CA2502359A1 - Susceptibility gene for myocardial infarction - Google Patents

Abstract

Linkage of Myocardial Infarction (MI) and a locus on chromosome 13q12 is disclosed. In particular, the FLAP gene within this locus is shown by association analysis to be a susceptibility gene for MI. Pathway targeting for drug delivery and diagnosis applications in identifying those have MI or at risk of developing MI, in particular are described.

Description

SUSCEPTIBILITY GENE FOR MYOCARDIAL INFARCTION
RELATED APPLICATION
This application claims the benefit of 60/419,432, filed October 17, 2002. The entire teachings of the above application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Myocardial infarction (MI) is one of the most common diagnoses in hospitalized patients in industrialized countries. Myocardial Infarction generally occurs when there is an abrupt decrease in coronary blood flow following a thrombotic occlusion of a coronary artery previously narrowed by atherosclerosis.
Infarction occurs when a coronary artery thrombus develops rapidly at a site a vascular injury, which is produced or facilitated by factors such as cigarette smoking, hypertension and lipid accumulation. In most cases, infarction occurs when an atherosclerotic plaque fissures, ruptures or ulcerates and when conditions favor thrombogenesis. In rare cases, infarction may be due to coronary artery occlusion caused by coronary emboli, congenital abnormalities, coronary spasm, and a wide variety of systemic, particularly inflammatory diseases.
Although classical risk factors such as smoking, hyperlipidemia, hypertension, 2o and diabetes are associated with many cases of coronary heart disease (CHD) and MI, many patients do not have involvement of these risk factors. In fact, many patients who exhibit one or more of these risk factors do not develop MI. Family history has long been recognized as one of the major risk factors. Although some of the familial clustering of MI reflects the genetic contribution to the other conventional risk factors, a large number of studies have suggested that there are sigiuficant genetic susceptibility factors, beyond those of the known risk factors (Friedlander Y, et al., By~
Heaf-t J. 1985; 53:382-7, Shea S. et al., ,I. Am. Coll. Ca3°diol. 1984;
4:793-801, and Hopkins P.N., et al., Am. J. Ca~~diol. 1988; 62:703-7). Major genetic susceptibility factors have not yet been identified.

SUMMARY OF THE INVENTION
As described herein, a locus on chromosome 13q12 has been identified as playing a major role in Myocardial Infarction (MI). The locus, herein after referred to as the MI locus, comprises nucleic acid that encodes 5-lipoxygenase activating protein (ALOXSAP or FLAP), herein after referred to as FLAP.
The present invention relates to isolated nucleic acid molecules comprising a portion or the entire human FLAP nucleic acid or a variant thereof. In one embodiment, the nucleic acid molecule has at least one polymorphism that is correlated with the incidence of myocardial infarction. The invention also relates to pathways targeting for drug delivery. A further embodiment of the invention is a method for the diagnosis of MI and a method for identification of susceptibility to myocardial infarction, by identifying polymorphisms in the FLAP nucleic acid, which identify those at risk. Also, described are haplotypes and SNPs that can be used to identify individuals with MI or at risk of developing MI. The polymorphism in the FLAP nucleic acid can be indicated by detecting the presence of a haplotype, comprising one or more of the markers: DGOOAAFIU, SG13S25, DGOOAAJFF, DGOOAAHII, DGOOAAHID, B SNP_310657, SG13S30, SG13S32, SG13S42, and SG13S35 at the 13q12 locus comprising a FLAP nucleic acid. The polymorphism further can comprise at least one of the polymorphisms as indicated in Table 3.
2o Identification of nucleic acids and polymorphisms in the MI locus can pave the way for a better understanding of the disease process, which in tunz can lead to improved diagnostic and therapeutic methods.
The invention further pertains to methods of diagnosing myocardial infarction or a susceptibility to myocardial infarction, comprising detecting an alteration in the expression or composition of a polypeptide encoded by a FLAP nucleic acid in a test sample, in comparison with the expression or composition of a polypeptide encoded by FLAP in a control sample, wherein the presence of an alteration in expression or composition of the polypeptide in the test sample is indicative of myocardial infarction or a susceptibility to myocardial infarction.
3o The invention also relates to an isolated nucleic acid molecule comprising a FLAP nucleic acid, wherein the FLAP nucleic acid has a nucleic acid sequence of SEQ m NO: 1 or SEQ ID NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID
NO: 3, wherein the nucleic acid molecule comprises a polymorphism as indicated in Table 3.
In another embodiment, the invention relates to an isolated nucleic acid molecule having a polymorphism as indicated in Table 3, which hybridizes under high stringency conditions to a nucleic acid sequence of SEQ ID NO: 1 or SEQ
ID
NO: 3, or the complement of SEQ m NO: 1 or SEQ m NO: 3.
In yet another embodiment, a method for assaying for the presence of a first nucleic acid molecule in a sample is described, comprising contacting said sample to with a second nucleic acid molecule, where the second nucleic acid molecule comprises a nucleic acid sequence of SEQ )D NO: 1 or SEQ ID NO: 3, and hybridizes to the first nucleic acid under high stringency conditions.
The invention also relates to a vector comprising an isolated nucleic acid molecule of the invention operably linked to a regulatory sequence, as well as to a 15 recombinant host cell comprising the vector. The invention also provides a method for preparing a polypeptide encoded by an isolated nucleic acid molecule comprising culturing the recombinant host cell under conditions suitable for expression of said nucleic acid molecule.
Also contemplated by the invention is a method of assaying a sample for the 2o presence of a polypeptide encoded by an isolated nucleic acid molecule of the invention, comprising contacting the sample with an antibody that specifically binds to the polypeptide.
The invention further provides a method of identifying an agent that alters expression of a FLAP nucleic acid, comprising: contacting a solution containing a 25 nucleic acid comprising the promoter region of the FLAP nucleic acid operably linked to a reporter gene with an agent to be tested; assessing the level of expression of the reporter gene; and comparing the level of expression with a level of expression of the reporter gene in the absence of the agent; wherein if the level of expression of the reporter gene in the presence of the agent differs, by an amount that is statistically 3o significant, from the level of expression in the absence of the agent, then the agent is a.n agent that alters expression of the FLAP nucleic acid. An agent identified by this method is also contemplated.
The invention additionally comprises a method of identifying an agent that alters expression of a FLAP nucleic acid, in which a solution containing a nucleic acid described herein or a derivative or fragment thereof is contacted with an agent to be tested, and expression of the nucleic acid, derivative or fragment in the presence of the agent is assessed and compared with expression of the nucleic acid, derivative or fragment in the absence of the agent. If expression of the nucleic acid, derivative or fragment in the presence of the agent differs, by an amount that is statistically to significant, from the expression in the absence of the agent, then the agent is an agent that alters expression of the FLAP nucleic acid. In certain embodiments, the expression of the nucleic acid, derivative or fragment in the presence of the agent comprises expression of one or more splicing variants) that differ in kind or in quantity from the expression of one or more splicing variants) the absence of the agent. Agents identified by this method are also contemplated. Representative agents include antisense nucleic acid to a FLAP nucleic acid; a FLAP polypeptide; a FLAP
nucleic acid receptor; a FLAP nucleic acid binding agent; a peptidomimetic; a fusion protein; a prodrug thereof; an antibody; and a ribozyme. A method of altering expression of a FLAP nucleic acid comprising contacting a cell containing a FLAP
2o nucleic acid with such an agent is also contemplated.
The invention further pertains to a method of identifying a polypeptide which interacts with a FLAP polypeptide, employing a yeast two-hybrid system that uses a first vector which comprises a nucleic acid encoding a DNA binding domain and a FLAP polypeptide, splicing variant, or a fragment or derivative thereof, and a second vector which comprises a nucleic acid encoding a transcription activation domain and a nucleic acid encoding a test polypeptide. If transcriptional activation occurs in the yeast two-hybrid system, the test polypeptide is a polypeptide which interacts with a FLAP polypeptide.
In a further embodiment, the invention relates to a myocardial infarction 3o therapeutic agent, such as a FLAP nucleic acid or fragment or derivative thereof; a 5-lipoxygenase nucleic acid or fragment or derivative thereof; a leulcotriene synthetase nucleic acid or fragment or derivative thereof; a polypeptide encoded by a FLAP
nucleic acid; a polypeptide encoded by a 5-lipoxygenase nucleic acid; a polypeptide encoded by a leukotriene synthetase nucleic acid; a FLAP receptor; a 5-lipoxygenase receptor; a leukotriene synthetase receptor; a FLAP nucleic acid binding agent; a 5-lipoxygenase binding agent; a leukotriene synthetase binding agent; a FLAP
nucleic acid binding agent; a 5-liopoxygenase nucleic acid biyding agent; a leukotriene synthetase nucleic acid binding agent; a peptidomimetic; a fusion protein; a prodrug;
an antibody; an agent that alters FLAP nucleic acid expression; an agent that alters activity of a polypeptide encoded by a FLAP nucleic acid, a 5-lipoxygenase nucleic to acid, or a leukotriene synthetase nucleic acid; an agent that alters posttranscriptional processing of a polypeptide encoded by a FLAP nucleic acid, a 5-lipoxygenase nucleic acid or a leukotriene synthetase nucleic acid; an agent that alters interaction of a FLAP nucleic acid with a FLAP nucleic acid binding agent; an agent that alters interaction of a 5-lipoxygenase nucleic acid with a 5-lipoxygenase nucleic acid binding agent; an agent that alters interaction of a leukotriene synthetase nucleic acid with a leukotriene synthetase nucleic acid binding agent; an agent that alters transcription of splicing variants encoded by a FLAP nucleic acid, a 5-lipoxygenase nucleic acid, or a leukotriene synthetase nucleic acid; or ribozymes; and pharmaceutical compositions comprising at least one myocardial infarction 2o therapeutic agent.
The invention also pertains to a method of treating a disease or condition associated with FLAP in an individual, comprising administering a myocardial infarction therapeutic agent to the individual, in a therapeutically effective amount. In certain embodiments, the myocardial infarction therapeutic agent is a FLAP
nucleic acid agonist or a FLAP nucleic acid antagonist.
A transgenic animal comprising a nucleic acid of the invention such as an exogenous FLAP nucleic acid or a nucleic acid encoding a FLAP polypeptide is also contemplated.
In yet another embodiment, the invention relates to a method for assaying a sample for the presence of a FLAP nucleic acid, by contacting the sample with a nucleic acid comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the sequence of said FLAP nucleic acid, under conditions appropriate for hybridization, and assessing whether hybridization has occurred between a FLAP nucleic acid and said nucleic acid, wherein if hybridization has occurred, a FLAP nucleic acid is present in the nucleic acid. In certain embodiments, the contiguous nucleic acid sequence is completely complementary to a part of the sequence of said FLAP nucleic acid and in other embodiments; amplification is of at least part of said FLAP nucleic acid.
In certain embodiments, the contiguous nucleic acid sequence is 100 or fewer nucleotides in length and is either: a) at least 80% identical to a contiguous sequence of nucleotides of SEQ m NO: 1 or SEQ m NO: 3; b) at least 80% identical to the complement of a contiguous sequence of nucleotides in of SEQ ID NO: 1 or SEQ m NO: 3; or c) capable of selectively hybridizing to said FLAP nucleic acid.
The invention also pertains to a reagent for assaying a sample for the presence of a FLAP nucleic acid, the reagent comprising a nucleic acid comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the nucleic acid sequence of said FLAP nucleic acid. The reagent can comprise a contiguous nucleotide sequence which is completely complementary to a part of the nucleic acid sequence of said FLAP nucleic acid. A reagent kit for assaying a sample for the presence of a FLAP nucleic acid is also described, including (e.g., in separate 2o containers), one or more labeled nucleic acids comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the nucleic acid sequence of said FLAP nucleic acid; and reagents for detection of said label.
The labeled nucleic acid can comprise a contiguous nucleotide sequence which is completely complementary to a part of the nucleic acid sequence of said FLAP
nucleic acid. Also described herein is a reagent kit for assaying a sample for the presence of a FLAP nucleic acid, comprising one or more nucleic acids comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the nucleic acid sequence of said FLAP nucleic acid, and which is capable of acting as a primer for said FLAP nucleic acid when maintained under conditions for primer extension.

The invention also provides for the use of a nucleic acid for assaying a sample for the presence of a FLAP nucleic acid, in which the nucleic acid is 100 or fewer nucleotides in length and is either: at least 80% identical to a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; at least 80% identical to the complement of a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID
NO: 3; or capable of selectively hybridizing to said FLAP nucleic acid.
In yet another embodiment, the use of a first nucleic acid for assaying a sample for the presence of a FLAP nucleic acid that has at least one nucleotide difference from the first nucleic acid is described, in which the first nucleic acid is to 100 or fewer nucleotides in length and which is either: at least 80%
identical to a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 or one of the sequences shown in Table 3; at least 80% identical to the complement of a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 one of the sequences shown in Table 3; or capable of selectively hybridizing to said FLAP nucleic acid.
The invention also relates to a method of diagnosing a susceptibility to myocardial infarction in an individual, comprising determining the presence or absence in the individual of certain "haplotypes" (combinations of genetic markers);
the presence of the haplotype is diagnostic of susceptibility to myocardial infarction.
In one embodiment, a haplotype associated with a susceptibility to myocardial 2o infarction comprises markers DGOOAAFILT, SG13S25, DGOOAAJFF, DGOOAAHII, SG13S32 and SG13S35 at the 13q12 locus. In one particular embodiment, the presence of the alleles T, G, G, G, A and G at DGOOAAFIU, SG13S25, DGOOAAJFF, DGOOAAHII, SG13S32 and SG13S35, respectively (the B6 haplotype), is diagnostic of susceptibility to myocardial infarction. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction comprises markers DGOOAAFILJ, SG13S25, DG00AAHII, SG13S30 and SG13S42 at the 13q12 locus.
In one particular embodiment, the presence of the alleles T, G, G, G and A at DGOOAAFIU, SG13S25, DGOOAAHII, SG13S30 and SG13S42, respectively (the BS
haplotype), is diagnostic of susceptibility to myocardial infarction. In a third 3o embodiment, a haplotype associated with a susceptibility to myocardial infarction comprises markers SG13S25, DGOOAAHII, SG13S30 and SG13S42 at the 13q12 _g_ locus. In one particular embodiment, the presence of the alleles G, G, G and A
at SG13S25, DGOOAAHII, SG13S30 and SG13S42 , respectively (the B4 haplotype), is diagnostic of susceptibility to myocardial infarction. In a fourth embodiment, a haplotype associated with a susceptibility to myocardial infarction comprises markers DGOOAAFIU, SG13S25, DGOOAAHID, B SNP-310657 and SG13S32 at the 13q12 locus. In one particular embodiment, the presence of the alleles T, G, T, G
and A at DGOOAAFIU, SG13S25, DGOOAAHID, B SNP_310657 and SG13S32, respectively (the A5 haplotype), is diagnostic of susceptibility to myocardial infarction.
In a fifth embodiment, a haplotype associated with a susceptibility to myocardial infarction to comprises markers SG13S25, DGOOAAHID, B SNP_310657 and SG13S32 at the 13q12 locus. In one particular embodiment, the presence of the alleles G, T, G
and A
at SG13S25, DGOOAAHID, B SNP 310657 and SG13S32, respectively (the A4 haplotype), is diagnostic of susceptibility to myocardial infarction. The presence or absence of the haplotype can be determined by various methods, including, for example, using enzymatic amplification, restriction fragment length polymorphism analysis, sequence analysis or electrophoretic analysis of nucleic acid from the individual.
The invention also relates to a method of diagnosing a susceptibility to myocardial infarction in an individual, comprising: obtaining a nucleic acid sample from said individual; and analyzing the nucleic acid sample for the presence or absence of a haplotype using markers DGOOAAFIU, SG13S25, DGOOAAJFF, DGOOAAHII, DGOOAAHID, B SNP_310657, SG13S30, SG13S32, SG13S42, and SG13S35 , with alleles T, G, G, G, T, G, G, A, A, G, respectively, at the 13q12 locus, wherein the presence of the haplotype is diagnostic for a susceptibility to myocardial infarction.
Also described herein is a method of diagnosing myocardial infarction or a susceptibility to myocardial infarction in an individual, comprising determining the presence or absence in the individual of a haplotype comprising one or more markers and/or single nucleotide polymorphisms as shown in Table 3 in the locus on chromosome 13q12 comprising a FLAP nucleic acid, wherein the presence of the haplotype is diagnostic of myocardial infarction or of a susceptibility to myocariial infarction.
A method for the diagnosis and identification of susceptibility to miocardial infarction in an individual is also described, comprising: screening for an at-risk haplotype in the FLAP nucleic acid that is more frequently present in an individual susceptible to myocardial infarction compared to an individual who is not susceptible to myocardial infarction wherein the at-risk haplotype increases the risk significantly.
In certain embodiments, the significant increase is at least about 20%, and in other embodiments, the significant increase is identified as an odds ratio of at least about 1.2.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention.
FIG. 1 shows the multipoint non-parametric LOD scores for a framework marker map on chromosome 13. A LOD score suggestive of linkage of 2.5 was found at marker D13S289. The maker map for chromosome 13 that was used in the linkage analysis is show~l in Table 1.
2o FIG. 2 shows LOD score results for the families after adding 14 markers to the candidate region. The inclusion of additional microsatellite markers increased the information on sharing by decent from 0.7 to 0.8, around the markers that gave the highest LOD scores. The marker map used in the second step of linkage anaysis is shown in Table 2.
FIG. 3A shows the results from a haplotype association analysis using 4 and 5 microsatellite markers. Thep-value of the association is plotted on the y-axis and position of markers on the x-axis. Only haplotypes that show association with ap-value < 10-5 are shown in the figure. The most significant microsatellite marker haplotype association is found using markers DG13S1103, DG13S166, DG13S1287, 3o DG13S1061 and DG13S301, with alleles 4, 0, 2, 14 and 3, respectively (p-value of 1.02 x 10-x). Carrier frequency of the haplotype is 7.3% in affected individuals and 0.3% in controls. These results are based on 437 patients and 721 controls.
The area that is common to all the haploytypes shown in the figure includes only one gene, FLAP.
FIG. 3B shows the alleles of the makers defining the most significant microsatellite marker haplotypes. The area defined with a black square is a common area to all the most significantly associated haplotypes. The FLAP nucleic acid is located between makers DG13S166 and D13S1238. Two marker haplotype involving alleles 0 and-2 for markers DG13S166 and S13S1238, respectively, is found in excess in patients. Carrier frequency of this haploype is 27% in patients and 15.4% in to controls (p-value 1 X 10-3) FIG. 4 shows the markers and genes around the FLAP (ALOXSAP) gene.
FIG. 5 shows the relative location of key SNPs and exons of the ALOXSAPIFLAP gene. Haplotype length varies between 33 to 68 kb.
FIGS. 6A-6Y4 show the genomic sequence of the FLAP gene (SEQ ID NO: 1).
FIG. 7A shows the amino acid sequence of FLAP (SEQ ID N0:2) and the mRNA of FLAP (SEQ ID NO: 3) FIGs. 7B-7V show the sequences of the FLAP nucleic acid flanking the SNPs that were identified by sequencing samples from patients (SEQ ID NOs: 398-535).
2o DETAILED DESCRIPTION OF THE INVENTION
Extensive genealogical information has been combined with powerful gene sharing methods to map a locus on chromosome 13q12 that is associated with myocardial infarction. Patients with myocardial infarction and controls were initially genotyped with microsatellite markers with an average spacing between markers of less than 100kb over the l2Mb candidate region. An epidemiological study of a population-based sample of MI patients demonstrated the relative risk for siblings of a female MI patient is significantly higher than the relative risk for siblings of a male proband (1.59 (CI 1.47 - 1.73) vs. 1.35 (CI 1.28 - 1.42)). The gender difference in risk of getting MI (males being more likely to get MI) also suggests somewhat 3o different etiology between males and females, where MI in females might represent a more extreme phenotype. This study stratified the population according to sex to determine the genetic causes of MI for males and females. The results of the genome wide search of genes that cause MI in Iceland is described. This linkage analysis resulted in linkage on chromosome 13q12.
Initial haplotype association analysis using 4 or 5 microsatellite markers that extended across the gene and were in excess in patients indicated that FLAP is a susceptibility gene for myocardial infarction. A region that is common to all the microsatellite haplotypes includes only one gene, the FLAP gene.
The FLAP nucleic acid encodes a 5-lipoxygenase activating protein, which, in combination with 5-lipoxygenase (5-LO), is required for leukotriene synthesis.
to Inhibitors of its function impede translocation of 5-lipoxygenase from the cytoplasm to the cell membrane and inhibit activation of 5-lipoxygenase. One other member of the leukotriene pathway, CysLT2 receptor, maps to chromosome 13q14.2 (53 cM on FIG. 2). The region of this gene shows excess sharing identical by decent (LOD
score=1) in female MI patients. This indicates that CysLT2 receptor might also play a role in the pathogenesis of MI.
Mutations andlor polymorphisms within the FLAP nucleic acid show association with the disease and can be used for methods of diagnosis.
Furthermore, the FLAP gene and other members of the leukotriene pathway, such as 5-LO, LTA4, LTB4, LTC4, LTD4 and CysLT2, are therapeutic targets for myocardial infarction.
NUCLEIC ACIDS OF THE INVENTION
FLAP Nucleic Acidr, Pof°tiohs afzd Ija~~iahts Accordingly, the invention pertains to isolated nucleic acid molecules comprising a human FLAP nucleic acid. The term, "FLAP nucleic acid," as used herein, refers to an isolated nucleic acid molecule encoding FLAP polypeptide.
The FLAP nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA. DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or 3o sense strand or the non-coding, or antisense strand. The nucleic acid molecule can include all or a portion of the coding sequence of the gene or nucleic acid and can further comprise additional non-coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example).
For example, a FLAP nucleic acid can consist of SEQ m NOs: 1 or 3 or the complement thereof, or to a portion or fragment of such an isolated nucleic acid molecule (e.g., cDNA or the nucleic acid) that encodes FLAP polypeptide (e.g., a polypeptide such as SEQ m NO: 2). In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ m NOs: 1 or 3, or their complement thereof.
Additionally, the nucleic acid molecules of the invention can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide. Such sequences include, but are not limited to, those that encode a glutathione-S-transferase (GST) fusion protein and those that encode a hemagglutinin A (HA) polypeptide marker from influenza.
An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleic acid sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by 2o recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC. In certain embodiments, an isolated nucleic acid molecule comprises at least about 50, ~0 or 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
For example, the isolated nucleic acid molecule can contain less than about 5 kb, 3o including but not limited to 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution.
"Isolated" nucleic acid molecules also encompass ifa vivo and i~a vity°o RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or l0 nucleic acid sequence can include a nucleic acid molecule or nucleic acid sequence that is synthesized chemically or by recombinant means. Therefore, recombinant DNA
contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleotide sequences include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
Iya vivo and in vitYO RNA transcripts of the DNA molecules of the present invention are also encompassed by "isolated" nucleotide sequences. Such isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by ih situ hybridization with chromosomes), or for detecting 2o expression of the nucleic acid in tissue (e.g., human tissue), such as by Northern blot analysis.
The present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a FLAP polypeptide (e.g., a polypeptide having an amino acid sequence comprising an amino acid sequence of SEQ ID NOs:
2), or another splicing variant of a FLAP polypeptide or polymorphic variant thereof.
Thus, for example, DNA molecules that comprise a sequence that is different from the naturally occurring nucleic acid sequence but which, due to the degeneracy of the genetic code, encode a FLAP polypeptide of the present invention are also the subjects of this invention. The invention also encompasses nucleotide sequences 3o encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of a FLAP polypeptide. Such variants can be naturally occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes.
Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably the nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of a FLAP polypeptide. In one preferred embodiment, the nucleotide sequences are fragments that comprise one or more polymorphic microsatellite markers. In another preferred embodiment, the nucleotide sequences to are fragments that comprise one or more single nucleotide polymorphisms in a FLAP
nucleic acid (e.g., the single nucleotide polymorphisms set forth in Table 3, below).
Other alterations of the nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules 2o include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleic acid sequence described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleic acid sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide). In one embodiment, the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of SEQ
ID
3o NOs: 1 or 3 or the complement thereof. In another embodiment, the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 2 or a polymorphic variant thereof.
In a preferred embodiment, the variant that hybridizes wider high stringency hybridizations has an activity of a FLAP.
Such nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions). "Specific hybridization," as used herein, refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher to similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed). "Stringency conditions" for hybridization is a term of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly 15 (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%, 95%). For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity. "High stringency conditions", "moderate stringency conditions" and "low stringency conditions"
for 20 nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols ih Molecular Biology (Ausubel, F.M. et al., "Curt°ent Protocols in Molecular Biology", John Wiley & Sons, (1998), the entire teachings of which are incorporated by reference herein). The exact conditions which determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2X
SSC, 25 O.1X SSC), temperature (e.g., room temperature, 42°C, 68°C) and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non-identical sequences. Thus, equivalent conditions 30 can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules.

