WO2008042762A2

WO2008042762A2 - Single nucleotide polymorphisms associated with cardiovascular disease

Info

Publication number: WO2008042762A2
Application number: PCT/US2007/079862
Authority: WO
Inventors: Qiny Wang; Eric J. Topol; Gong-Qing Shen; Lin Li; Shaoqi Rao
Original assignee: The Cleveland Clinic Foundation
Priority date: 2006-09-29
Filing date: 2007-09-28
Publication date: 2008-04-10
Also published as: WO2008042762A3

Abstract

A method is provided for identifying a subject who has an increased risk for developing a cardiovascular disease. The method can include detecting an alteration in a portion of a LRP8 gene. The alternation can be located in at least one of exon 9, exon 17, exon 19, or intron 6 of the LRP8 gene. The presence of the alteration can be correlated with an increased risk for developing the cardiovascular disease.

Description

SINGLE NUCLEOTIDE POLYMORPHISMS ASSOCIATED WITH CARDIOVASCULAR DISEASE

Technical Field

[0001] The present invention relates generally to methods for identifying and diagnosing cardiovascular diseases, and more particularly to single nucleotide polymorphisms for assessing the risk of developing cardiovascular diseases such as myocardial infarction and coronary artery disease.

Background of the Invention

[0002] Atherosclerotic coronary artery disease (CAD) and myocardial infarction (MI) are complex traits that account for the leading cause of death in the Western world and, by 2020, projected to be the number one cause of death and disability worldwide. Multiple prior studies have documented the heritability of CAD, and particularly its most acute manifestation of MI.

[0003] Genomewide linkage analysis is an unbiased approach that may lead to the identification of previously-unknown genetic loci or genes for CAD and MI. Genome- wide linkage scans with hundreds of sibling pairs have identified several major genetic susceptibility loci for CAD or MI. The MI susceptibility gene on chromosome 13ql2 has been identified as the ALOX5AP gene (encoding 5-lipoxygenase activating protein. For the chromosome 3q CAD locus, two candidate genes, GATA2 and Kalirin, have been associated with CAD. [0004] The LRP8 protein is also known as the apolipoprotein E receptor 2, ApoER2. It is a lipoprotein receptor from the low-density lipoprotein receptor (LDLR) family. The structure of LRP8 closely resembles LDLR and very low-density lipoprotein receptor. The LRP8 gene is highly expressed in the brain and testes, as well as in the heart, endothelial cells, vascular smooth muscle cells, and platelets. Binding of LDL to LRP8 activates the phosphorylation of LRP8, which further activates the p38 MAPK stress-response signaling pathway, leading to platelet aggregation and potentially thrombosis and MI.

Summary of the Invention

[0005] The present invention relates generally to methods for identifying and diagnosing cardiovascular diseases, and more particularly to single nucleotide polymorphisms for assessing the risk of developing cardiovascular diseases such as myocardial infarction and coronary artery disease. According to one aspect of the present invention, a method is provided for identifying a subject who has an increased risk for developing a cardiovascular disease. The method can include detecting an alteration in a portion of a LRP8 gene. The alternation can be located in at least one of exon 9, exon 17, exon 19, or intron 6 of the LRP8 gene. The presence of the alteration can be correlated with an increased risk for developing the cardiovascular disease.

[0006] According to another aspect of the present invention, a method is provided for diagnosing a subject who has a cardiovascular disease or an increased risk for developing a cardiovascular disease. One step of the method can include obtaining a polynucleotide sample from the subject. The polynucleotide sample can comprise a polynucleotide sequence corresponding to at least a portion of the LRP8 gene selected from the group consisting of exon 9, exon 17, exon 19 and intron 6. After obtaining the polynucleotide sample, a determination can be made as to whether the polynucleotide sequence corresponding to the at least one portion of the LRP8 gene is altered. The presence of the alteration can be correlated with an increased risk for developing the cardiovascular disease.

[0007] According to another aspect of the present invention, a method is provided for identifying a subject who has an increased risk for developing a cardiovascular disease. One step of the method can include detecting a LRP8 haplotype. The LRP8 haplotype can include at least two single nucleotide polymorphisms selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The presence of the LRP8 haplotype can be correlated with an increased risk for developing the cardiovascular disease.

[0008] According to another aspect of the present invention, a method is provided for assessing the risk of a cardiovascular disease in a subject. One step of the method can include assessing platelet reactivity in a blood sample of the subject and then assessing the level of a LRP8 gene alteration in the blood sample. The LRP8 gene alteration can be located in at least one of exon 9, exon 17, exon 19 or intron 6 of the LRP8 gene. The presence of the LRP8 gene alteration and an increase in platelet aggregation reactivity can be correlated with an increased risk for developing the cardiovascular disease.

[0009] According to another aspect of the present invention, a method is provided for identifying an agent useful in therapeutically or prophylactically treating a cardiovascular disease in a subject. One step of the method can include contacting a LRP8 gene variant or a LRP8 polypeptide variant with a candidate agent under conditions suitable to allow formation of a binding complex between the LRP8 gene variant or the LRP8 polypeptide variant and the candidate agent. The LRP8 gene variant and the LRP8 polypeptide variant can respectively include SEQ ID NO: 1 and SEQ ID NO: 6. After contacting the LRP8 gene variant or the LRP8 polypeptide variant with a candidate agent, the formation of the binding complex can be detected to identify the agent.

Brief Description of the Drawings

[0010] The foregoing and other features of the present invention will become apparent to those skilled in the art to which the present invention relates upon reading the following description with reference to the accompanying drawings, in which:

[0011] Fig. 1 is a series of graphs showing the relationship between LRP8 SNP R952Q and the chromosome lp34-36 premature myocardial infarction susceptibility locus. The X axis denotes the position of markers and the vertical axis is -log(P);

[0012] Figs. 2A-B show the effects of the R952Q SNP on phosphorylation of p38 MAPK induced by oxidized LDL (ox-LDL). Meg-01 cells were transfected with pEGFP-hLRP8 Wild type (WT) or pEGFP-hLRP8 mutant (R952Q) using Nucleofector. After 48h, cells were incubated with ox-LDL (2 μg/ml) for different times, and lysed. An equal amount of total cellular proteins (30 μg) was analyzed for phosphorylated and total p38 MAPK by Western blot analysis. A representative image of the blot is shown in Fig. 2A. The images -A-

were scanned and quantified. The data are shown as mean ± SD densitometric units in Fig.

2B. The experiments were replicated 6 times and the graph represents the data from 6 independent experiments; and

[0013] Fig. 3 is a bar graph showing the effects of LRP8 SNP R952Q on platelet aggregation.

Detailed Description

[0014] The present invention relates generally to methods for identifying and diagnosing cardiovascular diseases, and more particularly to single nucleotide polymorphisms for assessing the risk of developing cardiovascular diseases, such as myocardial infarction and coronary artery disease. The present invention is based on the discovery that genetic polymorphisms of the low density lipoprotein receptor-related protein 8 (LRP8) gene showed significant association with coronary artery disease (CAD) and myocardial infarction (MI). More particularly, the present invention is based on the discovery that the non- synonymous short nucleotide polymorphism (SNP) R952Q of LRP8 significantly increased the activation (i.e., phosphorylation) of p38 MAPK upon treatment with oxidized LDL. Based on this discovery, the present invention provides a method for identifying a subject who has an increased risk for developing a cardiovascular disease, a method of diagnosing a subject who has a cardiovascular disease or an increased risk for developing a cardiovascular disease, a method for assessing the risk of a cardiovascular disease in a subject, and a method for identifying an agent useful in therapeutically or prophylactically treating a cardiovascular disease in a subject.

[0015] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley- Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Edition, Springer- Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present invention. [0016] In the context of the present invention, the term "polypeptide" refers to an oligopeptide, peptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The term "polypeptide" also includes amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids. The term "polypeptide" also includes peptides and polypeptide fragments, motifs and the like, glycosylated polypeptides, and all "mimetic" and "peptidomimetic" polypeptide forms. [0017] As used herein, the term "polynucleotide" refers to oligonucleotides, nucleotides, or to a fragment of any of these, to DNA or RNA {e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acids, or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, siRNAs, microRNAs, and ribonucleoproteins. The term also encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.

[0018] As used herein, the term "complementary" refers to the capacity for precise pairing between two nucleobases of a polynucleotide and its corresponding target molecule. For example, if a nucleobase at a particular position of a polynucleotide is capable of hydrogen bonding with a nucleobase at a particular position of a target polynucleotide (the target nucleic acid being a DNA or RNA molecule, for example, then the position of hydrogen bonding between the polynucleotide and the target polynucleotide is considered to be complementary. A polynucleotide and a target polynucleotide are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which can be used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between a polynucleotide and a target polynucleotide. [0019] As used herein, the terms "effective," "effective amount," and "therapeutically effective amount" refer to that amount of a pharmaceutical composition that results in amelioration of symptoms or a prolongation of survival in a subject with a cardiovascular disease. A therapeutically relevant effect relieves to some extent one or more symptoms of a cardiovascular disease, or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the cardiovascular disease.

[0020] As used herein, the term "subject" refers to any warm-blooded organism including, but not limited to, human beings, rats, mice, dogs, goats, sheep, horses, monkeys, apes, rabbits, cattle, etc.

[0021] As used herein, the terms "allele" and "allelic variant" refer to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.

[0022] As used herein, the terms "homology," "identity," or "similarity" refer to sequence similarity between two polypeptides or between two polynucleotides. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0023] As used herein, the term "interact" as used herein is meant to include detectable interactions between molecules such as can be detected using, for examples, a hybridization assay. The term is also meant to include binding interactions between molecules.

Interactions may be, for example, polypeptide-polypeptide, polypeptide-polynucleotide, polypeptide- small molecule, or small molecule-polynucleotide in nature.

[0024] As used herein, the term "intron" refers to a segment of a polynucleotide, usually

DNA, that does not encode part of or all of an expressed polypeptide and which, in endogenous conditions, is transcribed into RNA but which is spliced out of the endogenous

RNA before the RNA is translated into a polypeptide. [0025] As used herein, the terms "intronic sequence" or "intronic polynucleotide sequence" refer to the polynucleotide sequence of an intron or portion thereof.

[0026] As used herein, the term "exon" refers to a segment of a polynucleotide, usually

DNA, that encodes part of or all of an expressed polypeptide.

[0027] As used herein, the terms "extronic sequence" or "extronic polynucleotide sequence" refer to the polynucleotide sequence of an exon or portion thereof.

[0028] As used herein, the term "modulation" refers to up-regulation (i.e., activation or stimulation), for example, by agonizing, and down-regulation (i.e., inhibition or suppression), for example, by antagonizing a bioactivity (e.g., expression of a gene).

[0029] As used herein, the term "polymorphism" refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e., two different polynucleotide sequences, is referred to as a "polymorphic region of a gene". A polymorphic region can be a single nucleotide, the identity of which differs in different alleles. A polymorphic region can also be several polynucleotides long.

[0030] As used herein, the terms "specifically hybridizes" or "specifically detects" refer to the ability of a polynucleotide or polypeptide to hybridize to a given number of consecutive nucleotides or amino acids, respectively.

[0031] As used herein, the term "haplotype" refers to a group of two or more SNPs that are functionally and/or spatially linked. Haplotypes define groups of SNPs that lie inside genes belonging to identical (or related metabolic) pathways and/or lie on the same chromosome. Often times, haplotypes can give better predictive/diagnostic information than a single SNP.

[0032] As used herein, the term "gene product" refers to all molecules derived from a gene, especially RNA and protein. Complementary DNA (cDNA) is also encompassed where, for example, the cDNA is made by naturally-occurring reverse transcriptase.

[0033] As used herein, the term "cardiovascular disease" refers to those diseases or disease states associated with high levels of Lp(a) in plasma as well as other lipoproteins, such as LDL. The term includes, without limitation, arteriosclerosis, atherosclerosis, CAD, peripheral artery disease, MI, stroke, restenosis, and bypass graft stenosis.

[0034] As used herein, the term "LRP8 gene variant" refers to an LRP8 gene having at least one genetic polymorphism, such as an insertion, deletion, point mutation, inversion, and/or splicing alteration, when compared to a wild-type LRP8 gene. [0035] As used herein, the term "LRP8 polypeptide variant" refers to a LRP8 polypeptide having at least one amino acid alteration as compared to a wild-type LRP8 polypeptide. [0036] Although it is not necessary to understand the mechanism in order to practice the present invention, and it is not intended that the present invention be so limited, it is shown by the present invention that SNP R952Q of the LRP8 gene increased the activation of p38 MAPK. Activation of p38 MAPK may influence the sensitization of platelets by LDL, resulting in formation of arachidonate metabolites and release of inflammatory molecules, which may increase the risk of atherosclerosis or atherothrombosis. The important role of p38 MAPK in inflammation has been well-established and overexpression of p38 MAPK has been shown to induce myocardial fibrosis and inflammation. It has also been shown that p38 MAPK becomes activated during ischemia and this activation leads to myocyte death and myocardial injury. These reported results are consistent with the present invention, i.e., the risk allele of SNP R952Q of LRP8 increased activation of p38 MAPK and susceptibility to CAD and MI. From this, it is believed that detecting LRP8 gene variants and/or LRP8 polypeptide variants will facilitate identification of subjects who have an increased risk for developing a cardiovascular disease.

