WO2006136791A1 - Polymorphisms and haplotypes in p2x7 gene and their use in determining susceptibility for atherosclerosis-mediated diseases - Google Patents

Abstract

This invention relates to polymorphisms and haplotypes in the human P2X7 gene and their use in determining susceptibility for atherosclerosis mediated disease. In particular, the invention relates to two polymorphisms in the coding sequence of the P2X7 gene, each of which lead to an amino acid change in the sequence of expressed protein; three single nucleotide polymorphisms (SNPs) in the promoter region; one SNP in the 5' UTR and seven SNPs in intronic sequence. The invention also relates to methods and materials for analysing allelic variation in the P2X7 gene, and to the use of P2X7 polymorphism in treatment of P2X7 -mediated diseases such as myocardial infarction and other atherosclerosis-associated diseases.

Description

POLYMORPHISMS AND HAPLOTYPES IN P2X7 GENE

AND THEIR USE IN DETERMINING SUSCEPTIBILITY FOR

ATHEROSCLEROSIS-MEDIATED DISEASES

This invention relates to polymorphisms and haplotypes in the human P2X7 gene and the discovery of their association with changes in the extent of atherosclerosis and atherosclerosis-mediated diseases (i.e. stroke, myocardial infarction and peripheral vascular disease). In particular, the inventors have found that a number of specific three- allele haplotypes are predictive of a reduced risk for atherosclerosis and atherosclerosis mediated diseases (and thus, can be termed "protective" haplotypes), conversely those patients that do not exhibit a "protective" haplotypes are, relative to those that do possess one of the "protective" haplotypes, at increased risk for atherosclerosis.

P2X7 is an ATP-gated ion channel found on monocytes and macrophages, as well as on several other pro-inflammatory cell types. In the art it is also referred to as P2X7 ATP receptor, P2Z, P2X7 receptor, P2RX7, P2X purinoreceptor 7 and MGC20Q89. When activated by high concentrations of extracellular ATP, P2X7 regulates release of the proinflammatory cytokines Interleukin-1 β and IL-18. Inflammation plays a key role in the response of the arterial vessel wall to the lipid-accumulation processes, which drive formation and rupture of atherosclerotic plaques and the consequent acute cardiac sequelae. Several risk factors for cardiovascular disease are well-known in the literature, some of which also have an inflammatory component. These risk factors include increases in plasma levels of triglycerides, DDL-cholesterol and IL-6; a lowering of plasma levels of HDL-cholesterol; a diagnosis of diabetes; smoking; male sex; and increasing age. Atherosclerosis is an arterial disease with typical progressive steps: ^1- endothelial dysfunction and fatty-streak formation; ²' advanced fibrous lesion formation;

³' plaque rupture followed by thrombosis.

A plaque is an atherosclerotic lesion in the intima of an arterial wall with morphology characterized by a lipid core covered by a fibrotic cap.

Rupture of an atherosclerotic plaque followed by thromboembolic events is considered the major pathophysiological mechanism leading to acute myocardial infarction and stroke. Identification of genetic markers that are closely associated with a predisposition to cardiovascular disease, such as atherosclerosis and atherosclerosis-mediated diseases, can be used to design diagnostic or prognostic genetic tests.

The invention also relates to methods and materials for analysing allelic variation in the P2X7 gene, and to the use of P2X7 polymorphisms and P2X7 haplotypes in the identification of an individuals' risk to develop atherosclerosis and atherosclerosis-mediated diseases such as myocardial infarction, stroke and peripheral vascular disease (PVD).

The SNPs and haplotypes of the invention also allow patient stratification. The sub- groups of individuals identified as having increased or decreased risk of developing atherosclerosis and atherosclerosis-mediated diseases, such as myocardial infarction, can be used, inter alia, for targeted clinical trial programs and possibly also pharmacogenetic therapies.

The human P2X7 gene (or rather cDNA) was initially cloned by Rassendren et al. (J. Biol. Chem. 272(9):5482-5486, 1997) from a human monocyte cDNA library using the rat P2X7 gene as a probe. The recovered cDNA encodes a predicted 595 amino acid protein that is 80% identical to the rat protein. On Northern blots, human P2X7 was expressed as a 6-kb mRNA in many tissues. Human P2X7 is located on chromosome 12 at locus 120033362-120086813 (NCBI build 35). The gene sequence is found in: AC079602.15.1.172008, Z98941.2.1.129192 & AC069209.38.1.157792 (these three, together with many others) overlap to form a contiguous sequence.

