compounds used in the screening assays may be from chemical compound libraries, peptide libraries and the like. The ITGB3 isofoπns used in the screening assays may be free in solution, affixed to a solid support, or expressed in an appropriate cell line. In some embodiments, the condition or disease associated with ITGB3 activity is coronary heart disease or other disorders of cholesterol metabolism. In another aspect of the invention, frequency data for ITGB3 genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and a ITGB3 genotype, haplotype, or haplotype pah. The trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment. In one embodiment, the method involves obtaining data on the frequency of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as hi a population exhibiting the trait. Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one or more of the methods described above. The haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as described above. In another embodiment, the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic fonn. For example, the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared, hi a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If the frequency of a particular ITGB3 genotype, haplotype, or haplotype pair is different in the trait population than in the reference population to a statistically significant degree, then the trait is predicted to be associated with that ITGB3 genotype, haplotype or haplotype pair. Preferably, the ITGB3 genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the genotypes and haplotypes shown in Tables 4 and 5, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
In a prefened embodiment of the method, the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drag targeting ITGB3 or response to a therapeutic treatment for a medical condition. As used herein, "medical condition" includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders. As used herein the term "clinical response" means any or all of the following: a quantitative measure of the response, no response, and/or adverse response (i.e., side effects).
In order to deduce a conelation between clinical response to a treatment and an ITGB3 genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population". This clinical data may be obtained by analyzing the results of a clinical trial that has already been ran and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials. As used herein, the term "clinical trial" means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.
It is prefened that the individuals included in the clinical population have been graded for the existence of the medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any conelation between haplotype and freatment outcome. This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pah and disease susceptibility or severity.
The therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetennined criteria. It is contemplated that hi many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the ITGB3 gene for each individual hi the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
After both the clinical and polymoφhism data have been obtained, coιτelations between individual response and ITGB3 genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their ITGB3 genotype or haplotype (or haplotype pair) (also refened to as a polymoiphism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymoφhism group are calculated.
These results are then analyzed to detemiine if any observed variation in clinical response between polymoφhism groups is statistically significant. Statistical analysis methods which may be used are described in L.D. Fisher and G. vanBelle, "Biostatistics: A Methodology for the Health Sciences", Wiley-Interscience (New York) 1993. This analysis may also include a regression calculation of which polymoiphic sites in the ITGB3 gene give the most significant contribution to the differences hi phenotype. One regression model useful in the invention is described in WO 01/01218, entitled "Methods for Obtaining and Using Haplotype Data". A second method for finding correlations between ITGB3 haplotype content and clinical responses uses predictive models based on en-or-minimizing optimization algorithms. One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry" in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997). Simulated annealing (Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K. Knight, "Artificial Intelligence", 2nd Edition (McGraw-Hill, New York, 1991, Ch. 18), standard gradient descent methods (Press et al., supra, Ch. 10), or other global or local optimization approaches (see discussion in Judson, supra) could also be used. Preferably, the correlation is found using a genetic algorithm approach as described in WO 01/01218. Correlations may also be analyzed using analysis of variation (ANOVA) techniques to detemiine how much of the variation in the clinical data is explained by different subsets of the polymoiphic sites in the ITGB3 gene. As described in WO 01/01218, ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10). From the analyses described above, a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of ITGB3 genotype or haplotype content. Preferably, the model is validated in one or more follow-up clinical trials designed to test the model.
The identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the ITGB3 gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the freatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug. The diagnostic method will detect the presence in an individual of the genotype, haplotype or haplotype pair that is associated with the clinical response and may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymoφhic sites in the ITGB3 gene), a serological test, or a physical exam measurement. The only requirement is that there be a good conelation between the diagnostic test results and the underlying ITGB3 genotype or haplotype that is in turn correlated with the clinical response. In a preferred embodiment, this diagnostic method uses the predictive haplotyping method described above.
Another embodiment of the invention comprises a method for reducing the potential for bias in a clinical trial of a candidate drug for treating a disease or condition predicted to be associated with ITGB3 activity. Haplotyping one or both copies of the ITGB3 gene in those individuals participating in the trial will allow the pharmaceutical scientist conducting the clinical trial to assign each individual fiom the tiial one of the ITGB3 haplotypes or haplotype pairs shown in Tables 5 and 4, respectively, or a ITGB3 sub-haplotype or sub-haplotype pair thereof. In one embodiment, the haplotypes may be determined directly, or alternatively, by a predictive genotype to haplotype approach as decribed above. In another embodiment, this can be accomplished by haplotyping individuals participating in a clinical trial by identifying, for example, in one or both copies of the individual's ITGB3 gene, the phased sequence of nucleotides present at each of PS 1 -PS44. Determining the ITGB3 haplotype or haplotype pair present in individuals participating in the clinical frial enables the pharmaceutical scientist to assign individuals possessing a specific haplotype or haplotype pair evenly to treatment and control groups. Typical clinical trials conducted may include, but are not limited to, Phase I, II, and III clinical trials. If the trial is measuring response to a drug for treating a disease or condition predicted to be associated with ITGB3 activity, each individual in the trial may produce a specific response to the candidate drug based upon the individual's haplotype or haplotype pair. To confrol for these differing drag responses in the trial and to reduce the potential for bias in the results that could be introduced by a larger frequency of a ITGB3 haplotype or haplotype pair in any particular freatment or control group due to random group assignment, each treatment and control group are assigned an even distribution (or equal numbers) of individuals having a particular ITGB3 haplotype or haplotype pair. To practice this method of the invention to reduce the potential for bias in a clinical trial, the phamiaceutical scientist requires no a priori knowledge of any effect a ITGB3 haplotype or haplotype pair may have on the results of the trial. Diseases or conditions predicted to be associated with ITGB3 activity include, e.g., Glanzman's thrombocytopenia and cardiovascular disease. It is also contemplated that the above described methods and compositions of the invention may be utilized in combination with identifying genotype(s) and/or haplotype(s) for other genomic regions.
Prefened embodiments of the invention are described hi the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herem. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and sphit of the invention being indicated by the claims that follow the examples. EXAMPLES
The Examples herein are meant to exemplify the various aspects of cany ing out the invention and are not intended to limit the scope of the invention in any way. The Examples do not include detailed descriptions for conventional methods employed, such as in the performance of genomic DNA isolation, PCR and sequencing procedures. Such methods are well-known to those skilled in the art and are described in numerous publications, for example, Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989).
Example 1 This example illustrates the clinical and biochemical characterization of 679 patients in the patient cohort.
A multicenter, 17-week, (16 weeks controlled), open-label, clinical discovery trial was designed to assess the relationship between genetic haplotype markers and treatment response associated with 4 different commercially available medications, all of which act as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (cerivastatin sodium [Baycol™], aton'astatin calcium [Lipitor®], simvastatin [Zocor®], and pravastatin sodium [Pravachol®]) in adult subjects with primaiy hypercholesterolemia. Study medications were packaged by their respective manufacturers and dispensed in a non-blinded fashion by a commercial phaπnacist. The cerivastatin sodium aim of the study was discontinued at the time of the withdrawal of the drug from the market by the manufacturer and therefore data from the partially completed arm are excluded from this analysis.
Prior to randomization, all subjects undenvent a screening and baseline period (up to 10 days). Then subjects were randomly assigned to the recommended starting dose, as stated in the package insert, of 1 of the 4 study medications. Following the initial 8 weeks of treatment, subjects proceeded to the highest allowed dose, as stated in the package insert, for an 8-week treatment period. As both periods incoiporate a fixed-dose design, dosing adjustments other than the Week 8 increase were not peimitted in this study. Thus, the total duration of therapy was maximally 16 weeks/subject from the point of randomization (8 weeks at the recommended starting dose; plus 8 weeks at the highest allowed dose as stated in the package insert).
Male or female outpatients aged 18 to 75 years with a diagnosis of type Ila or lib hypercholesterolemia who have been on the American Heart Association (AHA) Step I or Step II diet for at least 6 weeks prior to the onset of screening were eligible to participate. Subjects were either treatment-naϊve or previously treated for hypercholesterolemia with any approved medications. Previously treated subjects must have discontinued antihyperlipidemic medication 4 weeks prior to screening (8 weeks prior to screening if clofibrate [Atromid-S®] was in use) to be eligible. Subject inclusion criteria were based upon medical history assessments and laboratory determinations of cholesterol levels as described by the National Cholesterol Education Program (NCEP)-recommended goal for LDL-cholesterol (> 160 mg/dL for subjects with 0 to 1 coronary heart disease [CHD] risk factor, > 130 mg/dL for those with 2 or more CHD risk factors, or > 100 mg/dL for those with documented CHD or peripheral vascular disease) and had triglyceride levels < 400 mg/dL prior to randomization. Eligible subjects had an LDL-cholesterol level < 240 mg/dL at screening and baseline. Subjects had to demonstrate dietary compliance with the AHA Step I or Step II diet as measured by a food diary at baseline to be eligible for randomization. The entire patient cohort comprised 679 patients. Subjects were randomly assigned to 1 of 4 treatment groups: 0.4 mg/day cerivastatin sodium, 10 mg/day atorvastatin calcium, 20 mg/day simvastatin, or 10 mg/day pravastatin sodium at baseline. All medication was taken once daily hi the evening.
At the Week 8 visit, all subjects proceeded to the highest allowed dose, as stated hi the package insert, of their assigned medication. The doses for the treatment groups were as follows: 0.8 mg/day cerivastatin sodium, 80 mgday atoiYastatin calcium, 80 mg/day simvastatin, and 40 mg/day pravastatin sodium. All medication was taken once daily in the evening.
The primary phenotypic endpoint used in the association of treatment response to genetic variability was the percent change from baseline in LDL-cholesterol values after 8 weeks and after 16 weeks of treatment, separately. The final Week 8 value was defined as the mean of the last 2 measurements (Weeks 6 and 8) during the first 8 weeks (low dose) of therapy. The final Week 16 value was defined as the mean of the last 2 measurements (Weeks 14 and 16) during the final 8 weeks (high dose) of therapy. Baseline was defined as the mean of the measurements taken at screening and baseline. The patient cohorts for the diree completed statin arms were characterized with respect to statin taken in treatment as shown below.
Demographics, baseline characteristics, and lipid changes for the low-dose compliant population.
_,. . . AtoiYastatin Simvastatin Pravastatin Pooled
Characteristic (n=155) (n=168) (n=153) (n=4?6)
Male 65(41.9%) 91(54.2%) 64(41.8%) 220 (46.2%)
Caucasian 135(87.1%) 146(86.9%) 126(82.4%) 407(85.5%)
Smoker 27(17.4%) 34(20.2%) 31(20.3%) 92(19.3%)
Drinker 95(61.3%) 104(61.9%) 84(54.9%) 283(59.5%)
Age(yr)* 56.8±9.7' 56.0 ± 10.4 57.0±10.4 56.6±10.2
Height (cm)* 168 ±11 169 ±10 169 ±10 169 ±10
Weight (kg)* 81.8 ±16.6 84.1 ±17.3 84.1 ± 19.0 83.3 ±17.6
BMI(kg/m2)* 28.8 ±4.5 29.3 ±5.1 29.3 ± 6.0 29.1 ±5.2
LDL-C* BL (mg/dL) 172 ±27 175 ±25 173 ± 25 173 ±26
8-week%Δ -39.3 ±10.0 -35.8 ±11.0 -21.3 ±11.3 -32.3 ±13.2
16-week %Δf -52.2 ±11.9 -45. l± 11.4 -28.8 ±11.9 -42.0 ±15.2
HDL-C* BL (mg/dL) 50.6 ±13.7 47.3 ±11.2 48.9 ±12.4 48.9 ±12.5
8-week % Δ -0.3 ± 10.5 2.1 ±9.7 0.5 ± 8.6 0.9 ± 9.7
16-week Δt -3.3 ±10.4 1.6± 10.9 1.2±9.1 -0.1 ± 10.4
TGJ BL (mg/dL) 164(70,361) 173(60,384) 166(54,370) 167(54,384)
8-week %Δ -18 (-57, 52) -12 (-62, 234) -6 (-53, 183) -12 (-62, 234)
16-week % Δf -32 (-74, 45) -26 (-60, 70) -10 (-60, 300) -22 (-74, 300)
*Mean ± Standard Deviation shown; BL = baseline. f 16-week percent changes are based on the high-dose compliant population: pooled n=409. ^Median (Min, Max).
Statin
Ethnicity Lipitor® Zocor® Pravacol
Afr Am 4 (2%) 7 (4%) 12 (8%)
Am hid - 1 (0.6%) -
Asian 5 (3%) 3 (2%) 1 (0.6%)
Cauc 133 (79%) 135 (75%) 123 (77%)
Hisp-Lat 10 (6%) 9 (5%) 9 (6%)
Other 1 (0.6%) 2 (1%) 2 (1%)
Not Assigned 15 (9%) 24 (13%) 12 (8%)
Missing - 1 (0.6%) -
Example 2
This example illustrates examination of various regions of the ITGB3 gene in the 854 individuals of the experimental population, including the Index Repository and the patient cohort described in Example 1, for polymoiphic sites. The human individuals in the Index Repository included a reference population of 82 unrelated individuals, of which 79 were self-identified as belonging to one of four major population groups: Caucasian (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic Latino (18 individuals). In addition, the Index Repositoiy contains three unrelated indigenous American Indians (one from each of North, Cenfral and South America), one three- generation Caucasian family (from the CEPH LJtah cohort) and one two-generation African-American family AMPLIFICATION OF TARGET REGIONS
The following target regions of the ITGB3 gene were amplified using 'tailed' PCR primers, each of which includes a universal sequence fonning a noncomplemeiitary 'tail' attached to the 5' end of each unique sequence in the PCR primer pairs. The universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 ' (SEQ ED NO:224) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5'-AGGAAACAGCTATGACCAT-3r (SEQ ED NO:225). The nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and conespond to positions in SEQ ED NO:l (Figure 1).
PCR Primer Pairs
Fragment No. Forward Primer Reverse Primer PCR Product Fragment 1 1000-1022 complement of 1521-1499 522 nt Fragment 2 1039-1060 complement of 1515-1496 477 nt Fragment 3 1346-1367 complement of 1778-1758 433 nt Fragment 4 1590-1613 complement of 2235-2216 646 nt Fragment 5 4256-4278 complement of 4716-4693 461 nt Fragment 6 13179-13201 complement of 13723-13703 545 nt Fragment 7 14234-14256 complement of 14662-14642 429 nt Fragment 8 14495-14516 complement of 14858-14834 364 nt Fragment 9 16126-16150 complement of 16619-16597 494 nt Fragment 10 16930-16950 complement of 17414- 17392 485 nt Fragment 11 19241-19263 complement of 19644- 19622 404 nt Fragment 12 19748-19770 complement of 20177-20155 430 nt
Fragment 13 20537-20556 complement of 21009-20987 473 nt
Fragment 14 21731-21753 complement of 22207-22186 477 nt
Fragment 15 22002-22024 complement of 22412-22391 411 nt
Fragment 16 24385-24406 complement of 24930-24909 546 nt
Fragment 17 25559-25581 complement of 26029-26005 471 nt
Fragment 18 27822-27843 complement of 28255-28233 434 nt
Fragment 19 30265-30289 complement of 30754-30731 490 nt
Fragment 20 31300-31322 complement of 31718-31695 419 nt
These primer pairs were used in PCR reactions containing genomic DNA isolated from immortalized cell lines for each member of the Index Repositoiy. The PCR reactions were canied out under the following conditions:
Reaction volume = 10 μl
10 x Advantage 2 Polymerase reaction buffer (Clontech) = 1 μl
100 ng of human genomic DNA = 1 μl lO inM dNTP = 0.4 μl
Advantage 2 Polymerase enzyme mix (Clontech) = 0.2 μl
Forward Primer (10 μM) = 0.4 μl
Reverse Primer (10 μM) = 0.4 μl
Water = 6.6μl
Amplification profile: 97°C - 2 min. 1 cycle
97°C - 15 sec. 70°C - 45 sec. 10 cycles 72°C - 45 sec.
97°C - 15 sec. 64°C - 45 sec. 35 cycles 72°C - 45 sec.