Typically, conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined.
Exemplary conditions are described in Krause, M.H. and S.A. Aaronson, Methods ih Enzymology 200: 546-556 (1991), and in, Ausubel, et al., "Cu~~ent to Protocols ih Molecular Biology", John Wiley & Sons, (1998), which describes the determination of washing conditions for moderate or low stringency conditions.
Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each °C by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1 % in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in Tm of -17°C. Using these guidelines, the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch 2o sought.
For example, a low stringency wash can comprise washing in a solution containing 0.2X SSC/0.1 % SDS for 10 minutes at room temperature; a moderate stringency wash can comprise washing in a prewarmed solution (42°C) solution containing 0.2X SSC/0.1% SDS for 15 minutes at 42°C; and a high stringency wash can comprise washing in prewarmed (68°C) solution containing O.1X
SSC/0.1%SDS
for 15 minutes at 68°C. Furthermore, washes can be performed repeatedly or sequentially to obtain a desired result as known in the art. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between 3o the target nucleic acid molecule and the primer or probe used.

The percent homology or identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment).
The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = # of identical positions/total # of positions x 100). When a position in one sequence is occupied by the same nucleotide or amino acid residue as the corresponding position in the other sequence, then the molecules axe homologous at that position. As used herein, nucleic acid or amino to acid "homology" is equivalent to nucleic acid or amino acid "identity". In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, for example, at least 40%, in certain embodiments at least 60%, and in other embodiments at least 70%, 80%, 90% or 95% of the length of the reference sequence.
The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al., P~oc.
Natl.
Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).
Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABlOS
4(1):
11-17 (1988). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package (Acceliys, Cambridge, UK). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Co~aput. Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson and Lipman, Pr~oc. Natl. Acad. Sci. USA 85:2444-8 (1988).
In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package using either a BLOSUM63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP
program in the GCG software package using a gap weight of 50 and a length weight of 3.
to The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid sequence comprising SEQ ID NO: 1 or 3 or the complement of SEQ ID
NO: 1 or 3, and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid sequence encoding an amino acid sequence of the invention or polyrnorphic variant thereof.
The nucleic acid fragments of the invention are at least about 15, for example, at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, encoding antigenic polypeptides described herein are particularly useful, such 2o as for the generation of antibodies as described below.
Probes and PYime~s In a related aspect, the nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. "Probes" or "primers" are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. Such probes and primers include polypeptide nucleic acids, as described in Nielsen et al.(Sciehce 254:1497-1500 (1991)).
A probe or primer comprises a region of nucleic acid that hybridizes to at least about 15, for example about 20-25, and in certain embodiments about 40, 50 or 75, 3o consecutive nucleotides of a nucleic acid of the invention, such as a nucleic acid comprising a contiguous nucleic acid sequence of SEQ ID NOs: 1 or 3 or the complement of SEQ m Nos: 1 or 3, or a nucleic acid sequence encoding an amino acid sequence of SEQ m NO: 2 or polymorphic variant thereof. In preferred embodiments, a probe or primer comprises 100 or fewer nucleotides, in certain embodiments, from 6 to 50 nucleotides, for example, from 12 to 30 nucleotides.
In other embodiments, the probe or primer is at least 70% identical to the contiguous nucleic acid sequence or to the complement of the contiguous nucleotide sequence, for example, at least 80% identical, in certain embodiments at least 90%
identical, and in other embodiments at least 95% identical, or even capable of selectively hybridizing to the contiguous nucleic acid sequence or to the complement of the l0 contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
The nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein. For example, nucleic acid molecules can be amplified and isolated using the polymerase chain reaction and synthetic oligonucleotide primers based on one or more of SEQ m NOs: 1 or 3, or the complement thereof, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein. See generally PCR
Techfaology: Principles and Applications foY DNA A~aplificatioya (ed. H.A.
Erlich, Freeman Press, NY, NY, 1992); PC'R Protocols: A Guide to Methods and Applications (Eds. Innis et al., Academic Press, San Diego, CA, 1990); Mattila et al.,.
Nucl. Acids Res. 19:4967 (1991); Eckert et al., PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202.
The nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA
as a template, cloned into an appropriate vector and characterized by DNA
sequence analysis.
Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989), Landegren et al., Science 241:1077 (1988), transcription amplification (Kwon et al., P~oc. Natl. Acad. Sci. USA
86:1173 (1989)), and self sustained sequence replication (Guatelli et al., Pros. Nat.
Acad. Sci.
USA 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
The amplified DNA can be labeled, for example, radiolabeled, and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZIPLOX or other suitable vector. Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. For example, the direct analysis of the nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambroolc et al., Molecular CloyaifZg, A Laboratory Manz~al (2nd Ed., CSHP, New York 1989); Zyskind et al., Ree~mbinafat DNA Laboratory Manual, (Acad. Press, 1988)). Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of SEQ m NOs: 1 or 3 and/or the complement of one or more of SEQ ID NOs: 1 or 3 and/or a portion of one or more of SEQ m NOs: 1 or 3 or the 2o complement of one or more of SEQ ID NOs: 1 or 3 and/or a sequence encoding the amino acid sequences of SEQ m NOs: 2 or encoding a portion of one or more of SEQ
m NOs: 1 or 3 or their complement. They can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
For example, an antisense nucleic acid molecule (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, the antisense nucleic acid molecule can be produced biologically 3o using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).
The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify one or more of the disorders related to FLAP, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample. The nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses. Portions or fragments of the nucleotide sequences identified to herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to:
(i) map their respective genes on a chromosome; and, thus, locate gene regions or nucleic acid regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Additionally, the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization or therapeutic use, or as maakers for tissues in which the corresponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states.
The nucleic acid sequences can additionally be used as reagents in the screening 2o and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.
T~ecto~s Another aspect of the invention pertains to nucleic acid constructs containing a nucleic acid molecule of SEQ m NOs: 1 or 3 or the complement thereof (or a portion thereof). Yet another aspect of the invention pertains to nucleic acid constructs containing a nucleic acid molecule encoding an amino acid of SEQ m NO: 2 or polymorphic variant thereof. The constructs comprise a vector (e.g., an expression 3o vector) into which a sequence of the invention has been inserted in a sense or antisense orientation. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular double stranded DNA
loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain io vectors, such as expression vectors, are capable of directing the expression of genes or nucleic acids to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
Preferred recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for 2o expression, which is operably linked to the nucleic acid sequence to be expressed.
Within a recombinant expression vector, "operably linked" or "operatively linked" is intended to mean that the nucleic acid sequence of interest is linked to the regulatory sequences) in a manner which allows for expression of the nucleic acid sequence (e.g., in an iya vits~o transcription/translation system or in a host cell when the vector is introduced into the host cell). The teen "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, "Gene Expression Technology", Methods i~ Ehzy~aology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleic acid sequence in many types of host cell and those which direct expression of the nucleic acid sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of polypeptide desired. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides, encoded by nucleic acid molecules as described herein.
The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), to yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated ifz vitf~o, for example using T7 promoter regulatory sequences and polyrnerase.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may 2o not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E.
coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or tramsfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host 3o cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.
(supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene or nucleic acid that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene or nucleic acid of interest. Preferred selectable markers include those that confer resistance to drugs, such as G41 ~, hygromycin and methotrexate. Nucleic acid to molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene or nucleic acid will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic host cell or eukaryotic host cell in culture can be used to produce (i. e., express) a polypeptide of the invention.
Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector 2o encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid molecule of the invention has been introduced (e.g., an exogenous FLAP nucleic acid, or an exogenous nucleic acid encoding a FLAP polypeptide). Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homologous recombinant animals 3o in which endogenous nucleotide sequences have been altered. Such animals are useful for studying the function and/or activity of the nucleic acid sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians. 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, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an to "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
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. Patent Nos. 4,736,866 and 4,870,009, U.S.
Pat. No. 4,873,191 and in Hogan, Manipulatifzg the M~use Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Methods for constructing 2o homologous recombination vectors and homologous recombinant animals are described further in Bradley, CuYf°ent Opihiofa iu BioTeclahology 2:823-829 (1991) and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al., Nature 385:810-(1997) and PCT Publication Nos. WO 97/07668 and WO 97/07669.
POLYPEPTIDES OF THE INVENTION
The present invention also pertains to isolated polypeptides encoded by FLAP
nucleic acids ("FLAP polypeptides"), and fragments and variants thereof, as well as 3o polypeptides encoded by nucleotide sequences described herein (e.g., other splicing variants). The term "polypeptide" refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. 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 (e.g., in a "fusion protein") and still be "isolated" or "purified."
The polypeptides of the invention can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to 1o homogeneity are useful. The critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity.
In one embodiment, the language "substantially free of cellular material" includes preparations of the polypeptide having less than about 30% (by dry weight) other proteins (i. e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
When a polypeptide is recombinantly produced, 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.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of the polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes 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.
In one embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleic acid sequence 3o selected from the group consisting of SEQ ID NO: 1 or 3, or the complement of SEQ
ID NO: 1 or 3, or portions thereof, or a portion or polymorphic variant thereof.

However, the polypeptides of the invention also encompass fragment and sequence variants. Variants include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other splicing variants. Variants also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 or 3 or their complement, or portions thereof, or having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of nucleotide sequences encoding to SEQ >D NO: 2 or polymorphic variants thereof. Variants also include polypeptides substantially homologous or identical to these polypeptides but derived from another organism, i.e., an ortholog. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by recombinant methods.
As used herein, two polypeptides (or a region of the polypeptides) are substantially homologous or identical when the amino acid sequences are at least about 45-55%, in certain embodiments at least about 70-75%, and in other embodiments at least about 80-85%, and in others greater than about 90% or more 2o homologous or identical. A substantially homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid molecule hybridizing to SEQ m NO: 1 or 3 or portion thereof, under stringent conditions as more particularly described above, or will be encoded by a nucleic acid molecule hybridizing to a nucleic acid sequence encoding SEQ ID NO: 2 or a portion thereof or polymorphic variant thereof, under stringent conditions as more particularly described thereof.
The invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule of the 3o invention. Similarity is determined by conserved amino acid substitution.
Such substitutions are those that substitute a given amino acid in a polypeptide by another _28_ amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr.
Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).
A variant polypeptide can differ in amino acid sequence by one or more to substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. Further, variant polypeptides can be fully functional or can lack function in one or more activities. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
2o Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or iya vitro proliferative activity. Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al., Science 255:306-312 (1992)).
The invention also includes fragments of the polypeptides of the invention.
3o Fragments can be derived from a polypeptide encoded by a nucleic acid molecule comprising SEQ ID NO: 1 or 3, or the complement of SEQ ID NO: 1 or 3 (or other variants). However, the invention also encompasses fragments of the variants of the polypeptides described herein. As used herein, a fragment comprises at least 6 contiguous amino acids. Useful fragments include those that retain one or more of the biological activities of the polypeptide as well as fragments that can be used as an immunogen to generate polypeptide-specific antibodies.
Biologically active fragments (peptides which are, for example, 6, 9, 12, 15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) can comprise a domain, segment, or motif that has been identified by analysis of the polypeptide sequence using well-known methods, e.g., signal peptides, extracellular domains, one to or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, acylation sites, glycosylation sites, or phosphorylation sites.
Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment.
The invention thus provides chimeric or fusion polypeptides. These comprise 2o a polypeptide of the invention operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide. "Operatively linked" indicates that the polypeptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide. In one embodiment the fusion polypeptide does not affect function of the polypeptide pe~~ se. For example, the fusion polypeptide can be a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-terminus of the GST sequences. Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Such fusion 3o polypeptides, particularly poly-His fusions, can facilitate the purification of recombinant polypeptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a polypeptide can be increased using a heterologous signal sequence. Therefore, in another embodiment, the fusion polypeptide contains a heterologous signal sequence at its N-terminus.
EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
In drug discovery, for example, human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists. Bennett et al., .Iournal ofMoleculaf°Recoghitioh, 8:52-58 (1995) and Johanson et al., The.Iou~~ual of to Biological Che~raist~y, 270,16:9459-9471 (1995). Thus, this invention also encompasses soluble fusion polypeptides containing a polypeptide of the invention and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
A chimeric or fusion polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques.
In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of nucleic acid fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al., Cu~refzt Protocols ifz Molecular Biology, 1992).
Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.
The isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. In one embodiment, the polypeptide is 3o produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the polypeptide expressed in the host cell.
The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
The polypeptides of the present invention can be used to raise antibodies or to elicit an immune response. The polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the polypeptide or a molecule to which it binds (e.g., a ligand) in biological fluids. The polypeptides can also be used as markers for cells or tissues in which the corresponding polypeptide is preferentially expressed, either constitutively, during tissue differentiation, or in to diseased states. The polypeptides can be used to isolate a corresponding binding agent, e.g., ligand, such as, for example, in an interaction trap assay, and to screen for peptide or small molecule antagonists or agonists of the binding interaction.
For example, because members of the leukotriene pathway including FLAP bind to receptors, the leukotriene pathway polypeptides can be used to isolate such receptors.
ANTIBODIES OF THE INVENTION
Polyclonal and/or monoclonal aaltibodies that specifically bind one form of the polypeptide or nucleic acid product (e.g., a polypeptide encoded by a nucleic acid having a SNP as set forth in Table 3), but not to another form of the polypeptide or 2o nucleic acid product, are also provided. Antibodies are also provided which bind a portion of either polypeptide encoded by nucleic acids of the invention (e.g., SEQ m NO: 1 or SEQ ~ NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3), or to a polypeptide encoded by nucleic acids of the invention that contain a polymorphic site or sites. The invention also provides antibodies to the polypeptides and polypeptide fragments of the invention, or a portion thereof, or having an amino acid sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ

NOs: 1 or 3, or the complement thereof, or another variant or portion thereof.
The term "antibody" as used herein refers to irrununoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that 3o contain an antigen binding site that specifically binds an antigen. A
molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
Examples of immunologically active portions of immunoglobulin molecules include Flab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A
1o monoclonal antibody composition thus typically displays a single binding affinity fox a particular polypeptide of the invention with which it immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by I~ohler and Milstein, Nature 256:495-497 (.1975), the human B cell hybridoma technique (I~ozbor et al., Irnmunol.
Today 4:72 (1983)); the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancers The~~apy, Alan R. Liss, 1985, Iuc., pp. 77-96); or trioma techniques.
The technology for producing hybridomas is well known (see generally Cuf°f~efzt PnotocoZs in hramunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, NY).
Briefly, an immortal cell Iine (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and 3o the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody to a polypeptide of the invention (see, e.g., Cufweut Protocols in Immuf2ology, supra; Galfre et al., Nature 266:55052 (1977); R.H. Kenneth, in Mofzoclo~zal Antibodies: A New Dimef2sion In Biological Ah.alyses, Plentun Publishing Corp., New York, New York (1980); and Lerner, Yale J. Biol. Med. 54:387-402 (1981). Moreover, the ordinarily skilled worker will appreciate that there are many to variations of such methods that also would be useful.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia RecombihafZt Phage AsZtibody System, Catalog No. 27-9400-O1; and the Stratagene Sm fZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can 2o be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO
92118619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791;
PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., BiolTeclahology 9: 1370-1372 (1991); Hay et al., Hum. Ahtibod. Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); Griffiths et al., EMBO.I. 12:725-734 (1993).
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

In general, antibodies of the invention (e.g., a monoclonal antibody) can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation. A polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells. Moreover, an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for l0 example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase,13-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent 2o materials include luciferase, luciferin and aequorin, and examples of suitable radioactive material include l2sh 1311, ssS or 3H.
DIAGNOSTIC ASSAYS
The nucleic acids, probes, primers, polypeptides and antibodies described herein can be used in methods of diagnosis of MI or diagnosis of a susceptibility to MI or to a disease or condition associated with an MI gene, such as FLAP, as well as in kits useful for diagnosis of MI or a susceptibility to MI or to a disease or condition associated with FLAP. In one embodiment, the kit useful for diagnosis of MI or susceptibility to MI, or to a disease or condition associated with FLAP
comprises 3o primers as described herein, wherein the primers contain one or more of the SNPs identified in Table 3.

In one embodiment of the invention, diagnosis of MI or susceptibility to MI
(or diagnosis of or susceptibility to a disease or condition associated with FLAP), is made by detecting a polymorphism in a FLAP nucleic acid as described herein.
The polymorphism can be an alteration in a FLAP nucleic acid, such as the insertion or s deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift alteration; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several to nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of the gene or nucleic acid; duplication of all or a part of the gene or nucleic acid; transposition of all or a part of the gene or nucleic acid; or rearrangement of all or a part of the gene or nucleic acid. More than one such alteration may be present in a single gene or nucleic acid. Such sequence changes 15 cause an alteration in the polypeptide encoded by a FLAP nucleic acid. For example, if the alteration is a frame shift alteration, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a disease or condition associated with a FLAP nucleic acid or a 2o susceptibility to a disease or condition associated with a FLAP nucleic acid can be a synonymous alteration in one or more nucleotides (i.e., an alteration that does not result in a change in the polypeptide encoded by a FLAP nucleic acid). Such a polymorphism may alter splicing sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the nucleic acid. A FLAP
nucleic 25 acid that has any of the alteration described above is referred to herein as an "altered nucleic acid."
In a first method of diagnosing MI or a susceptibility to MI, hybridization methods, such as Southern analysis, Northern analysis, or iya situ hybridizations, can be used (see Curre~zt Ps-otocols in Molecular Biology, Ausubel, F. et al., eds., John 3o Wiley & Sons, including all supplements through 1999). For example, a biological sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed fox, or carrying a defect for, a susceptibility to a disease or condition associated with a FLAP nucleic acid (the "test individual"). The individual can be an adult, child, or fetus. The test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs. A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling.
The DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism to in a nucleic acid is present, and/or to determine which splicing variants) encoded by the FLAP is present. The presence of the polymorphism or splicing variants) can be indicated by hybridization of the nucleic acid in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A "nucleic acid probe", as used herein, can be a DNA
probe or an RNA probe; the nucleic acid probe can contain at least one polymorphism in a FLAP nucleic acid or contains a nucleic acid encoding a particular splicing variant of a FLAP nucleic acid. The probe can be any of the nucleic acid molecules described above (e.g., the nucleic acid, a fragment, a vector comprising the nucleic acid, a probe or primer, etc.).
To diagnose MI or a susceptibility to MT (or a disease or condition associated 2o with FLAP), the test sample containing a FLAP nucleic acid is contacted with at least one nucleic acid probe to form a hybridization sample. A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can be all or a portion of one of SEQ ID NOs: 1 and 3, or the complement thereof or a portion thereof;
or can be a nucleic acid encoding all or a portion of one of SEQ ID NO: 2. Other suitable probes for use in the diagnostic assays of the invention are described above (see e.g., probes and primers discussed under the heading, "Nucleic Acids of the Invention") The hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to a FLAP nucleic acid.
"Specific hybridization", as used herein, indicates exact hybridization (e.g., with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above.
hz a particularly preferred embodiment, the hybridization conditions for specific hybridization are high stringency.
to Specific hybridization, if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and FLAP nucleic acid in the test sample, then the FLAP has the polymorphism, or is the splicing variant, that is present in the nucleic acid probe. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic 15 acid probes is indicative of a polymorphism in the FLAP nucleic acid, or of the presence of a particular splicing variant encoding the FLAP nucleic acid, and is therefore diagnostic for a disease or condition associated with FLAP or a susceptibility to a disease or condition associated with FLAP (e.g., MI).
In Northern analysis (see Cu3"rel2t Protocols in Molecular Biology, Ausubel, F.
2o et al., eds., John Wiley & Sons, supra) the hybridization methods described above are used to identify the presence of a polymorphism or a particular splicing variant, associated with a disease or condition associated with or a susceptibility to a disease or condition associated with FLAP (e.g., MI). For Northern analysis, a test sample of RNA is obtained from the individual by appropriate means. Specific hybridization of 25 a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymorphism in a FLAP nucleic acid, or of the presence of a particular splicing variant encoded by a FLAP nucleic acid, and is therefore diagnostic for the disease or condition associated with FLAP, or for susceptibility to a disease or condition associated with FLAP (e.g., MI).
3o For representative examples of use of nucleic acid probes, see, for example, U.S. Patents No. 5,288,611 and 4,851,330.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described above. PNA is a DNA
mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or L~ attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P.E. et al., Biocoyzjugate Cheuzi~t~y 5, American Chemical Society, p. 1 (1994). The PNA probe can be designed to specifically hybridize to a nucleic acid having a polymorphism associated with a disease or condition associated with FLAP or associated with a susceptibility to a disease or condition associated With FLAP (e.g., MI). Hybridization of the PNA
l0 probe to a FLAP nucleic acid as described herein is diagnostic for the disease or condition or the susceptibility to the disease or condition.
In another method of the invention, mutation analysis by restriction digestion can be used to detect an altered nucleic acid, or nucleic acids containing a polymorphism(s), if the mutation or polymorphism in the nucleic acid results in the creation or elimination of a restriction site. A test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify a FLAP nucleic acid (and, if necessary, the flanking sequences) in the test sample of genomic DNA from the test individual. RFLP analysis is conducted as described (see C'uY~efzt Protocols izz Molecular Biology, supra). The digestion pattern of the relevant DNA fragment indicates the presence or absence of the alteration or polymorphism in the FLAP nucleic acid, and therefore indicates the presence or absence of a disease or condition associated with FLAP or the susceptibility to a disease or condition associated with FLAP (e.g., MI).
Sequence analysis can also be used to detect specific polyrnorphisms in the FLAP nucleic acid. A test sample of DNA or RNA is obtained from the test individual. PCR or other appropriate methods can be used to amplify the nucleic acid, and/or its flanking sequences, if desired. The sequence of a FLAP nucleic acid, or a fragment of the nucleic acid, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods. The sequence of the 3o nucleic acid, nucleic acid fragment, cDNA, cDNA fragment, mRNA, or mRNA
fragment is compared with the known nucleic acid sequence of the nucleic acid, cDNA (e.g., one or more of SEQ TD NOs: 1 or 3, andlor the complement of SEQ ID
NO: 1 or 3), or a nucleic acid sequence encoding SEQ m NO: 2 or a fragment thereof) or mRNA, as appropriate. The presence of a polymorphism in the FLAP
indicates that the individual has disease or a susceptibility to a disease associated with FLAP (e. g. , MI).
Allele-specific oligonucleotides can also be used to detect the presence of polymorphism(s) in the FLAP nucleic acid, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Sail~i, R. et al., Natuf°e 324:163-166 (1986)). An "allele-specific l0 oligonucleotide" (also referred to herein as an "allele-specific oligonucleotide probe") is an oligonucleotide of approximately 10-50 base pairs, for example, approximately 15-30 base pairs, that specifically hybridizes to a FLAP nucleic acid, and that contains a polymorphism associated with a disease or condition associated with FLAP or a susceptibility to a disease or condition associated with FLAP (e.g., Ml). An allele-specific oligonucleotide probe that is specific for particular polymorphisms in a FLAP
nucleic acid can be prepared, using standard methods (see Cuy~y-efzt Protocols iyi Molecular Biology, supra). To identify polymorphisms in the nucleic acid associated with disease or susceptibility to disease, a test sample of DNA is obtained from the individual. PCR can be used to amplify all or a fragment of a FLAP nucleic acid, and its flanking sequences. The DNA containing the amplified FLAP nucleic acid (or fragment of the nucleic acid) is dot-blotted, using standard methods (see Cur~e~zt Protocols in. Molecular Biology, supy~a), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified FLAP is then detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymorphism in the FLAP, and is therefore indicative of a disease or condition associated with FLAP
or a susceptibility to a disease or condition associated with FLAP (e.g., MI).
An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polyrnorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO
93/22456).
In another embodiment, arrays of oligonucleotide probes that are complementary to to target nucleic acid sequence segments from an individual, can be used to identify polymorphisms in a FLAP nucleic acid. For example, in one embodiment, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "GenechipsTM," have been generally described in the art, for example, U.S.
Pat. No.
5,143,854 and PCT patent publication Nos. WO 90/15070 and WO 92/10092. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science 251:767-777 (1991); Pirrung et al., U.S. Pat. 5,143,854; (see also PCT
Application WO 90/15070); Fodor et al., PCT Publication WO 92/10092; and U.S. Pat.
5,424,186, the entire teachings of each of which are incorporated by reference herein.
Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. 5,384,261, the entire teachings of which are incorporated by reference herein. In another example, linear arrays can be utilized.
Once an oligonucleotide array is prepared, a nucleic acid of interest is hybridized with the array and scanned for polymorphisms. Hybridization and scanning are generally carried out by methods described herein and also in, e.g., published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No.
5,424,186, the entire teachings of which are incorporated by reference herein.
In brief, a target nucleic acid sequence that includes one or more previously identified polyrnorphic markers is amplified using well-known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the two strands of the target sequence both upstream and downstream from the polymorphism. Asymmetric PCR techniques may also be used. Amplified target, generally incorporating a label, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array. In a reverse method, a 1o probe, containing a polymorphism, can be coupled to a solid surface and PCR
amplicons are then added to hybridize to these probes.
Although primarily described in terms of a single detection block, e.g., detection of a single polymorphism arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymorphisms. It will generally be i5 understood that detection blocks may be grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the array. For example, it may often be desirable to provide for the detection of those polyrnorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows 2o for the separate optimization of hybridization conditions for each situation.
Additional uses of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Patents Nos. 5,858,659 and 5,837,832, the entire teachings of which are incorporated by reference herein. Other methods of nucleic acid analysis can be used to detect polymorphisms in a nucleic acid described herein, 25 or variants encoded by a nucleic acid described herein. Representative methods include direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci.
USA
81:1991-1995 (1988); Sanger, F. et al., P~oc. Natl. Acad. Sci., USA 74:5463-(1977); Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent sequencing;
single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel 30 electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V.C. et al., P~oc. Natl. Acad. Sci. USA 86:232-236 (1989)), mobility shift analysis (Orita, M. et al., Py~oc. Natl. Acad. Sci. USA 86:2766-2770 (1989)), restriction enzyme analysis (Flavell et al., Cell 15:25 (1978); Geever, et al., Pf~oc. Natl.
Acad. Sci. USA
78:5081 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)); RNase protection assays (Myers, R.M. et al., Scieface 230:1242 (1985)); use of polypeptides which recognize nucleotide mismatches, such as E. coli mutS protein; allele-specific PCR, for example.
In one embodiment of the invention, diagnosis of a disease or condition associated with FLAP (e.g., MI) or a susceptibility to a disease or condition associated to with FLAP (e.g., MI) can also be made by expression analysis by quantitative PCR
(kinetic thermal cycling). This technique utilizing TaqMan ~ can be used to allow the identification of polymorphisms and whether a patient is homozygous or heterozygous. The technique can assess the presence of an alteration in the expression or composition of the polypeptide encoded by a FLAP nucleic acid or splicing variants encoded by a FLAP nucleic acid. Further, the expression of the variants can be quantified as physically or functionally different.
In another embodiment of the invention, diagnosis of MI or a susceptibility to MI (or of another disease or condition associated with FLAP) can also be made by examining expression and/or composition of a FLAP polypeptide, by a variety of 2o methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. A test sample from an individual is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a FLAP nucleic acid, or for the presence of a particular variant encoded by a FLAP nucleic acid. An alteration in expression of a polypeptide encoded by a FLAP nucleic acid can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by a FLAP nucleic acid is an alteration in the qualitative polypeptide expression (e.g., expression of an altered FLAP polypeptide or of a different splicing variant). In a preferred embodiment, diagnosis of disease or condition associated with FLAP or a susceptibility to a disease or condition associated with FLAP is made by detecting a particular splicing variant encoded by that FLAP variant, or a particular pattern of splicing variants.
Both such alterations (quantitative and qualitative) can also be present. An "alteration" in the polypeptide expression or composition, refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of polypeptide by a FLAP nucleic acid in a control sample. A
control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by the disease or a susceptibility to a disease or condition associated with a FLAP nucleic acid. An alteration in the to expression or composition of the polypeptide in the test sample, as compared with the control sample, is indicative of disease or condition associated with FLAP or a susceptibility to a disease or condition associated with FLAP (e.g., MI).
Similarly, the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, is indicative of a susceptibility to a disease or condition associated with a FLAP nucleic acid. Various means of examining expression or composition of the polypeptide encoded by a FLAP
nucleic acid can be used, including: spectroscopy, colorimetry, electrophoresis, isoelectric focusing and immunoassays (e.g., David et al., U.S. Pat. 4,376,110) such as 2o irmnunoblotting (see also Cur rent Protocols ifZ Molecular Biology, particularly Chapter 10). For example, in one embodiment, an antibody capable of binding to the polypeptide (e.g., as described above), preferably an antibody with a detectable label, can be used. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')a) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled 3o secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