The location of the polymorphisms can be precisely mapped by reference to published EMBL (or other sequence database) sequence accession numbers (i.e. see above), alternatively, the person skilled in the art can precisely identify the location of the polymorphism in the P2X7 gene simply by provision of flanking sequence adjacent the polymorphism sufficient to unambiguously locate the polymorphism. Provision of 10 or more nucleotides each side of the polymorphism should be sufficient to achieve precise location mapping of the particular polymorphism.

The use of knowledge of polymorphisms to help identify patients most suited to therapy with particular pharmaceutical agents is often termed "pharmacogenetics".

Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process. Polymorphisms are used in mapping the human genome and to elucidate the genetic component of diseases. The reader is directed to the following references for background details on pharmacogenetics and other uses of polymorphism detection: Linder et al. (1997), Clinical Chemistry, 43:254; Marshall (1997), Nature Biotechnology. 15:1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al, (1998), Nature Biotechnology. 16:33.

Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous. Thus, there is a need for improved approaches to pharmaceutical agent design and therapy. Furthermore, there are opportunities for clinical trial patient selection. Point mutations in polypeptides will be referred to as follows: natural amino acid

(using 1 or 3 letter nomenclature), position, new amino acid. For (a hypothetical) example "D25K" or "Asp25Lys" means that at position 25 an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one polypeptide will be shown between square brackets with individual mutations separated by commas. The presence of a particular base at a polymorphism position will be represented by the base following the polymorphism position. For (a hypothetical) example, the presence of adenine at position 300 will be represented as: 300A.

The present invention is based on the discovery of a genetic association between certain polymorphisms in the P2X7 gene and development of cardiovascular disease, in particular atherosclerosis, myocardial infarction and stroke. The inventors have found that a single nucleotide polymorphism is predictive of a reduced risk for atherosclerosis. The association link is further enhanced when this polymorphism is present as part of a haplotype. The inventors have also discovered that 2- or 3-allele haplotypes comprising at least one of two specific SNPs, show even greater association to myocardial infarction (a cardiovascular disease mediated by atherosclerosis). Various specific two- and three-allele haplotypes are shown to be particularly useful in the invention. Whilst certain SNPs and haplotypes are referred to herein as predictive or reduced risk for atherosclerosis, or "atherosclerosis preventing haplotypes", in practice, Conversely, subjects that do not exhibit such a "protective" haplotype, can be considered at increased risk of developing atherosclerosis, such that a clinical trial designed to test the efficacy of a test drug in treating or preventing atherosclerosis or an atherosclerosis mediated disease such as myocardial infarction may be designed to include clinical trial subjects that do not possess such protective haplotypes.

According to a first aspect of the invention there is provided a method of genotyping an individual in order to determine the individual's potential risk to develop atherosclerosis or an atherosclerosis-mediated disease, comprising determining the nucleotide present at each polymorphic position in an atherosclerosis-mediated haplotype on one or both copies of the relevant chromosome in a sample that has previously been removed from the individual, which haplotype includes the SNP rs 169631 or the SNP rs3751143 and at least one other SNP. Atherosclerosis-mediated diseases include: myocardial infarction (MI), stroke and peripheral vascular disease (PVD).

According to another aspect of the invention there is provided a method for screening an individual for a genetic predisposition to develop atherosclerosis and atherosclerosis mediated disease, comprising analysing the individual's nucleic acid in a sample removed from the individual for the presence or absence of an "atherosclerosis- susceptibility haplotype", which haplotype includes one or both of the P2X7 gene SNPs rs 169631 and rs3751143 and determining the status of the human by reference to the haplotype present.