SEQUENCING OF PCR PRODUCTS The PCR products were purified using a Whatman/Polyfiltronics 100 μl 384 well unifilter plate essentially according to the manufacturers protocol. The purified DNA was eluted in 50 μl of distilled water. Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol. The purified PCR products were sequenced in both directions using the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
ANALYSIS OF SEQUENCES FOR POLYMORPHIC SITES
Sequence infonnation was analyzed for the presence of polymoφhisms using the Polyphred program (Nickerson et al., Nucleic Acids Res. 14:2745-2751, 1997). The presence of a polymoφhism was generally confumed on both strands. The polymoφhisms and then locations in the ITGB3 reference genomic sequence Figure 1 (SEQ ED NO:l) are listed hi Table 3, presented following the examples. Example 3
This example illustrates analysis of the ITGB3 polymoφhisms identified in the Index Repositoiy and the patient cohort for human genotypes and haplotypes. The different genotypes containing these polymoφhisms that were observed in unrelated members of the reference population are shown in Table 4 below, following the examples, with the haplotype pair indicating the combination of haplotypes detemiined for the individual using the haplotype derivation protocol described below. In Table 4, homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. The haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented algorithm for assigning haplotypes to unrelated individuals in a population sample, as described in WO 01/80156.
By following this protocol, it was detemiined that the Index Repositoiy examined herein and, by extension, the general population contains the 98 human ITGB3 haplotypes shown in Table 5 below, wherein each of the ITGB3 haplotypes comprises a 5 ' - 3 ' ordered sequence of 44 polymoiphisms whose positions in SEQ ED NO:l and alleles are set forth in Table 5. h Table 5, the column labeled "Region Examined" provides the nucleotide positions in SEQ ID NO: 1 conesponding to sequenced regions of the gene. The columns labeled "PS No." and "PS Position" provide the polymoφhic site number designation (see Table 3) and the coiresponding nucleotide position of this polymoiphic site within SEQ ED NO: 1 or SEQ ID NO:226. The columns beneath the "Haplotype Number" heading are labeled to provide a unique number designation for each ITGB3 haplotype.
Table 6 below, following the Examples, shows the number of chromosomes characterized by a given ITGB3 haplotype for all unrelated individuals in the Index Repositoiy for which haplotype data was obtained. The number of these unrelated individuals who have a given ITGB3 haplotype pair is shown in Table 7 below, following the Examples. In Tables 6 and 7, the "Total" column shows this frequency data for all of these unrelated individuals, while the other columns show the frequency data for these unrelated individuals categorized according to their self-identified ethnogeographic origin. Abbreviations used in Tables 6 and 7 are AF = African Descent, AS = Asian, CA = Caucasian, HL = Hispanic-Latino, and AM = Native American. The size and composition of the Index Repositoiy were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population. For example, as described in Table 1 above, this repository contains approximately equal sample sizes of African-descent, Asian-American, European-American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background. The number of unrelated mdividuals in the Index Repositoiy provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population. Similarly, a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group. In addition, the size and composition of the Index Repositoiy means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the ITGB3 gene are likely to be similar to the relative frequencies of these ITGB3 haplotypes and haplotype pairs hi the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity obseiYed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.
Each ITGB3 haplotype shown in Table 5 defines an ITGB3 isogene. The ITGB3 isogene defined by a given ITGB3 haplotype comprises the examined regions of Figure 1 (SEQ ED NO: 1) indicated in Table 5, with the corresponding ordered sequence of nucleotides occurring at each polymorphic site within the ITGB3 gene shown in Table 5 for that defining haplotype.
Each ITGB3 isogene defined by one of the haplotypes shown in Table 5 will further correspond to a particular ITGB3 coding sequence variant. Each of these ITGB3 coding sequence variants comprises the regions of Figure 2 (SEQ ED NO:2) examined and is defined by the 5 ' - 3 ' ordered sequence of nucleotides occurring at each polymorphic site within the coding sequence of the ITGB3 gene, as shown in Table 8, following the Examples. In Table 8, the column labeled 'Region Examined' provides the nucleotide positions in Figure 2 (SEQ ID NO:2) corresponding to sequenced regions of the gene; the columns labeled 'PS No.' and 'PS Position' provide the polymoiphic site number designation (see Table 3) and the corresponding nucleotide position of this polymoiphic site within Figure 2 (SEQ ID NO:2). The columns beneath the 'Coding Sequence Number' heading are numbered to conespond to the haplotype number defining the ITGB3 isogene from which the coding sequence variant is derived. ITGB3 coding sequence variants that differ from the reference ITGB3 coding sequence are denoted in Table 8 by a letter (A, B. etc) identifying each unique novel coding sequence. The same letter at the top of more than one column denotes that a given novel coding sequence is present in multiple novel ITGB3 isogenes.
Similarly, each ITGB3 coding sequence represented in Table 8 encodes an ITGB3 protein variant. Each of the ITGB3 protein variants encoded by the 216 ITGB3 isogenes described herein comprises the regions of Figure 3 (SEQ ID NO:3) examined by sequencing and is defined by the N- tenυinus to C-tenninus sequence of amino acids resulting from the observed polymoφhisms at the polymoφhic sites within the coding sequence of the ITGB3 gene, as presented in Table 9, following the Examples. In Table 9, the column labeled 'Region Examined' provides amino acid positions in Figure 3 (SEQ ID NO:3) conesponding to sequenced regions of the gene. The columns labeled PS No. and PS Position provide the polymoφhic site number designation (see Table 3) and the corresponding amino acid position within Figure 3 (SEQ ED NO:3) affected by this polymoiphic site in the ITGB3 gene. The columns below the 'Protein Variants' heading are numbered to conespond to the haplotype number defining the ITGB3 isogene from which the protein variant is derived. ITGB3 protein variant sequences that differ from the reference ITGB3 protein sequence are denoted hi Table 9 by a letter (A, B, etc) identifying each unique protein variant sequence. The same letter at the top of more than one column denotes that the novel protein variant encoded by those particular ITGB3 isogenes are identical.
EXAMPLE 4 This example illustrates analysis of the ITGB3 haplotypes in Table 4 for association with individuals' response to Lipitor®.
All 679 randomized patients in the STRENGTH study were used to build HAPs. However, for analyses assessing the associations between genetic markers and difference in percent change in HDLC as a function of Lipitor® dose, the cohort was limited to patients taking Lipitor®. For low-dose analyses, patients who were not compliant in the taking of their statins (i.e., who had at least two consecutive visits with an answer "No" to the question of whether they had been between 80% and 120% compliant since the previous visit) during the low-dose period of the study were excluded; for the high-dose analyses, the patients who were not compliant during the low-dose period of the study were excluded as well as ti ose who were not compliant during the high-dose period by the same definition (i.e., had at least two consecutive visits with an answer "No" to the question of whether they had been between 80% and 120% compliant since the previous visit); patients with incomplete covariate information were excluded.
The percent change in HDL cholesterol and the difference in percent change in HDLC due to the increased Lipitor® dose during weeks 8-16 was detennined from the clinical data using the following foπnulas:
Low-dose percent change in HDLC = (Low-dose follow-up HDLC - Baseline HDLC)/(Baseline HDLC)* 100.
High-dose percent change in HDLC = (High-dose follow-up HDLC - Baseline HDLC)/(Baseline HDLC)* 100. Difference in percent change in HDLC between high dose and low dose (percent change in HDLC due to the increased Lipitor® dose during weeks 8-16) = High-dose percent change in HDLC
- Low-dose percent change in HDLC.
The parameters in these foπnulas were defined as follows: Baseline HDLC - the average of the screening and baseline visits' HDLC values, unless the screening and baseline LDL values were more than 15% apart from one another, in which case a second baseline sample of all lipids was collected, and baseline HDLC was the average of the two baseline HDLC values.
Low-dose follow-up HDLC - the average of the 6-week and 8-week values if both were available; otherwise the last single value from among the 4-week, 6-week, 8-week and early termination (if applicable) values was used.
High-dose follow-up HDLC - the average of the 14-week and 16-week values if both were available; otherwise the last single value from among the 12-week, 14-week, 16-week and early termination (if applicable) values was used.
Genetic variation in the clinical cohort was correlated with the above clinical endpoint to detemiine any statistically significant genetic associations present. In this context, a haplotype marker is defined as a prescription for the genotype at all the polymoφhic sites h a single copy of a particular gene. The prescription allows for a site to be either inelevant ('*'), or requires the genotype at the site to be one of the possible alleles (e.g., A or G). A particular marker can be expressed as a string of characters, one for each site. If a gene A has 5 known polymoφhic sites, for example, we can define a particular marker to be A:*A*CG. In order to have a copy of the example marker A:*A*CG, a person has to have a copy of the gene with A, C and G at polymoφhic sites 2, 4 and 5, respectively.
Only dominant and recessive markers were considered in this analysis. The number of haplotypes that a person has that match the marker is counted (0, 1, or 2 copies). These three-copy number groups are reduced to two-group markers by joining two of the copy number groups into one, using the conventional genetic models for recessive and dominant traits. A given population of patients with known genotypes at the 5 sites of the example *A*CG can then be divided into two groups, those that have the marker at the designated copy number (In-group), and those that don't have that copy number of the marker (Out-group). Recessive markers are those in which a copy number of 2 is designated as the hi group vs. (0 and 1) copies (Out group) and dominant markers are those in which copy numbers (1 and 2) are designated as the In group vs 0 copy (Out group). The 'In' group for a dominant marker shown in Table 10A below therefore represents members of the cohort who had 1 or 2 copies of the indicated marker while the 'Out' group represents members of the cohort with no copy of the indicated marker. Similarly, for a recessive marker in Table 10B below, the 'In' group represents members of the cohort with 2 copies of the marker while members of the 'Out' group possess 0 or 1 copy of the marker. A marker is associated with a phenotypic observation when there is a statistically significant difference in the observation between the different marker groups. All possible markers of the ITGB3 gene that satisfy' various conditions were enumerated. Polymoφhisms that had a frequency of less than 1% in the population were not considered. Only markers with a maximum of five polymorphisms were consideied. To account for covariates, the conelation of a variable with all its covariates was calculated and eliminated by substituting the residual of the variable with respect to the covariates in the marker association calculation. The association test was performed as a t-test of the phenotype between the in- group and the out-group of the tested marker. This t-test was performed for each enumerated marker and a ranked list of the top 100 markers is kept, with their t-test. The significance of any finding was quantified by perfonning the same test many times on randomly pemiuted data and obtaining the probability of the best marker p-value in the resulting distribution. This adjusted p-value was then used to rank the gene-marker- phenotype combinations by their likelihood of being associated.
In Tables 10A and B, the column labeled Sites indicates the number of polymoφhic sites present in the marker. The Marker column represents the sites by the polymoiphic site number (as per Table 3) and the nucleotide present at that site. For example, the marker IC, 42C indicates that the marker is composed of a cytosine at PSI and a cytosine at PS42; the identities of the nucleotides present at the other ITGB3 polymoφhic sites presented herein are irrelevant to the statistical significance of the association. The model column indicates whether the In group is defined by havmg 1 or 2 copies of the Marker (Dominant) or 2 copies of the Marker (Recessive). The In Mean and Out Mean columns present for the hi and Out groups, respectively, the mean difference in per cent change i HDLC relative to baseline after the 16 week high dose regimen vs. after the 8 week low dose regimen . hi Count and Out Count present the number of members of the Lipitor® cohort belonging to the hi and Out groups, respectively. The column labeled Delta presents the difference between the In mean and Out mean to illustrate the magnitude in the difference in response of members of the two groups. Raw P presents the p value calculated for the marker-pheiiotype association using the t-test, while Adjusted P presents the p value for the association detennined by the permutation test described above.
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
For each of the ITGB3 haplotypes shown m Table 10A analyzed using a dominant genetic model, the In group with 1 or 2 copies of that haplotype showed a mean difference in per cent change in HDLC from baseline aftei the week 16 determination at high dose of atorvastatin relative to per cent change in HDLC from baseline after the week 8 determination at low dose of atorvastatin that was worse, fiom a clinical perspective, than the mean difference in per cent change in HDLC from baseline ,at high dose lelative to low dose of atorvastatin hkel} to be observed m an individual having no copy of that haplotype. Conversely, when an ITGB3 haplotype shown in Table 10A was not present in an individual (0 copy; "Out group"), the individual was likely to experience a better adjusted mean per cent change in HDLC at high dose relative to low dose of atorvastatin than an individual having the haplotype. The mean difference observed for the hi group having at least one copy of the haplotypes in Table 10A ranged from -4.59?/o to -6.43%, while the mean difference observed for the Out group having zero copy of these haplotypes ranged from -0.115 to +1.53%. Hence, the HDLC levels of patients having at least one copy (i.e., the "In Group") of an ITGB3 haplotype shown in Table 10A were found to be more sensitive to atoiYastatin dose than patients having zero copy of these haplotypes (the Out group for these haplotypes). These patients in the hi group for the ITGB3 haplotypes shown in Table 10A responded to treatment with the high dose of atoiYastatin with a reduction in their HDLC relative to the effect on HDLC they experienced in response to treatment with the low dose of atorvastatin. HDLC levels for patients belonging to the Out group were found to be little changed or to increase upon freatment with the highest dose of atoiYastatin, compared to freatment with the lowest dose.
For an ITGB3 haplotype shown in Table 10B analyzed using a recessive genetic model, the Out group , comprising those with only 0 or 1 copies of that haplotype, showed a mean difference in per cent change in HDLC from baseline after the week 16 determination at high dose of atoiYastatin relative to per cent change in HDLC from baseline after the week 8 determination at low dose of aton'astatin that was worse, from a clinical perspective, than the mean difference in per cent change in HDLC from baseline at high dose relative to low dose of atorvastatin likely to be observed in individuals having two copies of that haplotype (In group). Conversely, the group of patients (In Group) with two copies of an ITGB3 haplotype shown in Table 10B experienced a better adjusted mean per cent change in HDLC at high dose relative to low dose of atorvastatin than the group of patients having 0 or 1 copy of the marker (Out group). The mean difference obsen'ed for the In group for the identified haplotypes in Table 10B ranged from +0.26% to +1.26%, while the mean difference observed for the Out group for these haplotypes ranged from —4.75% to -5.40%. Hence, the HDLC levels of patients having two copies of any ITGB3 haplotype shown in Table 10B were found to be more likely to show at least a small increase in change in HDLC with increased atoiYastatin dose than patients having only zero or one copy of any of these ITGB3 haplotypes. These patients in the In group responded to treatment with the high dose of atorvastatin with an increase in their HDLC relative to the effect on HDLC they experienced in response to treatment with the low dose of atorvastatin. HDLC levels for patients belonging to the Out group were found to decrease upon treatment with the highest dose of atorvastatin, compared to the HDLC level detennined after freatment with the lowest dose.
Thus any of these genetic markers or haplotypes in Tables 10A and 10 B are useful for identifying patients who may respond to higher doses of atorvastatin with a reduction in their HDLC levels rather than a small increase. Consequently, the haplotypes in Table 5, comprising all 44 polymoφhic sites identified by Applicants, that comprise any of these genetic markers are likewise useful for identifying these patients.
A reduced set of 13 polymoφhisms resulting in 91% of the haplotype diversity provided by the set of 44 polymoφhisms was also analyzed for correlations in HDLC response to dose of atorvastatin. All combinations of haplotypes including up to four polymoiphism were considered hi this analysis. Each unique haplotype with a frequency of >1% was tested for association with the clinical response. Each mdividual in the analysis cohort was classified as having 0, 1, or 2 copies of the haplotype. Only dominant (i.e., 1 or 2 copies of the marker vs. 0 copy of the marker) and recessive (i.e., 0 or 1 copy of the marker vs 2 copies) models were considered in this analysis. Analysis of covariance (ANCOVA) models with one degree of freedom for marker were used. The genetic analysis utilized as covariates age, gender, statin assignment (in the all statins combined model only), ethnicity, baseline level of LDLC, alcohol consumption, smoking status and body mass index (BME). Additional associations discovered by Applicants are tabulated below in Tables 11 A and 1 IB. In these tables, the column labeled LS Mean (LC, UC) for each of the two copy number groups (i.e., 0 copy vs. 1 or 2 copies for the dominant model; 0 or 1 copy vs. 2 copies for recessive markers) presents the mean difference in percent change in HDLC between high dose and low dose with lower and upper 95% confidence limits for the mean provided in the parentheses for that number of copies. The Count columns present the number of members of the statin treatment cohort belonging to each of these copy number groups. Marker. P value presents the p value calculated for the marker-phenotype association using the recessive or dominant ANCOVA model, as appropriate.
