WO2003091277A2 - Haplotypes du gene cetp - Google Patents

Haplotypes du gene cetp Download PDF

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Publication number
WO2003091277A2
WO2003091277A2 PCT/US2003/013288 US0313288W WO03091277A2 WO 2003091277 A2 WO2003091277 A2 WO 2003091277A2 US 0313288 W US0313288 W US 0313288W WO 03091277 A2 WO03091277 A2 WO 03091277A2
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cetp
seq
haplotype
guanine
group
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PCT/US2003/013288
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WO2003091277A3 (fr
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Alison E. Anastasio
Anne Chew
Amir Kazemi
Michael Lachowicz
Helen H. Lee
Katie E. Parks
Nathan Petersen
Eileen Rounds
Elizabeth Ann Sausker
Charles Tirrell
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2003239189A priority Critical patent/AU2003239189A1/en
Publication of WO2003091277A2 publication Critical patent/WO2003091277A2/fr
Publication of WO2003091277A3 publication Critical patent/WO2003091277A3/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q2600/156—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 genetic variants of the human cholesteryl ester transfer protein (CETP) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • CETP human cholesteryl ester transfer protein
  • Cardiovascular disease is a major health problem in the United States and worldwide (R. H. Knopp, N. Engl. J. Med. 341:498-511, 1999).
  • the major cause of cardiovascular disease is atherosclerosis, which results from the formation 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 (VCH 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).
  • TG plasma triglycerides
  • HDLC high-density lipoprotein cholesterol
  • LDL Apo B LDL apolipoprotein B
  • LDLC particles American Heart Association National Center, New Release NR 96-4430 (Circ/Apo B), August 1, 1996.
  • Risk factors for the development of cardiovascular disease have historically been divided into those that are modifiable (e.g., diet, exercise) or unmodifiable (e.g., family history).
  • the development of effective lipid-modifying drugs has enabled components of the lipid profile (high LDLC and low HDLC) to join the ranks of the modifiable risk factors.
  • Decreasing elevated LDLC and increasing low HDLC are both targeted goals for therapy in the current National Cholesterol Education Program guidelines for treatment of hypercholesterolemia.
  • Usual practice is to direct therapy toward elevated LDLC with treatment of low HDLC a secondary endpoint that is often managed by addition, after some weeks, of a second agent.
  • statins which function by inhibiting the activity of 3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for cholesterol formation in the liver and other tissues. Inhibition of HMG-CoA reductase results in a reduction in LDLC levels (Knopp, supra).
  • statins also secondarily increase HDLC with a mean increase of 5 - 10%, 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, msomnia, difficulty in concentrating, and elevation of creatine phosphokinase, which is correlated with rhabdomyolysis (Knopp, supra, Lupattelli G et al., Nucl. Med. Commun. 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 ® , respectively); atorvastatin calcium (sold as Lipitor ® by the Parke Davis Division of Pfizer); fluvastatin sodium (sold as Lescol ® by Novartis); and pravastatin 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).
  • cerivastatin sodium Extensive studies have been performed with cerivastatin sodium, atorvastatin calcium, simvastatin, and pravastatin sodium to determine efficacy in the treatment of hypercholesterolemia.
  • subjects with primary hypercholesterolemia experienced significantly reduced levels of total-cholesterol, LDLC, apolipoprotein B (apo B), triglycerides, total-cholesterol/HDLC ratio, and LDLC/HDLC ratios following treatment with cerivastatin sodium at 0.2 mg/day, 0.3mg/day or 0.4 mg/day for an 8-week period.
  • 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).
  • the mean LDLC decreases and HDLC increases for pravastatin 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).
  • HDLC was significantly and consistently increased by all doses of simvastatin.
  • 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.
  • atorvastatin Part of this basis may be genetic variation in proteins involved in lipid metabolism and atherogenecity (Kuivenhoven et al., supra).
  • CETP cholesteryl ester transfer protein
  • CETP has been associated with both proatherogenecity and antiatherogenecity (Stevenson et al., supra), and the exact role of CETP in the development of atherosclerosis is still largely unknown (Kakko et al., Eur. J. Clin. Invest. 30:18-25, 2000). Some researchers have reported an inverse relationship between CETP activity levels and HDLC levels; however, a number of other studies have failed to detect this association (Gudnason V. et al., European Journal of Clinical Investigation 29:116-128, 1999, internal citations omitted).
  • the cholesteryl ester transfer protein gene is located on chromosome 16ql3 and contains 16 exons that encode a 493 amino acid protein, including a 17 amino acid long leader peptide.
  • a reference sequence for the CETP gene is shown in the contiguous lines of Figure 1 (Genaissance Reference No. 7650429; SEQ ID NO: 1). Reference sequences for the coding sequence (GenBank Accession No.
  • NM_000078.1 and protein are shown in Figures 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3), respectively.
  • PS polymorphic sites
  • Polymorphisms at some of these PS are associated with baseline HDLC and CETP levels, and with the progression of atherosclerosis.
  • One common variation of guanine or adenine is located in the first intron at a position corresponding to nucleotide 22,128 of Figure 1 and is often referred to in the literature as the TaqlB polymorphism (Kuivenhoven et al., supra), but is referred to herein as PS22.
  • the Bl allele (guanrne) has been associated with lower baseline HDLC and higher CETP concentrations than seen in patients who do not have this allele.
  • a polymorphism of guanine or adenine at a position corresponding to nucleotide 43164 of Figure 1, and referred to herein as PS72 results in an amino acid variation of arginine (R) or glutamine (Q) at position 468 of the precursor polypeptide (i.e., position 451 of the mature protein).
  • the Q468 allele is associated with higher plasma CETP activity in men and lower total cholesterol in women than seen in individuals who do not have this allele (Kakko et al., supra; Kakko et al., Atherosclerosis 136:233-40, 1998).
  • a guanhie or adenine variation is located in the 3' UTR at a position corresponding to nucleotide 43507 of Figure 1, referred to herein as PS74.
  • PS74 a guanhie or adenine variation is located in the 3' UTR at a position corresponding to nucleotide 43507 of Figure 1, referred to herein as PS74.
  • the adenine polymorphism at PS74 is associated with low CETP activity (Tamminen M. et al., Atherosclerosis 124:237-247, 1996).
  • PS59 Another polymorphism of guanine or cytosine at nucleotide position 40936 of Figure 1, referred to herein as PS59, results in an amino acid variation of alanine or proline at position 390 in the precursor polypeptide, i.e., position 373 of the mature CETP protein (NCBI SNP Database Ref. SNP ID #5887, July 15, 1999). However, any effect of this polymorphism on CETP function or concentration has not been reported.
  • CETP gene Because of the demonstrated potential for variation in the CETP 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 CETP gene, as well as how such polymorphisms are combined in different copies of the gene. Such information could be applied for studying the biological function of CETP as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function.
  • CETP sub-haplotypes comprise (a) guanine at PS20 and guanine at PS47; (b) guanine at PS22 and guanine at PS47; (c) guanine at PS28 and guanine at PS47 (d) cytosine at PS32 and guanine at PS47; (e) cytosine at PS35 and guanine at PS47; (f) guanine at PS20 and guanine at PS46; (g) guanine at PS22 and guanine at PS46; (h) guanine at PS28 and guanine at PS46; (i) cytosine at PS32 and guanine at PS46; and (j) cytosine at PS35 and guanine at PS46.
  • a predictive statin response marker I was defined therein as comprising zero copy of any of haplotypes (a)-(j), while a statin response marker II was defined therein as comprising at least one copy of any of haplotypes (a)-(j), Consequently, the CETP haplotypes and haplotype pairs disclosed herein are useful in determining whether an individual has a statin response marker I or II. Such information 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 begin, combination therapy 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.