[0037] In an aspect of the present invention, a method is provided for identifying a subject who has an increased risk for developing a cardiovascular disease. The method includes detecting an alteration in a portion of a LRP8 gene located in at least one of exon 9, exon 17, exon 19 or intron 6 of the LRP8 gene. The presence of an LRP8 gene alteration can be correlated with an increased risk for developing a cardiovascular disease, such as MI or CAD, for example.

[0038] Alterations in the LRP8 gene can include insertions, deletions, point mutations, and inversion of polynucleotides in the LRP8 gene sequence. The alterations can occur at any position within the LRP8 gene including coding, non-coding, transcribed, non- transcribed, and regulatory regions. For example, the alteration can include a SNP in exon 19 having SEQ ID NO: 1 or SEQ ID NO: 2. Exon 19 can include the nucleotide bases 2996- 4507 of SEQ ID NO: 7. The SNP having SEQ ID NO: 1 can include an A^G or G^A transition at nucleotide 2997 of SEQ ID NO: 7. The SNP having SEQ ID NO: 2 can include a G^C or C^G transition at nucleotide 3896 of SEQ ID NO: 7. [0039] The alteration can also include a SNP in exon 17 having SEQ ID NO: 3. Exon 17 can include the nucleotide bases 2646-2818 of SEQ ID NO: 7. The SNP having SEQ ID NO:

3 can include a C->T or T->C transition at nucleotide 2764 of SEQ ID NO: 7.

[0040] The alteration can also include a SNP in exon 9 having SEQ ID NO: 4. Exon 9 can include the nucleotide bases 1395-1569 of SEQ ID NO: 7. The SNP having SEQ ID NO:

4 can include an A->C or C->A transition at nucleotide 1399 of SEQ ID NO: 7.

[0041] The alteration can also include a SNP SEQ ID NO: 5 in intron 6 of a wild-type LRP8 gene (GeneBank Accession No. AL355483). The SNP having SEQ ID NO: 5 can include an A->G or G-^A transition at nucleotide 8461 of the wild-type LRP8 gene. [0042] Other alterations that can be detected include polynucleotide modification such as methylation, gross rearrangement in the genome such as in a homogeneously- staining region, double minute chromosome or other extrachromosomal element, or cytoskeletal arrangement. [0043] In another aspect of the present invention, a method is provided for identifying a subject who has an increased risk for developing a cardiovascular disease. The method can include the step of detecting a LRP8 haplotype. The LRP8 haplotype can comprise at least two SNPs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The presence of the LRP8 haplotype can be correlated with an increased risk for developing cardiovascular disease.

[0044] The present invention also encompasses the detection of RNA transcribed from LRP8 gene variants. Detection of the RNA transcribed from the LRP8 gene variants can encompass alterations in copy number and polynucleotide sequence. Sequence changes can include insertion, deletion, point mutation, inversion, and splicing variation. Detection of LRP8 gene variant RNA can be indirectly accomplished by means of its cDNA. [0045] LRP8 gene variant DNA and RNA levels and gross rearrangement can be analyzed by any of the standard methods known in the art. For example, a polynucleotide can be isolated from a cell or analyzed in situ in a cell or tissue sample. For detecting alterations in polynucleotide levels or gross rearrangement, all, or any part, of the polynucleotide can be detected. Polynucleotide reagents derived from any desired region of a LRP 8 gene variant can be used as a probe or primer for these procedures. Copy number can be assessed by in situ hybridization or isolation of polynucleotides from the cell and quantified by standard hybridization procedures, such as Southern or Northern blot analysis, for example. Genes can be amplified in the forms of homogeneously- staining regions or double minute chromosomes. Accordingly, one method of detection involves assessing the cellular position of an amplified gene. This method encompasses standard in situ hybridization methods, or alternatively, detection of an amplified fragment derived from digestion with an appropriate restriction enzyme recognizing a sequence that is repeated in the amplified unit.

[0046] Identifying polynucleotide modifications, such as methylation, can be analyzed by any of the known methods in the art for digesting polynucleotides and analyzing modified polynucleotides, such as by BPLC, thin-layer chromatography, mass spectra analysis, and the like. Gross rearrangements in the genome can be detected by means of in situ hybridization, although this type of alteration can also be assessed by means of assays involving normal cellular components with which the genes are normally found, such as in specific membrane preparations.

[0047] Mutations in the LRP8 gene can be analyzed by any of the standard methods known in the art. For example, a polynucleotide can be isolated from a cell or analyzed in situ in a cell or tissue sample by means of specific hybridization probes designed to allow detection of the mutation. The portion of the polynucleotide that is detected can contain the mutation. It is to be understood that in some aspects of the present invention, as where the mutation affects secondary structure or other cellular association, distant regions affected by the mutation can be detected. The polynucleotide reagents can be derived from the mutated region of the LRP 8 gene to be used as a probe or primer for the procedures. However, as discussed above, polynucleotide reagents useful as probes can be derived from any position in the polynucleotide. RNA or cDNA can be used in the same way. [0048] In certain aspects of the present invention, detection of a mutation in the LRP8 gene can involve the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR, RACE PCR or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et ah, Science 241:1077-1080 (1988); and Nakazawa et ah, PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et ah, Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a subject, isolating a polynucleotide sample (e.g., genomic, mRNA, or both) from the cells of the sample, contacting the polynucleotide sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product or detecting the size of the amplification product and comparing the length to a control sample (e.g., a wild-type LRP8 polynucleotide). Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal (or wild- type) RNA or antisense DNA sequences.

[0049] In an example of the method, a SNP of the LRP8 gene can be genotyped using high throughout SNP analysis. High throughput SNP genotyping can be performed using the 5' nuclease allelic discrimination assay (TaqMan Assay) on an ABI PRISM 7900HT Sequence Detection System. The assay can include the forward target- specific PCR primer, the reverse primer, and the TaqMan MGB probes labeled with two special dyes-FAM and VIC. The probes can be purchased through TaqMan Assays-on-Demand or Assays-by-Design from ABI (Applied Biosystems, Foster City, CA, USA). Genotyping can be performed in a total of about 5 μl of PCR reaction volume containing about 2.5 μl of TaqMan Universal PCR Master Mix, about .25 μl of 20 x TaqMan MGB Assay Mix, and about 25 ng of genomic DNA. Automatic allele calling can be carried out by ABI PRISM 7900HT data collection and analysis software version 2.1.

[0050] Direct DNA sequence analysis of SNPs in about 24 DNA samples can be used to ensure the quality of SNP genotyping. Direct DNA sequence analysis can be performed using an ABI PRISM 3100 Genetic Analyzer (ABI, Foster City, CA, USA). A DNA fragment containing the SNP can be PCR-amplified in an about 25 μl volume containing about 2.5 μl of 10 x PCR buffer (about 1.5 mM MgCl₂, about 2.5 μl of about .2 mM dNTP, about .5 μM of each PCR primer, about 1 U of Taq polymerase, and about 50 ng of genomic DNA). PCR products can be separated from agarose gels, isolated and purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA, USA), and sequenced with both forward and/or reverse primers. The sequencing reaction can be performed using the BigDye Terminator vl.l Cycle Sequencing Kit (ABI, Foster City, CA, USA).

[0051] Alternatively, mutations in the LRP8 gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns as determined by gel electrophoresis. Further, sequence-specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method. Furthermore, sequence differences between a LRP8 gene variant and a wild- type LRP8 gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Biotechniques 19:448 (1995)), including sequencing by mass spectrometry (e.g., PCT International Publication No. WO 94/16101; Cohen et ah, Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0052] Other methods for detecting mutations in the LRP8 gene can include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al, Science 230:1242 (1985)); Cotton et ah, PNAS 85:4397 (1988); Saleeba et ah, Meth. Enzymol. 217:286-295 (1992)), where electrophoretic mobility of mutant and wild-type polynucleotide is compared (Orita et ah, PNAS 86:2766 (1989); Cotton et ah, Mutat. Res. 285:125-144 (1993); and Hayashi et ah, Genet. Anal. Tech. Appl. 9:73-79 (1992)), and/or movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et ah, Nature 313:495 (1985)). Examples of other techniques for detecting point mutations can include, but are not limited to, selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0053] Methods of detection of a mutation of the LRP8 gene can also include detection of a LRP8 polypeptide encoded by the LRP8 gene variant. For example, methods of detecting an LRP8 polypeptide variant having SEQ ID NO: 6 are included within the scope of the present invention. Additionally or alternatively, the methods of the present invention can include detection of polypeptides variants encoded by LRP8 gene variants having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. Detection can encompass assessing polypeptide levels, mutation, post-translational modification, and subcellular localization. Mutations can encompass deletion, insertion, substitution and inversion. Mutations at RNA splice junctions can result in polypeptide splice variants as well.

[0054] LRP8 polypeptide variant levels can be analyzed by any of the standard methods known in the art. For example, LRP8 polypeptide variants can be isolated from a cell or analyzed in situ in a cell or tissue sample. Quantification of LRP8 polypeptide variants can be accomplished in situ, for example by standard fluorescence detection procedures involving a fluorescently-labeled binding partner, such as an antibody or other protein with which the LRP8 polypeptide variants will bind. This could include a substrate upon which the polypeptide acts, or an enzyme which normally acts on the polypeptide. Quantification of isolated polypeptides can be accomplished by other standard methods for isolated protein, such as in situ gel detection, Western blot, or quantitative protein blot. Levels can also be assayed by functional means, such as the effects upon a specific substrate. In the case of a LRP8 polypeptide variant, this could involve the detection of an increase in p38 MAPK activation, for example.

[0055] One example of a functional means useful for detecting the presence of a LRP8 polypeptide variant and/or LRP8 gene variant can include a p38 MAPK activation assay. To conduct the p38 MAPK activation assay, a GFP-tagged human LRP 8 gene can be PCR- amplified with primers containing in-frame Hind III and BamH I restriction sites. The PCR fragment can be digested with Hind III and BamH I and sub-cloned into the pEGFP Cl vector cut with the same enzymes. Mutant LRP8-containing SNP R952Q can be generated by PCR-based site-directed mutagenesis. Both the wild-type and mutant LRP8 expression constructs can be verified by direct DNA sequence analysis of the entire inserts. [0056] Meg-01 cells (American Type Culture Collection, Rockville, MD, USA) can be grown in RPMI- 1640 medium containing about 10% fetal bovine serum and then maintained at a density of about 3 x 10⁵ cells/ml. In general, cells can be used for experiments after about 5 days of supplementation. Transfection of Meg-01 cells can be carried out with about 1 μg plasmid DNA/well with a Nucleofector device and corresponding kits (Amaxa, Inc., Cologne, Germany) in 6- well plates. About 48h after transfection, cells can be divided equally into five wells, and incubated with oxidized LDL for about 0, 20, 40, 60, and 120 minutes. Cells can then be lysed, an equal amount of total cellular proteins per sample separated with about 12% SDS-PAGE gels, and electro-transferred onto PVDF membranes. The blots can be blocked in about 5% non-fat milk powder in PBST for aboutl hour, washed briefly in TBST, and then incubated with a primary monoclonal antibody directed against phosphor-p38 MAPK (D-8, SC-7973, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a polyclonal antibody against total p38 MAPK (C-20 SC-535, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in about 5% non-fat milk /PBST overnight. Membranes can then be extensively washed in PBST (8 x 5 min) and incubated with about a 1:2000 dilution of HRP- conjugated anti-mouse IgM (Sigma, St. Louis, MO, USA) for about 1 hour at room temperature. Membranes can again be washed (3 x 5 min) in FBST, and ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA) can be used to visualize the protein signal.