Western blotting analysis, using an antibody as described above that specifically binds to a polypeptide encoded by an altered FLAP (e.g., by a FLAP
having a SNP as shown in Table 3), or an antibody that specifically binds to a polypeptide encoded by a non-altered nucleic acid, or an antibody that specifically binds to a particular splicing variant encoded by a nucleic acid, can be used to identify the presence in a test sample of a particular splicing variant or of a polypeptide encoded by a polymorphic or altered FLAP, or the absence in a test sample of a particular splicing variant or of a polypeptide encoded by a non-polyrnorphic or non-altered nucleic acid. The presence of a polypeptide encoded by a polymorphic or to altered nucleic acid, or the absence of a polypeptide encoded by a non-polymorphic or non-altered nucleic acid, is diagnostic for disease or condition associated with FLAP
or a susceptibility to a disease or condition associated with, as is the presence (or absence) of particular splicing variants encoded by the FLAP nucleic acid.
In one embodiment of this method, the level or amount of polypeptide encoded by a FLAP nucleic acid in a test sample is compared with the level or amount of the polypeptide encoded by the FLAP in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded 2o by the FLAP, and is diagnostic for disease or condition, or for a susceptibility to a disease or condition, associated with that FLAP. Alternatively, the composition of the polypeptide encoded by a FLAP nucleic acid in a test sample is compared with the composition of the polypeptide encoded by the FLAP in a control sample (e.g., the presence of different splicing variants). A difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample, is diagnostic for a disease or condition, or for a susceptibility to a disease or condition, associated with that FLAP. In another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample. A difference in the amount or level of the 3o polypeptide in the test sample, compared to the control sample; a difference in composition in the test sample, compared to the control sample; or both a difference in the amount or level, and a difference in the composition, is indicative of a disease or condition, or a susceptibility to a disease or condition, associated with FLAP (e.g., MI).
The invention further pertains to a method for the diagnosis and identification of susceptibility to myocardial infarction in an individual, by identifying an at-risk haplotype in FLAP. In one embodiment, the at-risk haplotype is one which confers a significant risk of MI. In one embodiment, sig~lificance associated with a haplotype is measured by an odds ratio. In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant risk is measured as an odds ratio of at to least about 1.2, including by not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. In a further embodiment, an odds ratio of at least 1.2 is significant. In a further embodiment, an odds ratio of at least about 1.5 is significant. In a further embodiment, a significant increase in risk is at least about 1.7 is significant. In a further embodiment, a significant increase in risk is at least about 20%, including but riot hmlted tO about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 98%. In a further embodiment, a significant increase in risk is at least about 50%. It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors.
The invention also pertains to methods of diagnosing myocardial infarction or a susceptibility to myocardial infarction in an individual, comprising screening for an at-risk haplotype in the FLAP nucleic acid that is more frequently present in an individual susceptible to myocardial infarction (affected), compared to the frequency of its presence in a healthy individual (control), wherein the presence of the haplotype is indicative of myocardial infarction or susceptibility to myocardial infarction.
Standard techniques for genotyping for the presence of SNPs and/or microsatellite markers that are associated with myocardial infarction can be used, such as fluorescent based techniques (Chen, et al., Geraonae Res. 9, 492 (1999), PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. In a preferred embodiment, the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in the FLAP nucleic acid that are associated with myocardial infarction, wherein an excess or higher frequency of the SNPs and/or microsatellites compared to a healthy control individual is indicative that the individual has myocardial infarction or is susceptible to myocardial infarction. See Table 3 that sets forth SNPs and markers for use as screening tools.
In one embodiment, the at-risk haplotype is characterized by the presence of polyrnorphism(s) represented in Table 3. For example, DGOOAAFICT at position 256047, where the SNP can be a "C" or a "T"; SG13S25 at position 283477, where the SNP can be a "G" or an "A"; DGOOAAJFF at position 287889, where the SNP
can be a "G" or an "A"; DGOOAAHII at position 294503, where the SNP can be a to "G" or an "A"; DGOOAAHID at position 296020, where the SNP can be a "T" or an "A"; B SNP-310657 at position 310657, where the SNP can be a_ "G" or an "A";
SG13S30 at position 312056, where the SNP can be a "G" or a "T"; SG13S32 at position 316763, where the SNP can be a "C" or an "A"; SG13S42 at position 320393, where the SNP can be a "G" or an "A"; and SG13S35 at position 324333, where the SNP can be a "G" or an "A". Kits (e.g., reagent kits) useful in the methods of diagnosis comprise components useful in any of the methods described herein, including for example, hybridization probes or primers as described herein (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies which bind to altered or to non-altered (native) FLAP polypeptide, means for amplification of nucleic acids comprising a FLAP, or means for analyzing the nucleic acid sequence of a nucleic acid described herein, or for analyzing the amino acid sequence of a polypeptide as described herein, etc. In one embodiment, a kit for diagnosing MI or susceptibility to MI can comprise primers for nucleic acid amplification of a region in the FLAP nucleic acid comprising an at-risk haplotype that is more frequently present in an individual having MI or susceptible to MI. The primers can be designed using portions of the nucleic acids flanking SNPs that are indicative of MI. In a particularly preferred embodiment, the primers are designed to amplify regions of the FLAP
nucleic acid associated with an at-risk haplotype for MI, or more particularly the 3o haplotypes defined by the following SNPs: DG00AAFIU, SG13S25, DGOOAAJFF, DGOOAAHII, DGOOAAHID, B SNP_310657, SG13S30, SG13S32, SG13S42, and SG13S35 , at the locus on chromosome 13q12.
SCREENING ASSAYS AND AGENTS IDENTIFIED THERBY
The invention provides methods (also referred to herein as "screening assays") for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the invention, as well as for identifying the presence of a polypeptide encoded by a nucleic acid of the invention. In one embodiment, the presence (or absence) of a nucleic acid molecule of interest (e.g., a nucleic acid that has significant homology to with a nucleic acid of the invention) in a sample can be assessed by contacting the sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a nucleic acid having the sequence of one of SEQ ID NOs: f or 3 or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of SEQ ID NO: 2, or a fragment or variant of such nucleic acids), under stringent conditions as described i5 above, and then assessing the sample for the presence (or absence) of hybridization.
In a preferred embodiment, high stringency conditions are conditions appropriate for selective hybridization. In another embodiment, a sample containing a nucleic acid molecule of interest is contacted with a nucleic acid containing a contiguous nucleic acid sequence (e.g., a primer or a probe as described above) that is at least partially 2o complementary to a part of the nucleic acid molecule of interest (e.g., a FLAP nucleic acid), and the contacted sample is assessed for the presence or absence of hybridization. In a preferred embodiment, the nucleic acid containing a contiguous nucleic acid sequence is completely complementary to a part of the nucleic acid molecule of interest.
25 In any of these embodiments, all or a portion of the nucleic acid of interest can be subjected to amplification prior to performing the hybridization.
In another embodiment, the presence (or absence) of a polypeptide of interest, such as a polypeptide of the invention or a fragment or variant thereof, in a sample can be assessed by contacting the sample with an antibody that specifically hybridizes 30 to the polypeptide of interest (e.g., an antibody such as those described above), and then assessing the sample for the presence (or absence) of binding of the antibody to the polypeptide of interest.
In another embodiment, the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes which alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein. For example, such agents can be agents which bind to polypeptides described herein (e.g., binding agent for members of the leukotriene pathway, such as FLAP binding agents); which have a stimulatory to or inhibitory effect on, for example, activity of polypeptides of the invention; or which change (e.g., enhance or inhibit) the ability of the polypeptides of the invention to interact with members of the leukotriene pathway binding agents (e.g., receptors or other binding agents); or which alter posttranslational processing of the leukotriene pathway member polypeptide, such as a FLAP polypeptide (e.g., agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.) In one embodiment, the invention provides assays for screening candidate or test agents that bind to or modulate the activity of polypeptides described herein (or 2o biologically active portions) thereof), as well as agents identifiable by the assays.
Test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S.,Anticahce~DrugDes. 12:145 (1997)).
In one embodiment, to identify agents which alter the activity of a FLAP
polypeptide, a cell, cell lysate, or solution containing or expressing a FLAP
polypeptide (e.g., SEQ ID NO: 2 or another splicing variant encoded by a FLAP

nucleic acid, such as a nucleic acid comprising a SNP as shown in Table 3), or a fragment or derivative thereof (as described above), can be contacted with an agent to be tested; alternatively, the polypeptide can be contacted directly with the agent to be tested. The level (amount) of FLAP activity is assessed (e.g., the level (amount) of FLAP activity is measured, either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity of the FLAP
polypeptide or active fragment or derivative thereof in the absence of the agent to be tested). If the level of the activity in the presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of a FLAP polypeptide. An increase in the level of FLAP activity in the presence of the agent relative to the activity in the absence of the agent, indicates that the agent is an agent that enhances (is an agonist of) FLAP activity. Similarly, a decrease in the level of FLAP activity in the presence of the agent, relative to the activity in the absence of the agent, indicates that the agent is an agent that inhibits (is an antagonist of) FLAP activity. In another embodiment, the level of activity of a FLAP polypeptide or derivative or fragment thereof in the presence of the agent to be tested, is compared with a control level that has previously been established. A statistically significant difference in the level of the activity in the presence of the agent from the control level indicates that the agent alters FLAP
activity.
The present invention also relates to an assay for identifying agents which alter the expression of a FLAP nucleic acid (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes; which alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the nucleic acid or which otherwise interact with the nucleic acids described herein, as well as agents identifiable by the assays. For example, a solution containing a nucleic acid encoding a FLAP polypeptide (e.g., a FLAP nucleic acid) can be contacted with an agent to be tested. The solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution that comprises elements necessary for transcription/translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired. The level and/or pattern of FLAP expression (e.g., the level and/or pattenl of mRNA or of protein expressed, such as the level and/or pattern of different splicing variants) is assessed, and is compared with the level and/or pattern of expression in a control (i.e., the level and/or pattern of the FLAP expression in the absence of the agent to be tested). If the level and/or pattern in the presence of the agent differ, by an amount or in a manner that is statistically significant, from the level and/or pattern in the absence of the agent, then the agent is an agent that alters the expression of the FLAP nucleic acid. Enhancement of FLAP expression indicates that the agent is an agonist of to FLAP activity. Similarly, inhibition of FLAP expression indicates that the agent is an antagonist of FLAP activity.
In another embodiment, the level and/or pattern of FLAP polypeptide(s) (e.g., different splicing variants) in the presence of the agent to be tested, is compared with a control level and/or pattern that have previously been established. A level and/or pattern in the presence of the agent that differs from the control level and/or pattern by an amount or in a manner that is statistically significant indicates that the agent alters FLAP expression.
In another embodiment of the invention, agents which alter the expression of a FLAP nucleic acid or which otherwise interact with the nucleic acids described 2o herein, can be identified using a cell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the FLAP nucleic acid operably linked to a reporter gene. After contact with an agent to be tested, the level of expression of the reporter gene (e.g., the level of mRNA or of protein expressed) is assessed, and is compared with the level of expression in a control (i.e., the level of the expression of the reporter gene in the absence of the agent to be tested). If the level in the presence of the agent differs, by an amount or in a mariner that is statistically significant, from the level in the absence of the agent, then the agent is an agent that alters the expression of the FLAP nucleic acid, as indicated by its ability to alter expression of a nucleic acid that is operably linked to the FLAP nucleic acid promoter.
3o Enhancement of the expression of the reporter indicates that the agent is an agonist of FLAP activity. Similarly, inhibition of the expression of the reporter indicates that the agent is an antagonist of FLAP activity. In another embodiment, the level of expression of the reporter in the presence of the test agent, is compared with a control level that has previously been established. A level in the presence of the agent that differs from the control level by an amount or in a manner that is statistically significant indicates that the agent alters expression.
Agents which alter the amounts of different splicing variants encoded by a FLAP nucleic acid (e.g., an agent which enhances activity of a first splicing variant, and wluch inhibits activity of a second splicing variant), as well as agents which are agonists of activity of a first splicing variamt and antagonists of activity of a second to splicing variant, can easily be identified using these methods described above.
In other embodiments of the invention, assays can be used to assess the impact of a test agent on the activity of a polypeptide relative to a FLAP binding agent. For example, a cell that expresses a compound that interacts with a FLAP nucleic acid (herein referred to as a "FLAP binding agent", which can be a polypeptide or other molecule that interacts with a FLAP nucleic acid, such as a receptor, or another molecule, such as 5-LO) is contacted with a FLAP in the presence of a test agent, and the ability of the test agent to alter the interaction between the FLAP and the FLAP
binding agent is determined. Alternatively, a cell lysate or a solution containing the FLAP binding agent, can be used. An agent which binds to the FLAP or the FLAP
2o binding agent can alter the interaction by interfering with, or enhancing the ability of the FLAP to bind to, associate with, or otherwise interact with the FLAP
binding agent. Determining the ability of the test agent to bind to a FLAP nucleic acid or a FLAP nucleic acid binding agent can be accomplished, for example, by coupling the test agent with a radioisotope or enzymatic label such that binding of the test agent to the polypeptide can be determined by detecting the labeled with l2sh 3sS, i4C
or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of 3o conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a test agent to interact with the polypeptide without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a test agent with a FLAP or a FLAP
binding agent without the labeling of either the test agent, FLAP, or the FLAP binding agent.
McConnell, H.M. et al., Sciefzce 257:1906-1912 (1992). As used herein, a "microphysiometer" (e.g., CytosensorTM) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between ligand and polypeptide.
Thus, these receptors can be used to screen for compounds that are agonists 1 o for use in treating a disease or condition associated with FLAP or a susceptibility to a disease or condition associated with FLAP, or antagonists for studying a susceptibility to a disease or condition associated with FLAP (e.g., MI). Drugs can be designed to regulate FLAP activation, that in turn can be used to regulate signaling pathways and transcription events of genes downstream or of proteins or polypeptides interacting with FLAP (e.g., 5-LO).
In another embodiment of the invention, assays can be used to identify polypeptides that interact with one or more FLAP polypeptides, as described herein.
For example, a yeast two-hybrid system such as that described by Fields and Song (Fields, S. and Song, O., Nature 340:245-246 (1989)) can be used to identify 2o polypeptides that interact with one or more FLAP polypeptides. In such a yeast two-hybrid system, vectors are constructed based on the flexibility of a transcription factor that has two functional domains (a DNA binding domain and a transcription activation domain). If the two domains are separated but fused to two different proteins that interact with one another, transcriptional activation can be achieved, and transcription of specific markers (e.g., nutritional markers such as His and Ade, or color markers such as lacZ) can be used to identify the presence of interaction and transcriptional activation. For example, in the methods of the invention, a first vector is used which includes a nucleic acid encoding a DNA binding domain and also a FLAP polypeptide, splicing variant, or fragment or derivative thereof, and a second 3o vector is used which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the FLAP polypeptide, splicing variant, or fragment or derivative thereof (e.g., a FLAP polypeptide binding agent or receptor). Incubation of yeast containing the first vector and the second vector under appropriate conditions (e.g., mating conditions such as used in the MatchmakerTM system from Clontech (Palo Alto, California, USA)) allows identification of colonies that express the markers of interest.
These colonies can be examined to identify the polypeptide(s) that interact with the FLAP
polypeptide or fragment or derivative thereof. Such polypeptides may be useful as agents that alter the activity of expression of a FLAP polypeptide, as described above.
In more than one embodiment of the above assay methods of the present i0 invention, it may be desirable to immobilize either the FLAP, the FLAP
binding agent, or other components of the assay on a solid support, in order to facilitate separation of complexed from uncomplexed forms of one or both of the polypeptides, as well as to accommodate automation of the assay. Binding of a test agent to the polypeptide, or interaction of the polypeptide with a binding agent in the presence and absence of a test agent, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein (e.g., a glutathione-S-transferase fusion protein) can be provided which adds a domain that allows a FLAP
nucleic acid or a FLAP binding agent to be bound to a matrix or other solid support.
2o In another embodiment, modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell, cell lysate, or solution containing a nucleic acid encoding a FLAP nucleic acid is contacted with a test agent and the expression of appropriate mRNA or polypeptide (e.g., splicing variant(s)) in the cell, cell lysate, or solution, is determined. The level of expression of appropriate mRNA or polypeptide(s) in the presence of the test agent is compared to the level of expression of mRNA or polypeptide(s) in the absence of the test agent. The test agent can then be identified as a modulator of expression based on this comparison.
For example, when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the test agent than in its absence, the test agent 3o is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
Alternatively, when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the test agent than in its absence, the test agent is identified as an inhibitor of the inRNA or polypeptide expression. The level of mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting mRNA or polypeptide.
In yet another embodiment, the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) the activity of a member of leukotriene pathway binding agent, such as a FLAP binding agent (e.g., 5-LO), as described herein. For example, to such agents can be agents which have a stimulatory or inhibitory effect on, for example, the activity of a member of leukotriene pathway binding agent, such as a FLAP binding agent; which change (e.g., enhance or inhibit) the ability a member of leukotriene pathway binding agents, (e.g., receptors or other binding agents) to interact with the polypeptides of the invention; or which alter posttranslational processing of the member of leukotriene pathway binding agent, (e.g., agents that alter proteolytic processing to direct the member of the leukotriene pathway binding agent from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more active binding agent is released from the cell, etc.).
2o For example, the invention provides assays for screening candidate or test agents that bind to or modulate the activity of a member of the leukotriene pathway (or enzymatically active portions) thereof), as well as agents identifiable by the assays. As described above, test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. Afatieayace~ Df°ug Des., 12:145 ( 1997)).

In one embodiment, to identify agents which alter the activity of a member of the leukotriene pathway (such as a FLAP binding agent), a cell, cell lysate, or solution containing or expressing a binding agent (e.g., 5-LO, or a leukotriene pathway member receptor), or a fragment (e.g., an enzymatically active fragment) or derivative thereof, can be contacted with an agent to be tested; alternatively, the binding agent (or fragment or derivative thereof) can be contacted directly with the agent to be tested. The level (amount) of binding agent activity is assessed (either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity in the absence of the agent to be tested). If the level of the activity in the to presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of the member of the leukotriene pathway. An increase in the level of the activity relative to a control, indicates that the agent is an agent that enhances (is an agonist of) the activity. Similarly, a decrease in the level of activity relative to a control, indicates that the agent is an agent that inhibits (is an antagonist of) the activity. W another embodiment, the level of activity in the presence of the agent to be tested, is compared with a control level that has previously been established. A
level of the activity in the presence of the agent that differs from the control level by an amount that is statistically significant indicates that the agent alters the activity.
2o This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
For example, an agent identified as described herein (e.g., a test agent that is a modulating agent, an antisense nucleic acid molecule, a specific antibody, or a polypeptide-binding agent) can be used in an animal model to determine the efficacy, toxicity,, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
Furthermore, this invention pertains to uses of novel agents identified by the 3o above-described screening assays for treatments as described herein. In addition, an agent identified as described herein can be used to alter activity of a polypeptide encoded by a FLAP nucleic acid, or to alter expression of a FLAP nucleic acid, by contacting the polypeptide or the nucleic acid (or contacting a cell comprising the polypeptide or the nucleic acid) with the agent identified as described herein.
PHARMACEUTICAL COMPOSITIONS
The present invention also pertains to pharmaceutical compositions comprising nucleic acids described herein, particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NO: 1 or 3 or the complement thereof, and/or comprising other l0 splicing variants encoded by a FLAP nucleic acid; and/or an agent that alters (e.g., enhances or inhibits) FLAP nucleic acid expression or FLAP polypeptide activity as described herein. For instance, a polypeptide, protein (e.g., a FLAP
receptor), an agent that alters FLAP nucleic acid expression, or a FLAP nucleic acid binding agent or binding partner, fragment, fusion protein or pro-drug thereof, or a nucleotide or nucleic acid construct (vector) comprising a nucleotide of the present invention, or an agent that alters FLAP polypeptide activity, can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit the mode of administration.
2o Suitable pharmaceutically acceptable Garners include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.