Single nucleotide polymorphisms (SNPs) represent one of the most common forms of genetic variation. These polymorphisms appear when a single nucleotide in the genome is altered (such as via substitution, addition or deletion). For example, if at a particular chromosomal location one member of a population has an adenine and another member has a thymine at the same position, then this position is a single nucleotide polymorphic site. Each version of the sequence with respect to the polymorphic site is referred to as an "allele" of the polymorphic site. SNPs tend to be evolutionarily stable from generation to generation and, as such, can be used to study specific genetic abnormalities throughout a population. IfSNPs occur in the protein coding region it can lead to the expression of a variant, sometimes defective, form of the protein that may lead to development of a genetic disease. Such SNPs can therefore serve as effective indicators of the genetic disease. Some SNPs may occur in non-coding regions, but nevertheless, may result in differential or defective splicing, or altered protein expression levels. SNPs can therefore be used as diagnostic tools for identifying individuals with a predisposition for certain diseases, genotyping the individual suffering from the disease in terms of the genetic causes underlying the condition, and facilitating drug development based on the insight revealed regarding the role of target proteins in the pathogenesis process.

A haplotype is a set of alleles found at linked polymorphic sites (such as within a gene) on a single (paternal or maternal) chromosome. If recombination within the gene is random, there may be as many as 2ⁿ haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs. One approach to identifying mutations or polymorphisms which are correlated with clinical response, is to carry out an association study using all the haplotypes that can be identified in the population of interest. The frequency of each haplotype is limited by the frequency of its rarest allele, so that SNPs with low frequency alleles are particularly useful as markers of low frequency haplotypes. As particular mutations or polymorphisms associated with certain clinical features, such as adverse or abnormal events, are likely to be of low frequency within the population, low frequency SNPs may be particularly useful in identifying these mutations (for examples see: Linkage disequilibrium at the cystathionine beta synthase (CBS) locus and the association between genetic variation at the CBS locus and plasma levels of homocysteine. Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, Chasse JF, London J, Stansbie D, Humphries SE, and Gudnason V; and Variation at the von willebrand factor (vWF) gene locus is associated with plasma vWF:Ag levels: identification of three novel single nucleotide polymorphisms in the vWF gene promoter. Blood (1999) 93:4277-83, Keightley AM, Lam YM, Brady JN, Cameron CL, Lillicrap D).

As used herein, an "atherosclerosis-susceptibility haplotype" is a haplotype that associates with increased or decreased susceptibility to develop atherosclerosis and atherosclerosis mediated disease. Such haplotypes include those identified herein, in particular those listed in Tables 2 to 5 with χ -test p values of less than or equal to 0.05, and thus have predictive value for detecting susceptibility to atherosclerosis and atherosclerosis-mediated disease in an individual. As will be apparent from the Examples disclosed herein, haplotypes that include one or both of the polymorphisms known in the art as rs 169631 and rs3751143 demonstrate significant association to myocardial infarction. Thus, an "atherosclerosis- susceptibility haplotype" is preferably one that includes either or both of these polymorphisms, or one in linkage disequilibrium above D' 0.9 with either. For example, the inventors have determined that SNPs that exhibit D' >0.9 to rsl69631 include: rs591874, rs656612, rs208288, rsl718125, rslO849851 and rs208293.

Examples of SNPs that exhibit D'>0.9 to rsl69631 or rs3751143 are also identified in Table 10. According to another aspect of the invention there is provided a method for haplotyping P2X7 gene in a human individual comprising the steps of: a) treating nucleic acid from a sample that has been removed from the individual so as to identify the nucleotides present at each allelic position of an "atherosclerosis- susceptibility haplotype", which haplotype includes one or both of the P2X7 gene SNPs rsl69631 and rs3751143, and b) assigning the individual a particular haplotype according to the nucleotides detected in step a).

The test sample (the nucleic acid containing sample) is conveniently a sample of blood, plasma, bronchoalveolar lavage fluid, salive, sputum, cheek-swab or other body fluid or tissue (such as a biopsy sample) obtained from an individual that comprise nucleic acid molecules. The nucleic acid containing sample that is to be analysed can either be a treated or untreated biological sample isolated from the individual. A treated sample, may be for example, one in which the nucleic acid contained in the original biological sample has been isolated or purified from other components in the sample (tissues, cells, proteins etc), or one where the nucleic acid in the original sample has first been amplified, for example by polymerase chain reaction. Thus, it will be appreciated that the test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis of allelic variation. For the avoidance of doubt, the methods of the invention do not involve diagnosis practised on the human body. The methods of the invention are preferably conducted on a sample that has previously been removed from the individual. The kits of the invention, however, may include means for extracting the sample from the individual.