[0057] Mutations in LRP8 polypeptides can be analyzed by any of the above or other standard methods known in the art. For example, a polypeptide can be isolated from a cell or analyzed in situ in a cell or tissue sample. Analytic methods can include assays for altered electrophoretic mobility, binding properties, tryptic peptide digest, molecular weight, antibody-binding pattern, isoelectric point, amino acid sequence, and any other of the known assay techniques useful for detecting mutations in a protein. Assays can include, but are not limited to, those discussed in Varlamov et al. (J. Biol. Chem. 271:13981 (1996)), which is incorporated herein by reference for teaching such assays. These can include C-terminal arginine binding, acidic pH optima, sensitivity to inhibitors, thermal stability, intracellular distribution, endopeptidase activity, effect on endopeptidase inhibitor, substrate affinity, enzyme kinetics, membrane association, posttranslational modification, active site confirmation, compartmentalization, binding to substrate, secretion, and turnover. Further assays for function can be found in Fricker, J. Cell Biochem. 38:279-289 (1988), and Manser et al., Biochem. J. 267:517-525, (1990), both of which are incorporated by reference for teaching specific functions that can be assayed for mutation in the LRP8 gene. [0058] In vitro techniques for detection of LRP8 polypeptide variants can include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Alternatively, LRP8 polypeptide variants can be detected in vivo in a subject by introducing into the subject a labeled anti-LRP8 polypeptide variant antibody. For example, the antibody can be directed against an epitope of the LRP8 polypeptide having SEQ ID NO: 6. Additionally, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. For detection of specific mutation in the protein, antibodies, or other binding partners, can be used that specifically recognize these alterations. Alternatively, mutations can be detected by direct sequencing of the polypeptide. [0059] Other alterations that can be detected include alterations in post-translational modification. Amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications. Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art. [0060] Known modifications can include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0061] Such modifications are well-known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins-Structure and Molecular Properties, 2^nd ed., TE Creighton, WH Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F. (Posttranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York 1-12 (1983)), Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)), and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)). [0062] In addition to detection methods that involve specific physical features, functional characteristics of a LRP8 polypeptide variant may also be useful for detection with known methods. These can include changes in biochemistry, such as substrate affinity, enzyme kinetics, membrane association, active site conformation, compartmentalization, forming a complex with substrates or enzymes that act upon the protein, secretion, turnover, pH optima, sensitivity to inhibitors, thermal stability, endopeptidase activity, effects on endopeptidase inhibitors, and any other such functional characteristic that is indicative of a mutation or alteration in post-translational modification. Specific assays can be found in the literature (see, e.g., Varlamov et al. (1996) J. Biol. Chem. 271:13981).

[0063] LRP 8 gene and polypeptide variants can be detected in a variety of systems. These include cell-free and cell-based systems in vitro, tissues, such as ex vivo tissues for returning to a subject, in a biopsy, and in vivo, such as in subjects being treated, for monitoring clinical trials, and in animal models. Cell-free systems can be derived from cell lines or cell strains in vitro, including recombinant cells, cells derived from subjects, subjects involved in clinical trials, and animal models, including transgenic animal models. In one aspect of the present invention, LRP8 gene and polypeptide variants can be detected in cell- based systems. This includes cell lines and cell strains in vitro, including recombinant lines and strains containing a LRP8 gene variant, expanded cells such as primary cultures, particularly those derived from a subject with CAD and/or MI, subjects undergoing clinical trials, and animal models of CAD and/or MI, including transgenic animals. [0064] LRP8 gene variants and/or LRP8 polypeptide variants can also be detected in tissues. These include tissues derived from subjects with CAD and/or MI, subjects undergoing clinical trials, and animal models. In one aspect of the present invention, the tissues can be those affected in CAD and/or MI. LRP8 gene variants and/or LRP8 polypeptide variants can also be detected in individual subjects with CAD and/or MI, subjects undergoing clinical trials, and in animal models of CAD and/or MI, including transgenic models. Examples of sources of detection include cell and tissue biopsies from subjects affected with CAD and/or MI, or at risk for developing CAD and/or MI. [0065] In addition to detecting LRP8 gene variants and/or LRP8 polypeptide variants directly, the present invention also encompasses the use of compounds that produce a specific effect on LRP8 gene or polypeptide variants as a further means of detecting or diagnosing a cardiovascular disease. This can include, for example, detection of binding partners, including binding partner(s) specific for LRP8 gene or polypeptide variants, and compounds that have a detectable effect on a function of LRP8 gene or polypeptide variants. For example, an increase in LRP8 polypeptide variant levels can be detected by a complex formed between the LRP8 polypeptide variants and a binding partner, or levels of free LRP8 polypeptide variant binding partner(s).

[0066] In another aspect of the present invention, a method for assessing the risk of a cardiovascular disease in a subject can comprise assessing platelet reactivity in a blood sample of the subject, and then assessing the level of a LRP8 gene alteration in the blood sample. The LRP8 gene alteration can include a portion of a LRP8 gene located in at least one of exon 9, exon 17, exon 19 or intron 6 of the LRP8 gene. The presence of the LRP8 gene alteration and an increase in platelet aggregation reactivity can be correlated with an increased risk for developing a cardiovascular disease, such as MI or CAD, for example. Alterations in the LRP8 gene can include insertions, deletions, point mutations, and inversion of polynucleotides in the LRP8 gene sequence. The alterations can occur at any position within the LRP8 gene including coding, non-coding, transcribed, non-transcribed, and regulatory regions. For example, the alteration can include a SNP in exon 19 having SEQ ID NO: 1 or SEQ ID NO: 2; a SNP in exon 17 having SEQ ID NO: 3; a SNP in exon 9 having SEQ ID NO: 4; and/or a SNP in intron 6 having SEQ ID NO: 5.

[0067] One example of a platelet aggregation assay can be performed within about 3 hours after collecting the blood sample from the subject. Aggregation can be measured by using an impedance method on a Chrono-log Whole Blood Impedance Aggregometer, for example. About 500 μl of blood can be mixed with about 500 μl of saline and the change in impedance of the sample can be measured in the presence of ADP during about a 6 minute test run and recorded on a computer with the Chrono-log AGGRO/LINK software. Impedance aggregation in ohms can be used to index the rate and the degree of platelet aggregation.

[0068] All these methods of detection can be used in procedures to screen subjects at risk for developing or having a cardiovascular disease such as CAD and/or MI, for example. Further, detection of LRP8 gene or polypeptide variants in subjects can serve as a prognostic marker for developing CAD and/or MI, for example, or a diagnostic marker for having CAD and/or MI when the subjects are not known to have CAD and/or MI or to be at risk for having CAD and/or MI. Diagnostic assays can be performed in cell-based systems, and particularly in cells associated with CAD or MI, in intact tissue, such as a biopsy, in nonhuman animals, and in humans in vivo. Diagnosis can be at the level of polynucleotide or polypeptide. [0069] The present invention also encompasses methods for modulating the level or activity of LRP8 gene or polypeptide variants. At the level of the gene, known recombinant techniques can be used to alter a gene in vitro or in situ. Excessive copies of, or all or part of, the LRP8 gene can be deleted. Deletions can be made in any desired region of the gene including transcribed, non-transcribed, coding and non-coding regions. Additional copies of a part or the entire gene can also be introduced into a genome. Finally, alterations in polynucleotide sequences can be introduced into the gene by recombinant techniques. Alterations can include deletions, insertions, inversions, and point mutation. Accordingly, CAD and/or MI that is caused by a LRP8 gene variant could be treated by introducing a functional (wild-type) LRP8 gene into a subject. Further, specific alterations could be introduced into the gene and function tested in any given cell type, such as in cell-based models for CAD and/or MI. Still further, any given mutation can be introduced into a cell and used to form a transgenic animal which can then serve as a model for CAD and/or MI testing.

[0070] The level and activity of a LRP8 gene RNA may also be subject to modulation. Polynucleotides corresponding to any desired region of the RNA can be used directly to block transcription or translation of LRP8 gene variants by means of antisense or ribozyme constructs. Thus, where a cardiovascular disease is characterized by abnormally high gene expression, these polynucleotides can be used to decrease expression levels. A DNA antisense polynucleotide can be designed to be complementary to a region of the LRP8 gene variant involved in transcription, thereby preventing transcription and hence production of the corresponding polypeptide variant. An antisense RNA or DNA polynucleotide may hybridize to the mRNA variant and thus block translation of mRNA into the polypeptide variant. An alternative technique can involve cleavage by ribozymes containing polynucleotide sequences complementary to one or more regions in the mRNA variant that attenuates the ability of the mRNA to be translated.

[0071] The present invention can also include the modulation of LRP8 gene variant expression using compounds that have been discovered by screening the effects of the compounds on LRP8 gene levels or function.

[0072] The present invention is further directed to methods for modulating LRP8 polypeptide variant levels or function. For example, antibodies can be prepared against specific polypeptide fragments containing sites required for function or against the intact polypeptide. Polypeptide levels can also be modulated by use of compounds discovered in screening techniques in which the polypeptide levels serve as a target for effective compounds. Finally, LRP8 polypeptide variants can be functionally affected by the use of compounds discovered in screening techniques that use an alteration of mutant function as an end point. [0073] Modulation can be in a cell-free system. In this context, for example, an assay could involve cleavage of substrate or other indicator of LRP8 polypeptide variant activity. Modulation can also occur in cell-based systems. These cells may be permanent cell lines, cell strains, primary cultures, recombinant cells, cells derived from affected individuals, and transgenic animal models of CAD and/or MI, among others. Modulation can also be in vivo, for example, in subjects having a cardiovascular disease, in subjects undergoing clinical trials, and animal models of CAD and/or MI, including transgenic animal models. Modulation could be measured by direct assay of a LRP8 polypeptide variant or by the results of LRP8 polypeptide variant function. All of these methods can be used to affect LRP8 polypeptide function in subjects having, or at risk for having, CAD and/or MI. Thus, the present invention can encompass the treatment of CAD and/or MI by modulating the levels or function of a LRP8 polypeptide variant.

[0074] The present invention can also encompass methods for identifying agents useful in therapeutically or prophylactically treating a cardiovascular disease in a subject. Additionally, the present invention can include methods for identifying agents that interact with LRP8 gene variants and/or LRP8 polypeptide variants, and particularly that modulate the level or function of LRP8 gene variants and/or LRP8 polypeptide variants. Modulation can be at the level of transcription, translation, or polypeptide function. Accordingly, where the levels of an LRP8 gene variant and/or a LRP8 polypeptide variant are abnormally high or low, agents can be screened for the ability to correct the level of expression. Alternatively, where a mutation affects the function of a LRP8 gene variants and/or LRP8 polypeptide variants, agents can be screened for their ability to compensate for or to correct the dysfunction. In this manner, LRP8 gene variants and/or LRP8 polypeptide variants can be used to identify agonists and antagonists useful for affecting LRP8 gene variant expression. These agents can then be used to affect LRP8 gene variant or LRP8 polypeptide variant expression or function in subjects with CAD and/or MI. Thus, these screening methods are useful to identify agents that can be used for treating CAD and/or MI. [0075] These agents may also be useful in a diagnostic context in that they can then be used to identify altered levels of LRP8 gene variants and/or LRP8 polypeptide variants in a cell, tissue, nonhuman animal, and human. For example, agents specifically interacting with an LRP8 gene variant or an LRP8 polypeptide variant to produce a particular result by producing that result in a cell, tissue, nonhuman animal, or human, can indicate that there is an alteration in the LRP8 gene or gene product.

[0076] Thus, modulators of gene expression can be identified in a method wherein a LRP8 gene variant or gene variant product is contacted with a candidate agent and the level or expression of gene or gene product is determined. The level or expression of gene or gene product in the presence of the candidate agent can be compared to the level or expression of gene or gene product in the absence of the candidate agent. The candidate agent can then be identified as a modulator of polynucleotide or polypeptide expression based on this comparison and then used, for example, to treat CAD and/or MI. When the level or expression of gene or gene product is statistically significantly greater in the presence of the candidate agent than in its absence, the candidate compound can be identified as a stimulator of levels or expression of the gene or gene product. When levels or product expression are statistically significantly less in the presence of the candidate agent than in its absence, the candidate agent may be identified as an inhibitor.

[0077] These agents can be used to test on model systems, including animal models of CAD and/or MI, human clinical trial subjects, cells derived from these sources, as well as transgenic animal models of CAD and/or MI. Accordingly, the present invention provides methods of treatment with the gene or gene product as a target, using an agent identified through drug screening as a modulator to modulate expression of the gene or gene product. Modulation includes both up-regulation (i.e., activation or agonization) or down-regulation (i.e., suppression or antagonization) of polynucleotide expression. [0078] Candidate agents can include, for example, peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et ah, Nature 354:82-84 (1991); Houghten et ah, Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et ah, Cell l2:161-ll% (1993)); antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries). [0079] Any of the biological or biochemical functions mediated by LRP8 gene variants and/or LRP8 polypeptide variants can be used in an endpoint assay. These can include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art.

[0080] A further aspect of the present invention involves pharmacogenomic analysis in the case of LRP8 polypeptide variants. The field of pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M., Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996), and Linder, M., Clin. Chem. 43(2):254-266 (1997). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain subjects or therapeutic failure of drugs in certain subjects as a result of individual variation in metabolism. Thus, the genotype of a subject can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes affects both the intensity and duration of drug action. Thus, the pharmacogenomics of a subject permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the subject's genotype. Accordingly, in one aspect of the present invention, natural variants of LRP8 polypeptide variants may be used to screen for agents that are effective against a given allele and are not toxic to a specific subject. Agents can thus be classed according to their effects against naturally occurring allelic variants. This allows more effective treatment and diagnosis of CAD and/or MI.