  • PS polymorphic sites
  • PS1 polymorphic sites
  • PS2 polymorphic sites
  • PS3 polymorphic sites
  • 4487 PS4
  • 4540 PS5
  • 4567 PS6
  • 4636 PS7
  • 4684 PS8
  • 4770 PS9
  • 4806 PS10
  • 5474 PS13
  • 5582 PS14
  • 5707 PS16
  • 5714 PS17
  • 5751 PS18
  • 5805 PS19
  • 5926 PS21
  • 12804 PS23
  • 12902 PS24
  • 12916 PS25
  • 13113 PS26
  • 14541 PS28
  • 14544 PS29
  • 14603 PS30
  • 14815 PS31
  • 15015 PS33
  • 15077 PS34
  • 15131 PS35
  • 15202 PS36
  • 15534 PS37
  • 15723 PS38
  • 15724 PS39
  • 15799 15799
  • the polymorphisms at these sites are cytosine or thymine at PS1, cytosine or thymine at PS2, thymine or cytosine at PS3, cytosine or guanine at PS4, gu--nine or adenine at PS5, cytosine or thymine at PS6, cytosine or thymine at PS7, thymine or cytosine at PS8, guanine or adenine at PS9, cytosine or adenine at PS10, guanine or adenine at PS13, cytosine or guanine at PS 14, cytosine or ademne at PS 16, guanine or adenine at PS 17, cytosine or adenine at PS18, thymine or cytosine at PS19, cytosine or thymine at PS21, adenine or guanine at PS23, cytosine or adenine at PS24, cytosine
  • the inventors have determined the identity of the alleles at these sites, as well as at the previously identified sites at nucleotide positions 4880 (PSll), 4882 (PS12), 5604 (PS15), 5858 (PS20), 5935 (PS22), 13375 (PS27), 14953 (PS32), 17005 (PS44), 17013 (PS45), 21588 (PS55), 24743 (PS59), 25197 (PS63), 25744 (PS66), 25779 (PS67), 26944 (PS71), 26971 (PS72), 27126 (PS73) and 27314 (PS74), in an experimental population of 854 human individuals.
  • This population included patients recruited into the study described in Example 1 and a reference population of 93 individuals.
  • the reference population included 83 unrelated individuals self- identified as belonging to one of four major population groups: African descent, Asian, Caucasian and
  • each CETP haplotype constitutes a genetic marker that defines the variant nucleotides that exist in the human population at this set of polymorphic sites in the CETP gene.
  • each CETP haplotype also represents a naturally-occurring isoform (also referred to herein as an "isogene") of the CETP gene.
  • the frequency of each haplotype and haplotype pair within the total reference population and within each of the four major population groups included in the experimental population was also determined.
  • the invention provides a method, composition and kit for genotyping the CETP gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS13, PS14, PS16, PS17, PS18, PS19, PS21, PS23, PS24, PS25, PS26, PS28, PS29, PS30, PS31, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43, PS46, PS47, PS48, PS49, PS50, PS51, PS52, PS53, PS54, PS56, PS57, PS58, PS60, PS61, PS62, PS64, PS65, PS68, PS69 and PS70 in both copies of the CETP gene from the individual.
  • the genotyping method may also comprise identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of 4880 (PSl l), 4882 (PS12), 5604 (PS15), 5858 (PS20), 5935 (PS22), 13375 (PS27), 14953 (PS32), 17005 (PS44), 17013 (PS45), 21588 (PS55), 24743 (PS59), 25197 (PS63), 25744 (PS66), 25779 (PS67), 26944 (PS71), 26971 (PS72), 27126 (PS73) and
  • a genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these CETP polymorphic sites.
  • a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel CETP polymorphic sites.
  • the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS1- PS74. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 4 below or has one of the haplotype pairs in Table 3 below.
  • the invention also provides a method for haplotyping the CETP gene in an individual.
  • the haplotyping method comprises determining, for one copy of the CETP gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS13, PS14, PS16, PS17, PS18, PS19, PS21, PS23, PS24, PS25, PS26, PS28, PS29, PS30, PS31, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43, PS46, PS47, PS48, PS49, PS50, PS51, PS52, PS53, PS54, PS56, PS57, PS58, PS60, PS61, PS62, PS64, PS65, PS68, PS69 and PS70.
  • the haplotyping method comprises determining whether one copy of the individual's CETP gene is defined by one of the CETP haplotypes shown in Table 4, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies of the individual's CETP gene are defined by one of the CETP haplotype pairs shown in Table 3 below, or a sub-haplotype pair thereof. Establishing the CETP haplotype or haplotype pair of an individual is useful for determining whether the individual has a statin response marker I or II.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate CETP as a candidate target for treating a specific condition or disease predicted to be associated with CETP activity. Determining for a particular population the frequency of one or more of the individual CETP haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue CETP as a target for treating the specific disease of interest.
  • variable CETP activity is associated with the disease
  • one or more CETP haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable CETP activity has little, if any, involvement with that disease.
  • the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any CETP haplotype or haplotype pair, apply the information derived from detecting CETP haplotypes in an individual to decide whether modulating CETP activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting CETP to treat a specific condition or disease predicted to be associated with CETP activity. For example, detecting which of the CETP haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the CETP isoforms present in the disease population, or for only the most frequent CETP 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 CETP gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with CETP 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 CETP haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute CETP haplotypes and/or haplotype pairs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of a CETP haplotype or haplotype pair that is associated with response to the drug being studied in the trial, 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 determining the phenotypic effect of any CETP haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and a CETP genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein.
  • the method comprises comparing the frequency of the CETP genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the CETP genotype or haplotype in a reference population.
  • a different frequency of the CETP genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the CETP 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 CETP haplotype is selected from the haplotypes shown in Table 4, or a sub-haplotype thereof.
  • Such methods have applicability in developing diagnostic tests and therapeutic treatments for cardiovascular disease (CVD), CVD risk factors and other disorders of cholesterol metabolism.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the CETP gene or a fragment thereof.
  • the reference sequence comprises the contiguous sequences shown in Figure 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of thymine at PSl, thymine at PS2, cytosine at PS3, guanine at PS4, adenine at PS5, thymine at PS6, thymine at PS7, cytosine at PS8, adenine at PS9, adenine at PS10, adenine at PS13, guanine at PS14, adenine at PS16, adenine at PS17, adenine at PS18, cytosine at PS19, thymine at PS21, guanine at PS23, adenine at PS24, guanine at PS25, cytosine at PS26, adenine at PS28
  • the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at PS 11 , cytosine at PS 12, adenine at PS15, adenine at PS20, adenine at PS22, thymine at PS27, thymine at PS32, thymine at PS44, cytosine at PS45, thymine at PS55, cytosine at PS59, thymine at PS63, guanine at PS66, adenine at PS67, guanine at PS71, adenine at PS72, adenine at PS73 and adenine at PS74.
  • additional polymorphisms selected from the group consisting of adenine at PS 11 , cytosine at PS 12, adenine at PS15, adenine at PS20, adenine at PS22, thymine at PS27, thymine at PS32, thymine at PS44, cytosine at PS45,
  • a preferred polymorphic variant is an isogene of the CETP gene.
  • a CETP isogene of the invention comprises cytosine or thymine at PSl, cytosine or thymine at PS2, thymine or cytosine at PS3, cytosine or guanine at PS4, guanine or adenine at PS5, cytosine or thymine at PS6, cytosine or thymine at PS7, thymine or cytosine at PS8, guanine or adenine at PS9, cytosine or adenine at PS10, cytosine or adenine at PSll, adenine or cytosine at PS 12, guanine or adenine at PS 13, cytosine or guanine at PS 14, cytosine or adenine at PS15, cytosine or adenine at PS16, guanine or adenine at PS17, cytosine or adenine at PS18,
  • a preferred novel CETP isogene encodes a CETP polypeptide with cholesteryl ester transfer activity.
  • the invention also provides a collection of CETP isogenes, referred to herein as a CETP genome anthology.
  • the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a CETP cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises at least one polymorphism selected from the group consisting of guanine at a position corresponding to nucleotide 44, adenine at a position corresponding to nucleotide 534, cytosine at a position corresponding to nucleotide 756, thymine at a position corresponding to nucleotide 804, adenine at a position corresponding to nucleotide 940, adenine at a position corresponding to nucleotide 1153, and guanine at a position corresponding to nucleotide 1161.
  • the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at a position corresponding to nucleotide 66, thymine at a position corresponding to nucleotide 861, cytosine at a position corresponding to nucleotide 869, cytosine at a position corresponding to nucleotide ' 1168, guanine at a position corresponding to nucleotide 1264, adenine at a position corresponding to nucleotide 1299, guanine at a position corresponding to nucleotide 1376, and adenine at a position corresponding to nucleotide 1403.
  • a preferred polymorphic cDNA variant is selected from the group consisting of CETP coding sequences A through W represented in Table 9.
  • a particularly preferred CETP coding sequence of the invention encodes a CETP polypeptide with cholesteryl ester transfer activity.
  • Polynucleotides complementary to these CETP genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the CETP gene will be useful in studying the expression and function of CETP, and in expressing CETP protein for use in screening for candidate drugs to treat diseases related to CETP activity.