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
S2
Figure imgf000084_0001
Figure imgf000084_0002
In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained.
For any and all embodiments of the present invention discussed herein, in which a feature is described in tenns of a Markush group or other giouping of alternatives, the iventors contemplate that such feature may also be described by, and that their invention specifically includes, any individual member or subgroup of members of such Markush group or other group.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be inteφieted as illustrative and not in a limiting sense.
All references cited in this specification, including patents and patent applications, are hereby incoφorated in then- entirety by reference. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants resen'e the right to challenge the accuracy and pertinency of the cited references.
TABLES 3-9
Table 3. Polymoφhic Sites Identified in the ITGB3 Gene
Polymoφhic Nucleotide Reference Variant CDS Variant AA
Site Number Poly Id(a) Position Allele Allele Position Variant
PSI 24417180 1118 C T
PS2 25338367 1202 A G
PS3 9118201 1773 G C
PS4 30508893 1875 C T
PS5 30508895 1911 G c
PS6 30508897 1957 G A 40 V14M
PS7 30508899 1974 G T 57 A19A
PSS 30508901 1975 C T 58 L20L
PS9 9118475 2048 C G
PS10 9118567 2087 G A
PS11 9118659 2117 G A
PS12 9118750 2157 C T
PS13 27891863 13273 G A
PS14 9120409 13334 C T
PS15 9120313 13384 T C 176 L59P
PS16 24693516 13405 T G 197 L66R
PS17 9120217 13550 T C 342 11141
PS18 24693513 13690 G A
PS19 9106887 16200 G T
PS20 9121666 17194 T C 882 P294P
PS21 9121571 17273 A G
PS22 9122668 19357 A G
PS23 9122572 19610 G C
PS24 9124881 19877 T C
PS25 27893045 20034 c T
PS26 9124691 20035 G A
PS27 24408349 20037 G A
PS28 9124595 20047 C ,
PS29 27893041 20105 C T
PS30 9114069 20615 G A
PS31 9113879 20704 A C 1143 V381V
PS32 9113783 20889 T G
PS33 25333428 21944 G A 1333 V445M
PS34 9386445 22144 A G 1533 E511E
PS35 9388001 22155 G A 1544 R515QorQ(PS36=A)
PS36 9386541 22156 G A 1545 R515RorQ(PS35=A)
PS37 9670067 25705 T C
PS38 9669970 25921 c T
PS39 9112541 27882 T c
PS40 9112348 28127 G A
PS41 9111389 30516 C T 2208 A736A
PS42 9111713 30618 C T
PS43 9111809 30662 T G
PS44 9112001 30729 c G
(a) Polyld is a unique identifier assigned to each PS by Genaissance Phaπnaceuticals, Inc.
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0002
Figure imgf000090_0001
Table 5(Part 1). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Examined(a) No.(b) Position(c) 1 2 3 4 5 6 7 8 9 10
1000-2235 1 11 18/30 C C C C C C C C C c
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 C c C c C C C C C C
1000-2235 4 1875/390 c c C c C C C C c C
1000-2235 5 1911/510 c G G G G G G G G G
1000-2235 6 1957/630 G A G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G T T
1000-2235 8 1975/870 C C C C C C C C T T
1000-2235 9 2048/990 G G C c G G G G G G
1000-2235 10 2087/11 10 G G G G G G G G G G
1000-2235 1 1 2117/1230 A A A G A A A A A A
1000-2235 12 2157/1350 C C C T C C C C C C
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C c C c C C C C C
13179-13723 15 13384/1710 T T T C T T T T T T
13179-13723 16 13405/1830 T T T T T T T T T T
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G T T G G
16930-17414 20 17194/2310 T T T T T T T T T T
16930-17414 21 17273/2430 A A A A A A A A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 G G G C G G G G G G
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 C C C c c c C C C C
19748-20177 26 20035/3030 G G G G G G G G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C C C C C T T C C
19748-20177 29 20105/3390 C C C C C C C c C C
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 A A A C A A C C A A
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 A A A G A A G G A A
21731-22412 35 22155/41 10 G G G G G G G G G G
21731-22412 36 22156/4230 G G G A G G A A G G
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T T T T T T T T
25559-26029 38 25921/4470 C c c c c c c c C C
27822-28255 39 27882/4590 c c T c c T c T c T
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 C c c C C C C C C c
30265-30754 42 3061S/4950 T T c T T C C C T c
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 c c c C c c C c G G
31300-31718 - - - - - - - - - - - -
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the 1 st position number refening to SEQ ID NO: 1 and the 2nd position number refening to SEQ ID NO : 226, a modified version of SEQ ID NO:l that comprises the context sequence of each polymoφhic site, PS1-PS44, to facilitate electronic searching of the haplotypes; (d) Alleles for ITGB3 haplotypes are presented 5' to 3 ' in each column. Table 5(Part 2). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Examined(a) No.(b) Position(c) 11 12 13 14 15 16 17 18 19 20
1000-2235 1 11 18/30 C C C C C C c C C c
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 G G G G G G G G G G
1000-2235 4 1875/390 C C C C C C C C C C
1000-2235 5 1911/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C C
1000-2235 9 2048/990 C C C C C C C C C C
1000-2235 10 2087/11 10 A A A A A A A G G G
1000-2235 1 1 21 17/1230 G G G G G G G G G G
1000-2235 12 2157/1350 C C C C C C C C C C
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G A A G
13179-13723 14 13334/1590 C C C C C C C C C C
13179-13723 15 13384/1710 C T T T T T T C T C
13179-13723 16 13405/1830 T T T T T T T T T G
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G _ G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G T G G G
16930-17414 20 17194/2310 T C C C T T T T T T
16930-17414 21 17273/2430 A G G G A A A A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 C G G G G G G C G C
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 c C c c C c C C C C
19748-20177 26 20035/3030 G A A A G G G G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C C C C C T C C C
19748-20177 29 20105/3390 c C C T C c C C C C
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 C C C C A A C C A C
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 G A A A A A G G A G
21731-22412 35 22155/41 10 G G G G G G G G G G
21731-22412 36 22156/4230 A G G G G G A A G A
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T C C C T T T T T T
25559-26029 38 25921/4470 c T T C c c c C C c
27822-28255 39 27882/4590 T c c c c c c T c T
27822-28255 40 28127/4710 G A G G G G G G G G
30265-30754 41 30516/4830 C C c C c c C C C c
30265-30754 42 30618/4950 C c c C c T c C T c
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 c c c C c c c C c c
31300-31718 - - - - - - - - - - - -
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the Imposition number refening to SEQ ID NO: 1 and the 2nd position number refening to SEQ ID NO:226, a modified \ersion of SEQ ID NO: l that comprises the context sequence of each polymoφhic site, PS 1-PS44, to facilitate electronic searching of the haplotypes; (d) Alleles for ITGB3 haplotypes are presented 5' to 3 ' in each column. Table 5(Part 3). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Exaιnined(a) No.(b) Position(c) 21 22 23 24 25 26 27 28 29 30
1000-2235 1 1118/30 C C C C C C C C C c
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 G G G G G G G G G G
1000-2235 4 1875/390 C C C C C C C C C C
1000-2235 5 191 1/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C C
1000-2235 9 2048/990 C C C C C C C C C C
1000-2235 10 2087/1 110 G G G G G G G G G G
1000-2235 1 1 2117/1230 G G G G G G G G G G
1000-2235 12 2157/1350 C C C C C C C C C C
4256-4716 - - - - - - - - -. - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C C C C C C C C C
13179-13723 15 13384/1710 C C C C T T T T T T
13179-13723 16 13405/1830 T T T T T T T T T T
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G G G G G
16930-17414 20 17194/2310 T T T T C C C C C C
16930-17414 21 17273/2430 A A A A A A G G G G
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 C C C C G G G G G G
19748-20177 24 19877/2790 T T T T T T C C T T
19748-20177 25 20034/2910 c c c T C c C C C c
19748-20177 26 20035/3030 G G G G G G G G A A
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C C C C C C C C C
19748-20177 29 20105/3390 C C c c C c C C C T
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 A C C C C C C C C C
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 G G G G A A A A A A
21731-22412 35 22155/41 10 G G G G G G G G G G
21731-22412 36 22156/4230 A A A A G G G G G G
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T T C T T T C C
25559-26029 38 25921/4470 C c c c T c C c T T
27822-28255 39 27882/4590 T c T T c c c T c c
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 C C C c c C C C C C
30265-30754 42 30618/4950 C T C c c C C C C C
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 c c c c c C C c c c
31300-31718 - - - - - - - - - - - -
(a) Region examined repr esents the nucleotide positions defining ; the start and stop positions within SEQ ID NO: 1 of the regions ! sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the 1 st position number refening to SEQ ID NO: 1 and the 2 position number refening to SEQ ID NO:226, a modified version of SEQ ID NO:l that comprises the context sequence of each polymoφhic site, PS1-PS44, to facilitate electronic searching of the haplotypes; (d) Alleles for ITGB3 haplotypes are presented 5' to 3' in each column. Table 5(Part 4). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Examined(a) No.(b) Position(c) 31 32 33 34 35 36 37 38 39 40
1000-2235 1 1118/30 C C C C C C C C C C
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 G G G G G G G G G G
1000-2235 4 1875/390 C C C C C C C C C c
1000-2235 5 1911/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 c C C C C C C C C C
1000-2235 9 2048/990 C C C C C C C C C C
1000-2235 10 2087/11 10 G G G G G G G G G G
1000-2235 11 2117/1230 G G G G G G G G G G
1000-2235 12 2157/1350 C C C C C c C C C C
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 c C C C C C C C C C
13179-13723 15 133S4/1710 T T T T T T T T T T
13179-13723 16 13405/1830 T T T T T T T T T T
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G , G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G G G G G
16930-17414 20 17194/2310 T T T T T T T T T T
16930-17414 21 17273/2430 A A A A A A A A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 G G G G G G G G G G
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 C C c C C c C C C C
19748-20177 26 20035/3030 G G G G G G G G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C C C C C C C C C
19748-20177 29 20105/3390 C C C C c C C C C C
20537-21009 30 20615/3510 A A A G G G G G G G
20537-21009 31 20704/3630 A A A A A A A A A A
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 A A A A A A A A A A
21731-22412 35 22155/4110 G G G G G G G G G G
21731-22412 36 22156/4230 G G G G G G G G G G
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T C T T T T T T
25559-26029 38 25921/4470 c C T T c c c C c c
27822-28255 39 27882/4590 c T c c c c c T T T
27822-28255 40 28127/4710 G G G G G G G A G G
30265-30754 41 30516/4830 C C C C c C C C C C
30265-30754 42 30618/4950 T C T C c T T C C T
30265-30754 43 30662/5070 T T T T T G T T T T
30265-30754 44 30729/5190 c C c c c C c C c c
31300-31718 - - - - - - - - - - - -
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions ; sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ 1 [D NO, with the
Im position number refening to SEQ ID NO: l and the 2m posit ion number refening to SEQ ID NO:226, ε i modified version of SEQ ID NO:l that comprises the context sequence < _f each polymoφhic site, PS1-PS44, to facilitate electronic searching of the s haplotypes; (d) Alleles for ITGB3 haplotypes are presentee 1 5' to 3 ' in < -ach column. Table 5(Part 5). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Examined(a) No.(b) Position(c) 41 42 43 44 45 46 47 48 49 50
1000-2235 1 1118/30 C C C c C C C C C C
1000-2235 9 1202/150 A A A A A A A A A A
1000-2235 1773/270 G G G G G G G G G G
1000-2235 4 1875/390 C C C C C C C C C C
1000-2235 5 1911/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C C
1000-2235 9 2048/990 C C C C C C C C C C
1000-2235 10 2087/11 10 G G G G G G G G G G
1000-2235 11 2117/1230 G G G G G G G G G G
1000-2235 12 2157/1350 C C C C C C C C C T
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C C C C C C C T C
13179-13723 15 13384/1710 T T T T T T T T T C
13179-13723 16 13405/1830 T T T T T T T T T . .
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G T T T T G G
16930-17414 20 17194/2310 T T T T T T T T T T
16930-17414 21 17273/2430 A A A A A A A A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 G G G G G G G G G C
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 C C C T c C c C c c
19748-20177 26 20035/3030 G G G G G G G G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C T C T T T T C C
19748-20177 29 20105/3390 C C C c c C c c C c
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 A C C A C C C C C C
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G A G G G G G
21731-22412 34 22144/3990 A A G A G G G G G G
21731-22412 35 22155/4110 G G G G G G G G G G
21731-22412 36 22156/4230 G G A G A A A A A A
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T T T T T T T T
25559-26029 38 25921/4470 T c c c c C c c c c
27822-28255 39 27882/4590 c c c c c c C T c c
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 C c c c C C c C c c
30265-30754 42 30618/4950 T T c T T C T C c T
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 C c c c c C c c c c
31300-31718 - - - - - - - - - - * -
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions t sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the
1st position number refeni ing to SEQ ID NO: 1 and the 2 position number refening to SEQ ID NO:226, ε i modified version of SEQ ID NO:l that comprises the context sequence < D-f each polymoφhic site, PS1-PS44, , to facilitate electronic searching of the haplotypes; (d) Alleles for ITGB3 haplotypes are presente I 5' to 2 \ ' in each column. Table 5(Part 6). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Examined(a) No.(b) Position(c) 51 52 53 54 55 56 57 58 59 60
1000-2235 1 1118/30 C C C C C C C c C C
1000-2235 1202/150 A A A A A A A ' A G G
1000-2235 3 1773/270 G G G G G G G G G G
1000-2235 4 1875/390 C C C C C T T T C C
1000-2235 5 191 1/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C c
1000-2235 9 2048/990 C C C C G C C C C C
1000-2235 10 2087/11 10 G G G G G A A G G G
1000-2235 11 2117/1230 G G G G G G G G G G
1000-2235 12 2157/1350 T T T T C C C C C T
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C T T C C C C C C
13179-13723 15 13384/1710 T T T T T T T C T T
13179-13723 16 13405/1830 T T T T T T T T T T '
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G G G G G
16930-17414 20 17194/2310 T T T T C C C T T T
16930-17414 21 17273/2430 A A A A G G G A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 G G G G G G G C G G
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 c C C C c C C C C C
19748-20177 26 20035/3030 G G G G A A A G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C c C c C C C C c
19748-20177 29 20105/3390 C C C C c C C C C C
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 A A A C C C C C A A
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 A A A G A A A G A A
21731-22412 35 22155/41 10 G G G G G G G G G G
21731-22412 36 22156/4230 G G G A G G G A G G
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T T C C C T T T
25559-26029 38 25921/4470 c c c c T C T c c C
27822-28255 39 27882/4590 c T c c c c c T T c
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 C C C c c C C C C c
30265-30754 42 30618/4950 T C T c c c C C C T
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 C c c c c c c C c c
31300-31718 - - - - - - - - - - - -
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions : sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the lsl position number refening to SEQ ID NO: 1 and the 2' 1 positioi number refening to SEQ ID NO:226, a i modified version of SEQ ID NO: l that comprises the context sequence of each polymoφhic site, PS1-PS44, , to facilitate electronic searching of the ! haplotypes; (d) Alleles for ITGB3 haplotypes are presentee I 5 ' to : 5 ' in each column. Table 5(Part 7). Haplotypes of the ITGB3 gene.
