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  Pharmaceutical Patents  

 

Title:  Use of 5-lipoxygenase activating protein (FLAP) gene to assess susceptibility for myocardial infarction
United States Patent: 
7,507,531
Issued: 
March 24, 2009

Inventors: 
Helgadottir; Anna (Reykjavik, IS)
Assignee: 
deCODE Genetics chf. (Reykjavik, IS)
Appl. No.: 
10/829,674
Filed: 
April 22, 2004


 

Pharm Bus Intell & Healthcare Studies


Abstract

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

Description of the Invention

SUMMARY OF THE INVENTION

As described herein, a locus on chromosome 13q12-13 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 (ALOX5AP or FLAP), herein after referred to as FLAP. The gene has also been shown to play a role in stroke.

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 or stroke. Identification of nucleic acids and polymorphisms in this locus can pave the way for a better understanding of the disease process, which in turn can lead to improved diagnostic and therapeutic methods.

The invention further pertains to methods of diagnosing a susceptibility to myocardial infarction or stroke, 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 a susceptibility to myocardial infarction or stroke.

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 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 13 (see Original Patent).

In another embodiment, the invention relates to an isolated nucleic acid molecule having a polymorphism as indicated in Table 13, 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.

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 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.

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 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 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 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 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. 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 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 variant(s) that differ in kind or in quantity from the expression of one or more splicing variant(s) 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 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.

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 ID NO: 1 or SEQ ID NO: 3; b) at least 80% identical to the complement of a contiguous sequence of nucleotides in of SEQ ID NO: 1 or SEQ ID 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 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 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 13; 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 13; or capable of selectively hybridizing to said FLAP nucleic acid.

The invention also relates to a method of diagnosing a susceptibility to myocardial infarction or stroke in an individual, comprising determining the presence or absence in the individual of certain single nucleotide polymorphisms (SNPs) or of certain "haplotypes" (combinations of genetic markers); the presence of the SNP or the haplotype is diagnostic of susceptibility to myocardial infarction or stroke. In one embodiment, a haplotype associated with a susceptibility to myocardial infarction or stroke comprises markers SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles T, G, G, G, A and G at SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35, respectively (the B6 haplotype), is diagnostic of susceptibility to myocardial infarction or stroke. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction or stroke comprises markers SG13S99, SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles T, G, G, G and A at SG13S99, SG13S25, SG13S106, SG13S30 and SG13S42, respectively (the B5 haplotype), is diagnostic of susceptibility to myocardial infarction or stroke. In a third embodiment, a haplotype associated with a susceptibility to myocardial infarction or stroke comprises markers SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles G, G, G and A at SG13S25, SG13S106, SG13S30 and SG13S42, respectively (the B4 haplotype), is diagnostic of susceptibility to myocardial infarction or stroke. In a fourth embodiment, a haplotype associated with a susceptibility to myocardial infarction or stroke comprises markers SG13S25, SG13S106, SG13S30 and SG13S32 at the 13q 12-13 locus. In one particular embodiment, the presence of the alleles G, G, G and A at SG13S25, SG13S106, SG13S30 and SG13S32, respectively (the Bs4 haplotype), is diagnostic of susceptibility to myocardial infarction or stroke.

In a fifth embodiment, a haplotype associated with a susceptibility to myocardial infarction or stroke comprises markers SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles T, G, T, G and A at SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32, respectively (the A5 haplotype), is diagnostic of susceptibility to myocardial infarction or stroke. In a sixth embodiment, a haplotype associated with a susceptibility to myocardial infarction or stroke comprises markers SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles G, T, G and A at SG13S25, SG13S114, SG13S89 and SG13S32, respectively (the A4 haplotype), is diagnostic of susceptibility to myocardial infarction or stroke. 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.

A further embodiment of the invention is a method for identification of susceptibility to myocardial infarction or stroke, by identifying haplotypes and/or SNPs that can be used to identify individuals at risk of developing MI or stroke. In certain embodiments, haplotypes can comprise, for example, at least one of the polymorphisms as indicated in Table 13, or as shown in the haplotypes in Table 5, Table 7, Table 9, Table 14, and/or Table 15. (see Original Patent) In certain additional embodiments, the haplotype can be one of haplotypes B4, BS4, B5, B6, A4, A5 or Hap B.