In certain embodiments of the invention, presence of an "atherosclerosis-susceptibility haplotype", indicates a lower predisposition to develop atherosclerosis and atherosclerosis mediated disease compared to a control. Representative examples of such haplotypes include those wherein the base at SNP rsl69631 is guanine (G) and/or SNP rs3751143 is adenine (A).

When specifying a particular nucleotide at an allele position it is important to appreciate which of the two complementary strands of nucleic acid the nucleotide resides on. For example, a G on the positive strand will correspond to a C on the negative (reverse) strand. The SNP alleles referred to herein, including the claims, are defined by the assays described in Tablel . For example, SNPs 1, 5 & 6 are on the reverse strand. The correct strand may also be deduced by the nucleotide sequence adjacent the allele, by reference to the sequence listings provided herein . The ability to identify patients that are at reduced risk of developing atherosclerosis or atherosclerosis mediated disease allows one, for example, to exclude such individuals from clinical trials designed to test the efficacy of drugs to prevent or ameliorate atherosclerosis or atherosclerosis mediated disease. The presence of specific "atherosclerosis reduced- susceptibility haplotypes" however does not mean that the individual will not suffer from atherosclerosis or an atherosclerosis mediated disease such as myocardial infarction. It merely suggests that the individual compared to the population as a whole has a lower risk of developing such conditions.

The inventors have noticed that those patients that have suffered a previous MI and possess an "atherosclerosis reduced-susceptibility haplotype" are less likely of suffering a relapse, or a further atherosclerosis-mediated disease. Thus, according to a particular embodiment the methods of the invention are performed on nucleic acid containing sample from an individual that has already suffered a myocardial infarction. In a further embodiment, the individual is male.

Whilst the SNP known as rs 169631 has been found to exhibit a genetic association to MI, association to MI with much greater statistical significance is observed with certain haplotypes that include either or both of the rs 169631 and rs3751143 polymorphisms. Certain triple-allele haplotypes proving to exhibit the greatest association when compared to the pair wise (double-allele) haplotypes. Thus, according to particular embodiments, the methods of the invention concern haplotypes that comprises 2, 3, or more alleles. Whilst the particular atherosclerosis-susceptibility haplotype to be measured can include, in addition to either or both of the rs 169631 and rs3751143 polymorphisms, polymorphisms from outwith the P2X7 gene, in certain embodiments of the invention, at least two of the SNPs making up the haplotype are located within the P2X7 gene. In another embodiment, all of the polymorphic loci making up the haplotype are located within the P2X7 gene. In particular embodiments, the haplotype includes both the rsl69631 and rs3751143 SNPs. As can be seen from Table 3, the pair wise haplotype that involves these two SNPs exhibited a χ²-test p values of 0.0001. As can be seen from Table 5, the three-way (triple-allele) haplotypes that include these two SNPs and one other SNP yielded χ²-test p values of: 0.0000089, 0.0000017, 0.00000029, 0.000000073, 0.000000027, 0.000000015 and 0.0000030.

Particularly useful haplotypes (atherosclerosis-susceptibility haplotypes) for use in any aspect of the present invention are haplotypes 1 - 15 (see Table 7).

According to a further aspect of the invention there is provided a diagnostic or prognostic method of predicting susceptibility to develop atherosclerosis or an atherosclerosis mediated disease, based on the detection of an "atherosclerosis- susceptibility haplotype" selected from the group consisting of: haplotypes 1 - 15, in an individual.

According to a further aspect of the invention there is provided a method of diagnosing or predicting susceptibility to developing cardiovascular disease, or late stage manifestations of cardiovascular disease, in particular atherosclerosis, in an individual, comprising determining the presence or absence in a sample from said individual of a disease-predisposing haplotype comprising a guanine at allele rsl 69631 (position 16 according to SEQ ID NO: 49), wherein the presence of said disease-predisposing haplotype is diagnostic or predictive of susceptibility to cardiovascular disease.