Region PS PS Hε iplotype Number(d)
Examined(a) No.(b) Position(c) 61 62 63 64 65 66 67 68 69 70
1000-2235 1 1118/30 T T T T T T T T T T
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 C C C C C C C C C C
1000-2235 4 1875/390 C c C C C C C C C C
1000-2235 5 191 1/510 c c C c C G G G G G
1000-2235 6 1957/630 G G G G G A A A A G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C C
1000-2235 9 2048/990 C G G G G G G G G C
1000-2235 10 2087/1110 G G G G G G G G G G
1000-2235 11 2117/1230 A A A A A A A A A A
1000-2235 12 2157/1350 C C C C C C C C C C
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C C C C C C C C C
13179-13723 15 13384/1710 T T T T T T T T T T
13179-13723 16 13405/1830 T T T T ' T T T T T T
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G G G T T
16930-17414 20 17194/2310 C C C C T C C T T T
16930-17414 21 17273/2430 A A A A A A G A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 G G G G G G G G G G
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 C C C C c C c C C c
19748-20177 26 20035/3030 G G G G G G G G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C C C C C C C T T
19748-20177 29 20105/3390 C C C c C C C C C c
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 C C C C A C C A C C
20537-21009 32 20889/3750 T T T T T G T T T T
21731-22412 33 21944/3870 G A G G G G G G G G
21731-22412 34 22144/3990 A A A A A A A A G G
21731-22412 35 22155/4110 G G A G G G G G G G
21731-22412 36 22156/4230 G G G G G G G G A A
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 C C C C T C T T C T
25559-26029 38 25921/4470 T T T T C T C C T c
27822-28255 39 27882/4590 c c c c c c c c c c
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 C C C C C T C C C c
30265-30754 42 30618/4950 C C c C T C C C C c
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 c c c C C c C C c c
31300-31718 - - - - - - - - - - - -
(a) Region examhied represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions : sequenced; (b) PS = polymoiphic site; (c) Position of PS within the indicated SEQ ID NO, with the ls'position number referring to SEQ ID NO: 1 and the 2nd position number referring to SEQ ID NO:226, a . modified version of SEQ ID NO:l that comprises the context sequence ι jfeach polyrnoφhic site, PS1-PS44, to facilitate electronic searching of the : haplotypes; (d) Alleles for ITGB3 haplotypes are presentee 1 5 ' to 2 V in < 2ach column. Table 5(Patt 8). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Numbered)
Exammed(a) No.(b) Position(c) 71 72 73 74 75 76 77 78 79 80
1000-2235 1 1 1 18/30 T T T T T T T T T T
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 C C C C C C C C C C
1000-2235 4 1875/390 C C C C C C c c c c
1000-2235 5 1911/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C C
1000-2235 9 2048/990 G G G G G G G G G G
1000-2235 10 2087/11 10 G G G G G G G G G G
1000-2235 1 1 21 17/1230 A A A A A A A A A A
1000-2235 12 2157/1350 C C C C C C C C C C
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C C C C C C C C C
13179-13723 15 13384/1710 C T T T T T T T T T
13179-13723 16 13405/1830 T T T T T T T T ,τ T
13179-13723 17 13550/1950 T c c T T T T T T T
13179-13723 I S 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G T T G G G G G G G
16930-17414 20 17194/2310 T T T C C C C C T T
16930-17414 21 17273/2430 A A A A A A A A A A
19241-19644 22 19357/2550 A A A A A A A G A A
19241-19644 23 19610/2670 C G G G G G G G C G
1974S-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 c C C C C c C c c C
19748-20177 26 20035/3030 G G G G G G G G G G
19748-20177 27 20037/3150 G G G A A G G G G G
19748-20177 28 20047/3270 C T T C C C C C C C
19748-20177 29 20105/3390 c C C C c C C C c C
20537-21009 30 20615/3510 G G G G G G G G G A
20537-21009 31 20704/3630 C C C C C C C C C A
20537-21009 32 20889/3750 T T T G G T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 G G G A A A A A G A
21731-22412 35 22155/4110 G G G G G G G G G G
21731-22412 36 22156/4230 A A A G G G G G A G
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T T T C T T T T
25559-26029 3S 25921/4470 c C c c C T c c c C
27822-28255 39 27882/4590 T c T c c c c c c T
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 C C C C C C C C C C
30265-30754 42 30618/4950 C C C C T C C C C c
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 c c C c C c c C c c
31300-31718 - - - - - - - - - - - -
(a) Region examined represents the nucleotide positions definmg the start and stop positions within SEQ ID NO: 1 of the regions sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the 1st position number refening to SEQ ID NO' 1 and the 2nd position number referring to SEQ ID NO:226, a modified version of SEQ ID NO: l that comprises the context sequence of each polymoφhic site, PS1-PS44, to facilitate electronic searching of the haplotypes, (d) Alleles for ITGB3 haplotypes are presented 5' to 3 ' in each column. Table 5(Part 9). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Nuιnber(d)
Examined(a) No.(b) Position(c) 81 82 83 84 85 86 87 88 89 90
1000-2235 1 1118/30 T T T T T T T T T T
1000-2235 2 1202/150 A A A A A A A A A A
1000-2235 3 1773/270 C C C C C C C C C C
1000-2235 4 1875/390 C C C C C C C C c C
1000-2235 5 1911/510 G G G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G G G
1000-2235 7 1974/750 G G G G G G G G G G
1000-2235 8 1975/870 C C C C C C C C C C
1000-2235 9 2048/990 G G G G G G G G G G
1000-2235 10 2087/1 1 10 G G G G G G G G G G
1000-2235 11 2117/1230 A A A A A A A A A G
1000-2235 12 2157/1350 C C C C C C C C C C
4256-4716 - - - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G G G
13179-13723 14 13334/1590 C C C C C C C C T C
13179-13723 15 13384/1710 T T T T T T T T T T
13179-13723 16 13405/1830 T T T T T T T T T T
13179-13723 17 13550/1950 T T T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G G G
14234-14858 - - - - - - - - - - - -
16126-16619 19 16200/2190 G G T T T T T T G T
16930-17414 20 17194/2310 T T T T T T T T T T
16930-17414 21 17273/2430 A A A A A A A A A A
19241-19644 22 19357/2550 A A A A A A A A A A
19241-19644 23 19610/2670 G G C G G G G G G G
19748-20177 24 19877/2790 T T T T T T T T T T
19748-20177 25 20034/2910 c c c C C C C c C C
19748-20177 26 20035/3030 G G G G G G G G G G
19748-20177 27 20037/3150 G G G G G G G G G G
19748-20177 28 20047/3270 C C T C T T T T C T
19748-20177 29 20105/3390 C C C C C c c c C c
20537-21009 30 20615/3510 G G G G G G G G G G
20537-21009 31 20704/3630 A A C C C C C C C C
20537-21009 32 20889/3750 T T T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G G G
21731-22412 34 22144/3990 A A G G G G G G G G
21731-22412 35 22155/4110 G G G G G G G G G G
21731-22412 36 22156/4230 G G A A A A A A A A
24385-24930 - - - - - - - - - - - -
25559-26029 37 25705/4350 T T T T C T T T T T
25559-26029 38 25921/4470 C C c c T c c C C C
27822-28255 39 27882/4590 c T c c c c c T c T
27822-28255 40 28127/4710 G G G G G G G G G G
30265-30754 41 30516/4830 c c c C C C C C C c
30265-30754 42 30618/4950 T c c C C C T c C c
30265-30754 43 30662/5070 T T T T T T T T T T
30265-30754 44 30729/5190 c c c C c c c c C c
31300-31718 - - - - - - - - - - - -
(a) Region examined represents the nucleotide positions defining ; the start and stop positions within SEQ ID NO: 1 of the regions £ sequenced; (b) PS = polymoφhic site; (c) Position of PS within the indicated SEQ ID NO, with the
1st position number referring to SEQ ID NO: l and the 2na position number referring to SEQ ID NO:226, a modified version of SEζ ) ID NO: 1 that comprises the context sequence of each polymoφhic site, PS1-PS44, to facilitate electronic seaπ ;hing of the haplotypes; (d) Alleles for ITGB3 haplotypes are presented 1 5 ' to - V in each column. Table 5(Part 10). Haplotypes of the ITGB3 gene.
Region PS PS Haplotype Number(d)
Examined(a) No.fb) Position(c) 91 92 93 94 95 96 97 98
1000-2235 1 1118/30 T T T T T T T T
1000-2235 2 1202/150 A A A A A A A A
1000-2235 3 1773/270 C C G G G G G G
1000-2235 4 1875/390 C C C C C C C C
1000-2235 5 1911/510 G G G G G G G G
1000-2235 6 1957/630 G G G G G G G G
1000-2235 7 1974/750 T T G G G G G G
1000-2235 8 1975/870 T T C C C C C C
1000-2235 9 2048/990 G G C C C C C C
1000-2235 10 ' 2087/11 10 G G A G G G G G
1000-2235 11 2117/1230 A A G G G G G G
1000-2235 12 2157/1350 C C C C C C C T
4256-4716 - - - - - - - - - -
13179-13723 13 13273/1470 G G G G G G G G
13179-13723 14 13334/1590 C C C C C C C T
13179-13723 15 13384/1710 T T T C T T T T
13179-13723 16 13405/1830 T T T T T T T T
13179-13723 17 13550/1950 T T T T T T T T
13179-13723 18 13690/2070 G G G G G G G G
14234-14858 - - - - - - - - - -
16126-16619 19 16200/2190 G G G G G G T G
16930-17414 20 17194/2310 C T C T T T T T
16930-17414 21 17273/2430 G A G A A A A A
19241-19644 22 19357/2550 A A A A A A A A
19241-19644 23 19610/2670 G C G C G G G G
19748-20177 24 19877/2790 T T T T T T T T
19748-20177 25 20034/2910 c c C c C C c C
19748-20177 26 20035/3030 G G A G G G G G
19748-20177 27 20037/3150 G G G G G G G G
19748-20177 28 20047/3270 C C C C C C T C
19748-20177 29 20105/3390 C c C C C C c C
20537-21009 30 20615/3510 G G G G G G G G
20537-21009 31 20704/3630 C C C C A A C C
20537-21009 32 20889/3750 T T T T T T T T
21731-22412 33 21944/3870 G G G G G G G G
21731-22412 34 22144/3990 A G A G A A G G
21731-22412 35 22155/4110 G G G G G G G G
21731-22412 36 22156/4230 G A G A G G A A
24385-24930 - - - - - - - - - -
25559-26029 37 25705/4350 C T C T T T T T
25559-26029 38 25921/4470 T C T C C C c c
27822-28255 39 27882/4590 c c c T c T c c
27822-28255 40 28127/4710 G G G G G G G G
30265-30754 41 30516/4830 c c C C c C c C
30265-30754 42 30618/4950 c c C C T C c c
30265-30754 43 30662/5070 T T T T T T T T
30265-30754 44 30729/5190 c c C c c C c c
31300-31718 - - - - - - - - - -
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions sequenced; (b) PS = polyir røφhic site; (c) Position of PS within the indicated SEQ ID NO, with the
1st osition number referring to SEQ ID NO: 1 and the 2nd posit ion number refening to SEQ ID NO:226, a modified version of SEQ ID NO: 1 that comprises 1 :he context sequence of each polymoφhic site, PS1-PS44, to facilitate electronic searching of the haplotypes; (d) Alleles for ITGB3 haplotypes are presentee 1 5 ' to . i ' in each column. Table 6. Frequency of Observed ITGB3 Haplotypes hi Unrelated Individuals
HAPNc . HAPID Total CA AP AS HL AM
1 9900058076 1 1 0 0 0 0
2 9900058053 1 0 1 0 0 0
3 9900058055 1 1 0 0 0 0
4 9900058068 1 0 1 0 0 0
5 9900058016 3 3 0 0 0 0
6 9900058011 5 4 0 0 1 0
7 9900058013 4 4 0 0 0 0
8 9900058038 1 0 0 0 1 0
9 9900058063 1 0 1 0 0 0
10 9900058066 1 0 0 0 0 0
11 9900058082 1 1 0 0 0 0
12 9900058031 2 0 0 0 2 0
13 9900057992 62 51 1 0 1 1
14 9900058061 1 0 0 0 0
15 9900058074 1 0 0 0 0
16 9900058040 1 0 0 0 0
17 9900058072 1 0 0 0 0
18 9900058083 1 0 0 0 0
19 9900058039 1 0 0 0 0
20 9900058010 5 4 0 0 0 0
21 9900058078 1 0 0 0 1 0
22 9900058043 1 1 0 0 0 0
23 9900057990 180 131 3 1 11 0
24 9900058077 1 0 0 0 1 0
25 9900058007 7 5 0 0 0 0
26 9900058029 9 0 2 0 0 0
27 9900058033 2 0 0 2 0 0
28 9900058025 0 0 2 0 0
29 9900058003 9 7 0 0 0 0
30 9900058070 1 1 0 0 0 0
31 9900057995 32 24 0 1 0
32 9900058012 3 1 0 0 1 0
33 9900058071 1 1 0 0 0 0
34 9900058015 4 3 0 0 0 0
35 9900058000 11 6 2 0 1 1
36 9900058034 2 0 2 0 0 0
37 9900057988 680 471 37 14 41 1
38 990005S021 3 3 0 0 0 0
39 9900057989 224 141 5 10 22 3
40 9900058014 4 4 0 0 0 0
41 9900058035 2 2 0 0 0 0
42 9900058080 1 1 0 0 0 0
43 9900058058 1 0 1 0 0 0
44 9900058075 1 1 0 0 0 0
45 9900058057 1 0 0 0 0 0
46 9900058002 10 3 3 2 1 0
47 9900058046 1 1 0 0 0 0
48 9900058022 3 0 1 0 1 1
49 9900058004 8 3 1 2 1 0
50 9900057999 18 12 0 0 0 0
51 9900058041 1 0 0 1 0 0
52 9900058052 1 1 0 0 0 0
53 9900058056 1 1 0 0 0 0 Table 6. Frequency of Observed ITGB3 Haplotypes In Unrelated Individuals
HAI 3 No. HAPID Total CA AF AS HL AM
54 9900057993 61 42 1 3 5 1
55 9900058067 1 0 0 0 1 0
56 9900058054 1 1 0 0 0 0
57 9900058085 1 1 0 0 0 0
58 9900058042 1 1 0 0 0 0
59 9900058030 2 1 0 0 0 0
60 9900058047 1 0 0 0 1 0
61 9900058044 1 0 0 1 0 0
62 9900058049 1 0 0 1 0 0
63 9900058028 2 0 0 2 0 0
64 9900057996 20 8 1 8 2 0
65 9900057998 18 14 0 2 0 0
66 9900058020 3 0 2 0 0 0
67 9900058062 1 1 0 0 0 0
68 9900058064 1 0 1 0 0 0
69 9900058060 1 0 1 0 0 0
70 9900058027 2 0 0 2 0 0
71 9900058009 8 1 4 0 0 0
72 9900058005 8 0 2 0 0 0
73 9900058032 2 0 1 0 0 0
74 9900058073 1 0 1 0 0 0
75 9900058048 1 0 1 0 0 0
76 9900058045 1 0 0 1 0 0
77 9900058069 1 0 1 0 0 0