  • the invention provides a recombinant expression vector comprising one of the polymorphic genomic and cDNA variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector. The recombinant vector and host cell may be used to express CETP for protein structure analysis and drug binding studies.
  • the invention provides a polypeptide comprising a polymorphic variant of a reference amino acid sequence for the CETP protein.
  • the reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymorphic variant comprises at least one variant amino acid selected from the group consisting of glycine at a position corresponding to amino acid position 15, lysine at a position corresponding to amino acid position 314, and methionine at a position corresponding to amino acid position 385.
  • the polymorphic variant also comprises at least one variant amino acid selected from the group consisting of proline at a position corresponding to amino acid position 290, proline at a position corresponding to amino acid position 390, valine at a position corresponding to amino acid position 422, glycine at a position corresponding to amino acid position 459, and glutamine at a position corresponding to amino acid position 468.
  • a preferred polymorphic variant comprises a CETP protein variant selected from the group consisting of A, B, D - J, M and N in Table 10.
  • a polymorphic variant of CETP is useful in studying the effect of the variation on the biological activity of CETP as well as on the binding affinity of candidate drugs targeting CETP for the treatment of cardiovascular disease (CVD), CVD risk factors and other disorders of cholesterol metabolism.
  • the present invention also provides antibodies that recognize and bind to the above polymorphic
  • CETP 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 CETP polymorphic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the CETP isogenes in vivo, for in vivo screening and testing of drugs targeted against CETP protein, and for testing the efficacy of therapeutic agents and compounds for cardiovascular disease (CVD), CVD risk factors and other disorders of cholesterol metabolism in a biological system.
  • CVD cardiovascular disease
  • the present invention also provides a computer system for storing and displaying polymorphism data determined for the CETP gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymorphism data.
  • the polymorphism data includes one or more of the following: the polymorphisms, the genotypes, the haplotypes, and the haplotype pairs identified for the CETP gene in a reference population.
  • the computer system is capable of producing a display showing CETP haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the CETP gene (Genaissance Reference No. 7650429; 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 polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • SEQ ID NO:286 is a modified version of SEQ ID NO:l that shows the context sequence of each polymorphic site, PS1-PS74, in a uniform format to facilitate electronic searching.
  • SEQ ID NO:286 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymorphic 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 CETP coding sequence (contiguous lines; SEQ ID NO:286
  • Figure 3 illustrates a reference sequence for the CETP protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymorphism(s) of Figure 2 positioned below the polymorphic site in the sequence.
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide or amino acid sequence, or one of the alternative polymorphisms found at a polymorphic site.
  • Candidate Gene - A gene wliich is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.
  • Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Full-genotype The unphased 5 ' to 3 ' sequence of nucleotide pairs found at all polymorphic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.
  • Sub-genotype The unphased 5' to 3' sequence of nucleotides seen at a subset of the polymorphic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.
  • Genotyping A process for determining a genotype of an individual.
  • Haplotype A 5' to 3' sequence of nucleotides found at one or more polymorphic sites in a locus on a single chromosome from a single individual.
  • haplotype includes a full-haplotype and/or a sub-haplotype as described below.
  • Full-haplotype The 5' to 3' sequence of nucleotides found at all polymorphic sites examined herein 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 the polymorphic sites examined herein in a locus on a single chromosome from a single individual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for determining 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 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 form of a gene, mRNA, cDNA, coding sequence or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One of the isoforms (e.g., alleles) of a gene found in a population.
  • An isogene (or allele) contains all of the polymorphisms present in the particular isoform 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 corresponding to a gene or a physical or phenotypic feature, where physical features include polymorphic sites.
  • Naturally-occurring A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
  • 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 polymorphic sites in a locus, phased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is known.
  • PS Polymorphic site
  • 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 polymorphism in the gene.
  • Polymorphism The sequence variation observed in an individual at a polymorphic site. Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Polymorphism data Information concerning one or more of the following for a specific gene: location of polymorphic sites; sequence variation at those sites; frequency of polymorphisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.
  • Polymorphism Database A collection of polymorphism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
  • 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%.
  • SNP Single Nucleotide Polymorphism
  • Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • Treatment A stimulus administered internally or externally to a subject.
  • Unphased As applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, unphased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is not known.
  • the present invention is based on the discovery of novel variants of the CETP gene.
  • the inventors herein discovered 216 isogenes of the CETP gene by characterizing the CETP gene found in genomic DNAs isolated from 854 individuals comprising individuals initially recruited into a statin study described in Example 1, as well as a reference population of 93 individuals.
  • the human individuals included a reference population of 82 unrelated individuals 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 Repository contains three unrelated indigenous American Indians (one from each of North, Central and South America), one three-generation Caucasian family (from the CEPH Utah cohort) and one two- generation African- American family.
  • the CETP isogenes present in experimental population are defined by 216 haplotypes for 74 bi- allelic polymorphic sites in the CETP gene.
  • the CETP polymorphic sites identified by the inventors are referred to as PS1-PS74 to designate the order in which they are located in the gene (see Table 2 below), with the 56 polymorphic sites believed to be novel referred to as PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS13, PS14, PS16, PS17, PS18, PS19, PS21, PS23, PS24, PS25, PS26, PS28, PS29, PS30, PS31, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43, PS46, PS47, PS48, PS49, PS50, PS51, PS52, PS53, PS54, PS56, PS57, PS58, PS60, PS61, PS62, PS64, PS65, PS68, PS69 and
  • the inventors herein Using the genotypes identified in the Experimental population for PS1-PS74 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the CETP gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the CETP gene include those shown in Tables 3 and 4, respectively.
  • the polymo ⁇ hism and haplotype data disclosed herein are useful for determining whether an individual has a statin response marker I or II as discussed above in Background.
  • haplotypes that comprise each of the predictive sub-haplotypes comprising a statin response marker I or II, as described in copending International Application US/03/XXXXX, filed April 28, 2003 are identified by haplotype ID number for each of the 10 sub-haplotypes (a)-(j).
  • the haplotype ID numbers can be correlated with the haplotype numbers based on the HAP number and HAP ID number information provided in Table 5. Additionaly, the polymorphism and haplotype data disclosed herein are useful for validating whether CETP is a suitable target for drugs to treat cardiovascular disease (CVD), CVD risk factors or other disorders of cholesterol metabolism, screening for such drugs and reducing bias in clinical trials of such drugs.
  • CVD cardiovascular disease
  • CVD risk factors or other disorders of cholesterol metabolism screening for such drugs and reducing bias in clinical trials of such drugs.
  • the invention also provides compositions and methods for detecting the novel CETP polymorphisms, haplotypes and haplotype pairs identified herein.
  • compositions comprise at least one oligonucleotide for detecting the variant nucleotide or nucleotide pair located at a CETP polymorphic site in one copy or two copies of the CETP gene.
  • oligonucleotides are referred to herein as CETP haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as CETP oligonucleotides.
  • a CETP haplotyping or genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, one of the novel polymorphic sites described herein.
  • oligonucleotide refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred 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 many factors that are routinely considered and practiced by the skilled artisan.
  • oligonucleotide 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. 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.
  • Haplotyping or genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a CETP polynucleotide.
  • the target region is located in a CETP isogene.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with another region in the CETP polynucleotide or with a non-
  • CETP polynucleotide under the same hybridizing conditions.
  • 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 polymorphisms in the CETP gene using the polymorphism information provided herein in conjunction with the known sequence information for the CETP gene 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 every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding 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, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D.
  • an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary 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.
  • Preferred haplotyping or genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • 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.
  • 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 polymorphic 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 nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO probe for detecting CETP gene polymorphisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • GCACCCTYGGTCATT SEQ ID NO 4 and its complement
  • ACATGCTYACTCAGT SEQ ID NO 5
  • CTGAGCCYTGGGAAA SEQ ID NO 6
  • GGTCAGCSCGGCTCC SEQ ID NO 7
  • CAAACTCRGGACTAG SEQ ID NO 8
  • TGAGAGGYGACTGAG SEQ ID NO 9
  • CCACATCYCTGCAGC SEQ ID NO 10 and its complement CACTTGGYCATCTGG
  • SEQ ID NO 11 and its complement ATAACACRTTCACAC SEQ ID NO 12 and its complement
  • AGACATTMCCCCTGC SEQ ID NO 13 and its complement GCTGGGCRGACATAC
  • GGCAATGSCCATGCC SEQ ID NO 15 and its complement TGGACACMCACTATG
  • SEQ ID NO 16 and its complement CCACTATRCCAGGAG SEQ ID NO 17 and its complement CTGAAGCMGGCTG
  • Apreferred ASO primer for detecting CETP gene polymorphism- comprises anucleotide sequence, listed 5' to 3', selected from the group consisting of:.