In one embodiment, in addition to the presence of a G at allele rsl 69631, the haplotype also includes a G at allele rs677112 and/or an A at allele rs3751143 (base identified according to the nucleic acid strand disclosed in the sequence listings herein). In a further embodiment the haplotype consists of or comprises a G at allele rs677112, a G at allele rsl69631 and an A at allele rs3751143 (base identified with reference to the nucleic acid strand disclosed in the sequence listings herein).

The inventors have identified that each of 9 specific SNPs within the P2X7 gene (one in 3 ' non-coding region and another in 5' non-coding region) are differentially associated with MI and atherosclerosis. These SNPs were found to be located in two LD blocks, and the SNPs showed linkage to each other in these two LD blocks. The first block encompassing SNPs with NCBI SNP ID numbers: rs677112, rs3900976, rsl69631 and rs208294 and the second block encompassing SNPs with NCBI SNP ID numbers: rs208298, rsl718119, rs891781, rs3751143 and rsl718161. Within each block SNPs had D' values above 0.90 in the case population, with the exception of SNP rs208298 which only showed linkage with D'>Q.9O to rs3751143, but which clearly belongs to the second block as it showed low linkage to the SNPs in the other LD-block (D' <0.4).

The inventors have found that stronger genetic association data is generated (and thus increased confidence of patient stratification) when at least one allele from each block forms part of the haplotype being tested. Of these, rs 169631 exhibits an association to myocardial infarction with a χ² p value of 0.01. The genetic association with atherosclerosis, extent of atherosclerosis and atherosclerosis-mediated diseases (such as MI and stroke) increases when this SNP forms part of a haplotype with one or more of the other SNPs identified herein.

Of the SNPs tested, SNPs rs2Q8298, «1718119 and rs3751143 result in amino acid substitutions. When assessing the coding rs3751143 SNP, the association data was much greater when this was combined with certain non-coding/silent SNPs. It is possible that these silent SNPs mediate an effect such as altered levels of expression.

In view of the statistically significant association between the presence of a guanine (G) at the rs 169631 SNP and myocardial infarction, the methods, uses, kits and nucleic acids of the invention are equally applicable to determination of this SNP on its own.

Thus, according to another aspect of the invention there is provided a method of diagnosing or predicting susceptibility to developing atherosclerosis or an atherosclerosis- mediated disease in an individual, comprising determining the presence or absence in a sample removed from said individual of a guanine (G) nucleotide at allele rs 169631 (position 16 according to SEQ ID NO: 49), wherein the presence of said nucleotide is diagnostic or predictive of susceptibility to cardiovascular disease.

The SNPs and haplotypes of the invention demonstrate significant association to myocardial infarction and atherosclerosis. However, the person skilled in the art will appreciate that a diagnostic test consisting solely of a haplotype of the invention will not be diagnostic of disease occurrence for any particular individual. Nevertheless, in line with future developments we envisage that the SNPs and haplotypes of the present invention could form part of a panel of markers that in combination will be predictive of disease or disease susceptibility for an individual, within normal clinical standards sufficient to influence clinical practice.

Because there are two copies of each chromosome (a maternal and paternal copy), at each chromosomal location the human may be homozygous for an allele or the human may be a heterozygote. If the individual is heterozygous the presence of both alternate polymorphisms will be present.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures, which may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. List 1 lists a number of mutation detection techniques, some based on the PCR. These may be used in combination with a number of signal generation systems, a selection of which is listed in List 2. Further amplification techniques are listed in List 3. Many current methods for the detection of allelic variation are reviewed by Nollau et ah, Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2^nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations:

List 1 - Mutation Detection Techniques

General: DNA sequencing, Sequencing by hybridisation

Scanning: PTT, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC,

Enzymatic mismatch cleavage

Hybridisation Based

Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays (DNA Chips).