[0081] Test systems for identifying agents can include both cell-free and cell-based systems derived from normal and affected tissue, cell lines and strains, primary cultures, animal CAD and/or MI models, including transgenic animals. Naturally- occurring cells can express abnormal levels of LRP 8 gene variants and/or LRP8 polypeptide variants. Alternatively, these cells can provide recombinant hosts for the expression of desired levels of LRP8 gene or gene product variants. A cell-free system can be used, for example, when assessing the effective agents on polynucleotide or polypeptide function. [0082] For example, in a cell-free system, competition binding assays can be designed to discover agents that interact with a LRP8 polypeptide variant. Thus, an agent can be exposed to the polypeptide under conditions that allow the agent to bind or to otherwise interact with the polypeptide. Soluble polypeptide can also be added to the mixture. If the test agent interacts with the soluble polypeptide, it can decrease the amount of complex formed or activity from the target. This type of assay is particularly useful in cases in which agents are sought that interact with specific regions of the polypeptide. Thus, the soluble polypeptide that competes with the target region can be designed to contain polypeptide sequences corresponding to a region of interest.

[0083] To perform cell-free drug screening assays, it may be desirable to immobilize either the polypeptide, fragment thereof, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the polypeptides, as well as to accommodate automation of the assay. Techniques for immobilizing polypeptides on matrices can be used in the drug screening assays. In one aspect of the present invention, a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, glutathione-S-transferase/LRP8 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione- derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate agent, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads can be washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of LRP8-binding polypeptide found in the bead fraction quantified from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art.

[0084] Alternatively, antibodies reactive with the polypeptide but which do not interfere with binding of the polypeptide to its target molecule can be derivatized to the wells of the plate, and the polypeptide trapped in the wells by antibody conjugation. Preparations of an LRP8-binding polypeptide and a candidate agent can be incubated in the LRP8 polypeptide- presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the LRP8 polypeptide target molecule, or which are reactive with the LRP8 polypeptide and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0085] Cell-based systems can include assays of individual cells or assays of cells in a tissue sample or in vivo. Drug screening assays can be cell-based or cell-free systems. Cell- based systems can be native, i.e., cells that normally express the protein, as a biopsy or expanded in cell culture. In one aspect of the present invention, however, cell-based assays can involve recombinant host cells expressing a LRP8 polypeptide variant. In vivo test systems include, not only subjects involved in clinical trials, but also animal CAD and/or MI models, including transgenic animals. Single cells can include recombinant host cells in which desired LRP8 gene variants can be been introduced. These host cells can express abnormally high or low levels of the LRP8 gene or gene product. Thus, the recombinant cells can be used as test systems for identifying agents that have the desired effect on the altered gene or gene product. Mutations can be naturally occurring or constructed for their effect on the course or development of CAD and/or MI, for example, determined by the model test systems discussed further below. Similarly, naturally-occurring or designed mutations can be introduced into transgenic animals which can then serve as an in vivo test system to identify compounds having a desired effect on a LRP8 gene or gene product.

[0086] In yet another aspect of the invention, LRP8 polypeptide variants can be used in a "two hybrid" assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) Oncogene 8:1693-1696; and WO94/10300) for isolating coding sequences for other cellular proteins which bind to or interact with LRP8 polypeptide variants. Briefly, the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins. In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first hybrid gene comprising the coding sequence for a DNA-binding domain of a transcriptional activator can be fused in frame to the coding sequence for a LRP8 polypeptide variant. The second hybrid protein can encodes a transcriptional activation domain fused in frame to a sample gene from a cDNA library. If the bait and sample hybrid proteins are able to interact, e.g., form a LRP8-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene can be detected and used to score for the interaction of LRP8 and sample proteins.

[0087] Modulators of LRP8 gene variants and/or LRP8 polypeptide variants identified according to these assays can be used to treat CAD and/or MI by treating cells that aberrantly express the gene or gene product. These methods of treatment can include the step of administering the modulators of polypeptide activity in a pharmaceutical composition (as described herein) to a subject in need of such treatment. The present invention thus provides a method for identifying an agent that can be used to treat autosomal CAD and/or MI. The method can include assaying the ability of the compound to modulate the expression of a LRP8 gene variant and/or LRP8 polypeptide variant to identify an agent that can be used to treat a cardiovascular disease such as MI or CAD, for example. [0088] LRP8 gene variants and/or LRP8 polypeptide variants may be useful in pharmaceutical compositions for diagnosing or modulating the level or expression of LRP8 gene or gene products in vivo, as in individual subjects treated for CAD and/or MI, subjects in clinical trials, animal CAD and/or MI models, and transgenic animal CAD and/or MI models. Thus, these pharmaceutical compositions may be useful for testing and treatment. LRP8 gene variants and/or LRP8 polypeptide variants may also be useful for otherwise modulating expression of the gene or gene product in cell-free or cell-based systems in vitro. They are further useful in ex vivo applications. LRP8 gene variants and gene variant products may also be useful for creating model test systems for CAD and/or MI, for example, recombinant cells, tissues, and animals. The genes and gene products are also useful in a diagnostic context as comparisons for other naturally-occurring variation in the LRP8 gene or gene product. Accordingly, these reagents can form the basis for a diagnostic kit. Further, specific variants (mutants) may be useful for testing agents that may be effective in the treatment or diagnosis of CAD and/or MI. Such mutants can also form the basis of a reagent in a test kit, particularly for introduction into a desired cell type or transgenic animal for drug testing. Accordingly, the present invention is also directed to isolated and purified LRP8 gene variants and LRP8 polypeptide variants.

[0089] The present invention thus also relates to compositions based on LRP8 gene variant or gene variant products. Compositions can also include, for example, polynucleotide primers derived from LRP8 gene variants, antisense polynucleotides derived from these variants, ribozymes based on the mutations, and antibodies specific for the variants. Compositions can further include recombinant cells containing any of the variants, vectors containing the variants, cells expressing the variants, fragments of the variants, and antibodies or other binding partners that specifically recognize the variants. These compositions can all be combined with a pharmaceutically acceptable carrier to create pharmaceutical compositions useful for detecting or modulating the level or expression of LRP8 gene variant or gene variant products and thereby diagnosing or treating CAD and/or ML

[0090] As used herein, a polypeptide is said to be "isolated" or "purified" when it is substantially free of cellular material when it is isolated from recombinant and non- recombinant cells or free of chemical precursors or other chemicals when it is chemically synthesized. A polypeptide, however, can be joined to another polypeptide with which it is not normally associated in a cell and still be considered "isolated" or "purified." The LRP8 polypeptide variants can be purified to homogeneity. It is understood, however, that preparations in which a polypeptide is not purified to homogeneity are useful and considered to contain an isolated form of the polypeptide. The preparation can allow for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the present invention can encompass various degrees of purity. [0091] In one aspect of the present invention, the language "substantially free of cellular material" can include preparations of a polypeptide having less than about 30% (by dry weight) of other proteins (i.e., contaminating protein), less than about 20% of other proteins, less than about 10% of other proteins, or less than about 5% other proteins. When an LRP8 polypeptide variant is recombinantly produced, for example, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.

[0092] The language "substantially free of chemical precursors or other chemicals" can include preparations of a polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one aspect of the present invention, the language "substantially free of chemical precursors or other chemicals" can include preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals. [0093] LRP8 polypeptide variants can be naturally- occurring or can be made by recombinant means or chemical synthesis to provide useful and novel characteristics for the polypeptide. This includes preventing immunogenicity from pharmaceutical formulations by preventing polypeptide aggregation. Useful variations can further include alteration of binding characteristics. A further useful variation at the same sites can result in a higher affinity for substrate. Useful variations also include changes that provide for affinity for another substrate. Another useful variation includes one that allows binding but which reduces cleavage of the substrate.

[0094] The present invention also provides antibodies that can selectively bind to LRP8 polypeptide variants. An antibody is considered to selectively bind even if it also binds to other proteins that are not substantially homologous with a LRP8 polypeptide variant. These other polypeptides can share homology with a fragment or domain of the polypeptide. This conservation in specific regions can give rise to antibodies that can bind to both polypeptides by virtue of the homologous sequence. In this case, it would be understood that antibody binding to the LRP8 polypeptide variant can still be selective.

[0095] To generate antibodies, an isolated polypeptide is used as an immunogen using standard techniques for polyclonal and monoclonal antibody preparation. Either the full- length polypeptide or antigenic polypeptide fragment can be used. Antibodies can be prepared from these regions or from discrete fragments in these regions. However, antibodies can be prepared from any region of the polypeptide as described herein. Antibodies can be developed against an entire polypeptide, such as the substrate binding domain, for example.

[0096] Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof, can be used. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances can include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes can include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes can include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials can include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. An example of a luminescent material can include luminal. Examples of bioluminescent materials can include luciferase, luciferin, and aequorin, and examples of suitable radioactive material can include ¹²⁵I, ¹³¹1, ³⁵S or ³H. [0097] An appropriate immunogenic preparation can be derived from native, recombinantly expressed polypeptide or chemically synthesized polypeptides. The antibodies can be used to isolate a LRP8 polypeptide variant, for example, by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the polypeptide from cells and recombinantly-produced polypeptide expressed in host cells.

[0098] The antibodies may be useful for detecting the presence of LRP8 polypeptide variants in cells or tissues to determine the pattern of expression of the polypeptides among various tissues in an organism. The antibodies can be used to detect the polypeptides in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. The antibodies can be used to assess abnormal tissue distribution or abnormal expression during development. Antibody detection of circulating fragments of LRP8 polypeptide variants can also be used to identify LRP8 polypeptide turnover. [0099] Further, the antibodies can be used to assess LRP 8 gene variant and/or LRP8 polypeptide expression in active stages of CAD and/or MI, or in a subject with a predisposition toward CAD and/or MI. For example, if a cardiovascular disease is characterized by a specific mutation in the LRP8 polypeptide, such as a polypeptide having SEQ ID NO: 6, antibodies specific for this mutant can be used to assay for the presence of the LRP8 polypeptide variant. Intracellularly-made antibodies ("intrabodies") which can recognize intracellular LRP8 polypeptide variants are also encompassed by the present invention.

[00100] The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Antibodies can be developed against the whole LRP8 polypeptide variants or portions thereof. The diagnostic uses can be applied not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting LRP8 polypeptide variant expression level or the presence of aberrant LRP8 polypeptide variants and aberrant tissue distribution or developmental expression, antibodies directed against LRP8 polypeptide variants or relevant fragments can be used to monitor therapeutic efficacy. The antibodies may also be useful for inhibiting LRP8 polypeptide variant function. These uses can also be applied in a therapeutic context.

[00101] An "isolated" LRP8 gene variant is one that is separated from other polynucleotides present in the natural source of the LRP8 gene variant. An "isolated" polynucleotide can be free of sequences which naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. However, there can be some flanking polynucleotide sequences, for example, up to about 5KB. The important point is that polynucleotide can be isolated from flanking sequences such that it can be subjected to the specific manipulations described herein, such as recombinant expression, preparation of probes and primers, and other uses specific to the polynucleotide sequences. [00102] An "isolated" polynucleotide, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the polynucleotide can be fused to other coding or regulatory sequences and still be considered isolated. For example, recombinant DNA molecules contained in a vector can be considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated polynucleotides according to the present invention can further include such molecules produced synthetically. [00103] LRP8 gene variants can encode the corresponding mature polypeptide variants plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a polypeptide from precursor to a mature form, facilitate polypeptide trafficking, prolong or shorten protein half-life, or facilitate manipulation of a polypeptide for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from a polypeptide by cellular enzymes.

[00104] LRP8 gene variants can include, but are not limited to, the polynucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. The LRP8 gene variants can also include the sequence encoding the corresponding mature polypeptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature polypeptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the gene variant may be fused to a marker sequence encoding, for example, a polypeptide that facilitates purification. [00105] Polynucleotides can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The polynucleotide, especially DNA, can be double- stranded or single-stranded. Single- stranded polynucleotides can be the coding strand (sense strand) or the non-coding strand (anti- sense strand).

[00106] The present invention also provides LRP8 gene variants encoding the polypeptide having SEQ ID NO: 6. Such polynucleotides may be naturally-occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. [00107] Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions. Furthermore, the present invention provides polynucleotides that comprise a fragment of the full length LRP8 gene. The fragments can be single- or double- stranded, and can comprise DNA or RNA. The fragment can be derived from either the coding or the non-coding sequence.

[00108] The present invention also provides LRP8 gene variants that encode epitope bearing regions of the LRP8 polypeptide variants described herein. The present invention also provides vectors containing LRP8 gene variants. The term "vector" refers to a vehicle, such as a polynucleotide, which can transport LRP8 gene variants. When the vector is a polynucleotide, a LRP8 gene variant can be covalently linked to the vector. With this aspect of the present invention, the vector can includes a plasmid, single- or double- stranded phage, a single- or double-stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, or MAC.