78 9900058001 11 0 8 0 1 0
79 9900058059 1 0 0 0 0 0
80 9900058024 4 3 0 0 0 0
81 9900057997 15 8 1 1 1 0
82 9900057994 38 28 0 0 1 0
83 9900058081 1 0 0 0 0 0
84 9900058079 1 1 0 0 0 0
85 9900058051 1 1 0 0 0 0
86 9900057991 120 78 1 9 6 0
87 9900058017 4 3 0 0 0 0
88 9900058036 2 0 1 0 1 0
89 9900058006 8 7 0 0 1 0
90 9900058008 7 0 5 0 2 0
91 9900058019 3 0 3 0 0 0
92 9900058065 1 0 1 0 0 0
93 9900058050 1 1 0 0 0 0
94 9900058084 1 1 0 0 0 0
95 9900058023 2 0 1 1 0 0
96 9900058037 9 1 0 0 0 0
97 9900058018 3 3 0 0 0 0
98 9900058026 2 0 0 1 1 0 •fc. 4^ to o
Figure imgf000103_0001
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Table 7. Number of Observed ITGB3 Haplotype Pairs In Unrelated Individuals
HAP 1 HAP2 Total CA AF AS HL AM
39 31 5 4 1 0 0 0
39 39 14 8 0 1 4 1
39 40 2 2 0 0 0 0
39 48 1 0 0 0 1 0
39 49 3 2 1 0 0 0
39 50 2 2 0 0 0 0
39 52 1 1 0 0 0 0
39 54 8 7 0 0 1 0
39 60 1 0 0 0 1 0
39 61 1 0 0 1 0 0
39 64 2 0 0 2 0 0
39 65 1 1 0 0 0 0
39 78 1 0 1 0 0 0
39 82 5 5 0 0 0 0
39 86 8 6 0 1 1 0
39 88 1 0 1 0 0 0
39 89 1 1 0 0 0 0
39 97 2 2 0 0 0 0
46 90 1 0 1 0 0 0
49 6 1 0 0 0 1 0
50 89 1 1 0 0 0 0
54 28 1 0 0 1 0 0
54 31 1 1 0 0 0 0
54 46 1 0 0 1 0 0
54 48 1 0 0 0 0 1
54 54 1 1 0 0 0 0
54 64 2 2 0 0 0 0
54 71 1 1 0 0 0 0
54 81 1 0 0 1 0 0
54 82 2 2 0 0 0 0
64 49 1 0 0 1 0 0
64 65 1 1 0 0 0 0
64 91 1 0 1 0 0 0
65 98 1 0 0 1 0 0
66 69 1 0 1 0 0 0
70 51 1 0 0 1 0 0
71 66 1 0 1 0 0 0
71 71 1 0 1 0 0 0
72 73 0 0 0 0 0 0
78 26 1 0 1 0 0 0
78 72 1 0 1 0 0 0
78 79 0 0 0 0 0 0
78 90 2 0 2 0 0 0
78 95 1 0 1 0 0 0
81 36 1 0 1 0 0 0
81 50 1 1 0 0 0 0
81 93 1 1 0 0 0 0
81 98 1 0 0 0 1 0
82 29 2 2 0 0 0 0
82 67 1 1 0 0 0 0
82 87 1 1 0 0 0 0
82 94 1 1 0 0 0 0
82 96 1 1 0 0 0 0 Table 7. Number of Observed ITGB3 Haplotype Pairs hi Unrelated Individuals HAP1 HAP2 Total CA AF AS HL AM
86 4 1 0 1 0 0 0
86 13 5 5 0 0 0 0
86 50 1 1 0 0 0 0
86 54 3 2 0 0 1 0
86 62 1 0 0 1 0 0
86 63 2 0 0 2 0 0
86 64 2 0 0 2 0 0
86 65 5 4 0 1 0 0
86 76 1 0 0 1 0 0
86 81 1 1 0 0 0 0
86 82 2 2 0 0 0 0
86 86 2 1 0 0 1 0
86 87 1 1 0 0 0 0
86 89 1 0 0 0 1 0
91 9 1 0 1 0 0 0
92 77 1 0 1 0 0 0
95 70 1 0 0 1 0 0
Table 8(Part 1). Nucleotides Present at Polymoφhic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d) Examined(a) No.(b)Position(c)l 2A 3 4B 5 6 7C 8C 9D 10D
1-2367 6 40 G A G G G G G G G G
1-2367 7 57 G G G G G G G G T T
1-2367 8 58 C C C C C C C C T T
1-2367 15 176 T T T C T T T T T T 1-2367 16 197 T T T T T T T T T T
1-2367 17 342 T T T T T T T T T T
1-2367 20 882 T T T T T T T T T T
1-2367 31 1143 A A A C A A C C A A
1-2367 33 1333 G G G G G G G G G G 1-2367 34 1533 A A A G A A G G A A
1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 G G G A G G A A G G
1-2367 41 2208 C C C C C C C C C C Table 8(Part 2). Nucleotides Present at Polymorphic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d)
Examined(a) No.(b)Position(c)llB 12E 13E 14E 15 16 17C 18B 19 20F
1-2367 6 40 G G G G G G G G G G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C C C C C C C C
1-2367 15 176 C T T T T T T C T C
1-2367 16 197 T T T T T T T T T G
1-2367 17 342 T T T T T T T T T T
1-2367 20 882 T C c c T T T T T T
1-2367 31 1143 C C c c A A C C A c
1-2367 33 1333 G G G G G G G G G G
1-2367 34 1533 G A A A A A G G A G
1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 A G G G G G A A G A
1-2367 41 2208 C C c c C C C C C C
Table 8(Part 3). Nucleotides Present at Polymoφhic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d)
Examhιed(a) No.(b)Position(c)21G22B 23B 24B 25E ' 26E 27E 28E 29E 30E
1-2367 6 40 G G G G G G G G G G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C c C C C C C C
1-2367 15 176 C C C C T T T T T T
1-2367 16 197 T T T T T T T T , T T
1-2367 17 342 T T T T T T T T T T
1-2367 20 882 T T T T c C c c C c
1-2367 31 1143 A c c C c C c c C c
1-2367 33 1333 G G G G G G G G G G
1-2367 34 1533 G G G G A A A A A A
1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 A A A A G G G G G G
1-2367 41 2208 C C c C C C C c C c Table 8(Part 4). Nucleotides Present at Polymoiphic Sites Withm the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d) Examined(a) No.(b)Position(c)31 32 33 34 35 36 37 38 39 40
1-2367 6 40 G G G G G G G G G G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C C C C C C C C
1-2367 15 176 T T T T T T T T T T 1-2367 16 197 T T T T T T T T T T
1-2367 17 342 T T T T T T T T T T
1-2367 20 882 T T T T T T T T T T
1-2367 31 1143 A A A A A A A A A A
1-2367 33 1333 G G G G G G G G G G 1-2367 34 1533 A A A A A A A A A A
1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 G G G G G G G G G G
1-2367 41 2208 C C C C C C C C C C Table 8(Part 5). Nucleotides Present at Polymoφhic Sites Withm the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d)
Examined(a) No.(b)Position(c)41 42H 43C 44 451 46C 47C 48C 49C 50B 1-2367 6 40 G G G G G G G G G G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C C C C C C C C
1-2367 15 176 T T T T T T T T T C
1-2367 16 197 T T T T T T T T T T 1-2367 17 342 T T T T T T T T T T
1-2367 20 882 T T T T T T T T T T
1-2367 31 1143 A C C A C C C C C C
1-2367 33 1333 G G G G A G G G G G
1-2367 34 1533 A A G A G G G G G G 1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 G G A G A A A A A A
1-2367 41 2208 C C C C C C C C C C
Table 8(Part 6). Nucleotides Present at Polymoφhic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Nunιber(d)
Examined(εi) No.(b): Position! (c)51 52 53 54C 55E 56E 57E 58B 59 60
1-2367 6 40 G G G G G G G G G G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C C C C C C C C
1-2367 15 176 T T T T T T T C T T
1-2367 16 197 T T T T T T T T T T
1-2367 17 342 T T T T T T T T T T
1-2367 20 882 T T T T C C C T T T
1-2367 31 1143 A A A C C C C C A A
1-2367 33 1333 G G G G G G G G G G
1-2367 34 1533 A A A G A A A G A A
1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 G G G A G G G A G G
1-2367 41 2208 C C C C C C C C C C Table 8(Part 7). Nucleotides Present at Polymoφhic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d) Examined(a) No.(b)Position(c)61E 62J 63K 64E 65 66L 67M 68A 69N 70C
1-2367 6 40 G G G G G A A A A G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C C C C C C C C
1-2367 15 176 T T T T T T T T T T 1-2367 16 197 T T T T T T T T T T
1-2367 17 342 T T T T T T T T T T
1-2367 20 882 C C C c T c C T T T
1-2367 31 1143 C C C c A c C A C C
1-2367 33 1333 G A G G G G G G G G 1-2367 34 1533 A A A A A A A A G G
1-2367 35 1544 G G A G G G G G G G
1-2367 36 1545 G G G G G G G G A A
1-2367 41 2208 C C C C C T C C C C Table 8(Part 8). Nucleotides Present at Polymoφhic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d)
Examined(a) No.(b) Positions (c)71B 720 730 74E 75E 76E 77E 78E 79C 80
1-2367 6 40 G G G G G G G G G G
1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C c c C C C C C C C
1-2367 15 176 C T T T T T T T T T
1-2367 16 197 T T T T T T T T T T
1-2367 17 342 T c c T T T T T T T
1-2367 20 882 T T T c c c c c T T
1-2367 31 1143 c c c c c c c c C A
1-2367 33 1333 G G G G G G G G G G
1-2367 34 1533 G G G A A A A A G A
1-2367 35 1544 G G G G G G G G G G
1-2367 36 1545 A A A G G G G G A G
1-2367 41 2208 c c C C C C C C C C
Table 8(Part 9). Nucleotides Present at Polymoφhic Sites Withm the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d)
Examined(a) No.(b)Position(c)81 82 83C 84C 85C 86C 87C 88C 89C 90C
1-2367 6 40 G G G G G G G G G G 1-2367 7 57 G G G G G G G G G G
1-2367 8 58 C C C C C C C C C C
1-2367 15 176 T T T T T T T T T T
1-2367 16 197 T T T T T T T T T T
1-2367 17 342 T T T T T T T T T T 1-2367 20 882 T T T T T T T T T T
1-2367 31 1143 A A C c c c c C C C
1-2367 33 1333 G G G G G G G G G G
1-2367 34 1533 A A G G G G G G G G
1-2367 35 1544 G G G G G G G G G G 1-2367 36 1545 G G A A A A A A A A
1-2367 41 2208 C C C c C C C C C C Table 8(Part 10). Nucleotides Present at Polymoφhic Sites Within the Observed ITGB3 Coding Sequences
Region PS PS Coding Sequence Number(d)
Examined(a) No.(b)Position(c)91P 92Q 93E 94B 95 96 97C 98C
1-2367 6 40 G G G G G G G G
1-2367 7 57 T T G G G G G G
1-2367 8 58 T T C C C C C C 1 1--22336677 1 155 1 17766 T T T T T T C T T T T
1-2367 16 197 T T T T T T T T
1-2367 17 342 T T T T T T T T
1-2367 20 882 C T c T T T T T
1-2367 31 1143 C c c c A A C C 1 1--22336677 3 333 1 1333333 G G G G G G G G G G G
1-2367 34 1533 A G A G A A G G
1-2367 35 1544 G G G G G G G G
1-2367 36 1545 G A G A G G A A
1-2367 41 2208 C C C C C C C C (a) Region examined represents the nucleotide positions in SEQ ID NO:2 defining the start and stop positions of the regions sequenced; (b) PS = polymoiphic site; (c) Position of PS within SEQ ID NO:2; (d) Alleles for ITGB3 codmg sequences are presented 5 ' to 3 ' in each column. The number at the top of each column designates the haplotype number of the ITGB3 isogene from which the coding sequence is derived. ITGB3 coding sequences that differ from the reference are denoted in this table by a letter following the isogene number.
Table 9(Part 1). Amino Acids Present at Polymoiphic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d)
Examined(a) No.(b)Position(c)l 2A 3 4B 5 6 7 8 9 10
0-788 6 14 V M V V V V V V V V
0-788 15 59 L L L P L L L L L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 2). Amino Acids Present at Polymoφhic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d) Examined(a) No.(b)Position(c)l lB 12 13 14 15 16 17 18B 19 20D
0-788 6 14 V V V V V V V V V V
0-788 15 59 P L L L L L L P L P
0-788 16 66 L L L L L L L L L R
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R Table 9(Part 3). Amino Acids Present at Polymoφhic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d) Examined(a) No.(b)Position(c)21B 22B 23B 24B 25 26 27 28 29 30
0-788 6 14 V V V V V V V V V V
0-788 15 59 P P P P L L L L L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 4). Amino Acids Present at Polymoiphic Sites Within the Observed ITGB3 Protein Sequences Region PS PS Protein Variants (d)
Examined(a) No.(b)Position(c)31 32 33 34 35 36 37 38 39 40
0-788 6 14 V V V V V V V V V V
0-788 15 59 L L L L L L L L L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 5). Amino Acids Present at Polymoφhic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d)
Examined(a) No.(b)Position(c ))4411 4422 43 44 45E 46 47 48 49 50B
0-788 6 14 V V V V V V V V V V
0-788 15 59 L L L L L L L L L P
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V M V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 6). Amino Acids Present at Polymoiphic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d)
Examined(a) No.(b)Position(c)51 52 53 54 55 56 57 58B 59 60
0-788 6 14 V V V V V V V V V V
0-788 15 59 L L L L L L L P L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 7). Amino Acids Present at Polymoiphic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d) Examined(a) No.(b)Position(c)61 62F 63G 64 65 66A 67A 68A 69C 70
0-788 6 14 V V V V V M M M M V
0-788 15 59 L L L L L L L L L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V M V V V V V V V V
0-788 35&36 515 R R Q R R R R R R R Table 9(Part 8). Amino Acids Present at Polymoφhic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d) Examined(a) No.(b)Position(c)71B 72 73 74 75 76 77 78 79 80
0-788 6 14 V V V V V V V V V V
0-788 15 59 P L L L L L L L L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 9). Amino Acids Present at Polymoφhic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d)
Examined(a) No.(b)Position(c)81 82 83 84 85 86 87 88 89 90
0-788 6 14 V V V V V V V V V V
0-788 15 59 L L L L L L L L L L
0-788 16 66 L L L L L L L L L L
0-788 33 445 V V V V V V V V V V
0-788 35&36 515 R R R R R R R R R R
Table 9(Part 10). Amino Acids Present at Polymoiphic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants (d)
Examined(a) No.(b)Position(c)91 92 93 94B 95 96 97 98
0-788 ' 6 14 V V V V V V V V
0-788 15 59 L L L P L L L L 0-788 16 66 L L L L L L L L
0-788 33 445 V V V V V V V V
0-788 35&36 515 R R R R R R R R
(a) Region examined represents the amino acid positions in SEQ ID NO:3 defining the start and stop positions of the regions sequenced; (b) PS = polymoφhic site; (c) Position of PS within SEQ ID NO:3; (d) Alleles for ITGB3 protein sequences are presented from N-terminus to C-tenninus in each column. The number at the top of each column designates the haplotype number of the ITGB3 isogene from which the protein sequence is derived. ITGB3 protein sequences that differ from the reference are denoted in this table by a letter following the isogene number.