Solution phase hybridisation: Taqman™ - US-5210Q15 & US-5487972 (Hoffmann-La Roche), Molecular Beacons - Tyagi et al (1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health Inst., New York)

Extension Based: ARMS™, ALEX™ - European Patent No. EP 332435 Bl (Zeneca Limited), COPS - Gibbs et al (1989), Nucleic Acids Research, 17, 2347. Incorporation Based: Mini-sequencing, APEX Restriction Enzyme Based: RFLP, Restriction site generating PCR Ligation Based: OLA Other: Invader assay List 2 - Signal Generation or Detection Systems

Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998 (Zeneca Limited)

Other: Chemiluniinescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric, Hybridisation protection assay, Mass spectrometry

List 3 - Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA

List 4 - Protein variation detection methods Immunoassay Immunohistology Peptide sequencing

Thus, the presence or absence of a disease-predisposing SNP or haplotype useful in the invention can be determined, for example, using enzymatic amplification of nucleic acid from the individual. In one embodiment, the presence or absence of a particular disease-predisposing SNP or haplotype is determined using polymerase chain reaction (PCR). In a further embodiment the PCR is performed with allele-specific oligonucleotide primers capable of discriminating between the different bases at a particular allele. Such as using amplification refractory mutation system (ARMS™-allele specific amplification). In a further embodiment, the PCR is performed using one or more fluorescently labelled probes or using one or more probes which include a DNA minor groove binder. The presence or absence of a particular disease-predisposing haplotype can also be determined, for example, by sequence analysis.

The nucleic acid sequence method for diagnosis is preferably one which is determined by a method selected from amplification refractory mutation system, restriction fragment length polymorphism and primer extension. In another embodiment, the nucleotide present at each polymorphic position is determined by sequence analysis, such as by dideoxy sequencing.

Preferred mutation detection techniques include ARMS™-allele specific amplification, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques. Immunoassay techniques are known in the art e.g. A Practical Guide to ELISA by D M Kemeny, Pergamon Press 1991 ; Principles and Practice of Immunoassay, 2^nd edition, C P Price & D J Newman, 1997, published by Stockton Press in USA & Canada and by Macmillan Reference in the United Kingdom. Particularly preferred methods include ARMS™-allele specific amplification, OLA and RFLP based methods. The allele specific amplification technique known in the art as ARMS™-allele specific amplification is an especially preferred method.

ARMS™-allele specific amplification (described in European patent No. EP-B- 332435, US patent No. 5,595,890 and Newton et al. (Nucleic Acids Research, Vol. 17, p.2503; 1989)), relies on the complementarity of the 3' terminal nucleotide of the primer and its template. The 3' terminal nucleotide of the primer being either complementary or non-complementary to the specific mutation, allele or polymorphism to be detected. There is a selective advantage for primer extension from the primer whose 3 ' terminal nucleotide complements the base mutation, allele or polymorphism. Those primers which have a 3 ' terminal mismatch with the template sequence severely inhibit or prevent enzymatic primer extension. Polymerase chain reaction or unidirectional primer extension reactions therefore result in product amplification when the 3 ' terminal nucleotide of the primer complements that of the template, but not, or at least not efficiently, when the 3' terminal nucleotide does not complement that of the template. In a further aspect, the diagnostic methods of the invention are used to assess the pharmacogenetics of a drug acting at P2X7.

Individuals who carry particular allelic variants of the P2X7 gene may therefore exhibit differences in their ability to regulate protein biosynthesis under different physiological conditions and will display altered abilities to react to different diseases. In addition, differences arising as a result of allelic variation may have a direct effect on the response of an individual to drug therapy. The diagnostic methods of the invention may be useful both to predict the clinical response to such agents and to determine therapeutic dose.

In a further aspect, the detection/diagnostic methods of the invention, are used to assess the predisposition and/or susceptibility of an individual to diseases mediated by atherosclerosis. This may be particularly relevant in the development of myocardial infarction and stroke and the present invention may be used to recognise individuals who are particularly at risk from developing these conditions.

In a further aspect, the diagnostic methods of the invention are used in the development of new drug therapies which selectively target one or more allelic variants of the P2X7 gene. Identification of a link between a particular allelic variant and or a haplotype and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs. Drugs may be designed to regulate the biological activity of variants implicated in the disease process whilst minimising effects on other variants. In a further diagnostic aspect of the invention the presence or absence of variant nucleotides is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes. The person of ordinary skill will be able to design and implement diagnostic procedures based on the detection of restriction fragment length polymorphism due to the loss or gain of one or more of the restriction sites due to the presence of a polymorphism.