_57_ The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard Garners such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
Methods of introduction of these compositions include, but are not limited to, intradermal, intrasnuscular, intraperitoneal, intraocular, intravenous, subcutaneous, io topical, oral and intranasal. Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devices ("gene guns") and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings.
For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
2o Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or Water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sots, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The agent may be i incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
Agents described herein can be formulated as neutral or salt forms.
to Pharmaceutically acceptable salts include those fornzed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The agents are administered in a therapeutically effective amount. The amount of agents which will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, ih vitYO
or is~ vivo assays may optionally be employed to help identify optimal dosage ranges. The 2o precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from ih vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval 3o by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like.
The pack or kit may also include means for reminding the patient to take the therapy.
The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. Agents assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
to METHODS OF THERAPY
The present invention also pertains to methods of treatment (prophylactic and/or therapeutic) for MI or a susceptibility to MI, using an MI therapeutic agent.
An "MI therapeutic agent" is an agent that alters (e.g., enhances or inhibits) FLAP
polypeptide activity and/or FLAP nucleic acid expression, as described herein (e.g., a nucleic acid agonist or antagonist). MI therapeutic agents can alter FLAP
polypeptide activity or nucleic acid expression by a variety of means, such as, for example, by providing additional FLAP polypeptide or upregulating the transcription or translation of the FLAP nucleic acid; by altering posttranslational processing of the FLAP
polypeptide; by altering transcription of FLAP splicing variants; or by interfering with 2o FLAP polypeptide activity (e.g., by binding to a FLAP polypeptide), or by downregulating the transcription or translation of a FLAP nucleic acid.
Representative MI therapeutic agents include the following:
nucleic acids or fragments or derivatives thereof described herein, particularly nucleotides encoding the polypeptides described herein and vectors comprising such nucleic acids (e.g., a gene, nucleic acid, cDNA, and/or mRNA, such as a nucleic acid encoding a member of the leukotriene pathway, such as a FLAP polypeptide or active fragment or derivative thereof, or an oligonucleotide; for example, one of SEQ ID Nos. 1 or 3 or the 3o complement thereof, or a nucleic acid encoding SEQ ID NO: 2, or fragments or derivatives thereof);

polypeptides described herein and/or other splicing variants encoded by a FLAP nucleic acid, or fragments or derivatives thereof);
other polypeptides (e.g., receptors of members of the leukotriene pathway, such as LTB4 receptors, LTC4 receptors, LTD4 receptors, Cys LT1 receptors, Cys LT2 receptors); binding agents of the leukotriene pathway, such as FLAP
binding agents (e.g., 5-LO); peptidomimetics; fusion proteins or prodrugs thereof; antibodies (e.g., an antibody to an altered FLAP polypeptide, or an to antibody to a non-altered FLAP polypeptide, or an antibody to a particular splicing variant encoded by a FLAP nucleic acid, as described above);
ribozymes; other small molecules; and other agents that alter (e.g., enhance or inhibit) a member of the leukotriene 15 pathway gene expression, such as FLAP nucleic acid expression or polypeptide activity, or that regulate transcription of FLAP splicing variants (e.g., agents that affect which splicing variants are expressed, or that affect the amount of each splicing variant that is expressed.
2o More than one MI therapeutic agent can be used concurrently, if desired.
An MI nucleic acid therapeutic agent that is a nucleic acid is used in the treatment of a susceptibility to MI. The term, "treatment" as used herein, refers not only to ameliorating symptoms associated with the disease, but also preventing or 25 delaying the onset of the disease, and also lessening the severity or frequency of symptoms of the disease. The therapy is designed to alter (e.g., inhibit or enhance), replace or supplement activity of a FLAP polypeptide in an individual. For example, an MI nucleic acid therapeutic agent can be administered in order to upregulate or increase the expression or availability of the FLAP nucleic acid or of specific splicing 3o variants of FLAP nucleic acid, or, conversely, to downregulate or decrease the expression or availability of the FLAP nucleic acid or specific splicing variants of the FLAP nucleic acid. Upregulation or increasing expression or availability of a native FLAP nucleic acid or of a particular splicing variant could interfere with or compensate for the expression or activity of a defective nucleic acid or another splicing variant; downregulation or decreasing expression or availability of a native FLAP nucleic acid or of a particular splicing variant could minimize the expression or activity of a defective nucleic acid or the particular splicing variant and thereby minimize the impact of the defective nucleic acid or the particular splicing variant.
The MI therapeutic agents) are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease, such as by ameliorating 1o symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease).
The amount which will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques. In addition, ih vitYo or ih vivo assays may optionally be employed to help identify optimal dosage ranges.
The precise dose to be employed in the fornmlation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
In one embodiment, a nucleic acid of the invention (e.g., a nucleic acid encoding a FLAP polypeptide, such as one of SEQ ID NO: 1 or 3 or the complement thereof; or another nucleic acid that encodes a FLAP polypeptide or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding SEQ ID NO: 2, can be used, either alone or in a pharmaceutical composition as described above. For example, a FLAP nucleic acid or a cDNA encoding a FLAP polypeptide, either by itself or included within a vector, can be introduced into cells (either iya vitro or ih vivo) such that the cells produce native FLAP polypeptide. If necessary, cells that have been transformed with the nucleic acid or cDNA or a vector comprising the 3o nucleic acid or cDNA can be introduced (or re-introduced) into an individual affected with the disease. Thus, cells which, in nature, lack native FLAP expression and activity, or have altered FLAP expression and activity, or have expression of a disease-associated FLAP splicing variant, can be engineered to express the FLAP
polypeptide or an active fragment of the FLAP polypeptide (or a different variant of the FLAP polypeptide). In a preferred embodiment, nucleic acid encoding a FLAP
polypeptide, or an active fragment or derivative thereof, can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal. Other nucleic acid transfer systems, including viral and nonviral transfer systems, can be used. Alternatively, nonviral nucleic acid transfer methods, such as calcium phosphate coprecipitation, mechanical techniques to (e.g., microinjection); membrane fusion-mediated transfer via liposomes; or direct DNA uptake, can also be used.
Alternatively, in another embodiment of the invention, a nucleic acid of the invention; a nucleic acid complementary to a nucleic acid of the invention; or a portion of such a nucleic acid (e.g., an oligonucleotide as described below), can be used in "antisense" therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA of an MI nucleic acid is administered or generated ih situ. The antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the FLAP polypeptide, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic 2o acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
An antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA
that encodes the FLAP polypeptide. Alternatively, the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of the FLAP. In one embodiment, the oligonucleotide probes are modified oligonucleotides 3o that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable ifa vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs ofDNA (see also U.S. Pat. Nos. 5,176,996, 5,264,564 and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy are also described, for example, by Van der Krol et al.
(Biotechfaiques 6:958-976 (1988)); and Stein et al. (Cahce~ Res. 48:2659-2668 (1988)). With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site are preferred.
To perform antisense therapy, oligonucleotides (mRNA, cDNA or DNA) are designed that are complementary to mRNA encoding the FLAP. The to antisense oligonucleotides bind to FLAP mRNA transcripts and prevent translation.
Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of an RNA, as referred to herein, indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above.
Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA
it may contain and still form a stable duplex (or triplex, as the case may be).
One 2o skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
The oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotides can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Ps°oc. Natl.
Acad. Sci. USA
86:6553-6556 (1989); Lemaitre et al., PYOC. Natl. Acad. Sci. USA 84:648-652 (1987);
3o PCT International Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT International Publication No. WO 89110134), or hybridization-triggered cleavage agents (see, e.g., I~rol et al., BioTec7araiques 6:958-976 (1988)) or intercalating agents.
(See, e.g., Zon, Pharm.Res. 5: 539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent).
The antisense molecules are delivered to cells that express FLAP ih vivo. A
number of methods can be used for delivering antisense DNA or RNA to cells;
e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell to surface) can be administered systematically. Alternatively, in a preferred embodiment, a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g., pol III or pol II). The use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that will form 15 complementary base pairs with the endogenous FLAP transcripts and thereby prevent translation of the FLAP mRNA. For example, a vector can be introduced iya vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed 2o by recombinant DNA technology methods standard in the art and described above.
For example, a plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.
Alternatively, viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
25 Endogenous FLAP expression can also be reduced by inactivating or "knocking out" FLAP or its promoter using targeted homologous recombination (e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-(1987); Thompson et al., Cell 5:313-321 (1989)). For example, an altered, non-functional FLAP (or a completely unrelated DNA sequence) flanked by DNA
3o homologous to the endogenous FLAP (either the coding regions or regulatory regions of FLAP) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the FLAP ira vivo.
Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the FLAP. The recombinant DNA constructs can be directly administered or targeted to the required site ih. vivo using appropriate vectors, as described above.
Alternatively, expression of non-altered FLAPs can be increased using a similar method:
targeted homologous recombination can be used to insert a DNA construct comprising a non-altered functional FLAP, or the complement thereof, or a portion thereof, in place of an altered FLAP in the cell, as described above. In another embodiment, targeted homologous recombination can be used to insert a DNA construct comprising a to nucleic acid that encodes an MI polypeptide variant that differs from that present in the cell.
Alternatively, endogenous FLAP expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of a FLAP
(i.e., the FLAP promoter and/or enhancers) to form triple helical structures that prevent transcription of the FLAP in target cells in the body. (See generally, Helene, C., AtzticahceY Drug Des., 6(6):569-84 (1991); Helene, C. et al., AsZf2. N. Y.
Acad. Sci.
660:27-36 (1992); and Maher, L. J., Bioassays 14(12):807-15 (1992)). Likewise, the antisense constructs described herein, by antagonizing the normal biological activity of one of the FLAP proteins, can be used in the maupulation of tissue, e.g., tissue 2o differentiation, both ih vivo and fog ex vivo tissue cultures. Furthermore, the anti-sense techniques (e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to an MI nucleic acid RNA or nucleic acid sequence) can be used to investigate the role of FLAP in normal cellular function. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
In yet another embodiment of the invention, other MI therapeutic agents as described herein can also be used in the treatment or prevention of a susceptibility to a disease or condition associated with FLAP. The therapeutic agents can be delivered in a composition, as described above, or by themselves. They can be administered 3o systemically, or can be targeted to a particular tissue. The therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; irZ vivo production (~,g., a transgenic animal, such as U.S. Pat.
No.
4,873,316 to Meade et al.), for example, and can be isolated using standard means such as those described herein.
A combination of any of the above methods of treatment (e.g., administration of non-altered FLAP polypeptide in conjunction with antisense therapy targeting altered FLAP mRNA; administration of a first splicing variant encoded by a FLAP in conjunction with antisense therapy targeting a second splicing encoded by a FLAP) can also be used.
The present invention is now illustrated by the following Exemplification, to which is not intended to be limiting in any way.
EXEMPLIFICATION
SUBJECTS AND METHODS
Study population Patients entering the study were defined from an infarction registry that includes all MIs (over 8,000 patients) in Iceland 1981-2000. This registry is a part of the World Health Organization MONICA Project (The World Health Organization MONICA Project (monitoring trends and determinants in cardiovascular disease):
a major international collaboration. WHO MONICA Project Principal Investigators.
J
ClifZ. Epidemiol. 1988; 41:105-14). Diagnosis of all patients in the registry follow strict diagnostic rules based on symptoms, electrocardiograms, cardiac enzymes, and necropsy findings.
Blood samples from 1342 MI patients, both cases with a family history and sporadic cases were collected. For each patient that participated, blood was collected from 2 relatives (unaffected or affected). Their genotypes were used to help with construction of haplotypes. In addition, blood samples from 624 unrelated controls were collected.

Lizzlzage an.al~sis Extended families (pedigrees) by clustering related female MI patients were constructed into families such that each patient is related to at least one other patient within and including six meiotic events. The information regarding the relatedness of patients was obtained from an encrypted genealogy database that covers the entire Icelandic nation (Gulcher et al., Euz°. J. Hum. Genet. 8: 739=742 (2000)). A
genomewide scan was performed using a framework map of 1000 microsatellite markers, using protocols described elsewhere (Gretarsdottir S., et al. Am. J.
Huzzz.
to Gezzet., 70: 593-603, 2002)). The marker order and positions where obtained from a modified version of the Marshfield genetic map (Center for Medical Genetics, Marshfield Medical Research Foundation), using genetic mapping based on our own data, and from deCODE genetics high resolution genetic map (Kong A., et al., Nat.
genet., 31: 241-247 (2002)). The population-based allele frequencies were constructed from a cohort of more than 30,000 Icelanders who have participated in genetic studies of various disease projects. Additional markers were genotyped within the locus on chromosome 13 to increase the information on identity by descent within the families. For those markers at least 180 Icelandic controls were genotyped to derive the population allele frequencies.
2o For statistical analysis, multipoint, affected only allele-sharing methods were used to assess evidence for linkage. All results, both the LOD and the non-parametric linkage (NPL) score, were obtained using the program ALLEGRO (Gudbjartsson D.F., et al., Nat Genet., 25: 12-13(2000)). The baseline linkage analysis (Gretarsdottir S., et al., Am. J. Hum. Genet. 70: 593-603, (2002)) uses the Spairs scoring function (Whittermore AS, and Haplern J A., Biometrics 50: 118-127 (1994)) and Kruglyak et al., Am. J. Hum. Genet., 58:1347-1363 (1996)) the exponential allele-sharing model (Kong A., and Cox N.J., Azzz. J. Hum. Genet. 61:1179-1188 (1997)), and a family weighting scheme which is halfway, on the log-scale, between weighing each affected pairs equally and weighing each family equally.

Ultf~a-fine mapping anal haplotype analysis A candidate susceptibility locus was defined as the region under the LOD
score curve where the score was one lower than the highest lod score ((peak lod score -1)\one lod drop). This region (approx. l2Mb) was ultra-fmemapped with microsatellite markers with an average spacing between markers of less than 100kb.
All usable microsatellite markers that found in public databases and mapped within that region were used. In addition, microsatellite markers identified within the deCODE genetics sequence assembly of the human genome were used.
Haplotype analysis The frequencies of haplotypes in the patient and the control groups using an expectation-maximization algorithm were estimated (Dempster A.P. et al., J. R.
Stat.
Soc. B. 39: 1-389 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase was used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies.
Using a likelihood approach, an alternative hypothesis where a candidate at-risk haplotype is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups was tested. Likelihoods are maximized separately under both hypothesis and a corresponding 1-df likelihood ratio statistics is used to evaluate the statistic significance.
To look for at-risk-haplotypes in the 1-lod drop, association of all possible combinations of genotyped marlcers was studied, provided those markers spanned a region of size less than 1000 kb. Due to a certain amount of testing, thep-values were adjusted using simulations. The combined patient and control groups were randomly divided into two sets, equal in size to the original group of patients and controls. The haplotype analysis was then repeated and the most significantp-value registered was observed. This randomisation scheme was repeated over 100 times to construct an empirical distribution ofp-values.

SNP haplotype association to MI
In an effort to identify SNP haplotypes that associate with MI we have typed SNPs identified mainly by sequencing the FLAP gene and the region flanking the gene. We genotyped a total number of 45 SNPs in 1343 patients and 624 unrelated controls. The largest subset of unrelated patients (related no closer than 4 meioses) was 921. We observed two correlated series of SNP haplotypes in excess in patients, denoted as A and B in Table 7. The length of the haplotypes varies between 33 and 69 I~b and cover one or two blocks of linkage disequilibrium. Both series of haplotypes contain the common allele 2 of the SNP SG13S25. All haplotypes in the to A series contain the SNP DGOOAAHID, while all haplotypes in the B series contain the SNP DGOOAAHII. In the B series, the haplotypes B4, B5, and B6 have a relative risk (RR) greater than 2 and allelic frequencies above 10% (Table 1). The haplotypes in the A series have slightly lower RR and p-values, but higher allelic frequency (15-16%), and as such we also consider them interesting. The haplotypes in series B and A are strongly correlated, i.e. the B haplotypes define a subset of the A
haplotypes.
Hence, B haplotypes are more specific than A haplotypes. However, A haplotypes are more sensitive, i. e. they capture more individuals with the putative mutation, as is observed in the population attributable risk which is less for B than for A.
Furthermore, these haplotypes show similar risk ratios and allelic frequency for early-onset patients (defined as onset of first MI before the age of 55). In addition, analyzing various groups of patients with known risk factors, such as hypertension, high cholesterol, smoking and diabetes, did not reveal any significant correlation with these haplotypes.
In conclusion, we have identified a series of correlated MI disease risk haplotypes, consisting of 4-6 SNPs, with relative risk greater than 2 and allelic frequency in MI patients greater than 10%. The length of the haplotypes varies between 39-68 kb. These haplotypes are carried by 19% (B5) to 29% (A4) of MI
patients. Our results suggest that the 'at risk' haplotypes in the FLAP gene represent a new major independent risk factor for MI.

Discussion In a genome wide search for susceptibility nucleic acids for MI, a locus to 13q12 was mapped. This locus was ultra-fine mapped with microsatellite markers.
Haplotype analysis strongly suggested a nucleic acid for FLAP (ALOXSAP), as a susceptibility gene for MI.
The FLAP gene encodes for a protein that is required for leukotriene s5nlthesis (LTA4, LTB4, LTC4, LTD4). Inhibitors of its function impede translocation of 5-lipoxygenase from the cytoplasm to the cell membrane and inhibit activation of to lipoxygenase. The leukotrienes are potent inflammatory lipid mediators derived from arachidonic acid that can potentially contribute to development of atherosclerosis and destabilization of atherosclerotic plaques through lipid oxidation and/or proinflammatory effects. Allen et al., (Circulation. 97: 2406-2413(1998)) described a novel mechanism in which atherosclerosis is associated with the appearance of a leukotriene receptors) capable of inducing hyperreactivity of human epicardial coronary arteries in response to LTC4 and LTD4. Allen et al. show a photomicrograph of a section of human atherosclerotic coronary artery a positive staining of a number of members of the leukotriene pathway, including FLAP.
Mehrabian et al. described the identification of 5-Lipoxygenase (5-LO) as a major 2o gene contributing to atherosclerosis susceptibility in mice. Mehrabian et al. described that heterozygous deficiency for the enzyme in a knockout model decreased the atherosclerotic lesion size in LDL-/- mice by about 95%. Mehrabian et al show that the enzyme is expressed abundantly in macrophage-rich regions of atherosclerotic lesions, and suggested that 5-LO and/or its products might act locally to promote lesion development (Mehrabian et al., Circulation Research. 91:120 (2002)).
Studies of FLAP inhibition in animal models of atheroscerosis are scarce.
However, in a rabbit model of acute MI assesssed 72 hours after coronary artery ligation the FLAP-inhibitor BAYx1005 madedly reduced mortality, from 65% to 25%, and blocked the increase in CPIs and neutrophil accumulation as well as the 3o ECG-changes observed in sham treated animals (J. Plzai°fuacol. Exp.
They., 276:332 (1996)).