I l l

Claims

What is claimed is:
1. A kit for detennining whether an individual has a statm response marker I or a statin response marker II, the kit comprising a set of oligonucleotides designed for identifying at least one of the alleles at each polymoφhic site (PS) hi a set of polymoφhic sites (PSs), wherein the set of polymoiphic sites comprises (1) PSI and PS42; (2) PS19 and PS42; (3) PS3, PS12, and PS42; (4)
PSI, PS12, and PS42; (5) PS3, PS19. and PS42; (6) PSI. PS4, and PS42; (7) PS3, PS19. and PS39:
(8) PSI, PS37, and PS42; (9) PS5, PS19, and PS42; (10) PSI, PS3, PS12, and PS42; (11) PS3, PS4,
PS12, and PS42; (12) PSI, PS12, PS37 and PS42; (13) PS3, PS12. PS37 and PS42; (14) PSI. PS4,
PS12, and PS42; (15) PS3, PS12, PS19 and PS42; (16) PS3, PS12, PS16 and PS42; (17) PSI, PS12, PS26 and PS42; (18) PSI, PS3, PS19, and PS42; (19) PS3, PS12, PS26 and PS42; (20) PS3, PS4,
PS19, and PS42; (21) PSI, PS10, PS12 and PS42; (22) PS3. PS10, PS12, and PS42; (23) PSI, PS12, PS16 and PS42; (24) PSI, PS12, PS21 and PS42; (25) PS3, PS12, PS21 and PS42; (26) PSI, PS12, PS20 and PS42; (27) PS3, PS12, PS20 and PS42; (28) PS3, PS16, PS19 and PS42; (29) PS3, PS19. PS37 and PS42; (30) PSI, PS3, PS19 and PS42; (31) PSI, PS3, PS4, PS12, and PS42; (32) PSI, PS12, PS21, PS37, and PS42; (33) PS3, PS12, PS21, PS37, and PS42; (34) PSI, PS3, PS12, PS37, and PS42; (35) PSI, PS4, PS12, PS37, and PS42; (36) PS3, PS4, PS12, PS37, and PS42; (37) PSI, PS12, PS20, PS37, and PS42; (38) PSI, PS3, PS12, PS19 and PS42; (39) PS3, PS12, PS20, PS37, and PS42; (40) PS3, PS4, PS12, PS19 and PS42; (41) PS3, PS12, PS19, PS37, and PS42; (42) PSI, PS3. PS12. PS16 and PS42; (43) PS3, PS4, PS12, PS16 and PS42; (44) PSI, PS4, PS12. PS19 and PS42; (45) PSI, PS3, PS12, PS26 and PS42; (46) PSI, PS4, PS12, PS26 and PS42; (47) PS3, PS4, PS12, PS26, and PS42; (48) PSI, PS12, PS16, PS37, and PS42; (49) PSI, PS3, PS4, PS19 and PS42; (50) PSI, PS12, PS19, PS37, and PS42; (51) PS3, PS12, PS16, PS37 and PS42; (52) PSI, PS3, PS10, PS12 and PS42; (53) PSI, PS4, PS10, PS12 and PS42; (54) PS3, PS4, PSIO, PS12, and PS42; (55) PSI, PS3, PS12, PS21 and PS42; (56) PSI, PS4, PS12, PS12 and PS42; (57) PSI, PS4, PS12, PS16 and PS42; (58) PS3, PS4, PS12, PS21, and PS42; (59) PS3, PS12, PS16, PS19 and
PS42; (60) PSI, PS3, PS12, PS20 and PS42; (61) PSI, PS4, PS12, PS20 and PS42; (62) PS3, PS4, PS12, PS20, and PS42; (63) PS3, PS12, PS19, PS26 and PS42; (64) PS3, PS10, PS12, PS19. and PS42; (65) PSI, PS12, PS16, PS19, and PS42; (66) PS21 and PS39; (67) PS10 and PS39; (68) PS26 and PS39; (69) PS20 and PS39; (70) PS15, PS21 and PS39; (71) PS10, PS15 and PS39; (72) PS15, PS26 and PS39; (73) PS15, PS20 and PS39; (74) PS21, PS30 and PS39; (75) PS10, PS30 and PS39; (76) PS5, PS21 and PS39; (77) PS26, PS30 and PS39; (78) PS20, PS30 and PS39; (79) PS21, PS37 and PS39; (80) PS4, PS15 and PS39; (81) PS5, PS10 and PS39; (82) PS15, PS37 and PS39; (83) PS5, PS26 and PS39; (84) PS15, PS21 and PS39; (85) PS10, PS15 and PS39; (86) PS5, PS15, PS21 and PS39; (87) PS15, PS26, PS30 and PS39; (88) PS15, PS20, PS30 and PS39; (89) PS15, PS21, PS37 and PS39; (90) PS5, PS21, PS30 and PS39; (91) PS5, PS10, PS15 and PS39; (92) PS5, PS15, PS20 and PS39; (93) PS5, PS15, PS26 and PS39; (94) PS15, PS20, PS37 and PS39; (95) PS5, PS10, PS30 and PS39; (96) PS5, PS15, PS21 and PS39; (97) PS5, PS10, PS15, PS30 and PS39; (98) PS5, PS15, PS20, PS30 and PS39; (99) PS5, PS15, PS26, PS30 and PS39; (100) PS15, PS21, PS30, PS37 and PS39; (101) PS3 and PS42; (102) PSI, PS3 and PS42; (103) PSI, PS28 and PS42; (104) PS12, PS16 and PS42; (105) PS30 and PS39; (106) PS3 and PS39; (107) PS39; (108) PS28, PS30 and PS39; (109) PSI and PS39; (110) PS28, PS30 and PS42; (111) PS28 and PS39; (112) PS12, PS28, PS30 and PS42; (113) PS3, PS16 and PS42; (114) PSI, PS38 and PS42; (115) PS28 and PS42; (116) PSI, PS16 and PS42; (117) PS12, PS28 and PS42; (118) PS12, PS28, PS38 and PS42; (119)
PS3, PS38 and PS42; (120) PS16, PS30 and PS39; (121) PS3, PS16 and PS39; (122) PS16 and PS39; (123) PS16, PS28. PS30 and PS39; (124) PSI, PS16 and PS39; (125) PS16, PS28, and PS39; (126) PS28, PS38 and PS42; (127) PSI, PS16, PS38 and PS42; (128) PS16, PS28 and PS42; (129) a set of polymoiphic sites comprising a linked haplotype to any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; or (130) a set of polymoφhic sites comprising a substitute haplotype for any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; wherein the nucleotide position of each polymoiphic site corresponds to the following nucleotide position in SEQ ID NO:l: 1118 (PSI), 1773 (PS3), 1875 (PS4), 1911 (PS5), 1957 (PS6), 2087 (PS10), 2157 (PS12), 13384 (PS15), 13405 (PS16), 16200 (PS19), 17194 (PS20), 17273
(PS21), 20035 (PS26), 20047 (PS28), 20615 (PS30), 21944 (PS33), 22155 (PS35), 25705 (PS37), 25921 (PS38), 27882 (PS39), and 30618 (PS42).
2. The kit of claim 1, wherein the set of oligonucleotides is designed for identifying both alleles at each polymoiphic site of the selected set of polymoφhic sites.
3. The kit of claim 1, wherein the set of PSs comprises PS3, PS12 and PS42; PSI, PS12 and PS42; PS3 and PS42; PSI and PS42; PSI, PS3, PS12 and PS42; or PS39
4. The kit of claim 3, wherein the set of PS is PS3, PS12 and PS42.
5. The kit of claim 1, wherein the individual is Caucasian.
6. The kit of claim 1, which further comprises a manual with instructions for perfonning one or more reactions on a human nucleic acid sample to identify the allele(s) present in the individual at each polymoφhic site in the set of polymoφhic sites and determining if the individual has a statin response marker I or a statin response marker II based on the identified allele(s).
7. The kit of claim 1, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table
IC has Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
8. The kit of claim 7, wherein Δ2 for the linkage disequilibrium between the linked haplotype and any of haplotypes 201, 205, 209, 214, 225, or 288 in Table IB is at least 0.95.
9. The kit of claim 1, wherein the linkage disequilibrium between the allele at a substitute polymoiphic site in the substitute haplotype and the allele in a replaced polymoφhic site in any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC has Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
10. The kit of claim 9, wherein the replaced polymorphic site is in any of haplotypes 201, 205, 209, 214, 225, or 288 in Table IB and Δ2 is at least 0.95.
11. The kit of claim 1, wherein at least one of the oligonucleotides in the set of oligonucleotides is an allele-specific oligonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:4, 6-9, 13, 15, 18-19, 22-23, 29, 31, 33, 36, 38, 40-42, and 45, the complements of SEQ ID NOS:4, 6 7-9, 13, 15, 18-19, 22-23, 29, 31, 33, 36, 38, 40-42, and 45, and SEQ ID NOS:48,
49, 52-59, 66, 67. 70, 71, 76-79, 84-89, 98, 99, 102, 103, 106, 107, 112, 113, 116, 117, 120, 121- 125, 130-131.
12. The kit of claim 1 1, wherein the set of polymoiphic sites is PSS, PS12, and PS42 and the set of oligonucleotides comprises first, second and third allele-specific oligonucleotide (ASO) probes, wherein the first ASO probe comprises SEQ ID NO:6, or its complement, and S in SEQ ID NO:6 is guanine; the second ASO probe comprises SEQ ID NO: 15, or its complement, and Y in SEQ ID NO: 15 is cytosine, and the third ASO probe comprises SEQ ID NO:45, or its complement, and Y in SEQ ED NO:45 is cytosine.
13. The kit of claim 1, wherein at least one of the oligonucleotides in the set of oligonucleotides is a primer extension oligonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 136- 137, 140-147, 154-155, 158-159, 164-167, 172-177, 186-187, 190- 191, 194-195, 200-201, 204-205, 208-213, 218-219.
14. The kit of claim 13, wherein the set of polymoiphic sites is PS3, PS12 and PS42 and the set of oligonucleotides comprises first, second and third primer-extension oligonucleotides, wherein the first primer extension oligonucleotide comprises SEQ ID NO: 140 or 141 , the second primer extension oligonucleotide comprises SEQ ID NO: 158 or 159 and the third primer extension oligonucleotide comprises SEQ ID NO:218 or 219.
15. A method for determining whether an individual has a statin response marker I or a statin response marker π, the method comprising deteπnining the copy number in the individual of a haplotype selected from the group consisting of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; a linked haplotype to any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101-194 in Table 1A. haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and wherein if the selected haplotype is one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB, a linked haplotype to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute haplotype for any one of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, then the individual has a statin response marker I if the individual has at least one copy of the selected haplotype and a statin response marker II if the individual has zero copy of the selected haplotype; and if the selected haplotype is one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, then the individual has a statin response marker I if the individual has zero or one copy of the selected haplotype and a statin response marker II if the individual has two copies of the selected haplotype.
16. The method of claim 15, wherein the individual is a candidate for treatment with a statin.
17. The method of claim 15, wherein the detennining step comprises genotyping each polymoφhic site hi a set of polymoiphic sites comprising the selected haplotype; and using the results of the genotyping step to identify for the set of polymorphic sites the haplotype pair present in the individual.
18. The method of claim 15, wherein the deteπnining step comprises consulting a data repository that provides information on the copy number present in the individual for the selected haplotype.
19. The method of claim 18, wherein the data repositoiy is the individual's medical records or a medical data card.
20. The method of claim 15, wherein the selected haplotype is one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
21. The method of claim 20, wherein the selected haplotype is haplotype 201, 205, or 209 in Table IB.
22. The method of claim 21, wherein the selected haplotype is haplotype 201.
23. The method of claim 15, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
24. The method of claim 23, wherein Δ2 for the linkage disequilibrium between the linked haplotype and haplotype 201, 205, 209, 214, 225, or 288 in Table IB is at least 0.95.
25. The method of claim 15, wherein the linkage disequilibrium between the allele at a substitute polymoiphic site in the substitute haplotype and the allele at a replaced polymoφhic site in any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
26. The method of claim 25, wherein the replaced polymoiphic site is in haplotype 201, 205, 209, 214, 225, or 288 in Table IB and Δ2 is at least 0.95.
27. The method of claim 15, wherein the individual is Caucasian.
28. A method for assigning an individual to a first or second statin response marker group, the method comprising: detennining the copy number in the individual of a haplotype selected from the group consisthig of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; a linked haplotype to any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in
Table IB and haplotypes 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and if the selected haplotype is one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-
463 in Table IB, then assigning the individual to the first statin response marker group if the individual has at least one copy of the selected haplotype and to the second statin response marker group if the individual has zero copy of the selected haplotype; if the selected haplotype is one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotjpe for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, then assigning the individual to the first statin response marker group if the individual has zero or one copy of the selected haplotype and to the second statin response marker group if the individual has two copies of the selected haplotype.
29. The method of claim 28, wherein the determining step comprises: genotyping each polymoφhic site hi a set of polymoφhic sites comprising the selected haplotype; and using the results of the genotyping step to identify for the set of polymoiphic sites the haplotype pair present in the individual.
30. The method of claim 28, wherein the deteπnining step comprises consulting a data repository that provides information on the copy number present in the individual for the selected haplotype.
31. The method of claim 30, wherein the data repositoiy is the individual's medical records or a medical data card.
32. The method of claim 28, wherein the selected haplotype is one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
33. The method of claim 28, wherein the individual is Caucasian.
34. The method of claim 28, wherein the individual is diagnosed as having Type Ila or Type lib hypercholesterolemia.
35. The method of claim 28, wherein the individual is a candidate for treatment with a statin.
36. The method of claim 28,wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
37. The method of claim 36, wherein Δ2 for the linkage disequilibrium between the linked haplotype and haplotypes 201, 205. 209, 214, 225, or 288 in Table IB is at least 0.95.
38. The method of claim 28, wherein the linkage disequilibrium between the allele at a substitute polymoφhic site in the substitute haplotype and the allele at a replaced polymoφhic site in any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 hi Table IB and haplotypes 501-515 in
Table IC has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
39. The method of claim 38, wherein the replaced polymoφhic site is in haplotypes 201, 205, 209, 214, 225, or 288 in Table IB and Δ2 is at least 0.95.
40. A method of selecting a statin therapy to provide an optimal High Density Lipoprotein Cholesterol (HDLC) response in an individual, the method comprising: determining whether the individual has a statin response marker I or a statin response marker II; and selecting a statin therapy based on the results of the detennining step.
41. The method of claim 40, wherein if the individual has a statin response marker II, then the selected statin therapy comprises any dose of atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid and if the individual has a statin response marker I, then the selected statin therapy comprises a low dose of atoiΥastatin or a phannaceutically acceptable salt of atorvastatin acid, a higher dose of atorvastatin or a phannaceutically acceptable salt of atorvastatin acid in conjunction with an HDLC-modulating therapy, or another statin.
42. The method of claim 40, wherein the detennining step comprises: identifying the copy number present in the individual of a haplotype selected from the group consisting of: haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; a linked haplotype to any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101-194 in
Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC. wherein if the selected haplotype is one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1A and haplotypes 201-463 in Table IB, then the individual has a statin response marker I if the individual has at least one copy of the selected marker and a statin response marker II if the individual has zero copy of the selected marker; and if the selected haplotype is one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, then the mdividual has a statin response marker I if the individual has zero or one copy of the selected haplotype and a statin response marker II if the individual has two copies of the selected haplotype.
43. The method of claim 42, wherein the selected haplotype is haplotype 201, 205, or 209 in Table IB.
44. The method of claim 40, wherein the determining step comprises consulting a data repository that states whether the individual has a statin response marker I or a statin response marker II.
45. The method of claim 40, wherein the individual is Caucasian.
46. The method of claims 40, wherein the individual has been diagnosed as having Type Ila or lib hypercholesterolemia.
47. A method for predicting an individual's High Density Lipoprotein Cholesterol (HDLC) response to treatment with a statin, the method comprising: detennining whether the individual has a statin response marker I or a statin response marker II; and making a response prediction based on the results of the detennining step; wherein the statin is atorvastatin or a phannaceutically acceptable salt of atorvastatin acid.
48. The method of claim 47, wherein if the individual is detennined to have a statin response marker I, then the response prediction is that the individual will experience an unfavorable HDLC response if treated with a statin dose comparable to a dose of atorvastatin calcium greater than about 10 mg/day; and if the individual is detennined to have a statin response marker II, then the response prediction is that the individual will experience a favorable HDLC response if treated with a statin dose comparable to a dose of atorvastatin calcium ranging from about 10 to about 80 mg/day.
49. The method of claim 47, wherein the statin is atorvastatin calcium.
50. The method of claim 47, wherein the determining step comprises consulting a data repository that states whether the individual has a statm response marker I or a statin response marker II.
51. The method of claim 50, wherein the data repository is the individual's medical records or a medical data card.
52. The method of claim 47, wherein the deteπnining step comprises: identifying the copy number present in the individual of a haplotype selected from the group consisting of: haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; a linked haplotype to any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; and a substitute haplotype for any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC; wherein if the selected haplotype is one of haplotypes 101-159 in Table 1A and haplotypes 201- 463 in Table IB, a linked marker to any one of haplotypes 101-159 in Table 1A and haplotypes 201-
463 in Table IB, or a substitute marker for any one of haplotypes 101-159 in Table 1 A and haplotypes 201-463 in Table IB, then assigning the individual to the first statin response marker group if the individual has at least one copy of the selected haplotype and to the second statm response marker group if the mdividual has zero copy of the selected haplotype; and if the selected haplotype is one of haplotypes 160-194 in Table 1A and haplotypes 501-515 in Table IC, a linked haplotype to any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 hi Table IC, or a substitute haplotype for any one of haplotypes 160-194 in Table 1A and haplotypes 501-515 hi Table IC, then assigning the individual to the first statin response marker group if the individual has zero or one copy of the selected haplotype and to the second statin response marker group if the individual has two copies of the selected haplotype.
53. The method of claim 52, wherein the selected haplotype is one of haplotypes 201, 205, 209, 214, 225, or 288 in Table IB.
54. The method of claim 52,wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
55. The method of claim 52, wherein the linkage disequilibrium between the allele at a substitute polymoφhic site in the substitute haplotpe and the allele at a replaced polymoφhic site in any one of haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least
0.90, at least 0.95, and 1.0.
56. The method of claim 47, wherein the individual is Caucasian.
57. The method of claims 47, wherein the individual has been diagnosed as having Type Ila or ub hypercholesterolemia.
58. An article of manufacture, comprising a phaπnaceutical foπnulation and at least one indicium identifying a population for whom the pharmaceutical formulation is indicated, wherein the phannaceutical formulation comprises a statin as at least one active ingredient and the identified population is partially or wholly defined by having a statin response marker I, wherein a trial population having the statin response marker I exhibits a better HDLC response to the phannaceutical foπnulation than to treatment with atorvastatin or a phannaceutically acceptable salt of atorvastatin acid.
59. The article of claim 58, wherein marketing of the phannaceutical foπnulation is regulated and the indicium comprises the approved label for the phannaceutical foπnulation, wherein the approved label states that the phaπnaceutical fomiulation provides a better HDLC response in a population having the statin response marker I.
60. The article of claim 59, wherein the approved label describes the mean percent change in HDLC expected for the identified population.
61. The article of claim 58, wherein marketing of the phannaceutical foπnulation is regulated and the indicium comprises the approved label for the pharmaceutical fonmulation, wherein the approved label states that the phamiaceutical foπnulation is indicated for mdividuals having the statin response marker I.