[00109] A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the LRP8 gene variant. Alternatively, the vector may integrate into the host cell genome and produce additional copies of LRP8 gene variants when the host cell replicates. The present invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of LRP 8 gene variants. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

[00110] Expression vectors can contain ds-acting regulatory regions that are operably linked in the vector to LRP8 gene variants such that transcription of the variants is allowed in a host cell. The LRP8 gene variants can be introduced into a host cell with a separate second polynucleotide capable of affecting transcription. Thus, the second polynucleotide may provide a trans-acting factor interacting with the ds-regulatory control region to allow transcription of the LRP8 gene variants from the vector. Alternatively, a trαns-acύng factor may be supplied by the host cell. Finally, a transacting factor can be produced from the vector itself.

[00111] It should be understood, however, that in some aspects, transcription and/or translation of LRP8 gene variants can occur in a cell-free system. The regulatory sequence(s) to which the LRP8 gene variants described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[00112] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers. [00113] In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[00114] A variety of expression vectors can be used to express LRP8 gene variants. Such vectors can include chromosomal, episomal, and virus-derived vectors, for example, and vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements including yeast artificial chromosomes, viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources, such as those derived from plasmid and bacteriophage genetic elements, e.g., cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[00115] The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art. [00116] LRP8 gene variants can be inserted into a vector by well-known methodology. Generally, a DNA sequence that will ultimately be expressed can be joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art. [00117] The vector containing the appropriate LRP8 gene variant can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells can include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells can include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[00118] As described herein, it may be desirable to express LRP8 polypeptide variants as fusion proteins. Accordingly, the present invention provides fusion vectors that allow for the production of LRP8 polypeptide variants. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired polypeptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors can include pGEX (Smith et al, Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, Gene 69:301-315 (1988)) and pET 1 Id (Studier et al, Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)). [00119] Recombinant protein expression can be maximized in a host bacterium by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) 119-128)). Alternatively, the sequence of the polynucleotide of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli (Wada et al, Nucleic Acids Res. 20:2111-2118 (1992)).

[00120] LRP8 polypeptide variants can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast, such as S. cerevisiae, can include pYepSecl (Baldari et al, EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al, Cell 30:933-943(1982)), pJRY88 (Schultz et al, Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, CA).

[00121] LRP8 polypeptide variants can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells {e.g., Sf 9 cells) include the pAc series (Smith et al, MoI. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al, Virology 170:31-39 (1989)).

[00122] In certain aspects of the present invention, the polypeptides described herein can be expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors can include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al, EMBO J. 6:187-195 (1987)). [00123] The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express LRP8 polypeptide variants. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the polynucleotides described herein. These are found, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[00124] The present invention also relates to recombinant host cells containing the vectors described herein. Host cells can therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells. The recombinant host cells can be prepared by introducing vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These can include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). [00125] Host cells can contain more than one vector. Thus, different LRP8 gene variants can be introduced on different vectors of the same cell. Similarly, LRP8 gene variants can be introduced either alone or with other polynucleotides that are not related to the LRP8 gene variants such as those providing trans-acting factors for expression vectors. [00126] In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication can occur in host cells providing functions that complement the defects.

[00127] Vectors can generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the LRP8 gene variants described herein or may be on a separate vector. Markers can include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait can be effective. While the mature corresponding polypeptides can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these polypeptides using RNA derived from the DNA constructs described herein.

[00128] Where secretion of a LRP8 polypeptide variant is desired, appropriate secretion signals can be incorporated into the vector. The signal sequence can be endogenous to the LRP8 polypeptide variant or heterologous to these polypeptides. Where the polypeptide is not secreted into the medium, the polypeptide can be isolated from the host cell by standard disruption procedures including, for example, freeze thaw, sonication, mechanical disruption, use of lysing agents, and the like. The polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, and high performance liquid chromatography, for example.

[00129] It should be appreciated that depending upon the host cell in recombinant production of the LRP8 polypeptide variants described herein, the polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process.

[00130] The host cells expressing the polypeptides described herein, and particularly recombinant host cells, can have a variety of uses. First, the cells can be useful for producing LRP8 polypeptide variants that can be further purified to produce desired amounts of LRP8 polypeptide variants or fragments. Thus, host cells containing expression vectors may be useful for polypeptide production. Host cells may also be useful for conducting cell-based assays involving LRP8 polypeptide variants. Thus, a recombinant host cell expressing a LRP8 polypeptide variant can be useful to assay for agents that modulate LRP8 gene function and/or affect MI and/or CAD.

[00131] In one aspect of the present invention, a host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing an LRP8 gene variant. Alternatively, the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See Thomas et ah, Cell 51:503 (1987) for a description of homologous recombination vectors. The vector can be introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous LRP8 gene may be selected (see, e.g., Li, E. et ah, Cell 69:915 (1992)). The selected cells may then be injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos. WO 90/11354, WO 91/01140, and WO 93/04169. [00132] The genetically engineered host cells can be used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example, or a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals may be useful for studying the function of LRP8 polypeptide variants and/or identifying and evaluating modulators of LRP8 polypeptide variant activity. Other examples of transgenic animals can include non- human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[00133] In one example, a host cell can be fertilized oocyte or an embryonic stem cell into which a LRP8 gene variant has been introduced. A transgenic animal can be produced by introducing the gene variant into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the corresponding LRP8 polypeptide variant to particular cells. [00134] Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals caring a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein. [00135] Transgenic animals containing recombinant cells that express LRP8 polypeptide variants described herein may be useful for conducting the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, LRP8 gene variant activation, and signal transduction, may not be evident from in vitro cell free or cell based assays. Accordingly, it may be useful to provide non-human transgenic animals to assay in vivo LRP8 gene function, the effect of specific LRP8 gene or polypeptide variants on LRP8 gene function, and the effect of chimeric LRP8 polypeptides. It may also be possible to assess the effect of null mutations; that is, mutations that substantially or completely eliminate one or more LRP8 gene functions. [00136] The LRP8 gene variants, LRP8 polypeptide variants (particularly fragments, such as the domains that interact with other cellular components), modulators of the LRP8 gene and polypeptide variants, and binding partners (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions can comprise the polynucleotide, polypeptide, or modulator and a pharmaceutically acceptable carrier. [00137] As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, (e.g., intravenous, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[00138] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, the composition can include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00139] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a LRP8 polypeptide or anti-LRP8 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, one method of preparation can include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00140] Oral compositions can generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00141] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant (e.g., a gas such as carbon dioxide) or a nebulizer.

[00142] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00143] In one aspect of the present invention, the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00144] Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention can be dictated by, and be directly dependent upon, the unique characteristics of the active compound, the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of subjects.

[00145] The LRP8 gene variants of the present invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al., PNAS 91:3054-3057 (1994)). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors) the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[00146] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The present invention may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will fully convey the present invention to those skilled in the art. Many modifications and other aspects of the present invention will come to mind in one skilled in the art to which the present invention pertains having the benefit of the teachings presented in the foregoing description. [00147] The following examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto.

Example 1

Materials and Methods

Study Populations

[00148] The GeneQuest population consists of 1,613 individuals from 428 multiplex families with premature CAD and MI. (Wang et al., Am J. Hum. Genet. 74(2):262-271 (2004)). Each family has at least two affected sibs. The details, including diagnostic criteria for CAD and MI, have been described previously. (Wang et al., Am J. Hum. Genet. 74(2):262-271 (2004)). For the case-control association study, the probands from the GeneQuest families were grouped together as the case group. Only 381 Caucasian patients with DNA samples available were selected, among which 183 were MI patients. Patients of other ethnic origins were excluded to avoid the confounding effects of population admixture. The 560 controls were selected from more than 9,800 individuals who underwent coronary angiography in Cardiac Catheterization Laboratories at Cleveland Clinic (Cleveland GeneBank), and only Caucasian individuals without detectable atherosclerotic lesions by angiography were included. 1,231 MI patients were selected from the Cleveland GeneBank and used as a replication cohort. [00149] The second familial and premature CAD/MI population, GeneQuest II, was ascertained using the identical criteria (Wang et ah, Am J. Hum. Genet. 74(2):262-271 (2004)) as defined in the original GeneQuest population at Center for Cardiovascular Genetics, Cleveland Clinic. A total of 22 Caucasian families with 441 family members were enrolled. The average size of the GeneQuest II families is 20 ± 14, and the number of affected individuals is 140.

[00150] The Italian cohort was enrolled in Verona, Italy and details about this study population were described previously. (Girelli et ah, N. Engl. J. Med. 343(11):774-780 (2000)). The cohort consisted of 416 unrelated individuals with MI (248 with family history) and 308 controls with no detectable stenosis by coronary angiography. [00151] This study was approved by local Institutional Review Boards on Human Subjects, and informed consent was obtained from the participants. Whole blood was drawn from each participant and genomic DNA was isolated from the blood using standard protocols.

Genotyping of SNPs

[00152] High throughput SNP genotyping was performed using the 5' nuclease allelic discrimination assay (TaqMan Assay) on an ABI PRISM 7900HT Sequence Detection System. The assay includes the forward target- specific PCR primer, the reverse primer, and the TaqMan MGB probes labeled with two special dyes-FAM and VIC. The probes were purchased through TaqMan Assays-on-Demand or Assays-by-Design from ABI (Applied Biosystems, Foster City, CA, USA). Genotyping was performed in a total 5 μl of PCR reaction volume containing 2.5 μl of TaqMan Universal PCR Master Mix, .25 μl of 20 x TaqMan MGB Assay Mix, and 25 ng of genomic DNA. Automatic allele calling was carried out by ABI PRISM 7900HT data collection and analysis software version 2.1. [00153] Direct DNA sequence analysis of SNPs in 24 DNA samples was used to ensure the quality of SNP genotyping. Direct DNA sequence analysis was performed using an ABI PRISM 3100 Genetic Analyzer (ABI, Foster City, CA, USA). A DNA fragment containing the SNP was PCR-amplified in a 25 μl volume containing 2.5 μl of 10 x PCR buffer (1.5 mM MgCl₂, 2.5 μl of .2 mM dNTP, .5 μM of each PCR primer, 1 U of Taq polymerase, and 50 ng of genomic DNA). PCR products were separated from agarose gels, isolated and purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA, USA), and sequenced with both forward and/or reverse primers. The sequencing reaction was performed using the BigDye Terminator vl.l Cycle Sequencing Kit (ABI, Foster City, CA, USA).

Activation Assay ofp38 MAPK

[00154] The GFP-tagged human LRP8 gene was PCR-amplified with primers containing in-frame Hind III and BamH I restriction sites. The PCR fragment was digested with Hind III and BamH I and sub-cloned into the pEGFP Cl vector cut with the same enzymes. Mutant LRP8 containing SNP R952Q was generated by PCR-based site-directed mutagenesis as described. (Chen et al., Nature 392(6673):293-296 (1998)). Both the wild type and mutant LRP8 expression constructs were verified by direct DNA sequence analysis of the entire inserts.

[00155] Meg-01 cells (American Type Culture Collection, Rockville, MD, USA) were grown in the RPMI- 1640 medium containing 10% fetal bovine serum and were maintained at a density of 3 x 10 cells/ml. In general, cells were used for experiments after 5 days of supplementation. Transfection of Meg-01 cells was carried out with 1 μg plasmid DNA/well with a Nucleofector device and corresponding kits (Amaxa, Inc., Cologne, Germany) in 6- well plates. 48h after transfection, cells were divided equally into five wells, and incubated with oxidized LDL (Podrez et al., J. Biol. Chem. 277(41):38517-38523 (2002)) for 0, 20, 40, 60, and 120 min. Cells were lysed, and an equal amount of total cellular proteins per sample was separated with 12% SDS-PAGE gels, and electro-transferred onto PVDF membranes. The blots were blocked in 5% non-fat milk powder in PBST for 1 h, washed briefly in TBST, and then incubated with an primary monoclonal antibody directed against phosphor-p38 MAPK (D-8, SC-7973, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a polyclonal antibody against total p38 MAPK (C-20 SC-535, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 5% non-fat milk /PBST overnight. Membranes were then extensively washed in PBST (8 x 5 min) and incubated with a 1:2000 dilution of HRP-conjugated anti-mouse IgM (Sigma, St. Louis, MO, USA) for 1 h at room temperature. Membranes were again washed (3 x 5 min) in FBST, and ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA) were used to visualize the protein signal. A prestained low molecular mass protein ladder (Bio-Rad, Hercules, CA, USA) was run in adjacent lanes. The images from Western blots were scanned, and quantified. Relative signal density of phosphorylated p38 MAPK vs. total p38 MAPK was calculated. Platelet Aggregation Assays

[00156] Platelet aggregation assays were performed within 3 hours after blood collection. Aggregation was measured by using an impedance method on a Chrono-log Whole Blood Impedance Aggregometer. 500 μl of blood was mixed with 500 μl of saline and the change in impedance of the sample was measured in the presence of ADP during a 6 minutes test run and recorded on a computer with the Chrono-log AGGRO/LINK software. Impedance aggregation in ohms was used to index the rate and the degree of platelet aggregation. [00157] In order to avoid the confounding effects of anti-platelet medications, we conducted statistical analysis of the data from the platelet aggregation assay only on 56 well- characterized MI patients who were verified for being absent of any history of taking antiplatelet drugs (e.g. aspirin). In order to reveal the relationship between platelet aggregation and LRP8 SNPs, an autosomal dominant model (i.e. Mean _ΛΛ = Mean . ≠ Mean ) was evaluated in the framework of general linear model (SAS

Ver 9.00).