_71_ Mutations and Jor polymorphisms within the FLAP nucleic acid, and other members of the same pathway (i.e., 5-lipoxygenase, LTA4, LTB4, LTC4, and CysLT2 receptor), that show association with the disease, can be used as a diagnostic test. The members of the 5-LO pathway in particular are valuable therapeutic targets for myocardial infarction.
Table 1 The marker map for chromosome 13 used in the linkage analysis.
Location Marker Location (cM) Marker (cM) 6 D13S175 63.9 D13S170 9.8 D13S1243 68.7 D13S265 13.5 D13S1304 73 D13S167 17.2 D13S217 76.3 D13S1241 21.5 D13S289 79.5 D13S1298 25.1 D13S171 81.6 D13S1267 28.9 D13S219 84.7 D13S1256 32.9 D13S218 85.1 D13S158 38.3 D13S263 87 D13S274 42.8 D13S326 93.5 D13S173 45.6 D 13 S 153 96.7 D 13 5778 49.4 D13S1320 102.7 D13S1315 52.6 D13S1296 110.6 D13S285 55.9 D13S156 115 D13S293 59.8 D13S1306 Table 2 Marker Map for the second step of Linkage Analysis Location Marker Location (cM) Marker (cM) 1.758 D13S175 42.585 D13S1248 9.235 D13S787 44.288 D13S1233 11.565 D13S1243 44.377 D13S263 16.898 D13S221 45.535 D13S325 17.454 D13S1304 45.536 D13S1270 18.011 D13S1254 45.537 D13S1276 18.59 D13S625 49.149 D13S326 19.308 D13S1244 49.532 D13S1272 19.768 D13S243 52.421 D13S168 22.234 D13S1250 52.674 D13S287 22.642 D13S1242 60.536 D13S1320 22.879 D13S217 64.272 D13S1296 25.013 D13S1299 71.287 D13S156 28.136 D13S289 76.828 D13S1306 28.678 D13S290 77.86 D13S170 29.134 D13S1287 82.828 D13S265 30.073 D13S260 91.199 D13S1241 31.98 D13S171 93.863 D13S1298 32.859 D13S267 97.735 D13S779 33.069 D13S1293 100.547 D13S1256 33.07 , D13S620 102.277 D13S274 34.131 D13S220 111.885 D13S173 36.427 D13S219 112.198 D13S796 39.458 D13S1808 115.619 D13S778 40.441 D13S218 119.036 D13S1315 41.113 D13S1288 126.898 D13S285 41.996 D13S1253 131.962 D13S293 Table 3 shows the five exons with positions that encode the FLAP protein, marl~ers and SNPs identified within the genomic sequence by the methods described herein.
Of the six SNPs, one SNP, B SNP_302465, is in the coding region. The polymorphism, SNP 302465, does not change the amino acid sequence in the protein.
Table 3 Size(bp) Exons/markers/SNPs Position(bp) SNPs Exon1 293736 Exon2 302512 B SNP 302465 302465 ~ hetero ous C-T
3%

1 heterozygous A-C

B SNP 302524 302524 55%

homozygous A-A

(22.5%) homozygous C-C

(22.5%) B SNP 302560 302560 1 heteroz ous A-G
2%

1 heterozygous C-T

B SNP 302617 302617 37%

homoz ous T-T
59%

homoz ous C-C
4%

Exon3 310475 B SNP 310657 310657 1 hetero ous A-G
6%

Exon4 314378 1 heterozygous G-C

B SNP 314500 314500 24%

homoz ous C-C
6%

Homozygous G-G

70%

ExonS 322459 SNP13B 81028729 ' 1 rs1028729) 145600 homozygousC-C
(11 %), heteroz gous homoz gous T-T
47%

rs1323898 * 151047 homozygousG-G(38%) heteroz gous G-A 47%

homoz ous A-A
15%

rs912392 * 193119 homozygousC-C
(13I) heteroz gous C-T 46%

homoz ous T-T

rs1556428 * 117676 homozygousG-G(1%) heteroz ous G-A 18%

homoz ous A-A
80%

rs22485654 * 227629 homozygousT-T
(75%) heteroz ous T-A 23%

homoz gous A-A
2%

DG00AFJT 293754 1 Homoz ousC-C
45% , heterozygous C-A 45%), homoz ous A-A
10%

DGOOAAHII 294503 1 homoz oust-G
44% , heterozygous G-A 46% , homoz ous A-A
10%

DG00AAHID 296020 1 homoz ousT-T
43% , heteroz ous T-A 45% , homoz ous A-A
12%

DGOOAAHIJ 298098 i homoz oust-G
60% , heterozygous G-A(35% , homoz ous A-A
6%

DGOOAAHIH 298188 1 homoz oust-G
32% , heterozygous G-A 48% , -, h omozygous A-A
(19%) ~DGOOAAHIE homozygous C-C

I rs3885907 * 298379 (23%), heterozygous C-A 48% , homoz gous A-A
29%

DGOOAAHIG 304334 1 homoz ousC-C
21% , heterozygous C-T 49% , homoz ous T-T

DGOOAAHIF 324849 1 homoz oust-G
54%), heterozygous G-C 39%), homoz gous C-C

7%

DGOOAAHOI 325651 1 homoz gousG-G
59% , heterozygous G-A(36%), homoz ous A-A
5%

~FLA298379 298379 1 _77_ FLA320393iSG13S42 320393 1 _~$_ * indicates a ublicly available SNP.

Table 4 Most significant 4 microsatellite marker haplotypes. Length=length of haplotype in Mb. P-val=p-value. RR=Relative,risk. N a~Number of patients. P
al=allelic frequency of haplotype. P ca =carrier frequency of haplotype. N ct=
number of controls. Alleles= alleles in the haplotype. Markers= markers in the haplotype.

markers: pos.rr-frqgt1 pert Allele lengthp-val RR N P P N P P s Markers of al ca ct al ca 0.88 4.71 6.23428 0.0650.125721 0.0110.0220 -12-6O DG13S163 0.82 8.60E-061NF438 0.0320.062720 0 0 0 4 2 14 DG13S1061 g_g 0.67 6.98E-061 435 0.030.059721 0.0020.0038 6 0 8 DG13S1061 26.7 D13S1287 0.7674.85E-062 436 0.0480.094721 0.0020.0040 0 2 12 DG13S1061 0.5151.93E-061NF422 0.0480.094721 0 0 2 0 0 6 DG13S1061 0.8641.68E-061NF424 0.0240.048717 0 0 0 2 0 16 DG13S293 0.9275.38E-061NF435 0.0340.067720 0 0 4 2 143 DG13S301 Table 5 Most significant 5 microsatellite marker haplotypes. Length=length of haplotype in Mb. P-val=p-value. RR=Relative risk. N a~Number of patients. P
al=allelic frequency of haplotype. P ca =carrier frequency of haplotype. N ct=
number of controls. Alleles= alleles in the haplotype. Markers= markers in the haplotype 5markers pos.rr-frqgt1 perc length p-valRR N P P N P P Alleles Markers of al ca ct al ca 0.851 7.45E-0615.43413 0.0340.067715 0.0020.0050 180 0 DG13S166 0.964 8.07E-061NF437 0.0230.045721 0 0 0 -126 8 DG13S163 0.964 2.38E-061NF437 0.0260.052720 0 0 0 6 0 8 DG135163 0.931 7.05E-065.8429 0.0680.131721 0.0120.0250 -600 -2D1351238 0.964 8.13E-061NF434 0.0210.041721 0 0 0 3 82 6 DG13S163 0.597 9.78E-064.58428 0.0740.143717 0.0170.034-6 0 00 -2D1351238 0.896 6.92E-06NF 428 0.0260.051721 0 0 -12 -60-2 2 D135290 I

0.722 2.18E-061NF 453 0.0260.051738 0 0 -6 0 0-2 2 D135290 0.982 7.88E-06NF 437 0.0280.055721 0 0 0 0 42 14DG1351061 I

0.841 8.88E-06NF 438 0.0320.062720 0 0 0 0 42 14D13S1287 I

0.841 9.67E-07NF 435 0.0290.057721 0 0 0 8 6 0 8 DG13S1061 I

0.982 7.90E-0618.63437 0.0260.052721 0.0010.0030 4 0 2 14DG13S1061 0.841 3.52E-0628.52436 0.0480.094721 0.0020.0040 0 0 2 12DG13S1061 0.705 5.28E-061NF435 0.0270.053721 0 0 0 8 6 0 8 DG13S1061 0.841 A..21E-061NF422 0.0480.093721 0 0 0 2 0 0 6 DG13S1061 0.767 4.02E-0628.11436 0.0490.095721 0.0020.0040 0 0 2 12DG13S1061 0.767 1.29E-0631.07436 0.0470.092721 0.0020.0030 0 0 2 12DG13S1061 0.705 4.25E-071NF422 0.0480.093721 0 0 0 2 0 0 6 DG13S1061 0.683 6.58E-061NF437 0.0290.056721 0 0 0 4 0 2 14DG13S1061 0.767 2.85E-0632.43436 0.0440.087721 0.0010.0030 0 0 2 12DG13S1061 0.8 9.58E-0618.39451 0.0230.045739 0.0010.0030 0 2 2 -16DG13S293 _ D13S289 0.865 5.08E-061NF453 0.0190.038739 0 0 0 0 2 0 -16DG13S293 0.927 1.02E-0727.65437 0.0370.073721 0.0010.0034 0 2 143 DG13S301 Table 6 Position (Mb) of microsatellite markers sequence assembly (SAS), primers and size of the markers.
mb marker orward r everse size f 25.09 ACGGTGATGACGCCTACATT CACATGGACCAATTACCTAGA
T

2042DG13S2101SEQ ID NO:. 4 A SEO ID NO: 5) 188 25.09 CAAATTTCAGATGTGCCAACC ACGGTGATGACGCCTACATT(S

2042DG13S48 SEQ ID NO: 6 EQ ID NO: 7 214 25.39 ACCAGCCTTTGCTTAGGA(SEQ ACATTCTAGTGCTACAGGGTA

6504D13S1304D NO: 8) CTC SEQ ID NO: 9 133 I

25.39 TGTTCTGCACACGAACATTCT(SETCCTGAGTCCTCTCCACCTG(S

6535DG13S2105Q ID NO: 10 EQ ID NO: 11 ) 104 25.44 TGGGAATTAATGAAGAACAACAACATGTTTCGAAGAACTCAAGA

5511DG13S2106A SEQ ID NO: 12 GG SEQ ID NO: 13 428 25.54 AAATTACTTCATCTTGACGATAACCTATTGGGGACTGCAGAGAG

4920D13S1254A SEQ ID NO: 14 SEQ ID NO: 15 218 25.54 GGGACTGCAGAGAGCAGAAG CAAGAAGGGAAATTCCTACGC

4925DG13S2107SEQ ID NO: 16 SEQ ID NO: 17 95 25.56 AGCCAGTGTCCACAAGGAAG GAGGGTGAGACACATCTCTGG

5956DG13S55 SEQ ID NO: 18) SEQ ID NO: 19 283 25.60 AATCGTGCCTCAGTTCCATC CCACCAGGAACAACACACAC

5793DG13S54 SEQ 1D NO: 20 SEQ ID NO: 21 156 25.61 TTGCTCTCCAGCCTGGGC (SEQTTCCTCTGGCTGCCTGCG

9693D13S625 ID NO: 22 ( SEQ ID NO: 23) 185 25.68 TTTGATTCCGTGGTCCATTA TTATTTGGTCGGTGCACCTTT

7422DG13S1479(SEQ ID NO: 24) SEQ ID N0.25) 339 25.74 GGTAGGTTGAAATGGGCTAACA TCATGACAAGGTGTTGGATTT

9344DG13S1440(SEQ ID NO: 26) SEQ ID NO: 27) 153 25.90 CCTCCTCTGCCATGAAGCTA CTATTTGGTCTGCGGGTfGT

1212DG13S1890(SEQ ID NO: 28) SEQ ID NO: 29) 418 25.92 TTTGAGCCCAGATCTAAGCAA AAATGTTAATGTCACCGACAAA

8081DG13S1879SEQ ID NO: 30 SEQ ID NO: 31 443 25.93 TACTGGGTTATCGCCTGACC CCAATGGACCTCTTGGACAT

2609DG13S1540SEQ lD NO: 32 SEQ ID NO: 33 152 25.94 TTTGAATGTTCATATATTTGTGGTCCCTCGTAATGAAACCTATTTG

6743DG13S1889G SEQ ID NO: 34 A SEQ ID NO: 35 222 25.94 TTTCGGCACAGTCCTCAATA CAGGGTGTGGTGACAT (SEQ

8679DG13S59 SEQ ID NO: 36 ID NO: 37 228 25.95 TGTTTCTTTCTTTCTCTCTCTCTTAAATGAGTTCAATGAGTTGTGG

2347DG13S1894TC SEQ ID NO: 38 TT SEQ ID NO: 39 209 25.98 CAGAGAGGAACAGGCAGAGG AGTGGCTGGGAAGCCTTATT

8360DG13S1545SEQ ID NO: 40 SEQ ID N0:41 394 26.07 AGGTGAGAGAACAAACCTGTCTTGCCTTCCTTCTAAGGCCAAC

1866DG13S1524SEQ ID NO: 42 SEQ ID NO: 43 115 26.18 TGTTATACATTTCAATTTCACCTCGTACTCCAGCCGGGCAAC

3492DG13S1491A SEQ ID NO: 44 SEQ ID NO: 45 286 26.23 TTGTTCAGTGCTCTATAGTTACAAGGTCACAAAGCTATGCGATTA

6289DG13S62 AGT SEQ ID NO: 46 SEQ lD NO: 47 158 26.27D13S1244TCAACAAGTGGATTAAGAAACTGCTGTTTATGGCTGAGAAGTATG~

(SEQ (SEQ
ID ID
NO: N0:49) 48) 26.28 TAGCAGGGTGCAGTCTA ACCATACCACCACCACCATC
(SEQ
ID

6935DG13S64 N0:50 SEQ 247 ID
NO:

26.31 ACTGTACTTCTGCCTGGGC TTTTGTAATGCCTCAACCATG
(SEQ

NO: ID
52 NO:

26.32 CTGTAGACTTTATCCCTGACTTA
CAATGAATGATGAAGATTCCAC

7184DG13S1529CTG SEQ ID NO: 54 TC 132 SEQ
ID
NO:

26.33 TGACACCATGTCTTACTGTTTGC
GAGGATACAATGAGAACCAAA

ID SEQ
NO: ID
56 NO:

26.38 CCACAGAATGCTCCAAAGGT GAGTTCAAGTGATGGATGACG

ID SEQ
NO: ID
58 N0.59 26.43 CAGATAGATGAATAGGTGGATGG
CACTGTTCCAAGTGCTTTGC

5811DG13S1444A SEQ ID NO: 60 SEQ 193 ID
NO:

26.48 GCAGGGCAAACTGCCTTAT TTTGGTGAAATGTCTGTTTATG
(SEQ

6657DG13S1458ID N0:62 G 402 SEQ
ID
NO:
63) 26.50 CTCAACCTGGCTTCTACT(SEQ TACTCCTTAATAAACTCCCC

4545D13S252 ID (SEQ 338 NO: ID
64 NO:

26.50 TATGCGTTGTGTGTGTG GGGCCTTAGATTCTTGTAGTG

(SEQ
ID

8231DG13S66 NO:66 G 217 SEQ
ID
NO:
67) 27.11 CTCGCATCTCGCTTCTCACT CTCAAGGGTCCAGTGGTTTG

ID ID
NO: NO:
68) 69) 27.14 TGTCCAGACTGCCTCCTACA TGCAACACCTGGTTCACAAT

0675DG13S1907SEQ (SEQ 131 ID ID
NO:70) NO:
71) 27.14 CACAGTGAGACTCTATCTCAAAA
TCAGACTGGCTTAGACTGTGG

5842D13S802 A (SEQ 150 SEQ ID
ID NO:
NO: 73 27.24 AAATTCCAAAGGCCAGGTG CCATACAGTTTCCTAGGTTCTG
(SEQ

0616DG13S1892D NO: 74 G 373 I SEQ
ID
NO:

27.25 CACCTGGCCAAATGTTTGTT TGCTTGAATCCAGAGACTGC

ID ID
NO: NO:
76 77) 27.27 TTTGCGAGTCCTTGTGGAGT ACAGTCCGCTCCCTCCTAAT

ID ID
NO: NO:

27.28 ATGCTTGGCCCTCAGTTT TTGGCAACCCAAGCTAATATG

(SEQ

0461DG13S69 ID (SEQ 296 NO: ID
80 N0:81 27.48 CTCCACAGTGACAGTGAGG GAGAGGTTCCCAATCCC

(SEQ

ID NO:
N0:82 83) 27.61 CATCAACCTCCCCACCAC(SEQ
TATTTTTTCAGTCCCACAGTTA

NO: SEQ

N0:85 27.61 CAGCTCCTGGCCATATTTCT GAGCCATTTCTCTGGGTCTG

ID ID
NO: N0:87 27.64 GGTCCGTGTCAACCCTTAGA CAGGTTGATGGGAGGGAAA

ID ID
NO: NO:

27.66 CGGGAAATGACAGTGAGACC TGCCTAGATTCTCCCGTAAG

ID ID
NO: NO:

27.70 GTGCCCAGCCAGATTC GCCCCCAGTCAGGTTT

(SEQ (SEQ
ID

5347D13S1242NO: ID 198 92 NO:

27.88 TTTCTCTCTCCACGGAATGAA AACCCATTCTCACAGGGTGTA

ID ID
NO:94 NO:

27.89 AGGAGTGTGGCAGCTTTGAG TGGATTCCCGTGAGTACCAG

ID ID
NO: NO:
96 97) 27.93 ATGCTGGGATCACAGGC AACCTGGTGGACTTTTGCT

(SEQ
ID

2154D13S217 NO: SEQ 170 98) ID
NO:
99) 28.08~DG13S581IAGCATTTCCAATGGTGCTTT CATGTTGATATGCCTGAAGGA

0632( SEQ ID NO: 100) ( SEQ ID N0:101 ) 28.16 CACTGTCTGCTGCCACTCAT AGAGATTATGTGATGTACCCTC

5348DG13S1471SEQ ID N0:102 TCTAT SEQ ID N0:103 267 28.30 CAAGCCTGGGACACAGAAAT TTTGCAGACACCACAACACA

3252DG13S583SEQ ID NO: 104 ( SEQ ID NO: 105 264 28.30 ATGACCTAGAAATGATACTGGC CAGACACCACAACACACATT

3256D13S120 SEQ ID NO: 106 SEQ ID NO: 107 175 ( 28.38 TGGTTTAAAAACCTCATGGG ATCCCAAACTCTGTACTTATGT

5566D13S1486SEQ ID NO: 108 AGG SEQ ID NO: 109 151 28.41 TTTGCACATACACATAAGCGAACCACAAATCCCGTGCACTAAA

5530DG13S1024SEQ ID NO: 110 SEQ ,ID NO: 111 139 28.41 ATTCCTGGGCTCATGGTACA TGCCGTCATCTGCTTTAGAA

5530DG13S1510SEQ ID NO: 112) SEQ 1D NO: 113 390 28.43 CCTTGGCTGTTGTGACTGGT CACTCAGGTGGGAGGATCAC

0308DG13S1495SEQ ID N0:114 SEQ ID NO: 115 285 28.51 GCTGTTTCCTTGGCTTCTTCT GCGATACTTGAGATGACCATG

7541DG13S1482SEQ ID NO: 116 A SEQ ID NO: 117) 291 28.55 CACTTTGCCAGTAGCCTTGA TTGGGAAAGTTAACCCAGAGA

1060DG13S1845SEQ ID N0:118 SEQ ID NO: 119 284 28.63 TTTGGGAAGAGCCATGAGAC CTCTGGGCATTGGAGGATTA

4903DG13S1030(SEQ ID NO: 120) (SEQ ID NO: 121 ) 354 28.63 TTTGGGAAGAGCCATGAGAC AATGCCCATGTGCACTGTAG

4903DG13S1467(SEQ ID NO: 122) (SEQ ID NO: 123) 231 28.68 GGGAGACAAGTCAGGTGAGG CTGAGTATGGAGTCTTCATCAT

6607DG13S584SEQ ID NO: 124) TATC (SEQ ID NO: 125)151 28.79 TCGTCTCGAAGAAAGAAAGAAGACACCATGGGTTAATTGCACA

4032DG13S1519SEQ ID N0:126 SEQ ID NO: 127) 286 28.87 TGACGTGGGTTCAGGTTGTA AGTGCATTGGTGCCTTCTCT

6156DG13S77 (SEQ ID NO: 128 SEQ fD NO: 129 220 28.97 GGACTGCCAATTCTACAGCA TTTCCATGGGAAATTTGGTC

0723DG13S586SEQ lD NO: 130 SEQ ID NO: 131 151 28.97 TGCTACTAGATTTGACCAACCA GACTTGTAAAGGATTTAGTGAT

5641DG13S79 SEQ ID NO: 132 TTCG SEQ ID NO: 133 128 29.05 GTGGAAGGCCTCTCTTG TGCTTCTTGAGGGAAAGCAT

9394DG13S80 SEQ ID NO: 134) SEQ ID NO: 135 233 29.12 CACGTGGTTCACCTCTCTAGG TTGGCCACTTATTTGTG

6152DG13S82 SEQ ID NO: 136 SEQ ID NO: 137 302 29.15 CGATGAGTGACAGGGCT (SEQ CCTCGTGGGTGGAATAA
ID

4691D13S1299NO: 138 SEQ ID NO: 139 225 29.15 TTGGCCATTAGCAATTAGCA CGTGGGTGGAATAAATCAGG

4737DG13S85 SEQ ID NO: 140 SEQ ID NO: 141 '153 29.15 GTTGAGGCAAGAGAATCACT GCACATTTACACCAGGGTG

8462D13S629 SEQ ID NO: 142 SEQ ID N0.143 145 29.22 CCTTCAGAGGATTTCCCTTTC CTGGTTTGACTCCAGCTTCA

4060DG13S1934SEQ ID NO: 144 SEQ ID NO: 145 431 29.24 TGTTCAAACCTAAGGTGCTTCA GAAACAACAACAACAACAACAA

5462DG13S1098SEQ ID NO: 146 CA SEQ ID NO: 147 416 29.25 CCTGGCACGGAATAGACACT GGCCTCCTTTGCTCTGAAG

9840DG13S1104SEQ ID NO: 148 SEQ 1D NO: 149 378 29.29 CATCCCTGTGGCTGATTAAGA AACAGTTCCAGCCCGTTCTA

4436DG13S1097SEQ 1D NO: 150) (SEQ ID NO: 151) 162 29.30DG13S1110TTTCAAAGGAATATCCAAGTGC TGGCGTACCATATAAACAGTTC265 9700( SEQ ID NO: 152) TC (SEQ ID NO: 153) 29.30 TTTCAAAGGAATATCCAAGTGC AAACGTGACACTTCCACACA