  • GATGGTACATGCTYA (SEQ ID NO 62) ; CCCCTCACTGAGTRA SEQ ID NO: 63) ;
  • ATGTCTCTGAGCCYT (SEQ ID NO 64) ; TCACTGTTTCCCARG SEQ ID NO: 65) ;
  • AGTGAGGGTCAGCSC (SEQ ID NO 66) ; ACGTGGGGAGCCGSG SEQ ID NO: 67) ;
  • CAGGTGCAAACTCRG (SEQ ID NO 68) ; CCTGCCCTAGTCCYG SEQ ID NO: 69) ;
  • AGGCAGCACTTGGYC (SEQ ID NO 74) ; CTGTGACCAGATGRC SEQ ID NO : 75 ) ;
  • TGAAAGATAACACRT (SEQ ID NO 76); TGAGTTGTGTGAAYG SEQ ID NO: 77) ;
  • TGCCACAGACATTMC (SEQ ID NO 78) ; GGCGGAGCAGGGGKA SEQ ID NO:79) ;
  • GTGGGGGCTGGGCRG (SEQ ID NO 80) ; GTATATGTATGTCYG SEQ ID NO : 81 ) ;
  • CTGCTGGGCAATGSC (SEQ ID NO 82) ; GGAGCAGGCATGGSC SEQ ID NO:83) ;
  • TTTTCATGGACACMC (SEQ ID NO 84) ; TCCTGGCATAGTGKG SEQ ID NO : 85 ) ;
  • GGACACCCACTATRC (SEQ ID NO 86) ; GGGAGGCTCCTGGYA SEQ ID NO : 87 ) ;
  • TGACATGTTGGGTRA (SEQ ID NO 94) ; CCTGCAAAT ⁇ TGTYA SEQ ID NO: 95) ;
  • AGCTGATGCCCCAYG SEQ ID NO 100
  • AGAGGGCCAGGGCRT SEQ ID NO:101
  • CACACTAGGCGCTYC (SEQ ID NO 104)
  • CTGTGCATCCATGRA (SEQ ID NO:105)
  • TGGCTCTGACACTYG SEQ ID NO 112
  • ATAACTAATCATCRA SEQ ID NO:113
  • GTCCAAGAGGACTYC SEQ ID NO 116
  • CAGGAGAATCTTGRA SEQ ID NO:117
  • CTCCTCCGCATTCYT (SEQ ID NO 124) .
  • CCTCGCAGCATCARG (SEQ ID NO:125) .
  • CTGAAGCTGGACCYG SEQ ID NO 126)
  • CCCTACTGGGCTCRG SEQ ID NO:127
  • TCCCCACCTTCTCKC (SEQ ID NO 130) , CCAGCAGTGTGGGMG (SEQ ID NO-131) ,
  • GGGGCTGGGCTGCKA SEQ ID NO 132
  • ATCTGGATCCCCTMG SEQ ID NO: 133 .
  • AGGGAGGAAACTCRG (SEQ ID NO 138)
  • ACCACCAAGTTTCYG (SEQ ID NO:139)
  • GGTTGGGAGTTGCRT SEQ ID NO 140
  • ACCCCTCCTCAGAYG SEQ ID NO-141
  • TCAGGCAGTGCTGRA SEQ ID NO 142
  • AAGCCCCAGGTCTYC SEQ ID NO:143
  • AGCTCCTACTCGGKT SEQ ID NO 144
  • TCCATGCCGAACAMC SEQ ID NO : 145
  • CTACCCCGAGCTAYT SEQ ID NO 148 CTACCCCGAGCTAYT SEQ ID NO 148; TGGGGAAAAGGAART (SEQ ID NO : 149 )
  • TGAAGAGGTGAGGYG SEQ ID NO 150 TGAAGAGGTGAGGYG SEQ ID NO 150
  • TCTCCCTGCACCCRC SEQ ID NO - 151
  • ATATCGTGACTACSG SEQ ID NO 154
  • AGGAGGCCTGGACSG SEQ ID NO : 155
  • GGGTGCCGAGGCTRA SEQ ID NO 166
  • TGGGAAGCTCTGTYA SEQ ID NO : 167
  • CATCTCCCACTACYC SEQ ID NO 170 CATCTCCCACTACYC SEQ ID NO 170
  • CTCTGCCACCCTGRG SEQ I D NO : 171
  • oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymorphic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymorphisms described herein and therefore such oligonucleotides are referred to herein as "primer- extension oligonucleotides".
  • the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymorphic site.
  • GGGGCACCCT SEQ ID NO 172) GGCAATGACC SEQ ID NO 173) ;
  • GGTACATGCT SEQ ID NO 174) CTCACTGAGT SEQ ID NO 175) ;
  • AAGATAACAC SEQ ID NO 188) GTTGTGTGAA SEQ ID NO 189) ;
  • ATGGGCTGAG SEQ ID NO 202) ATGGGCTGAG SEQ ID NO 202 ; TGACAGCTCC SEQ ID NO 203) ;
  • CATGTTGGGT SEQ ID NO 206) GCAAATATGT SEQ ID NO 207) ;
  • CACACTAGGC SEQ ID NO 214) ATCCATGGAG SEQ ID NO 215) ;
  • GCTGCACTAT SEQ ID NO 222) CCCAAGGCCA SEQ ID NO 223) ;
  • CTCTGACACT SEQ ID NO 224) ACTAATCATC SEQ ID NO 225) ; GGGCTGCAGC SEQ ID NO 226) , CAGCTTGTGA SEQ ID NO 227),
  • CAAGAGGACT SEQ ID NO 228) GAGAATCTTG SEQ ID NO 229) ,
  • AAGCTGGACC SEQ ID NO 238) TACTGGGCTC SEQ ID NO 239) ,
  • TGTGTGCACA SEQ ID NO 248) CTCCCCATGC SEQ ID NO 249) ,
  • CTCGGTTGTT SEQ ID NO 258) ACTCCATGCC SEQ ID NO 259) ,
  • a composition contains two or more differently labeled CETP oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs at two or more polymorphic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymorphic site.
  • CETP oligonucleotides 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 polymorphism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized CETP oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymorphisms in multiple genes at the same time. >
  • the invention provides a kit comprising at least two CETP oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
  • CETP genotype and “CETP haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymorphic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymorphic sites in the CETP gene.
  • the additional polymorphic sites may be currently known polymorphic sites or sites that are subsequently discovered.
  • One embodiment of a genotyping method of the invention involves examining both copies of the individual's CETP gene, or a fragment thereof, to identify the nucleotide pair at one or more polymorphic sites selected from the group consisting of PS1-PS74 in the two copies to assign a CETP genotype to the individual.
  • the polymorphic site(s) are selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS13, PS14, PS16, PS17, PS18, PS19, PS21, PS23, PS24, PS25, PS26, PS28, PS29, PS30, PS31, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43, PS46, PS47, PS48, PS49, PS50, PS51, PS52, PS53, PS54, PS56, PS57, PS58, PS60, PS61, PS62, PS64, PS65, PS68, PS69 and PS70.
  • the PS are selected from the group consisting of PS5, PS8, ' PS12, PS14, PS21, PS22, PS28, PS36, PS41, PS45, PS47, PS51, PS57, PS59, PS63, PS66, PS71, PS72, and PS74 or the group consisting of PS14, PS45, PS51, PS57, PS59, PS66, PS71, and PS72.
  • "examining a gene” may include examining one or more of: DNA containing the gene, mRNA transcripts thereof, or cDNA copies thereof.
  • the two “copies" of a gene, mRNA or cDNA (or fragment of such CETP molecules) in an individual may be the same allele or may be different alleles.
  • the identity of the nucleotide pair at one or more of the polymorphic sites selected from the group consisting of PSl 1, PS12, PS15, PS20, PS22, PS27, PS32, PS44, PS45, PS55, PS59, PS63, PS66, PS67, PS71, PS72, PS73 and PS74 is also determined.
  • a genotyping method of the invention comprises determining the identity of the nucleotide pair at each of PS1-PS74.
  • the genotyping method comprises determining the identity of the nucleotide pair at a subset of the 74 polymorphic sites chosen to provide 95% of the diversity, as measured by the Shannon- Weiner equation (R. Judson, et al. 2002. Pharmacogenomics. 3 (3): 379-391), observed in the set of haplotypes for the complete set of 74 polymorphic sites disclosed herein: PS5, PS8, PS12, PS14, PS21, PS22, PS28, PS36, PS41, PS45, PS47, PS51, PS57, PS59, PS63, PS66, PS71, PS72, and PS74.