According to a further aspect of the invention there is provided the use of an "atherosclerosis-susceptibility haplotype", selected from the group consisting of those comprising marker 3 (rsl69631) and/or 8 (rs3751143), as a tool for the prediction of atherosclerosis risk in a population and/or an individual. The invention further provides nucleotide primers which detect the P2X7 gene polymorphisms of the invention. Such primers can be of any length, for example between 8 and 100 nucleotides in length, but will preferably be between 12 and 50 nucleotides in length, more preferable between 17 and 30 nucleotides in length.

According to another aspect of the present invention there is provided an allele specific primer capable of detecting a P2X7 gene polymorphism, preferably at one or more of the positions as defined herein.

An allele specific primer is used, generally together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position e.g. as used for ARMS™-allele specific amplification assays. The allele specific primer is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides. An allele specific primer preferably corresponds exactly with the allele to be detected but derivatives thereof are also contemplated wherein about 6-8 of the nucleotides at the 3' terminus correspond with the allele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the primer. Often the nucleotide at the -2 and/or -3 position (relative to the 3' terminus) is mismatched in order to optimise differential primer binding and preferential extension from the correct allele discriminatory primer only.

Primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in standard textbooks, for example "Protocols for Oligonucleotides and Analogues; Synthesis and Properties," Methods in Molecular

Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1^st Edition. If required the primer(s) may be labelled to facilitate detection.

According to another aspect of the present invention there is provided an allele- specific oligonucleotide probe capable of detecting a P2X7 gene polymorphism, preferably at one or more of the positions defined herein.

The allele-specific oligonucleotide probe is preferably 17-50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.

The design of such probes will be apparent to the molecular biologist of ordinary skill. Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8-15 bases in length. In general such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene. However, if required one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected. The probes of the invention may carry one or more labels to facilitate detection, such as in Molecular Beacons. Single stranded oligonucleotides corresponding to SEQ ID NOs: 2 -10 or their complement, could be used as probes to detect the particular polymorphism at the central position. The probe would bind more efficiently to a target sequence that possessed the particular complementary polymorphism base at this central (polymorphism) location than one with a base mismatch. According to another aspect of the present invention there is provided an allele specific primer or an allele specific oligonucleotide probe capable of detecting a P2X7 gene polymorphism at one of the positions defined herein. According to another aspect of the present invention there is provided an allele specific primer or an allele specific oligonucleotide probe capable of detecting either of the SNPs selected from the group consisting of: rsl69631 and rs3751143.

According to another aspect of the invention there is provided a kit for screening for a genetic predisposition to atherosclerosis or an atherosclerosis mediated disease, which kit comprises:

(i) reagents for detecting the presence of an "atherosclerosis-susceptibility haplotype", which haplotype includes one or both of the P2X7 gene SNPs rsl69631 and rs3751143, and optionally, (ii) means for collecting a nucleic acid sample or nucleic acid containing sample.

According to another aspect of the invention there is provided an in vitro diagnostic kit for determining the identity of SNPs rs 169631 and/or rs3751143 in the human P2X7 gene, said kit comprising components for the determination of the nucleotide present at said SNP locations. In particular embodiments of the invention, the kit components for determining said

SNPs include allele-specific amplification primers or allele-specific hybridisation probes capable of determining the identity of the nucleotide bases at the SNP locations.

According to another aspect of the invention there is provided a kit comprising one or more diagnostic primer(s) and/or one or more allele-specific oligonucleotide probes(s) capable of determining the identity of the nucleotide present at SNPs rs 169631 and rs3751143, in the human P2X7 gene.

The diagnostic kits may comprise appropriate packaging and instructions for use in the methods of the invention. Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example taq polymerase. Such kits may also comprise companion primers and/or control primers or probes. A companion primer is one that is part of the pair of primers used to perform PCR. Such primer usually complements the template strand precisely.

According to another aspect of the invention there is provided the use of a diagnostic kit capable of haplotyping P2X7 gene at at least three allele positions, at least one of which is rsl69631 or rs3751143, for stratifying individuals into particular haplotype groups. One particular application of this is for selecting members from one or more of the stratified groups for inclusion in a clinical trial to determine the efficacy of a drug. In a particular embodiment, the clinical trial is measuring the efficacy of the drug at treating atherosclerosis or an atherosclerosis mediated disease.