Statistical Analysis

[00158] All SNPs were tested for Hardy- Weinberg equilibrium among controls using the Haploview software version 3.0 package. All SNPs were in Hardy- Weinberg equilibrium (P > .05).

[00159] Association of SNPs with the disease was assessed using Pearson's 2 x 2 contingency table Chi-square test or Fisher's exact test (SAS Ver 9.00). Odds ratios and 95% confidence intervals were estimated using the Chi-square test (SAS Ver 9.00). Multivariate analysis was performed by incorporating age and gender as covariates using multivariate logistic regression or in combination with additional covariates including total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride levels. Smoking was not included in the analysis due to lack of detailed phenotypic data. Genotyping data were additionally analyzed for association with CAD or MI using the Z-score method. (Skol et al., Nat. Genet. 38(2):209-213 (2006)). Empirical P-values were calculated, using 10,000 Monte Carlo simulations, by the CLUMP program (Sham and Curtis, Am. Hum. Genet. 59(Pt l):97-105 (1995)). [00160] Sib-TDT (Transmission Disequilibrium Test) analysis was carried out using the TDT/S-TDT program 1.1. (Spielman et al., Am. J. Hum. Genet. 52(3):506-516 (1993); Spielman and Ewens, Am. J. Hum. Genet. 59(5):983-989 (1996); Spielman and Ewens, Am. J. Hum. Genet. 62(2):450-458 (1998)). The sib-TDT was performed fro CAD only as the sample size for MI was small for sib-TDT analysis.

[00161] To remove the affects of genetic variant R952Q in LRP8 from the original Ip34- 36 linkage signal, we used multiple regression models with the LRP8 SNP. IBD was considered as a covariate to demonstrate its influence on the original linkage profile. In detail, the new H-E regression uses the following form of a general linear model:

where y is the quadratic form of the siblings 's phenotypes (here the mean-corrected

trait cross-product),

is the intercept, the subscripts m, s and k represent the original micro satellite marker, the LRP SNP and covariate gender respectively, d is the dominant genetic variance due to the marker or the SNP, a is the additive genetic variance due to the marker or the SNP, c^ is a nuisance parameter accounting for the effect of mean-

corrected cross-product of gender and the residual error.

are the probabilities of mean IBD sharing and exact 2

alleles IBD sharing respectively. Note that SAGE5.3 only reports 8 decimals instead of 12 decimals as with the previous version used to analyze the original GeneQuest pedigrees (hence, peak P-value is 2 x 10^~7 instead of the originally reported < 10^~12). (S.A.G.E., Statistical Analysis for Genetic Epidemiology (2003), A computer program package available from statistical solutions (Cork, Ireland)).

Example 2

Results

SNP R952Q in the LRP8 Gene Confers Risk of CAD and MI in the GeneQuest Population

[00162] Initially, we employed a population-based case-control association study design to characterize the candidate genes for MI at the lp34-36 locus. The 381 CAD cases were the Caucasian probands, one per family, from the GeneQuest families with familial, premature CAD and MI. (Wang et al., Am J. Hum. Genet. 74(2):262-271 (2004)). Among them, 183 had well-characterized MI and served as the MI cases. We excluded individuals of other ethnic origins to minimize the confounding effects of population admixture. The 560 controls were independent Caucasian individuals who were enrolled at Cleveland Clinic and showed no detectable stenosis by coronary angiography. The clinical features of cases and controls are shown in Table 1.

TABLE 1

Clinical characteristics of study populations and unaffected controls

Note: Data are shown as mean ± SD. Age, age-at-onset for cases and age-at-examination for controls. HDL = high-density lipoprotein; N/A, not applicable.

P < .001, compared with controls.

P < .01, compared with controls.

P <.O5, compared with controls.

BMI measured as body weight in kilograms divided by the square height in meter.

[00163] We employed a more systematic approach to further analyze candidate genes across the entire lp34-36 region (4.0-59.1 Mb) based on a gene's position and function. For the genetic interval under the linkage peak (13.2-34.1 Mb), one gene every 1-3 Mb was selected. For the genetic interval outside of the peak linkage (4.0-13.2 and 34.1-59.1 Mb), one gene every 3-6 Mb was selected. Genes with physiological functions relevant to those involved in the development of CAD or MI were prioritized. Twelve candidate genes were identified for analysis in this manner (Table 2). TABLE 2

Analysis of candidate genes at the lp34-36 locus for association with CAD and MI

Note: the entire lp34-36 locus spans a region from 4.0 Mb (CAT015) to 59.14 Mb

(GATA26G09P) with the plateau of genowide79significance from 13.2 Mb (GATA27E01) to

34.1 Mb (ATA79C10) (information on the markers can be found at Marshfield Genotyping

Service). a Positions as shown in the UCSC Genome Browser. cP-value for association with CAD. dP-value for association with ML

[00164] One SNP was studied for each gene based on its availability by the ABI assay-on- demand, minor allele frequency of > 30% (based on the assumption that common disease may be associated with evolutionally old, common variants), exonic position and nonsynonymous nature if possible. Of the twelve tested, only a single SNP, the non- synonymous R952Q variant located within the LRP8 gene (rs5174), showed significant association with CAD and MI (P = .003 for CAD and .004 for MI, table 2; P = .036 for CAD and .048 for MI after adjustment by approximate Bonferroni correction) (Table X). [00165] Permutation testing also showed a significant empirical P value for the association between SNP R952Q and CAD (P-emp = .003) or MI (P-emp = .004) (Table 3). TABLE 3

Association of SNP R952Q of LRP 8 with family-based CAD and MI

a Allele A at the nucleotide level corresponds to variant 952Q at the protein level. b P value for Hardy- Weinberg disequilibrium analysis. c Uncorrected P value. d P value obtained after adjustment for sex and age. Multivariant analysis was also performed for plasma total cholesterol levels, triglyceride levels, hypertension, and diabetes in addition to age and sex for GeneQuest CAD (P= .006), Italian MI (P= .041), and combined (P=.0005) populations. e Permutation P value calculated using 10,000 Monte Carlo simulations. f Combined population comprised 381 CAD cases from GeneQuest population, 22 MI probands from GeneQuest II population, and 248 Italian cases from familial MI population.

[00166] Multivariate analysis was performed to examine the possible confounding effects of age, gender, and other factors and found that SNP R952Q can be considered as an independent risk factor for CAD (P value adjusted for age and gender P-adj = .009 or .04 after adjustment for age, gender, total cholesterol, HDL, LDL cholesterol, and triglyceride levels) and MI (P-adj = .010, and .013, respectively) (Table 3). We also used the Z-score method (Skol et al., Nat. Genet. 38(2):209-213 (2006)) to analyze the SNP data; the R952Q variant was significantly associated with CAD and MI (Table 4). These results suggest that SNP R952Q in the LRP8 gene is associated with premature CAD and MI in an American Caucasian population. TABLE 4

Comparison of two different statistics in analyzing the association of LRP8 SNP

R952Q with CAD and MI

[00167] To determine whether SNP R952Q in LRP8 that was associated with CAD and MI is relevant to the previous lp34-36 MI linkage, we investigated the covariate effects of the LRP8 SNP on the original chromosome 1 linkage profile. SNP R952Q appeared to explain a significant amount of the original linkage signals (particularly the additive component) (Table 1).

Variant Associated with Risk of CAD and MI in the LRP8 Section ofHap-Block LD5

[00168] The LRP 8 gene contains 19 exons spanning ~ 60 kb. SNP R952Q showing positive association in the above study is located in exon 19. Analysis of the HapMap data showed that LRP8 was composed of five linkage disequilibrium (LD) blocks (LDl to LD5 from 5' to 3' end of the gene) among individuals of European- American ancestry. SNP R952Q is in LD5 at the 3'-terminus of the LRP8 gene. To test the association of other LD blocks of LRP8 with CAD and MI, one SNP was selected from each block based on the criteria defined earlier (LDl, rs3820198; LD2, rsl 288480; LD3, rs867884; LD4, rsl2039021) and used for fine-scale association mapping in LRP8. No association was detected between these SNPs and CAD or MI (P = .16- .94, Table 5). These data suggest that only LD5, the most 3' block of LRP8, be associated with risk of CAD and MI. TABLE 5

Analysis of association between SNPs in blocks LD1-LD4 of the LRP8 gene and

CAD and MI

Association with CAD

GeneQuest CAD population (381 cases) and 560 controls. b GeneQuest MI population (183 cases and 560 controls.

[00169] Further analysis of SNP genotyping data from the HapMap data revealed that LD5 of LRP8 extended beyond the 3' end of the LRP8 gene and spanned additional genes, MAGOH and FLJ20580. MAGOH encoding a component of the multiprotein exon junction complex, resides at 14.6 kb from the 3' end of LRP8. TagSNPs capturing MAGOH (rs6673692, rsl 0788949) were selected and tested, but we did not find any association with CAD (P = .61- .95) or MI (P = .13- .58) (Table 7). TABLE 6

Analysis of association between two additional genes in LD5 of LRP8 and CAD and

MI

shown as bases located all the UCSC database. P-value for association with CAD. P-value for association with MI.

[00170] Additionally, we tested two SNPs in FLJ20580 (rslO56424, rsl 134688), which is the gene distal to MAGOH (30 kb from LRP8), but again did not find any association with CAD (P = .74- .93) or MI (P = .57- .96). These data suggest that MAGOH and FLJ20580 are not associated with CAD or MI and that functional variants conferring risk of CAD and MI reside within LRP8.

[00171] Similar to the HapMap data, in the GeneQuest control population LRP8 SNP R952Q is in a continuous haplotype block (LD5) with rs6673692 and rs 10788949 in the MAGOH gene, and rslO56425 and rsl 134688 in the FLJ20580 gene (Fig. 8). However, in the CAD and MI populations, LD5 was disrupted and SNP R952Q was cut off from the block (if LD significance is equal to D'>0.8). These results suggest that disruption of an LD block in a disease population may be considered as supportive evidence for its association with the disease.

Family-Based TDT

[00172] Sib-TDT (Transmission Disequilibrium Test) was used to determine whether the risk allele of a SNP was preferentially transmitted to affected offspring. If a specific allele is transmitted more frequently to affected offspring, the allele is both linked and associated with CAD. (Spielman and Ewens, Am. J. Hum. Genet. 62(2):450-458 (1998)). Sib-TDT is also an effective strategy to minimize the confounding effects of population admixture. SNP R952Q of LRP8 SNP was genotyped in the full GeneQuest cohort (Table 7), including probands and other family members. Sib-TDT of the genotyping data revealed that R952Q was significantly associated with CAD (P = .005, Table 8). Together, these data suggest that SNP R952Q in LRP8 is associated with CAD and MI using both population- and family- based study designs in GeneQuest.

TABLE 7

Demographic features of the GeneQuest and GeneQuest and GeneQuest II family- based study populations

TABLE 8

Sib-TDT analysis in the GeneQuest and GeneQuest II populations

Y: the number minor alleles among affected sibs over all sibships; allele A at the nucleotide level corresponds to variant 952Q at the protein level.

Association ofLRP8 SNP R952Q with CAD in the GeneQuest II Families

[00173] Recently, we enrolled 441 individuals from 22 large Caucasian families (the GeneQuest II replication population, average pedigree size = 20, Table 7) to further test the association of LRP8 with CAD/ML Selection criteria for GeneQuest II were identical to the original GeneQuest cohort. (Wang et al., Am. J. Hum. Genet. 74(2):262-271 (2004)). Briefly, each proband must have presented with "premature" CAD, which was defined as any previous or current evidence of significant atherosclerotic CAD occurring to males of < 45 years of age and females of < 50 years of age. For recruitment, each proband was required to have at least one other living sibling meeting the same criteria. SNP R952Q was genotyped in the GeneQuest II cohort. Sib-TDT of the genotyping data showed that SNP R952Q was significantly associated with CAD (P = .009, Table 8). These data confirm that LRP8 SNP R952Q is associated with CAD in American, Caucasian families with premature CAD and MI.