  • the genotyping method comprises determining the identity of the nucleotide pair at one or more PS selected from the PS for which the nucleic acid variation results in a nonsynonymous variation in the polypeptide sequence: PS14, PS45, PS51, PS57, PS59, PS66, PS71, and PS72.
  • One method of examining both copies of the individual's CETP gene is by isolating from the individual a nucleic acid sample comprising the two copies of the CETP gene, mRNA transcripts thereof or cDNA copies thereof, or a fragment of any of the foregoing, that are present in the individual.
  • the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the CETP gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymorphisms located in introns or in 5 ' and 3 ' untranslated regions if not present in the mRNA or cDNA. If a CETP gene fragment is isolated, it must contain the polymorphic site(s) to be genotyped.
  • One embodiment of a haplotyping method of the invention comprises examining at least one copy of the individual's CETP gene, or a fragment thereof, to identify the phased sequence of nucleotides at two or more polymorphic sites selected from the group consisting of PS1-PS74, comparing the phased sequence to CETP haplotypes in a reference population, and assigning a CETP haplotype to the individual that is consistent with the phased sequence.
  • the method further comprises examining the second copy of the individual's CETP gene to identify the phased sequence of nucleotides at the selected PS for the second copy, comparing the second phased sequence to CETP haplotypes in a reference population, and assigning to the individual a CETP haplotype that is consistent with the second phased sequence.
  • the nucleotide at each of PS1-PS74 is identified.
  • the haplotyping method comprises determining the identity of the nucleotide at a subset of the 74 polymorphic sites chosen to provide 95% of the diversity observed in the set of haplotypes for the complete set of 74 polymorphic sites disclosed herein: PS5, PS8, PS12, PS14, PS21, PS22, PS28, PS36, PS41, PS45, PS47, PS51, PS57, PS59, PS63, PS66, PS71, PS72, and PS74.
  • the haplotyping method comprises determining the identity of the nucleotide at a set of PS selected from the PS for which the nucleic acid variation results in a nonsynonymous variation in the polypeptide sequence: PS14, PS45, PS51, PS57, PS59, PS66, PS71, and PS72.
  • one PS is selected from the group consisting of PS20, PS22, PS28, PS32, and PS35 and a second PS is selected from the group consisting of PS46 and 47.
  • the CETP haplotype assigned to the individual is selected from the group consisting of the CETP haplotypes shown in Table 4.
  • Another embodiment comprises comprises examining both copies of the individual's CETP
  • "examining a gene” may include examining one or more of: DNA containing the gene, mRNA transcripts thereof, or cDNA copies thereof.
  • One method of examining one copy of the individual's CETP gene is by isolating from the individual a nucleic acid sample containing only one of the two copies of the CETP gene, mRNA or cDNA, or a fragment of such CETP molecules, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS13, PS14, PS16, PS17, PS18, PS19, PS21, PS23, PS24, PS25, PS26, PS28, PS29, PS30, PS31, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43
  • the CETP haplotype is assigned to the individual by also identifying the nucleotide at one or more polymorphic sites selected from the group consisting of PSl 1, PS12, PS15, PS20, PS22, PS27, PS32, PS44, PS45, PS55, PS59, PS63, PS66, PS67, PS71, PS72, PS73 and PS74.
  • the nucleotide at each of PS1-PS74 is identified.
  • the haplotyping method comprises determining whether an individual has one or more of the CETP haplotypes shown in Table 4. This can be accomplished by identifying the phased sequence of nucleotides present at PS1-PS74 for at least one copy of the individual's CETP gene, comparing the phased sequence to the CETP haplotypes in Table 4 and assigning to that copy a CETP haplotype from Table 4 that is consistent with the phased sequence. This identifying step does not necessarily require that each of PS1-PS74 be directly examined. Typically only a subset of PS1-PS74 will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 4.
  • a CETP haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at two or more polymorphic sites selected from the group consisting of PS1-PS74 in each copy of the CETP gene that is present in the individual, comparing the phased sequence to CETP haplotypes or haplytype pairs observed in a reference population, and assigning a haplotype to each copy that is consistent with the phased sequence.
  • the two or more PS are selected from the group consisting of: PS5, PS8, PS12, PS14, PS21, PS22, PS28, PS36, PS41, PS45, PS47, PS51, PS57, PS59, PS63, PS66, PS71, PS72, or PS74.
  • the two or more PS are selected from the group consisting of: PS14, PS45, PS51, PS57, PS59, PS66, PS71, and PS72.
  • one PS is selected from the group consisting of PS20, PS22, PS28, PS32, and PS35 and a second PS is selected from the group consisting of PS46 and 47.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS74 in each copy of the CETP gene.
  • the haplotypes observed in a reference population are those in Table 4 and the haplotype pairs are those in Table 3.
  • the haplotyping method comprises determining whether an individual has one of the CETP haplotype pairs shown in Table 3.
  • One way to accomplish this is to identify the phased sequence of nucleotides at PS1-PS74 for each copy of the individual's CETP gene, comparing the sequence to haplotype or haplotype pair data for a reference population and assigning to the individual a CETP haplotype pair that is consistent with each of the phased sequences,
  • the CETP haplotype pairs shown in Table 3 are preferred haplotype pairs for a reference population and the CETP haplotypes in Table 4 are preferred haplotypes for a reference population.
  • the identifying step does not necessarily require that each of PS1-PS74 be directly examined. As a result of linkage disequilibrium, typically only a subset of PS1-PS74 will need to be directly examined to assign to an individual a haplotype pair shown in Table 3.
  • the nucleic acid used in the above haplotyping methods of the invention may be isolated using any method capable of separating the two copies of the CETP gene or fragment such as one of the methods described above for preparing CETP isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will typically only provide haplotype information on one of the two CETP gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional CETP 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 CETP gene in an individual.
  • the haplotype for the other allele may be inferred if the individual has a known genotype for the polymorphic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
  • the identifying step is preferably performed with each copy of the gene being placed in separate containers.
  • the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container.
  • 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 polymorphic site(s), then detecting a combination of the first and third dyes would identify the polymorphism in the first gene copy while detecting a combination of the second and third dyes would identify the polymorphism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymorphic site(s) may be determined by amplifying a target region(s) containing the polymorphic site(s) directly from one or both copies of the CETP gene, or a fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymorphic 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 polymorphism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both gua ine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guamne/guanine).
  • the target region(s) 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 including transcription- based amplification systems (U.S. Patent No.
  • a polymorphism 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 pair 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 polymorphic 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 polymorphic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed 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, streptavidin 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.
  • the genotype or haplotype for the CETP gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, mRNA, cDNA or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each of the polymorphic sites to be included in the genotype or haplotype.
  • polymorphisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter 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).
  • riboprobes Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • variant alleles can be identified by single strand conformation polymorphism (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)
  • SSCP single strand conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the pofymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) 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 containing a polymo ⁇ hism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17:8392, 1989; Ruano 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 identity of the allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by haplotyping or genotyping the allele(s) at another polymo ⁇ hic site that is in linkage disequilibrium with the allele at the polymo ⁇ hic site of interest.
  • Polymo ⁇ hic sites with alleles in linkage disequilibrium with the alleles of presently disclosed polymo ⁇ hic sites may be located in 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 allele(s) of novel polymo ⁇ hic 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.
  • an individual's CETP haplotype pair is predicted from its CETP genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a CETP genotype for the individual at two or more CETP polymo ⁇ hic sites PS1-PS74 described herein, accessing data containing CETP haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the individual's CETP genotype.
  • the reference haplotype pairs include the CETP haplotype pairs shown in Table 3.
  • the reference haplotype pairs include all CETP haplotype pairs that may be formed from pairing the haplotypes of Table 4.
  • the method comprises determining the genotype at a subset of the 74 polymo ⁇ hic sites chosen to provide 95% of the diversity observed in the set of haplotypes for the complete set of 74 polymo ⁇ hic sites disclosed herein: PS5, PS8, PS12, PS14, PS21, PS22, PS28, PS36, PS41, PS45, PS47, PS51, PS57, PS59, PS63, PS66, PS71, PS72, and PS74.
  • the method comprises determining the genotype at two or more PS selected from the PS for which the nucleic acid variation results in a nonsynonymous variation in the polypeptide sequence: PS14, PS45, PS51, PS57, PS59, PS66, PS71, and PS72.