9909DG13S86 SEQ ID NO: 154 SEQ ID NO: 155 177 29.35 TTCAATGAAGGTGCCGAAGT TGTCTATCCCAAAGCAA(SEQ

9961DG13S87 SEQ ID NO: 156 I D NO: 157 218 29.52 GCAAGACTCTGTTGAAGAAGAAGTCCCTCTGTTTGAGTTTCTCG

2443DG13S1111A SEQ ID NO: 158 SEQ ID NO: 159 110 29.57 AGGCACAGTCGCTCATGTC AAACTTTAGCTAATGGTGGTCA
(SEQ

4665DG13S1101D NO: 160 AA SEQ ID NO. 161 333 I

29.62 TGTGATTCCAGGGAGCTATCA TAGGTGTGTGGAGGACAGCA

2755DG13S1106SEQ ID NO. 162 SEQ ID NO. 163 416 29.65 CCAGTTTCAGTTAGCCAAGTCTGGAGAGGGAATGAATGCAGGA

8910DG13S172SEQ ID NO: 164 SEQ ID NO: 165 267 29.66 GAGCATGTGTGACTTTCATATTCAGTGGCTATTCATTGCTACAG

5709D13S1246AG SEQ ID NO: 166 G SEQ ID NO: 167 177 29.67 TTGCTGGATGCTGGTTTCTA(SEQAAAGAGAGAGAGAAAGAGAAA

2561DG13S1103D NO: 168 GAAAGA SEQ ID NO: 264 29.82 CTGGTTGAGCGGCATT(SEQ TGCAGCCTGGATGACA(SEQ
ID

5975D13S289 NO: 170) ID NO: 171 260 29.82 CCTATGGAAGCATAGGGAAGAA(CCCACTTCTGAGTCTCCTGAT( 6631DG13S166SEQ ID NO: 172) SEQ ID NO: 173) 395 29.90 GGGATGCAGAAAGGATGTGT(SEAAGAATGCTGGCCAACGTAA(S

6689DG13S164Q ID NO: 174) EQ ID NO: 177 218 29.90 CTCTCAGCAGGCATCCA(SEQ GCCAACGTAATTGACACCA(SE
ID

6700D13S1238NO: 178) Q ID N0:179) 129 30.03 CCTTAGGCCCCATAATCT(SEQ CAAATTCCTCAATTGCAAAAT(S
ID

1378D13S290 NO: 180 EQ ID N0:181 176 30.08 GGTCATTCAGGGAGCCATTC(SECCATTATATTTCACCAAGAGGC

6303D13S1229Q ID NO: 182 TGC SEQ ID NO: 183 119 30.19 TGCCTGGTCATCTACCCATT(SEQTCTACTGCAGCGCTGATCTT(S

2847DG13S1460ID NO: 184) EQ ID NO: 185 264 30.21 CATTTATGAATGGAGGTGAAGC(ATGGGAGCTCAAAGGGAAAT(S

7670DG13S1933SEQ ID NO: 186 EQ ID NO: 187 186 30.30 CAGCAGGAAGATGGACAGGT(SECACACTGCATCACACATACCC( 3213DG13S1448Q ID NO: 188 SEQ ID NO: 189 136 30.31 TATGCCAGTATGCCTGCT(SEQ GTCACATCAGTCCATTTGC(SE

7871D13S1287NO: 190 Q ID NO: 191 232 30.34 CCAAAGCAAGTAACCTCCTCA(SAAACAATCACTGCCCTCTGG(S

2102DG13S1061EQ ID NO: 192 EQ ID NO. 193 227 30.57 TGATGAAATTGCCTAGTGATGC(SGGATCCAATCGTACGCTACC(S

1837DG13S1904EQ ID NO: 194 EQ ID NO. 195 136 30.64 CGAATGGGTGACTAACAGCA(SECTGGAGTGCAGGGACATGA(S

3438DG13S882Q ID NO: 196 EQ ID NO: 197 378 30.66 AAAGAAATATTCCAAGAAGAAAGTTGCACAACTTTGTGTAGAGCA

5937DG13S295AAA SEO ID NO: 198 T SEQ ID NO: 199 279 30.67 GGGTATGTCTTTATTCTCGGCAGGTGCATTCACAGACCAGTCATT

4468D13S1226TA SEQ ID NO: 200 (SEQ ID NO: 201 219 30.69 GGGCTTGAAGGCACTAAATGT(SCCAAGCAGTAATTCCTTCCTCA

0959DG13S293EQ ID NO: 202 SEQ ID N0:203) 313 30.71 ACCTAAACACCACGGACTGG(SECAGGTATCGACATTCTTCCAAA

2468DG13S1490Q ID NO: 204) (SEQ ID NO: 205 418 30.82DG13S93 TGGGAAGCCAGTAAAGTAGGAA(AAAGAGACTCCACACATCCATT190 448 3 SEQ ID NO: 206) T(SEQ ID NO: 207) 30.82 AGGGCTATTCCTCAAGGTGTT(STGCTAACACTACCCTCGCAAT( 4859DG13S94 EQ ID NO: 208 SEQ ID NO: 209) 332 30.92 GGGCAGGAATCTCTGAAGTG CTCCACTGAGAAGCCAAGGA(S

8429DG13S1534SEQ ID NO: 210 EQ ID NO. 211 382 30.94 AGGCCAAGCTGGTCCATAG(SEQTCTCTCAAAGCCTCGCTCTC(S

0369DG13S95 ID N0:212 EQ ID NO: 213 126 30.97 CCTTTGAGGCTGGATCTGTT(SETTTCCTTATCATTCATTCCCTC

0238DG13S96 Q ID NO: 214 A SEQ ID NO: 215 218 31.03 AGATATTGTCTCCGTTCCATGA(SCCCAGATATAAGGACCTGGCT

8874D13S260 EQ ID NO: 216 A SEQ ID NO: 217 163 31.09 TTTAAGCCCTGTGGAATGTATTT(GACATTGCAGGTCAAGTAGGG

2294DG13S17 SEQ ID NO: 218 SEQ ID NO: 219 157 31.20 TGCATAAGGCTGGAGACAGA(SECACAGCAGATGGGAGCAAA(S

7844DG13S306 Q ID NO: 220 EQ ID N0:221 158 31.26 GTGCATGTGCATACCAGACC(SEGGCAAGATGACCTCTGGAAA(S

0521DG13S18 Q ID NO: 222 EQ ID NO: 223 31g 31.29 GTCCACTGCAGCACACAGAG(SEGCACTGGTAGATACATGCTAA

9720DG13S1905Q ID NO: 224 CG SEQ ID NO: 225 383 31.35 GGGTATCTTGGCCAGGTGT(SEQTGGCTAAGCACAATCCCTTT(S

3230DG13S307 ID NO: 226) EQ ID NO: 227 403 31.35 TTTGTGTTCCAGGTGAGAATTG(SGAACCATATCCCAAGGCACT(S

5135DG13S1062EQ ID NO: 228 EQ ID NO: 229) 120 31.41 AACCCAAATCAACAAACCAGA(SEAATGAATTCTGGGTCACATGC( 4329DG13S1874Q ID NO: 230) SEQ ID NO: 231) 404 31.42 TTGTTCCCACATTCATTCTACA(STTAAACTCGTGGCAAAGACG(S

9562DG13S1093EQ ID NO: 232 EQ ID NO: 233 273 31.62 CACCATGCCTGGCTCTTT(SEQAACTTCTCCAGTTGTGTGGTTG
ID

6502DG13S1059NO: 234 (SEQ ID NO: 235) 330 31.72 AGCTGAGCTCATGCCACT(SEQCAAGACCTTGTGCATTTGGA(S

3749DG13S1086ID NO: 236 EQ ID NO: 237 155 31.74 AGCCAGACATGGTAGTGTGC(SEGCAATAACTCACACATCAGCAA

6074DG13S1515Q ID NO: 238 SEQ ID N0:239) 417 31.85 CCTACCATTGACACTCTCAG(SETAGGGCCATCCATTCT(SEQ
ID

5732D13S171 Q ID NO: 240 NO: 241 231 31.91 ACCAAGATATGAAGGCCAAA(SECCTCCAGCTAGAACAATGTGA

7332DG13S1092Q ID NO: 242 A SEQ ID NO: 243 176 32.00 TGTCCATAGCTGTAGCCCTGT(SCTCAATGGGCATCTTTAGGC(S

2852DG13S1449EQ ID NO: 244 EQ ID NO: 245 27g 32.07 TGTAATTCAACGACTGGTGTCC(SAGCTTCTGATGGTTGCTGGT(S

2957DG13S1489EQ ID NO: 246 EQ ID NO: 247 130 32.08 CAAACAAACAAACAAGCAAACC(TGGACGTTTCTTTCAGTGAGG( 3989DG13S312 SEQ ID NO: 248 SEQ ID NO: 249 349 32.12 TGATAACTTACCAGCATGTGAGCTCACCTCACCTAAGGATCTGC( 5177DG13S1511SEQ ID NO: 250 SEQ ID NO: 251 314 32.18 CATGCAATTGCCCAATAGAG(SETTGGGCTTGTCTACCTAGTTCA

3547DG13S314 Q ID NO: 252 SEQ ID NO: 253 335 32.19 TGGGTTCCTCATACTGGAGTG(SGCCTGAGCTCCAAGCTCTTT(S

5358DG13S1090EQ ID NO: 254 EQ ID NO: 255 169 32.25 GCTGCACGTATTTGTTGGTG(SEAAACAGCAGAAATGGGAACC(S

1038DG13S1071Q ID NO: 256 ) EQ ID NO: 257 239 ~ DG13S1068CCGTGGGCTATCAATTTCTG(SEAAGATGCAATCTGGTTTCCAA(238 32.35~~ ~

6895 Q ID NO: 258 ) SEQ ID NO: 259 ) 32.37 CCCAAGACTGAGGAGGTCAA(SEGCTGACGGAGAGGAAAGAGA( 3040DG13S1077Q ID NO: 260 SEQ ID NO: 261 374 32.42 TGACAAGGGTGTGGTTATGG CCGCACTTTCTCTTCTGGAC

2780DG13S1906SEQ ID NO: 262 SEQ ID N0:263 425 32.51 TGAGAAGCCTGGGCATTAAG ACAAGCTCATCCAGGGAAAG

1590DG13S316SEQ ID NO: 264 SEQ ID NO: 265 243 32.61 TTGGAAAGGAAGAAAGGAAGG TTGAAACCTAAATGCCACCTG

0517DG13S317SEQ ID NO: 266 ( SEQ ID N0:267 215 32.61 ACCTGTTGTATGGCAGCAGT GGTTGACTCTTTCCCCAACT

0713D13S1493SEQ ID NO: 268 SEQ ID NO: 269 248 ( 32.78 AGAGCTGATCTGGCCGAAG GGTGGACACAGAATCCACACT

9894DG13S1558SEQ ID N0:270 SEQ ID NO: 271 399 ( 32.86 GGCCTGAAAGGTATCCTC (SEQTCCCACCATAAGCACAAG

5950D13S267 D N0:272 SEQ ID NO: 273 160 I

32.96 TCAACCTAGGATTGGCATTACA TCTAGGATTTGTGCCTTTCCA

1410DG13S1478SEQ ID NO: 274 SEQ ID NO: 275) 387 33.00 GACGTCTTAGGATTGACTTCTGCCCAAATACACATTCTTAAAGGG

9922DG13S1513SEQ ID NO: 276 AAA SEQ ID NO: 277 173 33.12 GACTGCAGATCGTGGGACTT TTCTCCAGAGAAACCAAACCA

5696DG13S1461(SEQ ID NO: 278) (SEQ ID NO: 279 148 33.16 ATTCGTGCAGCTGTTTCTGC GCATGACATTGTAAATGGAGG

8468DG13S1551(SEQ ID NO. 280) A SEQ ID N0:281 263 33.25 GGTGGGAATGTGTGACTGAA CCAGGTACAACATTCTCCTGAT

4989DG13S1884(SEQ ID NO. 282) (SEQ ID N0:283) 123 33.34 TGCAGGTGGGAGTCAA (SEQ AAATAACAAGAAGTGACCTTCC
ID

0124D13S1293NO. 284 TA SEQ ID NO: 285 129 33.34 TGTTCTCCTCACCCTGCTCT TTTCAGGCTAGGAAGATCCTTT

6908DG13S326SEQ ID NO: 286 (SEQ ID NO: 287 261 33.39 AAAGGATGCATTCGGTTAGAG ACTGTCCTGTGCCTGTGCTT

2629DG13S1518SEQ ID NO: 288 SEQ ID NO: 289 375 33.40 CCTGAATAGGTGGAATTAAGATCTCAAGGAGCATACACACACAC

5527DG13S23 AA SEQ ID NO: 290 A SEQ ID NO: 291 107 33.43 GTCCACCTAATGGCTCATTC CAAGAAGCACTCATGTTTGTG

1536D13S620 SEQ ID NO: 292 SEQ ID NO: 293 185 33.43 AGCCTGTGATTGGCTGAGA GGCTTACAGCTGCCTCCTTT
(SEQ

7092DG13S1866ID NO: 294 SEQ ID NO: 295 410 33.49 CCCACAGAGCACTTTGTTAGA GCCTCCCTTAAGCTGTTATGC

5718DG13S1927SEQ ID NO: 296 SEQ ID NO: 297 401 33.50 CACTCTTTACTGCCAATCACTCCGCCGTGTGGGTGTATGAAT

3440DG13S1503SEQ ID N0:298 SEQ ID NO: 299 226 33.56 TTGTACCAGGAACCAAAGACAA CACAGACAGAGGCACATTGA

8100DG13S332SEQ 1D NO: 300 SEQ ID NO: 301 176 33.67 GCTCTGGTCACTCCTGCTGT CATGCCTGGCTGATTGTTT

5841DG13S333SEQ ID NO: 302) SEQ ID NO: 303 446 33.77 CCAACATCGGGAACTG (SEQ TGCATTCTTTAAGTCCATGTC
ID

1389D13S220 NO: 304 SEQ ID NO: 305 ~ 191 33.81 CAGCAACTGACAACTCATCCA CCTCAATCCTCAGCTCCAAC

8041DG13S1919SEQ ID NO: 306 SEQ ID N0.307 255 33.87 TCCTTCACAGCTTCAAACTCA AGTGAGAAGCTTCCATACTGG

3614DG13S1439SEQ ID NO: 308 ) T (SEQ ID NO: 309) 239 33.90DG13S335GCCAACCGTTAGACAAATGA CTACATGTGCACCACAACACC~
~ 201 _87_ 6065 (SEQ ID NO: 310) (SEQ ID NO: 311) 33.92 AGTTTATTGCCGCCGAGAG ACCCACCACATTCACAAGC
(SEQ