Validation of Association ofLRP8 SNP R952Q with MI in an Italian Cohort

[00174] In order to further replicate the association of LRP8 with MI, we studied a separate Caucasian population with familial MI from Italy. Out of a total of 416 MI cases, 60% (248) had a family history of MI and were selected for further study. We genotyped SNP R952Q in the Italian cohort (248 MI cases and 308 controls, Table 1). The SNP showed significant association with CAD and MI (Table 3) and were further confirmed by permutation testing, yielding a significant empirical P-value. These results were also significant after adjusting for age and gender (Table 3). Significant association was also identified when all 416 Italian MI cases were analyzed together (P = .008). These results provide the second replication of our finding of association between LRP8 SNP R952Q and CAD/ML

Assessment of Association of LRP 8 SNP R952Q with CAD in a Combined White Cohort

[00175] To provide a comprehensive assessment of the association of LRP8 SNP R952Q with CAD, we performed an analysis of all familial Caucasian CAD populations combined (651 cases and 868 controls) as suggested by Skol et al. (Skol et al., Nat. Genet. 38(2):209- 213 (2006)). The significant P values for association of CAD with the positive LRP8 SNP R952Q were improved by > 10-fold (P = .0003 to .0006) (Table 3).

SNP R952Q Affects the Function ofLRP8

[00176] LRP8 SNP R952Q is a non-synonymous substitution of a positively-charged arginine residue by a negatively charged glutamine residue. To clarify whether this SNP can affect the function of the LRP8 protein, we transfected mammalian expression constructs with either wild type LRP8 or mutant LRP8 gene with the R952Q variant into Meg-01 cells. Cells were treated with oxidized LDL (ox-LDL) for various time points and activation of p38 MAPK was assayed. Six replicate experiments showed that the peak phosphorylation level of p38 MAPK by mutant LRP8 with R952Q was greater than wild-type (P = .009) and the high level of phosphorylation of p38 MAPK was maintained for a much longer time (P = .001 at 120 min) (Figs. 2A-B). These results suggest that the R952Q variant of LRP8 be a functional SNP that results in the increased phosphorylation (activation) of p38 MAPK. [00177] We further studied the correlation of LRP8 SNP R952Q with activity of platelet aggregation in 56 MI patients who did not take any anti-platelet medications. As shown in Table 3, the R952Q variant was associated with a small, but significant increase (14%) of platelet activity at two concentrations of the ADP agonist (P = .02 and .03).

Lack of Association ofLRP8 SNP R952Q with MI in Sporadic Disease

[00178] Since we identified a consistent association of LRP8 SNP R952Q with CAD and MI in three populations with familial and premature disease, we wanted to test if the association held in patients with sporadic and late-onset disease development. To this end, we studied one population with primarily sporadic CAD and MI, the Cleveland GeneBank cohort (Table 1). It is interesting to note that one other major difference in this population versus the previous populations is markedly reduced total and LDL cholesterol and triglyceride levels (Table 1). We genotyped the Cleveland GeneBank cohort consisting of 1,231 patients mostly with late-onset, sporadic MI and compared allelic frequencies to the 560 normal controls. No significant association was identified (Table 9). These results suggest that the functional SNP R952Q in LRP 8 may be associated with familial, premature CAD/MI, but not with the sporadic form of the disease.

TABLE 9

Lack of association of LRP8 SNP R952Q with sporadic CAD and MI in the GeneBank population (1,231 cases) and 560 controls

OR, odds ratio; CL, confidence interval; P-obs, uncorrected P-value; P-adj, P-value obtained after adjustment for gender and age; allele A at the nucleotide level corresponds to variant 952Q at the protein level.

Example 3

[00179] Here we report the identification of association of a previously unknown susceptibility gene, LRP8, with familial and premature CAD and MI. Several lines of evidence strongly support this conclusion. (1) Using a population-based approach, SNP R952Q residing in the last LD block (LD5) at the 3 '-terminus of LRP8 showed a significantly increased risk of premature, familial CAD and MI in an American Caucasian population (GeneQuest). (2) LRP 8 SNPs in other four LD blocks (LDl to LD4) showed negative association with CAD and MI. (3) SNPs in other candidate genes under the lp34-36 linkage were not associated with CAD and MI, and these genes include GJA4, ECEl, EPHB2, MFAP2, PLA2G2A, HMGCL, LDLRAPl, NR0B2, SLC9A1, AK2, GJB5, CSF3R, CDC20, FAAH, MAGOH, and FLJ20580. (4) Family-based (TDT) analysis showed that the risk allele of SNP R952Q was preferentially transmitted to affected individuals in GeneQuest families and provided further support that the LRP8 variant conferred risk of CAD. (5) Family-based (TDT) analysis showed that SNP R952Q conferred risk of CAD in another, independent population with familial CAD/MI, the GeneQuest II American Caucasian families with premature CAD/ML (6) The other replication was made in an Italian, Caucasian population with familial MI using a population-based approach. (7) Further analysis with all familial CAD/MI populations combined showed highly significant association between LRP8 SNP R952Q and CAD/MI (P-emp = .0006). (8) Functional studies demonstrated that SNP R952Q had a pronounced effect on the function of LRP8. Together, these results suggest that LRP8 SNP R952Q conferred risk of familial and premature CAD and MI. [00180] Platelets are a critical component of the atherosclerotic process. (Vorchheimer and Becker, Mayo Clin. Proc. 81(l):59-68 (2006)). Platelets are known to secrete and express substances for coagulation and inflammation that may play roles in atherosclerosis. (Vorchheimer and Becker, Mayo Clin. Proc. 81(l):59-68 (2006)). The activation of platelets is a key risk factor for atherothrombosis. Interestingly, we found that the SNP R952Q in LRP8 associated with CAD and MI was also associated with a modest, but significant increase (14%) in platelet aggregation activity in 56 MI patients who were free of any antiplatelet therapy (P = .02- .03). Thus, one mechanism by which the LRP8 variant increases risk of CAD and MI may be through sensitization of platelets. [00181] LRP8 has been shown to be a receptor for LDL and acts by homodimerization or heterodimerization with other receptors (VLDLR, β2-glycoprotein I). (Korporaal et ah, J. Biol. Chem. 279(50):52526-52534 (2004); Lutters et ah, Lutters et ah, J. Biol. Chem. 278(36):33831-33838 (2003); Strasser et ah, MoI. Cell Biol. 24(3):1378-1386 (2004)) LDL can bind to LRP8 and the interaction induces tyrosine phosphorylation of LRP8 and activation of p38 MAPK in platelets. (Korporaal et ah, J. Biol. Chem. 279(50):52526-52534 (2004)). SNP R952Q of LRP8 increased the activation of p38 MAPK (Figs. 2A-B). Activation of p38 MAPK may influence the sensitization of platelets by LDL, resulting in formation of arachidonate metabolites and release of inflammatory molecules, which may increase the risk of atherosclerosis or atherothrombosis. The p38 MAPK is a stress-activated protein kinase that may play a role in the development of atherosclerosis. The p38 MAPK could affect leukocyte emigration and lead to increased leukocyte accumulation in ischemic - reperfused tissue. (Johns et ah, Pharmacol. Res. 51(5):463-471 (2005)). The important role of p38 MAPK in inflammation has been well-established and overexpression of p38 MAPK has been shown to induce myocardial fibrosis and inflammation. (Tenhumen et ah, Circ. Res. 99(5):485-493 (2006); Tenhumen et al, FASEB J. 20(11): 1907-1909 (2006)). Kumar et al. showed that the activation of p38 MAPK was responsible for endothelial cell apoptosis induced by gamma-irradiation. (Kumar et al, J. Biol. Chem. 279(41):43352-43360 (2004)). It has also been shown that p38 MAPK becomes activated during ischemia and this activation leads to myocyte death and myocardial injury. (Tanno et al, Circ. Res. 93(3):254-261 (2003)). An inhibitor of p38 MAPK catalytic site, SB203580, inhibited ischemia-induced phosphorylation of p38 MAPK and reduced myocardial infarction volume in mice. (Tanno et al, Circ. Res. 93(3):254-261 (2003)). These reported results are consistent with our finding that the risk allele of SNP R952Q of LRP8 increased activation of p38 MAPK and susceptibility to CAD and MI. Further, the potential roles of the LRP8 SNP in the pathogenic processes of CAD and MI may go beyond the p38 MAPK activation and platelet aggregation, and may involve endothelial cells, vascular smooth muscle cells, and cardiac cells where LRP8 is expressed. In addition, LRP8 is a receptor for apoE and can bind and internalize αpoE-containing lipid vesicles. Mice deficient in apoE develop atherosclerotic lesions and depressed expression of apoE has been associated with atherosclerosis in humans. (Greenow et al, J. MoI. Med. 83(5):329-342 (2005)). Future studies will more precisely define the mechanism by which LRP8 variants play a role in the pathogenesis of CAD and MI. [00182] To the best of our knowledge, this study is the first to employ the family-based SNP association design to identify a susceptibility gene for CAD and MI. Functional SNP R952Q of LRP '8 demonstrated significant association with CAD/MI in two independent populations, GeneQuest (P = .005) and GeneQuest II (P = .009) (Figs. 9 and 10). Interestingly, the R952Q SNP was not associated with sporadic CAD/ML This result likely reflects the sporadic nature of MI patients (mostly no family history) in the Cleveland GeneBank cohort in contrast to CAD and MI patients from the GeneQuest and GeneQuest II families and Italian cohort with their unique enrollment characteristics such as early onset of the disease (premature MI) and/or existence of family history. These results further stress the importance of using homogeneous populations as in the GeneQuest, GeneQuest II and Italian cohorts to identify the most reliable associations with MI with a case-control design. Furthermore, unlike the Italian cohort which was enrolled on the basis of MI, the patients in GeneBank were not identified on this basis, but rather were participants in a large 10,000 patient cardiovascular DNA repository. Accordingly, the criteria for enrolling a population may also be important. Overall, these results suggest that LRP8 is associated with early-onset and familial CAD and MI, but not with late-onset, sporadic CAD and ML These results also suggest that genetically, familial and premature CAD and MI may be distinctively different from the late-onset, sporadic form of the disease.

[00183] It is important to point out that as in other studies of common complex diseases or traits, we cannot exclude the possibilities: (1) Other SNPs in LRP8 may be in LD with SNP R952Q and also associated with CAD and MI; (2) LRP8 SNP R952Q may be in LD with variants in additional genes in the lp34-36 linkage interval which also confer risk of CAD and ML Such a case was reported for the 3q CAD linkage where two candidate genes, both GATAl and Kalirin, were associated with CAD. (Connelly et al., PLoS.Genet 2 (8) (2006); Hauser et al., Am. J. Hum. Genet. 75(3):436-447 (2004); Wang et al., Am. J. Hum. Genet. 80(4):650-663 (2007)). Nevertheless, our studies identified LRP8 SNP R952Q as a genetic marker for association with CAD and MI. Future studies employing a systematic approach to analyze each candidate gene under the lp34-36 linkage may identify other susceptibility genes for CAD and MI.

[00184] In conclusion, we have demonstrated that SNP R952Q in the LRP8 gene is significantly associated with familial and premature CAD/MI, but not with the sporadic and late onset form of the disease. Our results implicate a new, LRP8-mediated molecular pathway for susceptibility to familial CAD and MI.

Example 4

Four Other SNPs In LRP8 Were Associated With Risk Of CAD and MI In The GeneQuest Population

[00185] The LRP 8 gene contains 19 exons spanning approximately 60 kb. SNP R952Q (rs5174) is located in exon 19. To further determine whether LRP 8 is associated with CAD and MI, we studied four other SNPs close to R952Q in the gene. Three SNPs are located in exons (rs2297660, rs3737983 and rs5177) and one SNP is located in an intron (rs7546246). Allelic association was analyzed by contingency table Chi-square tests. All four SNPs showed significant association with CAD or MI (Table 10). TABLE 10 Association of five SNPs in the LRP 8 gene with CAD and MI

A. Association with CAD in the GeneQuest population

C. Association with MI in an Italian o ulation

*snpl, RS7546246; snp2, RS2297660; SNP3, RS3737983; SNP4, RS5174; SNP5, RS5177; OR, odds ratio; CL, confidence interval.

[00186] We also used the Z-score method to analyze the genotyping data; all four SNPs and the R952Q variant remained significantly associated with CAD and MI (Table 11). These results suggest that the LRP8 gene is a susceptibility gene for premature CAD and MI in an American Caucasian population.