  • the CETP haplotype pair can be assigned by comparing the individual's genotype with the genotypes corresponding to the haplotype pairs known to exist in the general population or in a specific population group, and determining which haplotype pair is consistent with the genotype of the individual. In some embodiments,- the comparing step may be performed by visual inspection (for example, by consulting Table 3).
  • frequency data (such as that presented in Table 6) may be used to determine which of these haplotype pairs is most likely to be present in the individual. This determination may also be performed in some embodiments by visual inspection, for example by consulting Table 6. If a particular CETP haplotype pair consistent with the genotype of the individual is more frequent in the reference population than others consistent with the genotype, then that haplotype pair with the highest frequency is the most likely to be present in the individual. 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 CETP haplotype pair frequency data determined 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 should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
  • a preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each of the four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.
  • the haplotype frequency data for each ethnogeographic group is examined to determine 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 errors in the genotyping process. If large deviations from ⁇ ardy-
  • the assigning step involves performing the following analysis.
  • each of the possible haplotype pairs is compared to the haplotype pairs in the reference population.
  • the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual.
  • only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual 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.
  • 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).
  • 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).
  • the invention also provides a method for determining the frequency of a CETP genotype, haplotype, or haplotype pair in a population.
  • the method comprises, for each member of the population, detemiining the genotype, haplotype or the haplotype pair for the novel CETP polymo ⁇ hic 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 treatment).
  • CETP 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 CETP protein as a candidate target for treating a medical condition predicted to be associated with CETP activity.
  • the method comprises comparing the frequency of each CETP haplotype shown in Table 4 in the trait population and in a control population and making a decision whether to pursue CETP 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.
  • CETP haplotype is present at a frequency in the trait population that is different from the frequency in the control population at a statistically significant level.
  • a decision to pursue the CETP protein as a target should be made.
  • the frequencies of each of the CETP haplotypes are not statistically significantly different between the trait and control populations, a decision not to pursue the CETP 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 CETP protein as a target. Any CETP haplotype not present in a population is considered to have a frequency of zero.
  • each of the trait and control 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 control populations may be comprised of just one ethnogeographic origin.
  • frequency data for CETP haplotypes are determined in a population having a condition or disease predicted to be associated with CETP activity and used in a method for screening for compounds targeting the CETP protein to treat such condition or disease.
  • frequency data are determined in the population of interest for the CETP haplotypes shown in Table 4.
  • 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 determined 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 form.
  • the frequency data may be present in a database that is accessible by a computer.
  • the CETP isoforms corresponding to CETP haplotypes occurring 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 CETP isoform.
  • the desired frequency for the haplotypes might be chosen to be the frequency of the most frequent haplotype, greater than or less than some cut-off value, such as 10% in the population, or the desired frequency might be determined 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 determined.
  • 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 CETP isoforms 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 CETP activity may be cardiovascular disease (CVD), CVD risk factors and other disorders of cholesterol metabolism.
  • CVD cardiovascular disease
  • frequency data for CETP 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 CETP genotype, haplotype, or haplotype pair.
  • 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 in 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 form.
  • 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.
  • the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If the frequency of a particular CETP 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 CETP genotype, haplotype or haplotype pair.
  • the CETP genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the genotypes and haplotypes shown in Tables 3 and 4, or from sub- genotypes and sub-haplotypes derived from these genotypes and haplotypes.
  • Sub-genotypes useful in the invention preferably do not include sub-genotypes solely for any one of PSl 1, PS12, PS15, PS20, PS22, PS27, PS32, PS44, PS45, PS55, PS59, PS63, PS66, PS67, PS71, PS72, PS73 and PS74 or for any combination thereof.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting CETP or response to a therapeutic treatment for a medical condition.
  • 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.
  • 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).
  • clinical population In order to deduce a correlation between clinical response to a treatment and a CETP 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 run 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 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 correlation between haplotype and 5 treatment 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 pair 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 predetermined criteria. It is .0 contemplated that in 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.
  • the CETP gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • correlations between L5 individual response and CETP genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their CETP genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
  • a second method for finding correlations between CETP haplotype content and clinical responses uses predictive models based on error-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, 0 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.
  • the correlation 5 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 determine how much of the variation in the clinical data is explained by different subsets of the polymo ⁇ hic sites in the CETP gene.
  • ANOVA analysis of variation
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of CETP 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 CETP gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, 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 CETP 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 CETP activity.
  • Haplotyping one or both copies of the CETP gene in those individuals participating in the trial will allow the pharmaceutical scientist conducting the clinical trial to assign each individual from the trial one of the CETP haplotypes or haplotype pairs shown in Tables 4 and 3, respectively, or a CETP 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 CETP gene, the phased sequence of nucleotides present at each of PS1-PS74. Determining the CETP haplotype or haplotype pair present in individuals participating in the clinical trial 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 CETP activity, 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 CETP haplotype or haplotype pair.
  • the pharmaceutical scientist requires no a priori knowledge of any effect a CETP haplotype or haplotype pair may have on the results of the trial.
  • Diseases or conditions predicted to be associated with CETP activity include, e.g., cardiovascular disease (CVD), CVD risk factors and other disorders of cholesterol metabolism.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the CETP gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
  • the nucleotide sequence of a variant CETP 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 PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS13, PS14, PS16, PS17, PS18, PS19, PS21, PS23, PS24, PS25, PS26, PS28, PS29, PS30, PS31, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS42, PS43, PS46, PS47, PS48, PS49, PS50, PS51, PS52, PS53, PS54, PS56, PS57,
  • nucleotide sequence of a variant fragment of the CETP 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 CETP gene (or other reported CETP sequences) or to portions of the reference sequence (or other reported CETP sequences), except for the haplotyping and genotyping oligonucleotides described above.
  • the location of a polymo ⁇ hism in a variant CETP gene or fragment is preferably identified by aligning its sequence against SEQ ID NO: 1.
  • the polymo ⁇ hism is selected from the group consisting of thymine at PSl, thymine at PS2, cytosine at PS3, guanine at PS4, adenine at PS5, thymine at PS6, thymine at PS7, cytosine at PS8, adenine at PS9, adenine at PS10, adenine at PS13, guanine at PS14, adenine at PS16, adenine at PS17, adenine at PS18, cytosine at PS19, thymine at PS21, guanine at PS23, adenine at PS24, guanine at PS25, cytosine at PS26, adenine at PS28, thymine at PS29, adenine at PS30, thymine at PS31, adenme at PS33
  • the polymo ⁇ hic variant comprises a naturally-occurring isogene of the CETP gene which is defined by any one of haplotypes 1- 216 shown in Table 4 below.
  • Preferred isogenes encode a CETP polypeptide with cholesteryl ester transfer activity.
  • Other preferred isogenes encode CETP polypeptides with conservative or moderately conservative amino acid changes, as defined by R. Grantham (1974 Science 185:862-4) or other systems well-known to practitioners in the art.
  • Additional preferred polynucleotides comprise a CETP isogene defined by a sub-haplotype selected from the group consisting of: (a) guanine at PS20 and guanine at PS47; (b) guanine at PS22 and guanine at PS47; (c) guanine at PS28 and guanine at PS47 (d) cytosine at PS32 and guanine at PS47; (e) cytosine at PS35 and guanine at PS47; (f) guanine at PS20 and guanine at PS46; (g) guanine at PS22 and guanine at PS46; (h) guanine at PS28 and guanine at PS46; (i) cytosine at PS32 and guanine at PS46; and (j) cytosine at PS35 and guanine at PS46.
  • a sub-haplotype selected from the group consisting of: (a) guanine at PS20 and gu
  • the CETP isogene is defined by sub-haplotype (a) or (b).
  • Haplotypes comprising any of one of sub-haplotypes (a) - (j) have their HAP ID numbers listed in Tables 11 or 12; the HAP ID numbers can be correlated with the haplotype numbers used herein using the information in Table 5.
  • Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the CETP gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymo ⁇ hic sites described herein.
  • Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • Any particular CETP variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
  • CETP isogenes, or fragments thereof may be isolated using any method that allows separation of the two "copies" of the CETP 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 (TIVC) 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.
  • TIVC 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.
  • CETP genome anthologies are collections of at least two CETP 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.
  • a CETP genome anthology may comprise individual CETP 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 CETP isogenes in the anthology may be stored in separate containers.
  • a preferred CETP genome anthology of the invention comprises a set ofisogenes defined by the haplotypes shown in Table 4 below. More preferably, the genome anthology comprises two or more isogenes selected from isogenes shown in Table 7 as comprising a variant CETP coding sequence or shown in Table 8 as encoding a variant CETP polypeptide.