62. The article of claim 58, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, or a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
63. The article of claim 62, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
64. The article of claim 62. wherein the linkage disequilibrium between the allele at a substitute polymoiphic site in the substitute haplotype and the allele at a replaced polymoiphic site in any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
65. The article of claim 58, wherein the statin is present in the phannaceutical formulation at an amount effective to reduce LDLC levels.
66. The article of claim 58, wherein the statin is simvastatin, a pharmaceutically acceptable salt of simvastatin acid, lovastatin, a pharmaceutically acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a pharmaceutically acceptable salt of rosuvastatin acid, pravastatin, or a phannaceutically acceptable salt of pravastatin acid.
67. The article of claim 66, wherein the statm is simvastatin or pravastatin sodium.
68. The article of claim 58, wherein the trial population having the statin response marker I exhibits a better HDLC response to the phannaceutical formulation than to treatment with a high dose of atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid.
69. The article of claim 68, wherein the trial population having the statm response marker I exhibits a better HDLC response to the pharmaceutical foπnulation than to treatment with about 80 mg/day of atorvastatin calcium.
70. An article of manufacture, comprising packaging material and a pharmaceutical foπnulation contained within said packaging material, wherein the pharmaceutical foπnulation comprises a statin as at least one separate active ingredient, and said packaging material comprises an approved label which states that the phaπnaceutical formulation is indicated for a population partly or wholly defined by having a statm response marker I, wherein a trial population having the statin response marker I exhibits a better HDLC response to the phannaceutical fonnulation than to treatment with atorvastatin or a phannaceutically acceptable salt of aton'astatin acid.
71. The article of claim 70, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, and a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
72. The article of claim 70, wherein the approved label describes the mean percent change in HDLC expected for the identified population.
73. The article of claim 70, wherein the approved label further states that individuals having the statin response marker I may be identified using a specified genetic test.
74. The article of claim 70, wherein the statin is simvastatm, a phannaceutically acceptable salt of simvastatin acid, lovastatin, a phannaceutically acceptable salt of lovastatin, fluvastatin, a pharmaceutically acceptable salt of fluvastatin acid, rosuvastatin, a phannaceutically acceptable salt of rosuvastatin acid, pravastatin, or a phannaceutically acceptable salt of pravastatin acid.
75. A method for-manufacturing a drug product comprising combining in a package a pharmaceutical formulation comprising a statin as at least one active ingredient and a label which states that the pharmaceutical fomiulation is indicated for treating a population partially or wholly defined by having a statin response marker I, wherein a trial population having the statin response marker I exhibits a better HDLC response to the pharmaceutical formulation than to treatment with atorvastatin or a phannaceutically acceptable salt of atorvastatin acid.
76. The method of claim 75, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, and a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
77. The method of claim 75, wherein the label further states that the indicated population is further defined as being Caucasian.
78. A method of seeking regulatory approval for marketing a pharmaceutical formulation for treating a disease or condition in a population partially or wholly defined by having a statin response marker I, the method comprising conducting at least one clinical trial which comprises first and second treatment groups of patients having the disease or condition, wherein each patient has a statin response marker I, and wherein each patient in the first treatment group is treated with the phannaceutical formulation and each patient in the second treatment group is treated with atorvastatin, a phannaceutically acceptable salt of atorvastatin, or a derivative thereof; demonstrating that the second treatment group exhibits a mean per cent change hi High Density Lipoprotein Cholesterol (HDLC) that is worse than the mean per cent change in HDLC exhibited by the fust treatment group; and filing with a regulatory agency an application for marketing approval of the pharmaceutical fonnulation with a label stating that the pharmaceutical formulation is indicated for treating the disease or condition in patients having the statin response marker I.
79. The method of claim 78, wherein the phannaceutically acceptable salt of atorvastatin comprises atorvastatin calcium.
80. The method of claim 78, wherein the disease or condition is a cardiovascular disorder and the statin is present in the phaniiaceutical fomiulation in an amount effective for lowering LDLC levels in each of the first and second treatment groups.
81. The method of claim 78, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, and a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
82. The method of claim 81, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
83. The method of 81, wherein the linkage disequilibrium between the allele at a substitute polymoiphic site in the substitute haplotype and the allele at a replaced polymoφhic site in any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
84. The method of claim 78. wherein the individual is Caucasian.
85. The method of claim 78, wherein the regulatory agency is the United States Food and Drug Administration or the European Agency for the Evaluation of Medicinal Products.
86. An article of manufacture, comprising a phamiaceutical fonnulation and at least one indicium identifying a population for whom the pharmaceutical fonnulation is indicated, wherein the phaπnaceutical foπnulation comprises as separate active ingredients atorvastatin or a phannaceutically acceptable salt of atorvastatin acid and an HDL cholesterol (HDLC) modulating agent. wherein the identified population is partially or wholly defined by having a statin response marker I, and wherein a trial population having the statin response marker I exhibits a worse mean HDLC response to treatment with only atorvastatin or a phannaceutically acceptable salt of atorvastatin acid than a trial population lacking the statin response marker I.
87. The article of claim 86, wherein the HDLC modulating agent is present in the pharmaceutical formulation in an amount effective to increase the mean HDLC levels in a population having the statin response marker I.
88. The article of claim 86, wherein marketing of the phannaceutical foπnulation is regulated and the indicium comprises the approved label for the phannaceutical fonnulation, wherein the approved label states that the phannaceutical fonnulation is indicated for individuals having the statin response marker I.
89. The article of claim 86, wherein marketing of the phaπnaceutical formulation is regulated and the indicium comprises the approved label for the phaπnaceutical formulation, wherein the label states that the phannaceutical fonnulation provides a better HDLC response in a population having the statin response marker I than a phannaceutical formulation comprising only atoiΥastatin or a pharmaceutically acceptable salt of atorvastatin as the active ingredient.
90. The article of claim 86, wherein marketing of the phannaceutical fomiulation is regulated and the indicium comprises the approved label for the pharmaceutical formulation, wherein the label states that the phannaceutical fonnulation is indicated for individuals who score positive for having the statm response marker I on a specified genetic test.
91. The article of claim 90, wherein the label describes the mean percent change in HDLC expected for the identified population.
92. The article of any of claims 86, wherein the HDLC modulating agent is niacin or a fibrate.
93. The article of claim 86, wherein the phamiaceutical formulation comprises atorvastatin calcium.
94. The article of claim 93, wherein the atoiΥastatin calcium is present in the pharmaceutical fonnulation at an amount effective to reduce mean LDL cholesterol (LDLC) levels.
95. The article of claim 94, wherein the amount effective to reduce mean LDLC ranges from about 10 to about 80 mg.
96. The article of claim 86, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, and a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
97. The article of claim 96, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
98. The article of claim 96, wherein the linkage disequilibrium between the allele at a substitute polymoiphic site in the substitute haplotype and the allele at a replaced polymoφhic site in any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
99. The article of clahn 86, wherein the phamiaceutical formulation is a sustained release foπnulation.
100. The article of claim 86, wherein the article further comprises an additional indicium identifying the indicated population.
101. The article of claim 100, wherein the phaπnaceutical formulation is a tablet or capsule and the additional indicium comprises the color or shape of the tablet or capsule.
102. The article of claim 100 wherein the pharmaceutical formulation is a tablet or capsule and the additional indicium comprises a symbol stamped on the tablet or capsule.
103. The article of claim 86, wherein the identified population is further defined as being Caucasian.
104. An article of manufacture, comprising packaging material and a pharmaceutical foπnulation contained within said packaging material, wherein the phaπnaceutical fonnulation comprises as separate active ingredients atoiYastatin or a phannaceutically acceptable salt of atoiYastatin acid and an HDL cholesterol (HDLC) modulating agent, and said packaging material comprises a label which states that the pharmaceutical fomiulation is indicated for a population partly or wholly defined by having a statm response marker I, and wherein a trial population having the statin response marker I exhibits a worse mean HDLC response to treatment with only atorvastatin or a phannaceutically acceptable salt of atoiYastatin acid than a trial population lacking the statin response marker I
105. The article of claim 104, wherein the HDLC modulating agent is present in the phamiaceutical fonnulation in an amount effective to increase the mean HDLC levels in a population havmg the statin response marker I.
106. The article of claim 104, wherein the statm response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, and a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
107. The article of claim 106, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
108. The article of claim 106, wherein the linkage disequilibrium between the allele at a substitute polymoφhic site in the substitute haplotype and the allele at a replaced polymoφhic site in any one of haplotypes 201, 205, 209. 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
109. The article of clahn 104, wherein atorvastatin calcium is present in an amount ranging from about 10 to about 80 mg.
110. The article of claim 104, wherein the label further states that a genetic test can be used to identify individuals having the statin response marker I.
111. A method for-manufacturing a drug product comprising combining in a package a pharmaceutical fonnulation comprising as separate active ingredients atorvastatin or a phannaceutically acceptable salt of atorvastatin acid and an HDL cholesterol (HDLC) modulating agent, and a label which states that the phamiaceutical fonnulation is indicated for treating a population partially or wholly defined by having a statin response marker I, wherein a trial population having the statin response marker I exhibits a worse mean HDLC response to treatment with only atorvastatin or a pharmaceutically acceptable salt of atorvastatin acid than a trial population lacking the statin response marker I.
112. The method of claim 111, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, or a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
113. The method of claim 112, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 201, 205, 20,9, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
114. The method of claim 112, wherein the linkage disequilibrium between the allele at a substitute polymorphic site in the substitute haplotype and the allele at a replaced polymoiphic site in any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
115. The method of claim 111, wherein the label further states that the indicated population is further defined as being Caucasian.
116. A method for marketing a drug product comprising a statm as at least one active ingredient for treating a disease or condition in a population partially or wholly defined by having a statin response marker I, the method comprising promoting to a target audience the use of the drug product for treating a disorder or condition in individuals having a statin response marker I; wherein a trial population having the statin response marker I exhibits a worse mean HDL cholesterol (HDLC) response to atorvastatin or a phannaceutically acceptable salt of atoiYastatin acid at high dose than to the drug product.
117. The method of claim 116, wherein the statin response marker I is at least one copy of any of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, a linked haplotype to any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB, or a substitute haplotype for any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB.
118. The method of clahn 117, wherein the linkage disequilibrium between the linked haplotype and any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
119. The method of claim 117, wherein the linkage disequilibrium between the allele at a substitute polymoiphic site in the substitute haplotype and the allele at a replaced polymoiphic site in any one of haplotypes 201, 205, 209, 214, 225, and 288 in Table IB has a Δ2 selected from the group consisting of at least 0.75, least 0.80, at least 0.85, at least 0.90, at least 0.95, and 1.0.
120. The method of claim 116, wherein the disorder or condition is dyslipidemia, hyperlipidemia, or hypercholesterolemia.
121. The method of clahn 116, wherein the target audience is selected from physicians, pharmacists, patients with the disorder, and a government regulator}' agency.
122. The method of claim 116, wherein the target audience comprises physicians and the promoting step comprises providing dosage and perfonnance infonnation from one or more clinical trials of individuals having the statin response marker I.
123. The method of claim 122, wherein the promoting step further comprises providing physicians with a supply of free diagnostic kits for identifying patients who have the statin response marker I.
124. The method of claim 116, wherein the individuals are Caucasian.
125. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
(a) a first nucleotide sequence which comprises an integrin, beta 3(ITGB3) isogene encoding a ITGB3 polypeptide, wherein the ITGB3 isogene is selected from the group consisting of isogenes 1- 38 and 40 - 98 defined by a correspondingly numbered haplotype represented in
Table 5, wherein each of the isogenes comprises nucleotides 1000-2235, 4256-4716, 13179- 13723, 14235-14858, 16126-16619, 16930-17414, 19241-19644, 19748-20177, 20537- 21009, 21731-22412, 24385-24930, 25559-26029, 27822-28255, 30265-30754, and 31300- 31718 of SEQ ID NO:l, except where substituted by the sequence of alleles for the coπespondingly numbered haplotype at the polymorphic sites whose nucleotide positions in
SEQ ID NO: 1 are set forth in Table 5; and
(b) a second nucleotide sequence which is complementary to the first nucleotide sequence.
126. The isolated polynucleotide of claim 125, wherein the ITGB3 isogene of the first nucleotide sequence is selected from the group consisting of isogenes 3, 6, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 38, 43, 46, 48, 49, 55, 56, 57, 58, 59, 93, 94, 96, and 97.
127. The isolated polynucleotide of claim 125, wherein the ITGB3 isogene of the first nucleotide sequence is selected from the isogenes defined by a haplotype selected from the group consisting of: haplotypes 101-194 in Table 1A, haplotypes 201-463 in Table IB and haplotypes 501-515 in Table IC.
128. The isolated polynucleotide of claim 127, wherein the selected haplotype is haplotypes 201, 205, 209, 214, 225, or 288 hi Table IB.
129. The isolated polynucleotide of clahn 125, wheiein the ITGB3 isogene encodes the human
ITGB3 polypeptide sequence of SEQ ID NO:3 and is selected from the group consisting of isogenes 1,3, 5-10, 12-17. 19, 25-38, 40-44, 46-49, 51-57, 59-61, 64-65, 70, 72-93 and 95-98.
130. A recombinant nonhuman organism transfoπned or transfected with the isolated polynucleotide of claim 125; wherein the organism expresses an ITGB3 polypeptide encoded by the selected ITGB3 isogene.
131. An isolated fragment of an integrin, beta 3(ITGB3) isogene, wherein the fragment comprises one or more polymoφhisms selected from the group consisting of thymine at PSI, guanine at PS2, cytosine at PS3, thymine at PS4, cytosine at PS5. adenine at PS6, thymine at PS7, thymine at PS8, guanine at PS9, adenine at PS10, adenine at PSI 1. thymine at PS 12, adenine at PS 13, guanine at PS16, adenine at PS18, thymine at PS19, guanine at PS21, guanine at PS22, cytosine at PS23, cytosine at PS24, thymine at PS25, adenine at PS26, adenine at PS27, thymine at PS29, adenine at PS30, cytosine at PS31, guanine at PS32, adenine at PS33, adenine at PS35, cytosine at PS37, thymine at PS38, cytosine at PS39, adenine at PS40, thymine at PS41, thymine at PS42, guanine at PS43 and guanine at PS44, wherein the selected polymoiphism has the following position in SEQ ID:1: 1118 (PSI), 1202 (PS2), 1773 (PS3), 1875 (PS4), 1911 (PS5), 1957 (PS6), 1974 (PS7), 1975
(PSS), 2048 (PS9), 2087 (PS10), 2117 (PS11), 2157 (PS12), 13273 (PS13). 13405 (PS16), 13690 (PS18), 16200 (PS19), 17273 (PS21), 19357 (PS22), 19610 (PS23), 19877 (PS24). 20034 (PS25), 20035 (PS26). 20037 (PS27), 20105 (PS29), 20615 (PS30), 20704 (PS31), 20889 (PS32), 21944 (PS33), 22155 (PS35), 25705 (PS37), 25921 (PS38), 27882 (PS39 , 28127 (PS40), 30516 (PS41), 30618 (PS42), 30662 (PS43) and 30729 (PS44), and wherein the fragment is at least 15 and up to about 1000 nucleotides in length.
132. A genome anthology for the integrin, beta 3(ITGB3) gene which comprises two or more ITGB3 isogenes selected from the group consisting of isogenes 1-98, wherein each of the selected isogenes is defined by a coιτespondingly numbered haplotype represented in Table 5, and wherein each of the isogenes comprises nucleotides 1000-2235, 4256-4716, 13179-13723, 14235-14858, 16126-16619,
16930-17414, 19241-19644. 19748-20177, 20537-21009, 21731-22412, 24385-24930, 25559- 26029, 27822-28255, 30265-30754, and 31300-31718 of SEQ ID NO:l except where substituted by the sequence of alleles for the correspondingly numbered haplotype at each of the polymoφhic sites whose nucleotide positions in SEQ ID NO: 1 are set forth in Table 5.
133. The genome anthology of method 132, wherein each of the ITGB3 isogenes is selected from the group consisting of isogenes 3, 6, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 38, 39, 43, 46, 48, 49, 55, 56, 57, 58, 59, 93, 94, 96, and 97.
134. The genome anthology of method 133, comprising the isogenes defined by a haplotype selected from the group consisting of haplotypes 201, 205, 209, 214, 225, or 288 in Table IB.
135. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of
(a) a first nucleotide sequence which comprises an ITGB3 coding sequence encoding a human ITGB3 polypeptide, wherein the coding sequence is selected from the group consisting of
ITGB3 coding sequences A, B, C, D, E, F, G, H, I, K, J, L, M, N, O, P and Q represented in Table 8, wherein the selected coding sequence comprises SEQ ID NO:2, except where substituted by the corresponding sequence of alleles for the selected coding sequence at the set of polymoiphic sites presented in Table 8, wherein each polymorphic site (PS) has the following nucleotide position in SEQ ID NO:2: PS6 (40), PS7(57), PS8 (58), PS15 (176), PS16 (197),
PS17 (342), PS20 (882), PS31.(1143), PS33 (1333), PS34 (1533), PS35 (1544), PS36 (1545) and PS41 (2208); and
(b) a second nucleotide sequence which is complementary to the first nucleotide sequence.
136. The isolated polynucleotide of claim 135, wherein the nucleotide sequence comprises the ITGB3 coding sequence encoding the human ITGB3 polypeptide with a sequence of SEQ ED NO:3 and the coding sequence is selected from the group consisting of ITGB3 coding sequences C, D, E, H, O, P and Q represented in Table 8.
137. A recombinant nonhuman organism transfonned or transfected with the isolated polynucleotide of claim 135, wherein the organism expresses a integrin, beta 3(ITGB3) polypeptide encoded by the selected coding sequence.
138. An isolated fragment of a ITGB3 coding sequence, wherein the fragment is at least 15 and up to about 1000 nucleotides, and wherein the fragment comprises one or more polymoφhisms selected from the group consisting of adenine at a position corresponding to nucleotide 40, thymine at a position coiresponding to nucleotide 57, thymine at a position corresponding to nucleotide 58, guanine at a position conesponding to nucleotide 197, cytosine at a position corresponding to nucleotide 1143, adenine at a position conesponding to nucleotide 1333, adenine at a position conesponding to nucleotide 1544, and thymine at a position conesponding to nucleotide 2208 hi SEQ ID NO:2.
139. A method for haplotyping the integrin, beta 3 (ITGB3) gene of an individual, which comprises obtaining a genotype for each polymoφhic site in a set of two or more polymoφhic sites selected from the group consisting of PS1-PS44, wherein the nucleotide position of each polymoφhic site coπesponds to the following nucleotide position in SEQ ID NO: 1 : 1118 (PS 1), 1202 (PS2), 1773 (PS3), 1875 (PS4), 1911 (PS5), 1957 (PS6), 1974 (PS7), 1975 (PSS), 2048 (PS9), 2087 (PSIO), 2117 (PSI 1), 2157 (PS 12), 13273 (PS13), 13334 (PS14), 13384 (PS15), 13405 (PS16), 13550 (PS17), 13690 (PS18), 16200 (PS19), 17194 (PS20), 17273 (PS21), 19357 (PS22), 19610 (PS23), 19877 (PS24), 20034 (PS25). 20035 (PS26), 20037 (PS27), 20047 (PS28), 20105 (PS29), 20615 (PS30), 20704 (PS31), 20889 (PS32), 21944 (PS33), 22144 (PS34), 22155 (PS35), 22156 (PS36), 25705 (PS37), 25921 (PS38), 27882 (PS39), 28127 (PS40), 30516 (PS41), 30618
(PS42), 30662 (PS43) and 30729 (PS44); and using the obtained genotypes to detemiine the phased sequence of nucleotides present at the selected set of polymoφhic sites in at least one copy of the individual's ITGB3 gene.
140. The method of claim 139, wherein at least one PS in the set of polymoiphic sites is selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PSS, PS9, PS10, PSI 1, PS12, PS13,
PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44.
141. The method of claim 139, wherein the set of PSs is selected from the group consist ig of: PSI, PS3. PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42.
142. The method of claim 141, wherein the set of PSs is selected from the group consisting of: PSI, PS3, PS 12, PS39, and PS42.
143. A method for assigning a haplotype pah- for the integrin, beta 3(ITGB3) gene to an individual comprising: (a) obtaining an ITGB3 genotype for the individual, wherein the genotype comprises the nucleotide pair at two or more polymoiphic sites (PS) selected from the group consisting of PSI to PS44, wherein the nucleotide position of each polymoφhic site corresponds to the following nucleotide position in SEQ ID NO:l: 1118 (PSI), 1202 (PS2), 1773 (PS3), 1875 (PS4), 191 1 (PS5). 1957 (PS6), 1974 (PS7), 1975 (PSS), 2048 (PS9), 2087 (PS10), 2117 (PSI 1), 2157 (PS12), 13273 (PS13), 13334 (PS14), 13384 (PS15), 13405 (PS16), 13550 (PS17), 13690
(PS18), 16200 (PS19), 17194 (PS20), 17273 (PS21), 19357 (PS22), 19610 (PS23), 19877 (PS24), 20034 (PS25), 20035 (PS26), 20037 (PS27), 20047 (PS28), 20105 (PS29), 20615 (PS30), 20704 (PS31), 20889 (PS32), 21944 (PS33), 22144 (PS34), 22155 (PS35), 22156 (PS36). 25705 (PS37), 25921 (PS38), 27882 (PS39), 28127 (PS40), 30516 (PS41), 30618 (PS42), 30662 (PS43) and 30729 (PS44);
(b) comparing the obtained genotypes to haplotype pair data for the ITGB3 gene, wherein the haplotype pair data comprise the haplotype pan- data set forth in Table 4; and
(c) assigning to the individual a haplotype pair that is consistent with the genotype of the individual and with the haplotype pair data.
144. The method of claim 143, wherein the two or more PS are selected from the group consisting of: PSI, PS2, PS3, PS4, PS5, PS6, PS7, PSS, PS9, PS10, PSI 1, PS12, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44.
145. The method of claim 143, wherein the two or more PS are selected from the group consisting of: PSI, PS3, PS4, PS5, PS6, PSIO, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42.
146. The method of claim 145, wherein the two or more PS are selected from the group consisting of PS1, PS3, PS12, PS39 and PS42.
147. A method for genotyping the integrin, beta 3(ITGB3) gene of an individual, comprising detenninmg for the two copies of the ITGB3 gene present in the individual the identity of the nucleotide pah- at one or more polymoiphic sites (PS) selected from the group consisting of PS 1 , PS2, PS3, PS4, PS5, PS6, PS7, PSS, PS9, PSIO, PSI 1, PS12, PS13, PS16, PS1S, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40,
PS41, PS42, PS43 and PS44, wherein the nucleotide position of each polymorphic site coπesponds to the following nucleotide position in SEQ ID NO: 1: 1118 (PSI), 1202 (PS2), 1773 (PS3), 1875 (PS4), 1911 (PS5), 1957 (PS6), 1974 (PS7), 1975 (PSS), 2048 (PS9), 2087 (PS10), 21 17 (PSI 1), 2157 (PS12), 13273 (PS13), 13405 (PS16), 13690 (PS18), 16200 (PS19), 17273 (PS21), 19357 (PS22), 19610 (PS23), 19877 (PS24), 20034 (PS25), 20035 (PS26), 20037 (PS27), 20105 (PS29),
20615 (PS30), 20704 (PS31), 20889 (PS32), 21944 (PS33), 22155 (PS35), 25705 (PS37), 25921 (PS38), 27882 (PS39), 28127 (PS40), 30516 (PS41), 30618 (PS42), 30662 (PS43) and 30729 (PS44).
148. The method of claim 147, wherein the one or more PS is selected from the group consisting of: PSI, PS3, PS4, PS5, PS6, PS10, PS12, PS16, PS19, PS21, PS26, PS30, PS33, PS35, PS37, PS38,
PS39 and PS42.
149. The method of claim 148, wherein the one or more PS is selected from the group consisting of: PSI, PS3, PS12, PS39, and PS42.
150. A method for identifying an association between a trait and at least one haplotype or haplotype pair of the integrin, beta 3(ITGB3) gene which comprises comparing the frequency of the haplotype or haplotype pah- in a population exhibiting the trait with the frequency of the haplotype or haplotype pair in a reference population, wherein the haplotype is selected from haplotypes 1-98 shown in Table 5 and wherein the haplotype pair is selected from the haplotype pairs shown in Table 4, wherein a different frequency of the haplotype or haplotype pair in the trait population than in the reference population indicates the trait is associated with the haplotype or haplotype pair.
151. The method of claim 150, wherein the haplotype is selected from the group consisting of haplotypes 3, 6, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34,'35, 38, 39, 43, 46, 48, 49, 55, 56, 57, 58, 59, 93, 94, 96, and 97 represented in Table 5.
152. A method for reducing the potential for bias a clinical trial of a candidate drug for treating a disease or condition predicted to be associated with ITGB3 activity, the method comprising detemiining which of the ITGB3 haplotypes in Table 5 or the ITGB3 haplotype pairs in Table 4 is present in each individual that is participating in the trial; and assigning each individual to a treatment group or a control group to produce an equal number of each of the determined ITGB3 haplotypes or haplotype pairs in the treatment group and the control group: 153. The method of claim 152, wherein the ITGB3 haplotypes are defined by the following set of polymoφhic sites: PSI, PS3, PS4, PS5, PS6, PSIO, PS12, PS15, PS16, PS19, PS20, PS21, PS26,
PS28, PS30, PS33, PS35, PS37, PS38, PS39 and PS42.
154. The method of claim 153, wherein the ITGB3 haplotypes are defined by a set of polymoφhic sites selected from the group consisting of: PS3, PS12 and PS42; PSI, PS12 and PS42; PS3 and
PS42; PSI and PS42; PSI, PS3, PS12 and PS42; or PS39.
155. An isolated polypeptide comprising a ITGB3 protein variant selected from the group consisting of protein variants A, B, C, D, E, F, and G represented in the table below and wherein the selected ITGB3 protein variant comprises SEQ ID NO:3, except where substituted by the corresponding sequence of amino acids whose positions and alleles are shown in the table below: Table 9. Amino Acids Present at Polymoφhic Sites Within the Observed ITGB3 Protein Sequences
Region PS PS Protein Variants of ITGB3 Examined(a) No.(b)Position(c)A B C D E F G
0-788 6 14 M V M V V V V
0-788 15 59 L P L P L L L
0-788 16 66 L L L R L L L
0-788 33 445 V V V V M M V
0-78S 35&36 515 R R R R R R Q
(a) Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO:3 of the regions sequenced; (b)PS = polymoiphic site; (c) Position of PS in SEQ ED NO:3.
156. An isolated monoclonal antibody specific for and immunoreactive with the selected ITGB3 protein variant comprising the isolated polypeptide of claim 155.
157. A method for screening for drugs targeting the selected ITGB3 protein variant comprising the isolated polypeptide of claim 155, the method comprising contacting the selected ITGB3 protein variant with a candidate agent and assaying for binding between the candidate agent and the selected ITGB3 protein variant.
158. An isolated fragment of an ITGB3 protein variant, wherein the fragment is at least 6 amino acids in length and comprises one or more variant amino acids selected from the group consisting of methionine at a position conesponding to amino acid position 14, arginine at a position conesponding to amino acid position 66, methionine at a position conesponding to amino acid position 445, and glutamine at a position conesponding to amino acid position 515 in SEQ DD
NO:3.
159. A method for screening for compounds targeting the ITGB3 protein to treat a condition or disease predicted to be associated with ITGB3 activity, the method comprising; determining the frequency of each of the ITGB3 haplotypes shown in Table 5 in a population having the disease; and if the frequency of the ITGB3 haplotype meets a desired cutoff frequency criterion, then screening for a compound that displays a desired agonist or antagonist activity for the ITGB3 isofonn defined by that haplotype:
160. A method for validating the ITGB3 protein as a candidate target for treating a medical condition predicted to be associated with ITGB3 activity, the method comprising: comparing the frequency of each of the ITGB3 haplotypes in Table 5 between first and second populations, wherein the first population is a group of individuals having the medical condition and the second population is a group of individuals lacking the medical condition; and making a decision whether to pursue ITGB3 as a target for treating the medical condition; wherein if at least one of the ITGB3 haplotypes is present in a frequency in the first population that is different from the frequency in the second population, then the decision is to pursue the ITGB3 protein as a target and if none of the ITGB3 haplotypes are seen in a different frequency, at a statistically significant level, between the first and second populations, then the decision is to not pursue the ITGB3 protein as a target.
161. An isolated oligonucleotide designed for detecting a polymoφhism in the integrin, beta 3 (ITGB3) gene at a polymoφhic site (PS) selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24,
PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44, wherein the oligonucleotide contains or is located one to several nucleotides downstream of the selected PS, wherein the oligonucleotide has a length of about 15 to about 100 nucleotides, and wherein the selected PS has the following nucleotide position in SEQ ED NO:l: 1118 (PSI), 1202 (PS2), 1773 (PS3), 1875 (PS4), 1911 (PS5), 1957 (PS6), 1974 (PS7), 1975 (PSS),
2048 (PS9), 2087 (PS10), 2117 (PS11), 2157 (PS12), 13273 (PS13), 13405 (PS16), 13690 (PS 18), 16200 (PS19), 17273 (PS21), 19357 (PS22), 19610 (PS23), 19877 (PS24), 20034 (PS25), 20035 (PS26), 20037 (PS27), 20105 (PS29), 20615 (PS30), 20704 (PS31), 20889 (PS32), 21944 (PS33), 22155 (PS35), 25705 (PS37), 25921 (PS38), 27882 (PS39), 28127 (PS40), 30516 (PS41), 30618 (PS42), 30662 (PS43) and 30729 (PS44).
162. The isolated oligonucleotide of claim 161, which is an allele-specific oligonucleotide that specifically hybridizes to an allele of the ITGB3 gene at a region containing the polymoiphic site.
163. The allele-specific oligonucleotide of claim 162, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:4-16, 19, 21-22, 24-30, 32-36, 38, 40-47, the complements of SEQ ID NOS:4-16, 19, 21-22, 24-30, 32-36, 38, 40-47, and SEQ ID NOS:48-73,
78-79, 82-85, 88-101, 104-113, 116-117, 120-135.
164. The isolated oligonucleotide of claim 161, which is a primer-extension oligonucleotide.
165. The primer-extension oligonucleotide of claim 164, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:136-161, 166-167, 170-173, 176-189, 192-201, 204-205, 208-223.
166. A kit for haplotyping the integrin, beta 3 (ITGB3) gene of an mdividual, the kit comprising a set of oligonucleotides designed for identifying at least one of the alleles at each polymoφhic site (PS) in a set of two or more polymoφhic sites (PSs), wherein the polymoφhic sites in the set of two br more PSs are selected from the group consisting of PSI to PS44, wherein the nucleotide position of each polymoφhic site conesponds to the following nucleotide position in SEQ ED NO: 1 : 1118 (PSI), 1202 (PS2), 1773 (PS3), 1875 (PS4), 1911 (PS5), 1957 (PS6), 1974 (PS7), 1975 (PS8), 2048 (PS9), 2087 (PS10), 2117 (PSI 1), 2157 (PS12), 13273 (PS13), 13334 (PS 14), 13384 (PS15), 13405 (PS16), 13550 (PS17), 13690 (PS 18), 16200 (PS19), 17194 (PS20), 17273 (PS21), 19357 (PS22),
19610 (PS23), 19877 (PS24), 20034 (PS25), 20035 (PS26), 20037 (PS27), 20047 (PS28), 20105 (PS29), 20615 (PS30), 20704 (PS31), 20889 (PS32), 21944 (PS33), 22144 (PS34), 22155 (PS35), 22156 (PS36), 25705 (PS37), 25921 (PS38), 27882 (PS39), 28127 (PS40), 30516 (PS41), 30618 (PS42), 30662 (PS43) and 30729 (PS44).
167. The kit of claim 166, wherein at least one PS in the set of two or more polymoiphic sites is selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12, PS13, PS16, PS18, PS19, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS29, PS30, PS31, PS32, PS33, PS35, PS37, PS38, PS39, PS40, PS41, PS42, PS43 and PS44.
168. The kit of claim 166, wherein the PSs are selected from the group consisting of: PSI, PSS, PS4, PS5, PS6, PS10, PS12, PS15, PS16, PS19, PS20, PS21, PS26, PS28, PS30, PS33, PS35, PS37,
PS38, PS39 and PS42
169. The kit of claim 168, wherein the PSs are selected from the group consisting of: PSI, PS3, PS 12, PS39, and PS42.
170. The kit of claim 169, wherein the set of oligonucleotides is designed for identifying both alleles at each polymoφhic site of the selected set of two or more polymoiphic sites.
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