TABLE I l Comparison of two different statistics in analyzing association of LRP8 SNPs with CAD and

MI: the Chi- square test and the Z-Score method

SNP GeneQuest CAD GeneQuest MI Italian MI

Chi-Square 6.46 5.61 10.01 rs7546246 P-Value 0.011 0.018 0.002

Z-Score 2.53 2.35 3.19

P-value 0.011 0.019 0.001

Chi-Square 6.08 7.63 14.33 rs2297660 P-Value 0.014 0.006 0.0002

Z-Score 2.46 2.74 3.83

P-Value 0.014 0.006 0.0001

Chi-Square 9.10 8.03 5.23 rs3737983 P-Value 0.003 0.005 0.022

Z-Score 3.01 2.81 2.30

P-Value 0.003 0.005 0.021

Chi-Square 9.05 8.16 6.95 rs5174 P-Value 0.003 0.004 0.008

Z-Score 3.00 2.82 2.66

P-Value 0.003 0.005 0.008

Chi-Square 3.93 4.75 7.08 rs5177 P-Value 0.048 0.029 0.008

Z-Score 2.00 2.16 2.68

P-Value 0.048 0.031 0.007

[00187] Haplotype analysis was carried out for genotyping data from the 5 SNPs in LRP8. A total of 24 different haplotypes were identified in the GeneQuest population and analyzed for their association with CAD or MI. We identified one haplotype, TACGC, that was associated with the risk allele of SNP rs2297660 and present in 3.1% of CAD patients and 4.2% of MI patients, but did not exist in the 560 controls. This haplotype confers high risk to CAD (P = 3.0 x 10^"10) and MI (P = 6.0 x 10^"10) (Fisher's exact test, uncorrected for the 26 multiple tests; odds ratio/OR = ∞ due to the absence of this risk haplotype in the control population; Table 12). TABLE 12

Risk and protective haplotypes associated with CAD and MI A. Identification of risk haplotypes in the GeneQuest CAD population

Type GeneQuest CAD

Case (%) Control (%) OR 95% C.I. P-value

TACGC 3.1 0.0 oo oo 3.0 X 10^"iυ

CCTAG

3.1 0.2 18.2 4.3-77.1 3.8 X 10^"8

< 1% risk haplotype 4.4 0.1 52.3 7.1-382.6 6.2 X 10^"13

AU risk haplotypes 16.0 1.7 11 6.7-18.1 1.1 X 10^"31

< 1% risk haplotype: CACGC, TACAC, TCTAG, TCCAC.

B. Identification of risk haplotypes in the GeneQuest MI population

Type GeneQuest MI

Case (%) Control (%) OR 95% C.I. P-value

TACGC 4.2 0 .0 oo oo 6.0 X 10^"iυ

CCTAG 3.8 0 .2 22.2 5.0-98.3 1.8 X lO^"7

AU risk 20.7 0 .7 36.4 17.4-76. 3 6.4 X 10^"4υ haplotypes*

*Other risk haplotypes (frequency): TCCAC (1.8%, TACAC (1.6%), CACGC (1.5%), TCTAG (1.4%), TATGC (1.1%), CCCGC (1.2%), AND TCTGC (0.8%)

C. Identification of risk ha lot es in the Italian MI o ulation

AU risk haplotype: CATAG, TACGC.

D. Identification of a rotective ha lot e associated with CAD and MI

[00188] One other haplotype CCTAG showed strong association with CAD (OR = 18.2, P = 3.8 x 10^~8) and MI (OR = 22.2, P = 0.0056) (Table 12). Several other haplotypes with a population frequency of < 1% also showed significant association with CAD and MI. An odds ratio of 11 and 36 were obtained for the combined risk haplotypes for CAD (P = 1.1 x 10^"31) and MI (P = 6.4 x 10^"40) (Table 12). Interestingly, we identified a haplotype, TCCGC, that protects against CAD (OR = 0.5, P = 4.0 x 10^"11) and MI (OR = 0.4, P = 6.5 x 10^"12) (Table 12).

[00189] Haplotype analysis revealed that the five SNPs we genotyped were in strong linkage disequilibrium (LD) (defined as block LD5 consisting of rs5177, rs5174, rs3737983, rs2297660, and rs7546246); thus, not surprisingly all five SNPs showed association with the disease. This result is consistent with the HapMap data that these five SNPs, among individuals of European- American ancestry, are in one LD block located at the 3 '-terminus of the LRP8 gene. One SNP was selected for each block (LDl, rs3820198; LD2, rsl288480; LD3, rs867884; and LD4, rsl2039021) and used for fine-scale association mapping in LRP8. No association was found between these SNPs and CAD or MI (P = 0.16-0.94, Table 5). These data suggest that functional variants that confer risk of CAD and MI are defined within the 3'-LD5 block of LRP8.

[00190] Analysis of SNP genotyping data from the HapMap database revealed that LD5 of LRP8 may extend beyond LRP8 and contain two other small genes, MAGOH encoding a component of the multiprotein exon junction complex and a hypothetical gene FLJ20580 (genomic sizes of 11.6 kb and 6.5 kb, respectively). This is confirmed by LD analysis of our genotyping data in the control population. However, two SNPs in MAGOH (rs6673692, rsl0788949) and two SNPs in FLJ20580 (rslO56424, rsl 134688) did not show any association with CAD (P = 0.61-0.95) or MI (P = 0.13-0.96) (Table 2). These data suggest that functional variants that confer risk of CAD and MI are defined within the LRP8 section of hap-block LD5.

Transmission Disequilibrium Test (TDT) Further Implicates The Association O/LRP8 SNPs And SNP Haplotypes With CAD

[00191] Sib-TDT analysis has been used to determine whether the risk allele of a SNP or a specific haplotype is preferentially transmitted to an affected offspring. If a specific allele or a specific haplotype is transmitted more frequently to affected offspring, the allele or haplotype is both linked and associated with the disease (MI) (Spielman, RS and Ewens, WJ, N. Engl. J. Med. 347, 1916-1923 (2002)). AU 5 LRP8 SNPs were genotyped in the full GeneQuest cohort, including the probands and other family members (Wang, Q et ah, Am. J. Hum. Genet. 74, 262-271 (2004)). Sib-TDT of the genotyping data revealed that four SNPs, rs7546246, rs3737983, rs5174 and rs5177 were significantly associated with CAD (P < 0.05, Table 13). Furthermore, sib-TDT also showed that the TACGC risk haplotype and the protective haplotype TCCGC were significantly associated with CAD (P < 0.05, Table 13). Sib-TDT could not be performed for MI due to lack of a sufficient sample size.

TABLE 13

S-TDT analysis of LRP8 SNPs and haplotypes associated with CAD and MI

A. Single SNPs S-TDT analysis

B. Risk-Protective Haplotype S-TDT analysis

[00192] We genotyped the five SNPs associated with CAD and MI in the GeneQuest population in an Italian cohort with 416 MI cases and 308 controls. All five SNPs showed significant association with CAD and MI (Table 10). The TACGC risk haplotype was identified in four MI patients, but none of the controls in the Italian cohort (P = 0.04) (Table 12). The CCTAG risk haplotype was not detected in the Italian cohort. One other risk haplotype was identified in the Italian cohort, CATAG (P = 0.0056) (Table 12). The protective haplotype TCCGC was identified in 57.6% cases and 64.6% controls (P = 0.007) (Table 12).

Further Definition Of The TACGC Risk Haplotype

[00193] To further characterize the TACGC risk haplotype, we genotyped 19 individuals carrying this haplotype with 4 SNPs upstream of the TACGC risk haplotype and 18 SNPs downstream. As shown in Table 14, the TACGC risk haplotype is defined between SNP rs 12039021 and rs6677126, a region spanning intron 2 of LRP 8 to 3 '-untranslated region.

TABLE 14

Definition of the boundaries of the TACGC haplotype

rs S 2567⁽534 V cc CC re CC CC

A [ID cc I

A Af.^"

Note: 19 CADMl patients (#i to #19) carrying the TACGC risk haplotype of LRPS were genotyped with SNPs upstream ami downstream of the haplotype. The results define^" SNP rs 12039021 as the tipstream flanking SNP for the risk hapiotype as individuals 7 and 10 do not showed recombination. SNP rs6677126 is the downstream Uaαking SNP as multiple individuals, ti-6. #13, #15, and #19 showed recombination at this position.

[00194] DNA sequence analysis of the coding regions and exon-intron boundaries of LRP8 in the 19 patients revealed 4 previously known SNPs (rs2297660 in exon 9, rs5173 in exon 17, rs3737983 in exon 17, and rs5174 in exon 19). Two new SNPs, c.89C>G in intron 2 and c.70T>C in intron 11 were identified (data not shown).

[00195] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims.

Claims

Having described the invention, we claim:

1. A method for identifying a subject who has an increased risk for developing a cardiovascular disease, the method comprising the step of: isolating a nucleic acid from a sample that has been removed from the subject; detecting an alteration in a portion of a LRP8 gene, the alteration being located in at least one of exon 9, exon 17, exon 19, or intron 6 of the LRP8 gene; wherein the presence of the alteration is correlated with an increased risk for developing the cardiovascular disease.

2. The method of claim 1, the alteration comprising a short nucleotide polymorphism (SNP) in at least one of exon 9, exon 17, exon 19 or intron 6 of the LRP8 gene that increases the activation of p38 MAPK.

3. The method of claim 1, the cardiovascular disease being selected from the group consisting of myocardial infarction and coronary artery disease.

4. The method of claim 2, the SNP in exon 19 having SEQ ID NO: 1 or SEQ ID NO: 2.

5. The method of claim 2, the SNP in exon 17 having SEQ ID NO: 3.

6. The method of claim 2, the SNP in exon 9 having SEQ ID NO: 4.

7. The method of claim 2, the SNP in intron 6 having SEQ ID NO: 5.

8. The method of claim 2, the SNP having SEQ ID NO: 1 resulting in a LRP8 polypeptide variant having SEQ ID NO: 6.

9. A method of diagnosing a subject who has a cardiovascular disease or an increased risk for developing a cardiovascular disease, the method comprising the steps of: obtaining a polynucleotide sample from the subject, the polynucleotide sample comprising a polynucleotide sequence corresponding to at least a portion of the LRP8 gene selected from the group consisting of exon 9, exon 17, exon 19 and intron 6; and determining if the polynucleotide sequence corresponding to the at least one portion of the LRP8 gene is altered such that the presence of the alteration is correlated with an increased risk for developing the cardiovascular disease.

10. The method of claim 9, the alteration comprising a short nucleotide polymorphism (SNP) in at least one of exon 9, exon 17, exon 19 or intron 6 of the LRP8 gene that increases the activation of p38 MAPK.

11. The method of claim 9, the cardiovascular disease being selected from the group consisting of myocardial infarction and coronary artery disease.

12. The method of claim 10, the SNP in exon 19 having SEQ ID NO: 1 or SEQ ID NO: 2.

13. The method of claim 10, the SNP in exon 17 having SEQ ID NO: 3.

14. The method of claim 10, the SNP in exon 9 having SEQ ID NO: 4.

15. The method of claim 10, the SNP in intron 6 having SEQ ID NO: 5.

16. The method of claim 10, the SNP having SEQ ID NO: 1 resulting in a LRP8 polypeptide variant having SEQ ID NO: 6.

17. A method for identifying a subject who has an increased risk for developing a cardiovascular disease, the method comprising the step of: isolating a nucleic acid from a sample that been removed from the subject; detecting a LRP8 haplotype, the LRP8 haplotype comprising at least two single nucleotide polymorphisms (SNPs) selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; wherein the presence of the LRP8 haplotype is correlated with an increased risk for developing the cardiovascular disease.

18. The method of claim 17, the LRP8 haplotype comprising a TACGC haplotype, the presence of the TACGC haplotype being associated with an increased risk of myocardial infarction and coronary artery disease.

19. A method for assessing the risk of a cardiovascular disease in a subject, the method comprising the steps of: assessing platelet reactivity in a blood sample of the subject; and assessing the level of a LRP8 gene alteration in the blood sample, the LRP8 gene alteration being located in at least one of exon 9, exon 17, exon 19 or intron 6 of the

LRP8 gene; wherein the presence of the LRP8 gene alteration and an increase in platelet aggregation reactivity are correlated with an increased risk for developing the cardiovascular disease.

20. The method of claim 19, the alteration comprising a short nucleotide polymorphism (SNP) in at least one of exon 9, exon 17, exon 19 or intron 6 of the LRP8 gene that increases the activation of p38 MAPK.

21. The method of claim 20, the SNP in exon 19 having SEQ ID NO: 1 or SEQ ID NO: 2

22. The method of claim 20, the SNP in exon 17 having SEQ ID NO: 3.

23. The method of claim 20, the SNP in exon 9 having SEQ ID NO: 4.

24. The method of claim 20, the SNP in intron 6 having SEQ ID NO: 5.

25. A method for identifying an agent useful in therapeutically or prophylactically treating a cardiovascular disease in a subject, the method comprising the steps of: contacting a LRP8 gene variant or a LRP8 polypeptide variant with a candidate agent under conditions suitable to allow formation of a binding complex between the LRP8 polypeptide variant and the candidate agent, the LRP8 gene variant and the LRP8 polypeptide variant respectively comprising SEQ ID NO: 1 and SEQ ID NO: 6; and detecting the formation of the binding complex; wherein the presence of the complex identifies the agent.