  • Another preferred genome anthology prises two or more isogenes selected from the isogenes defined by a omprising the isogenes defined by haplotypes with the following HAP ID numbers: 9900020900, 9900020905, 9900020906, 9900020915, 9900020918, 9900020922, 9900020925, 9900020926, 9900020935, 9900020938, 9900020951, 9900020953, 9900020955, 9900020959, 9900020960, 9900020962, 9900020965, 9900020966, 9900020967, 9900020969, 9900020971, 9900020972, 9900020979, 9900020989, 9900020996, 9900021003, 9900021006, 9900021017, 9900021019, 9900021022, 9900021030, 9900021036, 9900021063, 9900021069, 9900021081, 9900021085, 9900021089, 9900021098, 9900021103, 9900021
  • haplotypes with these HAP ID numbers are shown in copending International Application PCT/US03/XXXXX filed April 28, 2003 to have correlations between haplotype copy number and HDLC response to statins.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded CETP protein in a prokaryotic or a eukaryotic 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 in a given host cell.
  • 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 CETP sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
  • the recombinant expression vector may be introduced 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).
  • eukaryotic expression vectors that function in eukaryotic 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 eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282:1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • CETP 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 a CETP cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the CETP reference coding sequence shown in Figure 2.
  • the invention also provides CETP mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
  • SEQ ID NO:2 (or its corresponding RNA sequence) for SEQ ID NO:2 (as described in the Examples below), except for having one or more polymo ⁇ hisms selected from the group consisting of guanine at a position corresponding to nucleotide 44, adenine at a position corresponding to nucleotide 534, cytosine at a position corresponding to nucleotide 756, thymine at a position corresponding to nucleotide 804, adenine at a position corresponding to nucleotide 940, adenine at a position corresponding to nucleotide 1153, and guanine at a position corresponding to nucleotide 1161, and may also comprise one or more additional polymo ⁇ hisms selected from the group consisting of adenine at a position corresponding to nucleotide 66, thymine at a position corresponding to nucleotide 861, cytosine at a position corresponding to nu
  • a preferred polymo ⁇ hic cDNA variant also referred to as a CETP coding sequence variant, is selected from the group consisting of A through W represented in Table 9 (or Table 7); more preferably, the cDNA variant encodes a nonreference CETP polypeptide selected from the group of CETP polypeptides A-0 in Table 10 (or Table 8). Most preferably, the CETP coding sequence variant encodes a nonreference CETP polypeptide with cholesteryl ester transfer activity. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain one or more of the novel polymo ⁇ hisms described herein.
  • the invention specifically excludes polynucleotides identical to previously identified CETP mRNAs or cDNAs, and previously described fragments thereof.
  • Polynucleotides comprising a variant CETP 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 a CETP 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 CETP 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 polymo ⁇ hic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the CETP genomic, mRNA and cDNA variants described herein.
  • Polynucleotides comprising a polymo ⁇ hic gene variant or fragment of the invention may be useful for therapeutic pu ⁇ oses.
  • an expression vector encoding the isoform may be admimstered to the patient.
  • the patient may be one who lacks the CETP isogene encoding that isoform or may already have at least one copy of that isogene.
  • CETP isogene In other situations, it may be desirable to decrease or block expression of a particular CETP isogene.
  • Expression of a CETP 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 preferred.
  • 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. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense oligonucleotides may also be designed to block translation of CETP mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of CETP mRNA transcribed from a particular isogene.
  • the untranslated 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.
  • Such molecules may be formulated as a pharmaceutical composition for administration to the patient.
  • Oligoribonucleotides and/or ohgodeoxynucleotides 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 polymo ⁇ hic variant of (a) the reference CETP amino acid sequence shown in Figure 3 or (b) a fragment of this reference sequence.
  • the location of a variant amino acid in a CETP polypeptide or fragment of the invention is preferably identified by aligning its sequence against SEQ ID NO:3 (Fig. 3).
  • a CETP protein variant (or isoform) of the invention comprises an amino acid sequence identical to SEQ ID NO:3 (as described in the Examples below), except for having one or more variant amino acids selected from the group consisting ofglycine at a position corresponding to amino acid position 15, lysine at a position corresponding to amino acid position 314, and methionine at a position corresponding to amino acid position 385, and may also comprise one or more additional variant amino acids selected from the group consisting of proline at a position corresponding to amino acid position 290, proline at a position corresponding to amino acid position 390, valine at a position corresponding to ammo acid position 422, glycine at a position corresponding to amino acid position 459, and glutamine at a position corresponding to amino acid position 468.
  • a CETP protein fragment of the invention is any fragment of a CETP 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 CETP, including SEQ ID NO:3, and previously described fragments thereof.
  • CETP protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO: 3 and having any novel combination of amino acid variations described herein.
  • a CETP protein variant is selected from the group consisting of A through O represented in Table 8; more preferably, the CETP protein variant has cholesteryl ester transfer activity.
  • the CETP protein variant is selected from the group consisting of A, B, D - J, M and N.
  • a CETP 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 CETP peptide variants may be useful as antigens to generate antibodies specific for one of the above CETP isoforms.
  • the CETP peptide variants may be useful in drug screening assays.
  • a CETP variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing an appropriate variant CETP genomic or cDNA sequence described above.
  • the CETP protein variant may be isolated from a biological sample of an individual having a CETP isogene which encodes the variant protein.
  • a particular CETP isoform of the invention can be isolated by immunoaff ⁇ nity chromatography using an antibody which specifically binds to that particular CETP isoform but does not bind to the other CETP isoform.
  • CETP 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 isoform of the CETP protein or peptide as discussed further below.
  • CETP 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). In the case of immunoaffinity chromatography, antibodies specific for a particular polymo ⁇ hic variant may be used.
  • a polymo ⁇ hic variant CETP gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric CETP protein.
  • the non-CETP portion of the chimeric protein may be recognized by a commercially available antibody.
  • the cliimeric protein may also be engineered to contain a cleavage site located between the CETP and non-CETP portions so that the CETP protein may be cleaved and purified away from the non-CETP portion.
  • An additional embodiment of the invention relates to using a novel CETP protein isoform, or a fragment thereof, in any of a variety of drug screening assays.
  • Screen assays may be performed to identify agents that bind specifically to all known CETP 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 CETP protein or peptide variant may be free in solution or affixed to a solid support.
  • detecting the interaction between the agents and the CETP isoform is performed by measuring cholesteryl ester transfer activity by the CETP.
  • high throughput screening of compounds for binding to a CETP 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 CETP protein(s) of interest and then washed. Bound CETP protein(s) are then detected using methods well-known in the art.
  • a novel CETP protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the CETP protein.
  • a particular CETP haplotype or group of CETP haplotypes encodes a CETP protein variant with an amino acid sequence distinct from that of CETP protein isoforms encoded by other CETP haplotypes
  • detection of that particular CETP haplotype or group of CETP haplotypes may be accomplished by detecting expression of the encoded CETP protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan.
  • the invention provides antibodies specific for and immunoreactive with one or more of the novel CETP protein or peptide variants described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the CETP 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 CETP 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. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).
  • albumins e.g., human, bovine, fish, ovine
  • keyhole limpet hemocyanin Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk 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 CETP isoform expressed by that individual.
  • the antibody may be formulated as a pharmaceutical composition which includes a pharmaceutically acceptable carrier.
  • Antibodies specific for and immunoreactive with one of the novel protein isoforms described herein may be used to immunoprecipitate the CETP protein variant from solution as well as react with CETP protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
  • the antibodies will detect CETP protein isoforms 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 CETP protein variants described herein is used in immunoassays to detect this variant in biological samples.
  • an antibody of the present invention is contacted with a biological sample and the formation of a complex between the CETP 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; Current Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York).
  • Standard techniques known in 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 Current Protocols in 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 polymo ⁇ hisms identified herein on expression of CETP may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts of the CETP 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 CETP 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 CETP 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 CETP isogene, cDNA or coding sequence may be introduced into the cell in a vector such that the isogene, cDNA or coding sequence remains extrachromosomal.
  • the gene will be expressed by the cell from the extrachromosomal location.
  • the CETP isogene, cDNA or coding sequence is 5 introduced into a cell in such a way that it recombines with the endogenous CETP gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired CETP gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention.
  • cells into which the CETP 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 CETP isogene, cDNA or coding sequence.
  • continuous culture cells such as COS, CHO, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the CETP isogene, cDNA or coding sequence.