8653DG13S340ID NO. 312 SEQ ID NO: 313 373 34.01 CGATTGCCATGTCTCTTTGA GAGATCTGGCCTGGATI-fGT

9455DG13S1496SEO ID NO: 314 SEQ ID NO: 315 155 34.03 TGAGGCCAGCCTTACCTCTAT CCAGACATGGTGGCTTGT

4089DG13S342SEO ID NO: 316 SEQ ID NO: 317 366 34.06 GAAGGAAGGAAGGGAAGGAA AAGGATGAGAAGAGTCCATGC

1777DG13S344SEQ ID NOv 318 SEQ ID NO: 319 292 34.06 AAATACCCTTTGAACAGACACACTAGCTGAGCATGGTGGTACG

7239DG13S345SEQ ID NO: 320 SEQ ID NO: 321 201 34.07 AAAGACAAGACAGCAATCCAAA GCAGAACCCAGGCTACAGAT

7874DG13S346SEQ ID NO: 322 SEQ ID NO. 323 152 34.08 TCATTGTCAGCACAGAATGAACT(GGAGGGAGGGAAGAAAGAGA

4138DG13S347SEQ ID NO: 324 SEQ ID NO: 325 ) 338 34.08 GCAACACAGTGAAAGCCCA(SEQACAGGAGCATGCCACCATG(SE

4326D13S624 ID NO: 326 Q ID NO: 327 191 34.15 GGGAAGAGGAGATTGACTTGTT(GGAACACCATCATTCCAACC(S

6075DG13S339SEQ ID NO: 328 EQ ID NO: 329 232 34.19 TACAAGCTCCACCGTCCTTC(SETGAGTTGCTGCCTCTTCAAA(S

2478DG13S1926Q ID NO: 330 EQ ID NO: 331 ) 261 34.22 TGCTAATGGGCCAAGGAATA(SEGCTAAATGTCCTCATGAATAGC

0227DG13S1469Q ID NO: 332) C(SEQ ID NO: 333 ) 382 34.30 TGTCCTGCAGACAGATGGTC(SECCTCCGGAGTAGCTGGATTA(S

1448DG13S351Q ID NO: 334) EQ ID NO: 335 ) 294 34.38 GAGACTGGCCCTCATTCTTG(SEAAGAAGCCAGAGACAAAGAAA

7883DG13S26 Q ID NO: 336 TACA SEQ ID NO: 337 330 ) 34.53 CATCTATCTTTGGATTCAGTGGTTGCTCCCAACATCTTACCAG(S

5441DG13S30 G SEQ ID NO: 338 EQ ID NO: 339 388 34.56 TGTCCTCTGGTCATTTCTATGGT(CATGAATGAGAAGTGATGAAT

5594DG13S1435SEQ ID NO: 340 GG SEQ ID NO: 341 235 ) 34.65 AACACGGGAAATTCCAACAG(SETGAAGAACTGAAATTGCCAGTA

9858DG13S1446Q ID NO: 342 A SEQ ID NO: 343 379 34.71 CAGACACTGTAAACTGGCTTCG(GCCACATTGCTATCAGCGTA(S

2260DG13S356SEQ ID NO: 344 EQ ID NO: 345 212 34.73 TGTCATAGGCTTGCGGTATTT(SETTGGTAGGGTCCTTTCCTTT(S

8756DG13S357Q ID NO: 346 EQ ID NO: 347 202 34.77 GCCTGCTCACTGTTGTTTGA(SECGGTTATCAGAGACTGGTGGT( 0571DG13S1032Q ID NO: 348 SEQ ID NO: 349 211 34.79 GGCTTATTTCATGTACGGCTA(SEGGTTAAACTCTACTTAGTCCTG

9679DG13S1557Q ID NO: 350 ATGC SEQ ID NO: 351 158 34.88 GAACTCTGCAGGCACCTCTT(SECCTGAAGCGCTTGTACTGAA(S

2934DG13S1925Q ID NO: 352 EQ ID NO: 353 456 34.93 TGTTGCGTACTCAGCCCATA GACAGGTGTCAAACGGGTCT(S

2690DG13S1484SEQ ID N0:354 EQ ID NO: 355 246 34.94 TTGGCTTCTCGCTCTTTCTT(SEQAGCCATCAGTCACATGCAAA

2547DG13S360ID NO: 356 SEQ ID NO: 357 350 34.99 AGATCTCCAGGGCAGAGGAC(SECCTTCCTCCCTCCTTCTCTC(S

8979DG13S1522Q ID NO: 358 EQ ID NO: 359 355 35.07 CGTCATTGATCCCAATCATCT(SEGGCTGATAGCCTCCCTTGTA

4962DG13S1517Q ID NO: 360 ) (SEQ ID N0:361) 235 35.07DG13S1521GAGAGAGAGCAGCTTGCATGT(SGGCTGATAGCCTCCCTTGTA(S172 _88_ 4962 EQ ID N0:362) EQ ID N0:363) 35.12 ACCTTTCAAGCTTCCGGTTT(SEQTTCCATCCGTCCATCTATCC(S

6882DG13S364ID NO: 364 EQ ID NO: 365 172 35.32 TTAAAGTCACTTGTCTGTGGTCA(TTTGTAGGAATCAAGTCAAATA

8663DG13S1036SEQ ID NO: 366 ATGTA SEQ ID NO: 367 216 35.33 CAAACATCACACTGGGCAAA(SETGCTTTGGAATCTTTCTTGCT(S

5364DG13S367Q ID NO: 368) EQ ID NO: 369 301 35.37 CTGCCAGGATGTCAGCATT(SEQTCCACACTTTCTCATCACCTAA

1957DG13S1901ID NO: 370 A SEQ ID NO: 371 440 35.42 CTTTCGGAAGCTTGAGCCTA(SECCCAAGACCACTGCCATATT(S

0295DG13S1037Q ID NO: 372 EQ ID NO: 373) 269 35.42 TGACAGGTTTGGGTATATTGGA(TGCTTAATGTAGTGGCAGCA(S

5841DG13S1854SEQ ID NO: 374) EQ ID NO: 375 124 35.50 TCCTGCCTTTGTGAATTCCT(SEQGTTGAATGAGGTGGGCATTA(S

6053DG13S1038ID NO: 376 EQ ID NO: 377 334 35.54 CCATTTAATCCTCCAGCCATT(SEGCTCCACCTTGTTACCCTGA(S

7210DG13S1039Q ID NO: 378 EQ ID NO: 379 167 35.60 ACAACCCTGGAATCTGGACT(SEGAAGGAAAGGAAAGGAAAGAA

9252DG13S1840Q ID NO: 380 A SEQ ID NO: 381 217 35.61 TGACAAGACTGAAACTTCATCAG(GATGCTTGCTTTGGGAGGTA(S

9286DG13S369SEQ ID NO: 382) EQ ID NO: 383) 257 35.62 TTGAGGACCTGTCGTTACG TTATAGAGCAGTTAAGGCACA
(SEQ

7911D13S305 D NO: 384) (SEQ ID NO: 385) 394 I

35.65 TGAGGGTGGTAAGCCCTTATT(SGGAGTTGTGGCCTCTCTCTCT( 6659DG13S375EQ ID NO: 386) SEQ ID NO: 387) 192 35.76 AAGCAAATATGCAAAATTGC(SEQTCCTTCTGTTTCTTGACTTAAC

0368D13S219 D NO: 388 A SEQ ID NO: 389 125 I

35.82 TGCTAAGAGGGCAGATCTCA(SEGGCTCATAGCCAATl-fCTCC

5852DG13S378Q ID NO: 390 SEQ ID NO: 391 324 35.83 CGGCATTCTCAATAACCTCAA TCTTTGATGAGGATCAATTAGT

2127DG13S32 SEQ ID NO: 392 GG SEQ ID NO: 393 214 35.87 ACGCACACACACACACACAC TGCCTCTGTAATCCTGTGTAGC

2936DG13S1549SEQ ID NO: 394 SEQ ID N0:395 260 35.91 GCTCTAAGGTGGGTCCCAATA GGGAATGACAAGATCAGTTTA

2321DG13S1473SEQ ID N0:396 CC SEQ ID NO: 397 163 -~9-Table 7.
The selected SNP haplotypes and the corresponding p-values, relative rislc (RR), number of patients (#aff), allelic frequency in patients (aff.frq.), carrier frequency in patients (carr.frq.),number of controls (#con), allelic frequency in controls (con.frq.), population attributable risk (PAR). The patients used for this analysis were all unrelated within 4 meioses.
_ a ci~ ~ a n c n M ~ ~ ~ O M M n M
M

O _ QOO oO_ _ _ _ _ C~ M C~C~C~C~

-val RR #affaff.frcarr.fr#concon.frPAR
. . .

B44.8E-052.089030.1060.20 619 0.0540.11 2 2 2 0 B52.4E-052.209100.1010.19 623 0.0490.113 2 2 2 0 B61.8E-062.229130.1310.24 623 0.0630.143 2 2 2 0 2 A45.1 1.819190.1590.29 623 0.0950.14 2 3 2 0 A52.6E-061.919200.1500.28 624 0.0850.143 2 3 2 0 to All references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (44)

What is claimed is:

1. A method of diagnosing a susceptibility to myocardial infarction in an individual, comprising detecting a polymorphism in a FLAP nucleic acid, wherein the presence of the polymorphism in the nucleic acid is indicative of a susceptibility to myocardial infarction.

2. A method of diagnosing a susceptibility to myocardial infarction, comprising detecting an alteration in the expression or composition of a polypeptide encoded by a FLAP nucleic acid in a test sample, in comparison with the expression or composition of a polypeptide encoded by a FLAP nucleic acid in a control sample, wherein the presence of an alteration in expression or composition of the polypeptide in the test sample is indicative of a susceptibility to myocardial infarction.

3. The method of Claim 1 wherein the polymorphism in the FLAP nucleic acid is indicated by detecting the presence of a haplotype comprising one or more of the markers DG00AAFIU, SG13S25, DG00AAJFF, DG00AAHII, DG00AAHID, B_SNP_310657, SG13S30, SG13S32, SG13S42, and SG13S35 at the 13q12 locus comprising a FLAP nucleic acid.

4. The method of Claim 1 wherein the polymorphism comprises at least one of the polymorphisms as indicated in Table 3.

5. A method of diagnosing myocardial infarction, comprising detecting an alteration in the expression or composition of a polypeptide encoded by a FLAP nucleic acid in a test sample, in comparison with the expression or composition of a polypeptide encoded by a FLAP nucleic acid in a control sample, wherein the presence of an alteration in expression or composition of the polypeptide in the test sample is indicative of myocardial infarction.

6. An isolated nucleic acid molecule comprising a FLAP nucleic acid, wherein the FLAP nucleic acid has a nucleic acid sequence of SEQ ID NO: 1 or SEQ
ID NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the nucleic acid molecule comprises a polymorphism as indicated in Table 3.

7. An isolated nucleic acid molecule having a polymorphism as indicated in Table 3, which hybridizes under high stringency conditions to a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or the complement of SEQ ID
NO: 1 or SEQ ID NO: 3.

8. A method for assaying for the presence of a first nucleic acid molecule in a sample, comprising contacting said sample with a second nucleic acid molecule, where the second nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and hybridizes to the first nucleic acid under high stringency conditions.

9. A vector comprising an isolated nucleic acid molecule selected from the group consisting of:

a) a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or b) complement of a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID
NO: 3;

wherein the nucleic acid molecule is operably linked to a regulatory sequence.

10. A recombinant host cell comprising the vector of Claim 9.

11. A method for producing a polypeptide encoded by an isolated nucleic acid molecule having a polymorphism as indicated in Table 3, comprising culturing the recombinant host cell of Claim 10 under conditions suitable for expression of the nucleic acid molecule.

12. A method of assaying for the presence of a polypeptide encoded by an isolated nucleic acid molecule according to Claim 6 in a sample, the method comprising contacting the sample with an antibody which specifically binds to the encoded polypeptide.

13. A method of identifying an agent that alters expression of a FLAP nucleic acid, comprising:

a) contacting a solution containing a nucleic acid comprising the promoter region of the FLAP nucleic acid operably linked to a reporter gene with an agent to be tested;

b) assessing the level of expression of the reporter gene; and c) comparing the level of expression with a level of expression of the reporter gene in the absence of the agent; wherein if the level of expression of the reporter gene in the presence of the agent differs, by an amount that is statistically significant, from the level of expression in the absence of the agent, then the agent is an agent that alters expression of the FLAP nucleic acid.

14. An agent that alters expression of the FLAP nucleic acid, identifiable according to the method of Claim 13.

15. A method of identifying an agent that alters expression of a FLAP nucleic acid, comprising:

a) contacting a solution containing a nucleic acid of Claim 1 or a derivative or fragment thereof with an agent to be tested;

b) comparing expression with expression of the nucleic acid, derivative or fragment in the absence of the agent;

wherein if expression of the nucleotide, derivative or fragment in the presence of the agent differs, by an amount that is statistically significant, from the expression in the absence of the agent, then the agent is an agent that alters expression of the FLAP nucleic acid.

16. The method of Claim 15, wherein the expression of the nucleotide, derivative or fragment in the presence of the agent comprises expression of one or more splicing variants) that differ in kind or in quantity from the expression of one or more splicing variant(s) the absence of the agent.

17. An agent that alters expression of a FLAP nucleic acid, identifiable according to the method of Claim 15.

18. An agent that alters expression of a FLAP nucleic acid, selected from the group consisting of: antisense nucleic acid to a FLAP nucleic acid; a FLAP
polypeptide; a FLAP nucleic acid receptor; a FLAP nucleic acid binding agent; a peptidomimetic; a fusion protein; a prodrug thereof; an antibody; and a ribozyme.

19. A method of altering expression of a FLAP nucleic acid, comprising contacting a cell containing a FLAP nucleic acid with an agent of Claim 18.

20. A method of identifying a polypeptide which interacts with a FLAP
polypeptide, comprising employing a yeast two-hybrid system using a first vector which comprises a nucleic acid encoding a DNA binding domain and a FLAP polypeptide, splicing variant, or a fragment or derivative thereof, and a second vector which comprises a nucleic acid encoding a transcription activation domain and a nucleic acid encoding a test polypeptide, wherein if transcriptional activation occurs in the yeast two-hybrid system, the test polypeptide is a polypeptide which interacts with a FLAP polypeptide.

21. A transgenic animal comprising a nucleic acid selected from the group consisting of: an exogenous FLAP nucleic acid and a nucleic acid encoding a FLAP polypeptide.

22. A method for assaying a sample for the presence of a FLAP nucleic acid, comprising:

a) contacting said sample with a nucleic acid comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the sequence of said FLAP nucleic acid under conditions appropriate for hybridization; and b) assessing whether hybridization has occurred between a FLAP nucleic acid nucleic acid and said nucleic acid comprising a contiguous nucleotide sequence which is at least partially complementary to a part of the sequence of said FLAP nucleic acid;

wherein if hybridization has occurred, a FLAP nucleic acid is present in the nucleic acid.

23. The method of Claim 22, wherein said nucleic acid comprising a contiguous nucleic acid sequence is completely complementary to a part of the sequence of said FLAP nucleic acid.

24. The method of Claim 22, comprising amplification of at least part of said FLAP nucleic acid.

25. The method of Claim 22, wherein said contiguous nucleic acid sequence is 100 or fewer nucleotides in length and is either: a) at least 80% identical to a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; b) at least 80% identical to the complement of a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; or c) capable of selectively hybridizing to said FLAP nucleic acid.

26. A reagent for assaying a sample for the presence of a FLAP nucleic acid, said reagent comprising a nucleic acid comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the nucleic acid sequence of said nucleic acid.

27. The reagent of Claim 26, wherein the nucleic acid comprises a contiguous nucleotide sequence, which is completely complementary to a part of the nucleic acid sequence of said FLAP nucleic acid.

28. A reagent kit for assaying a sample for the presence of a FLAP nucleic acid, comprising in separate containers:
a) one or more labeled nucleic acids comprising a contiguous nucleotide sequence which is at least partially complementary to a part of the nucleic acid sequence of said FLAP nucleic acid; and b) reagents for detection of said label.

29. The reagent kit of Claim 28, wherein the labeled nucleic acid comprises a contiguous nucleotide sequences which is completely complementary to a part of the nucleic acid sequence of said FLAP nucleic acid.

30. A reagent kit for assaying a sample for the presence of a FLAP nucleic acid, comprising one or more nucleic acids comprising a contiguous nucleic acid sequence which is at least partially complementary to a part of the nucleic acid sequence of said FLAP nucleic acid, and which is capable of acting as a primer for said FLAP nucleic acid when maintained under conditions for primer extension.

31. The use of a nucleic acid which is 100 or fewer nucleotides in length and which is either: a) at least 80% identical to a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; b) at least 80% identical to the complement of a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; or c) capable of selectively hybridizing to said FLAP nucleic acid, for assaying a sample for the presence of a FLAP nucleic acid.

32. The use of a first nucleic acid which is 100 or fewer nucleotides in length and which is either:
a) at least 80% identical to a contiguous sequence of nucleotides of SEQ
ID NO: 1 or SEQ ID NO: 3;
b) at least 80% identical to the complement of a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; or c) capable of selectively hybridizing to said FLAP nucleic acid; for assaying a sample for the presence of a FLAP nucleic acid that has at least one nucleotide difference from the first nucleic acid.

33. The use of a nucleic acid which is 100 or fewer nucleotides in length and which is either:
a) at least 80% identical to a contiguous sequence of nucleotides in one of the nucleic acid sequences as shown in Table 3;
b) at least 80% identical to the complement of a contiguous sequence of nucleotides in one of the nucleic acid sequences as shown in Table 3;
or c) capable of selectively hybridizing to said FLAP nucleic acid;
for diagnosing a susceptibility to a disease or condition associated with a FLAP nucleic acid.

34. A method of diagnosing a susceptibility to myocardial infarction in an individual, comprising determining the presence or absence in the individual of a haplotype using one or more of the markers DG00AAFIU, SG13S25, DG00AAJFF, DG00AAHII, DG00AAHID, B_SNP_310657, SG13S30, SG13S32, SG13S42, and SG13S35, with alleles T, G, G, G, T, G, G, A, A and G at the 13q12 loci comprising a FLAP nucleic acid, wherein the presence of the haplotype is diagnostic of susceptibility to myocardial infarction.

35. The method of Claim 34, wherein determining the presence or absence of the haplotype comprises enzymatic amplification of nucleic acid from the individual.

36. The method of claim 35, wherein determining the presence or absence of the haplotype further comprises electrophoretic analysis.

37. The method of claim 34, wherein determining the presence or absence of the haplotype further comprises restriction fragment length polymorphism analysis.

38. The method of claim 34, wherein determining the presence or absence of the haplotype further comprises sequence analysis.

39. A method of diagnosing a susceptibility to myocardial infarction in an individual, comprising:
obtaining a nucleic acid sample from said individual; and analyzing the nucleic acid sample for the presence or absence of a haplotype using one or more of the markers DG00AAFIU, SG13S25, DG00AAJFF, DG00AAHII, DG00AAHID, B_SNP_310657, SG13S30, SG13S32, SG13S42, and SG13S35, with alleles T, G, G, G, T, G, G, A, A and G at the 13q12 loci comprising a FLAP nucleic acid, wherein the presence of the haplotype is diagnostic for a susceptibility to myocardial infarction.

40. A method of diagnosing myocardial infarction in an individual, comprising determining the presence or absence in the individual of a haplotype comprising one or more markers and/or single nucleotide polymorphisms as shown in Table 3 in the locus on chromosome 13q12 comprising a FLAP

nucleic acid, wherein the presence of the haplotype is diagnostic of myocardial infarction

41. A method of diagnosing a susceptibility to myocardial infarction in an individual, comprising determining the presence or absence in the individual of a haplotype comprising one or more markers and/or single nucleotide polymorphisms as shown in Table 3 in the locus on chromosome 13q12 comprising a FLAP nucleic acid, wherein the presence of the haplotype is diagnostic of a susceptibility to myocardial infarction.

42. A method for the diagnosis and identification of susceptibility to myocardial infarction in an individual, comprising: screening for an at-risk haplotype in the FLAP nucleic acid that is more frequently present in an individual susceptible to myocardial infarction compared to an individual who is not susceptible to myocardial infarction wherein the at-risk haplotype increases the risk significantly.

43. The method of Claim 42 wherein the significant increase is at least about 20%.

44. The method of Claim 42 wherein the significant increase is identified as an odds ratio of at least about 1.2.