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

An additional embodiment comprises methods for the diagnosis of increased risk of susceptibility to myocardial infarction or stroke in an individual, by screening for a SNP or an at-risk haplotype in the FLAP nucleic acid that is more frequently present in an individual susceptible to myocardial infarction or stroke (affected), compared to the frequency of its presence in a healthy individual (control). The presence of the SNP or at-risk haplotype is indicative of a susceptibility to myocardial infarction or stroke. In one embodiment, an at-risk haplotype has a p value <0.05. In certain other embodiments, the screening for the presence of an at-risk haplotype comprises screening for an at-risk haplotype within or near FLAP that significantly correlates with a haplotype such as a halotype shown in Table 5 (see Original Patent); a haplotype shown in Table 7 (see Original Patent); a haplotype shown in Table 9 (see Original Patent); a haplotype shown in Table 14 (see Original Patent); a haplotype shown in Table 15 (see Original Patent); haplotype B4; haplotype Bs4; haplotype B5; haplotype B6; haplotype A4; haplotype A5; or haplotype HapB. In other embodiments, screening for the presence of an at-risk haplotype comprises screening for an at-risk haplotype within or near FLAP that significantly correlates with susceptibility to myocardial infarction or stroke.

A further embodiment comprises methods of diagnosing FLAP-associated myocardial infarction or stroke in an individual who has had a myocardial infarction and/or a stroke, by detecting a polymorphism in a FLAP nucleic acid, or an alteration in the expression or composition of a polypeptide encoded by a flap nucleic acid, wherein the presence of the polymorphism in the nucleic acid or the alteration in expression or composition is indicative of FLAP-associated myocardial infarction or stroke. Additional embodiments of the invention include methods for identification of FLAP-associated myocardial infarction or stroke, by identifying haplotypes and/or SNPs associated with MI or stroke. The haplotypes can comprise, for example, at least one of the polymorphisms as indicated in Table 13, or as shown in the haplotypes in Table 5, Table 7, Table 9, Table 14, and/or Table 15. In certain additional embodiments, the haplotype can one of haplotypes B4, BS4, B5, B6, A4, A5 or Hap B.

DETAILED DESCRIPTION OF THE INVENTION

Extensive genealogical information has been combined with powerful gene sharing methods to map a susceptibility gene for myocardial infarction on chromosome 13q 12-13. A genome wide scan of 296 multiplex Icelandic families with 713 MI patients was performed. Through a suggestive linkage to a locus on chromosome 13q12-13 for female MI patients and early onset MI patients, and a microsatellite marker association analysis, the gene encoding the 5-lipoxygenase activating protein (FLAP) was identified.

Subsequent to the mapping of the MI susceptibility gene to chromosome 13q12-13 the candidate locus was finely mapped with microsatellite markers. Patients with myocardial infarction and controls were initially genotyped with microsatellite markers with an average spacing between markers of less than 100 kb over a 7.6 Mb candidate region. Initial haplotype association analysis that included these microsatellite markers revealed several extended haplotypes composed of 4 and 5 microsatellite markers that were significantly associated with female MI (see FIGS. 1 and 2, see Original Patent). A region common to all these extended haplotypes, is defined by markers DG13S166 and D13S1238. This region included only one gene, the FLAP nucleic acid sequence. The two marker haplotype involving alleles 0 and -2 for markers DG13S166 and D13S1238, respectively, was found in excess in patients.

This was the first evidence that the FLAP gene might be involved in the pathogenesis of myocardial infarction.

Subsequent haplotype analysis that included more microsatellite markers in the candidate region on chromosome 13q12-13, now with a marker density of 1 microsatellite marker per 60 kb, showed decreased significance of the original haplotype association. However, the haplotype association analysis using increased density of markers again pointed towards the FLAP gene. This analysis strongly suggested that a 300 kb region was involved in the susceptibility of myocardial infarction. This 300 kb region included only two genes, the gene encoding highly charged protein (D13S106E) and the gene encoding 5-lipoxygenase activating protein (FLAP).

In view of the association results described above and because of the known role of the 5-lipoxygenease pathway in inflammatory processes, FLAP was an attractive candidate and therefore further efforts were focused on this gene.