  • L5 cells can be used to compare the biological activities of the different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant CETP 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 embryonic stage, i.e., the one-cell stage, or generally not later than 0 about the eight-cell stage.
  • Transgenic animals carrying the constructs 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 5 method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells.
  • mice Examples of animals into which the CETP 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, 0 pages 254-272).
  • Transgenic animals stably expressing a human CETP isogene, cDNA or coding sequence and producing the encoded human CETP protein can be used as biological models for studying diseases related to abnormal CETP 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 5 disorders affected by expression or function of a novel CETP isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel CETP isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one of the novel CETP isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel CETP 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 CETP isogene is reduced and/or eliminated.
  • the composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered saline, 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 pharmaceutical 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, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • 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.
  • 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 determined 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 administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • 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 generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the CETP gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the CETP polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files).
  • polymo ⁇ hism 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.
  • This example illustrates examination of various regions of the CETP gene for polymo ⁇ hic sites.
  • the following target regions of the CETP gene were amplified using 'tailed' PCR primers, each of which includes a universal sequence forming a noncomplementary '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 ID NO:284) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5 '-AGGAAACAGCTATGACCAT-3 ' (SEQ ID NO:285).
  • the nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and correspond to positions in SEQ ID NO: 1 ( Figure 1).
  • 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 0 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.
  • Sequence information was analyzed for the presence of polymo ⁇ hisms using the Polyphred program (Nickerson et al., Nucleic Acids Res. 14:2745-2751, 1997). The presence of a polymo ⁇ hism was confirmed on both strands.
  • the polymo ⁇ hisms and their locations in the CETP reference genomic sequence are listed in Table 2 below.
  • This example illustrates analysis of the CETP polymo ⁇ hisms identified in the experimental population for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in unrelated members of the experimental population are shown in Table 3 below, with the haplotype pair indicating 5 the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 3 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides.
  • haplotype pairs shown in Table 3 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.
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites.
  • This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
  • the list of haplotypes was augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family).
  • each of the CETP haplotypes comprises a 5' - 3' ordered sequence of 74 polymo ⁇ hisms whose positions in SEQ ID NO: 1 and alleles are set forth in Table 4.
  • the column labeled "Region Examined” provides the nucleotide positions in SEQ ID NO:l corresponding to sequenced regions of the gene.
  • PS No. and PS Position provide the polymo ⁇ hic site number designation (see Table 2) and the corresponding nucleotide position of this polymo ⁇ hic site within SEQ ID NO: 1 or SEQ ID NO:286.
  • the columns beneath the "Haplotype Number” heading are labeled to provide a unique number designation for each CETP haplotype.
  • SEQ ID NO: 1 refers to Figure 1 , with the two alternative allelic variants of each polymo ⁇ hic site indicated by the appropriate nucleotide symbol.
  • SEQ ID NO:286 is a modified version of SEQ ID NO: 1 that shows the context sequence of each of PS1-PS74 in a uniform format to facilitate electronic searching of the CETP haplotypes.
  • SEQ ID NO:286 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 polymo ⁇ hic site is separated by genomic sequence whose composition is defined elsewhere herein.
  • Table 5 shows the number of chromosomes characterized by a given CETP haplotype for all unrelated individuals in the experimental population for which haplotype data was obtained.
  • the haplotype number and the haplotype ID number may be correlated with the information in Tables 11 and 12 to determine which of the haplotypes comprise any of the sub-haplotypes (a) - (j) for which copy number was found to be correlated with HDLC response to statin treatment.
  • the number of these unrelated individuals who have a given CETP haplotype pair is shown in Table 6.
  • 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 reference population were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population.
  • this reference population 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 individuals in the reference population 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 experimental population, weighted to Caucasians means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the CETP gene are likely to be similar to the relative frequencies of these CETP haplotypes and haplotype pairs in the Caucasian U.S. population and in the three other population groups represented in the experimental population.
  • the genetic diversity observed 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 CETP haplotype shown in Table 4 defines a CETP isogene.
  • the CETP isogene defined by a given CETP haplotype comprises the examined regions of SEQ ID NO: 1 indicated in Table 4, with the corresponding ordered sequence of nucleotides occurring at each polymo ⁇ hic site within the CETP gene shown in Table 4for that defining haplotype.
  • Each CETP isogene defined by one of the haplotypes shown in Table 4 will further correspond to a particular CETP coding sequence variant.
  • Each of these CETP coding sequence variants comprises the regions of SEQ ID NO:2 examined (i.e., the entire coding sequence) and is defined by the 5 ' - 3 ' ordered sequence of nucleotides occurring at each polymo ⁇ hic site within the coding sequence of the CETP gene, as shown in Table 7.
  • the column labeled 'Region Examined' provides the nucleotide positions in SEQ ID NO:2 corresponding to sequenced regions of the gene; the columns labeled 'PS No.' and 'PS Position' provide the polymo ⁇ hic site number designation (see Table 2) and the corresponding nucleotide position of this polymo ⁇ hic site within SEQ ID NO:2.
  • the columns beneath the 'Coding Sequence Number' heading are numbered to correspond to the haplotype number defining the CETP isogene from wliich the coding sequence variant is derived.
  • CETP coding sequence variants that differ from the reference CETP coding sequence are denoted in Table 7 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 CETP isogenes.
  • Table 9 summarizes the data in Table 7 for the unique novel CETP coding sequences, A through W, observed
  • each CETP coding sequence represented in Table 7 encodes a CETP protein variant.
  • Each of the CETP protein variants encoded by the 216 CETP isogenes described herein comprises the regions of SEQ ID NO:3 examined by sequencing (i.e., the entire polypeptide chain) and is defined by the N-terminus to C-terminus sequence of amino acids resulting from the observed polymo ⁇ hisms at the polymo ⁇ hic sites within the coding sequence of the CETP gene, as presented in Table 8.
  • the column labeled 'Region Examined' provides amino acid positions in SEQ ID NO: 3 corresponding to sequenced regions of the gene.
  • Polyld is a unique identifier assigned to each PS by Genaissance Phannaceuticals, Inc. (R) Reported previously.

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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne de nouveaux variants génétiques du gène de la protéine de transfert d'ester de cholestéryle (CETP). L'invention concerne également divers génotypes, haplotypes, et paires d'haplotypes existant dans la population des Etats-unis pour le gène CETP. L'invention concerne en outre des compositions et des méthodes d'haplotypage et/ou de génotypage du gène CETP chez un individu. L'invention concerne enfin des polynucléotides définis par les haplotypes mentionnés ci-dessus.
PCT/US2003/013288 2002-04-26 2003-04-28 Haplotypes du gene cetp WO2003091277A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003239189A AU2003239189A1 (en) 2002-04-26 2003-04-28 Haplotypes of the cetp gene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37579102P 2002-04-26 2002-04-26
US60/375,791 2002-04-26

Publications (2)

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WO2003091277A2 true WO2003091277A2 (fr) 2003-11-06
WO2003091277A3 WO2003091277A3 (fr) 2005-04-14

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PCT/US2003/013346 WO2003091698A2 (fr) 2002-04-26 2003-04-28 Marqueurs genetiques de la cetp reperant des modifications, imputables aux statines, affectant le cholesterol hdl
PCT/US2003/013288 WO2003091277A2 (fr) 2002-04-26 2003-04-28 Haplotypes du gene cetp

Family Applications Before (1)

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PCT/US2003/013346 WO2003091698A2 (fr) 2002-04-26 2003-04-28 Marqueurs genetiques de la cetp reperant des modifications, imputables aux statines, affectant le cholesterol hdl

Country Status (2)

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AU (2) AU2003239189A1 (fr)
WO (2) WO2003091698A2 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474796A (en) * 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI [Online] 29 March 2003 DOE JOINT GENOME INSTITUTE: 'Direct submission', XP002985097 Retrieved from STN Database accession no. AC012181 *
DATABASE NCBI [Online] September 2000 DOE JOINT GENOME INSTITUTE: 'Sequencing of human chromosome 16', XP002985096 Retrieved from STN Database accession no. AC010550 *

Also Published As

Publication number Publication date
AU2003239189A1 (en) 2003-11-10
AU2003228761A1 (en) 2003-11-10
WO2003091698A2 (fr) 2003-11-06
WO2003091277A3 (fr) 2005-04-14
AU2003239189A8 (en) 2003-11-10
WO2003091698A3 (fr) 2005-04-21
AU2003228761A8 (en) 2003-11-10

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