SNP Haplotype Association to MI, and Subsequently to Stroke

In an effort to identify haplotypes involving only SNP markers that associate with MI, additional SNPs were identified by sequencing the FLAP gene and the region flanking the gene. A total of 48 SNPs in 1343 patients and 624 unrelated controls were genotyped. Haplotype association analysis involving only these SNPs revealed two correlated series of SNP haplotypes that were in significant excess in patients, denoted as A and B in Table 7. The length of the haplotypes varied between 33 and 69 kb, and the haplotypes covered one or two blocks of linkage disequilibrium. Both series of haplotypes contained the common allele G of the SNP SG13S25. All haplotypes in the A series contain the SNP SG13S114, while all haplotypes in the B series contain the SNP SG13S106. In the B series, the haplotypes B4, B5, and B6 have a relative risk (RR) greater than 2 and with allelic frequencies above 10%. The haplotypes in the A series have slightly lower RR and lower p-values, but higher frequency (15-16%). The haplotypes in series B and A are strongly correlated, i.e., the haplotypes in B define a subset of the haplotypes in A. Hence, haplotypes in series B are more specific than A. However, haplotypes in series A 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 showed similar risk ratios and allelic frequencies for early-onset patients (defined as onset of first MI before the age of 55) and for both genders. 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, suggesting that the haplotypes in the FLAP gene represent an independent genetic susceptibility factor for MI.

The product of the FLAP gene is involved in an important inflammatory pathway, and could thus be a predisposing factor for plaque rupture in MI. Since MI and stroke are both considered to be atherothrombotic diseases the MI at-risk haplotypes were assessed to determine whether they also were associated with stroke. Nineteen of the SNPs that defined the MI at-risk haplotypes (A and B series) were evaluated in stroke patients and unrelated controls. In the analysis, a subset of patients that did not have MI and were unrelated within 4 meiosis was used. The results from the haplotype association analysis are summarized in Table 9. The frequency of the at-risk haplotypes in all stroke patients was very similar to that of the MI patients and the haplotype conferred a similar relative risk. The B4 haplotype, previously described for MI, is carried by 19% of all stroke patients and 11% of controls. Carriers of this haplotype have nearly twofold risk (RR=1.95, P=1.6 10-4) of having a stroke. Adding the fifth SNP (SG13S35) to the B4 haplotype increases the relative risk to 2.04 (p-value 5.8 10-5). The allelic frequency of this haplotype is 10.2% in stroke patients and 5.3% in controls. Also shown in Table 9 is a 4 SNP haplotype defined as Bs4 that is highly correlated with the B4 haplotype (r2=0.93). Bs4 haplotype has a RR of 2.01, carrier frequency in patients of 19% and population attributable risk of 10%. This haplotype was tested with different subtypes of stroke (Table 9). Of interest is that all stroke subtypes have a considerably higher frequency of the `at-risk` haplotype than controls resulting in the increased relative risk.

The FLAP nucleic acid encodes a 5-lipoxygenase activating protein, which, in combination with 5-lipoxygenase (5-LO), is required for leukotriene synthesis. FLAP acts coordinately with 5-LO to catalyze the first step in the synthesis of leukotrienes from arachidonic acid. It catalyzes the conversion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE), and further to the allylic epoxide 5 (S)-trans7,9 trans 11,14-cis-eicosatetraenoic acid (leukotriene A4, LTA4).

The leukotrienes are a family of highly potent biological mediators of inflammatory processes produced primarily by bone marrow derived leukocytes such as monocytes, macrophages, and neurophils. Both FLAP and 5-LO are detected within atherosclerosis lesions (Proc Natl Acad Sci USA. 2003 Feb. 4; 100(3):1238-43.), indicating that the vessel itself can be a source of leukotrienes. Inhibitors of FLAP function impede translocation of 5-LO from the cytoplasm to the cell membrane and inhibit activation of 5-LO and thereby decrease leukotriene synthesis.

Independent confirmation of FLAP association to MI was obtained in a British cohort of patients with sporadic MI. These findings support FLAP as the first specific gene isolated that confers substantial risk of the complex traits of MI and stroke.

As a result of these discoveries, genetic tests can now be developed to identify those that are at increased risk of developing myocardial infarction (MI) or stroke.
 

Claim 1 of 4 Claims

1. A method of assessing susceptibility to myocardial infarction (MI) in a human individual, the method comprising: screening nucleic acid of the individual to determine whether the nucleic acid comprises a 5-lipoxygenase activating protein (FLAP) haplotype that comprises polymorphisms SG13S114, allele T; SG13S32, allele A; SG13S25, allele G; and SG13S89, allele G; wherein the presence of the haplotype in the nucleic acid of the individual identifies the individual as having elevated susceptibility to MI, and wherein the absence of the haplotype in the nucleic acid of the individual identifies the individual as not having the elevated susceptibility to MI due to the haplotype.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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