According to another aspect of the invention there is provided a method for selecting individuals for participation in a clinical trial to assess the efficacy of a drug for treating atherosclerosis or an atherosclerosis-mediated disease, comprising: a) individually haplotyping the P2X7 gene at two or more allele positions from a nucleic acid containing sample already isolated from each individual, wherein at least one of the two or more allele positions is selected from the group consisting of: is rsl69631 and rs3751143, b) grouping the individuals according to the particular P2X7 gene haplotype that each individual belongs to, and c) selecting individuals from one or more haplotype groups for inclusion in the clinical trial.

The SNPs and haplotypes of the invention represent a valuable information source with which to characterise individuals in terms of, for example, their identity and susceptibility to disease development or susceptibility to treatment with particular drugs. These SNPs and/or haplotypes, including nucleotide sequences related to these may be stored in a computer readable medium. The polymorphism and haplotypes referred to herein are particularly useful as components in databases useful for sequence identity, genome mapping, pharmacogenetics and other search analyses. Generally, the sequence information relating to the nucleic acid sequences and polymorphisms of the invention may be reduced to, converted into or stored in a tangible medium, such as a computer disk, preferably in a computer readable form. For example, chromatographic scan data or peak data, photographic scan or peak data, mass spectrographic data, sequence gel (or other) data.

The computer readable medium may be used, for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis. The computer readable medium can be any composition of matter used to store information or data, including, for example, floppy disks, tapes, chips, compact disks, digital disks, video disks, punch cards and hard drives.

According to another aspect of the present invention there is provided a method of treating a human in need of treatment with a small molecule drug acting on the P2X7 protein or an anti-sense oligonucleotide or ribozyme acting against the P2X7 mRNA, in which the method comprises: i) determining the identity of SNPs rs 169631 and rs3751143 in the human P2X7 gene ii) determining the status of the human by reference to SNPs present at alleles rsl69631 and rs3751143; and, iii) administering an effective amount of the drug.

According to another aspect of the present invention there is provided a pharmaceutical pack comprising a drug acting as a P2X7 antagonist and instructions for administration of the drug to humans diagnostically tested for a polymorphism therein, preferably at one or more of the positions defined herein.

According to another aspect of the present invention there is provided an antibody specific for an allelic variant of human P2X7 polypeptide as described herein.

Antibodies can be prepared using any suitable method. For example, purified polypeptide may be utilized to prepare specific antibodies. The term "antibodies" is meant to include polyclonal antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F(ab')2, Fab and single chain Fv. Antibodies are defined to be specifically binding if they bind the allelic variant of P2X7 with a K_a of greater than or equal to about 10⁷ M^'1. Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. KY. Acad. ScL, (1949) 51:660.

Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, antigen is administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme- linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530. Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980). The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology (1990) 3:1-9, which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, (1989) 7: 394.

Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example "A Practical Guide to ELISA" by D. M. Kemeny, Pergamon Press, Oxford, England. According to another aspect of the invention there is provided a diagnostic kit comprising an antibody of the invention.

The invention will now be illustrated but not limited by reference to the following Examples. All temperatures are in degrees Celsius.

In the Examples below, unless otherwise stated, the following methodology and materials have been applied.

AMPLITAQ™ , available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.

General molecular biology procedures can be followed from any of the methods described in "Molecular Cloning - A Laboratory Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or "Current Protocols in Molecular Biology Volumes 1-3 ,Edited by FM Asubel, R Brent, RE Kingston pub John Wiley 1998

Electropherograms were obtained in a standard manner: data was collected by ABI377 data collection software and the wave form generated by ABI Prism sequencing analysis (2.1.2). EXAMPLE 1

The present invention is directed to the discovery of disease-predisposing SNPs and haplotypes comprising said SNPs that are closely associated with atherosclerosis and atherosclerosis-mediated diseases or conditions such as myocardial infarction. As disclosed herein, genetic linkage approaches were used to identify a strong association between certain disease-predisposing haplotypes and myocardial infarction or atherosclerosis Subjects fthe SCARF study)

During the period of January 1996 to December 2000, all patients less than 60 years of age who were admitted for acute myocardial infarction to the coronary care units of three hospitals in the northern part of Stockholm (Danderyd Hospital, Karolinska Hospital and Norrtalje Hospital) were screened for inclusion in the study. A total of 755 patients were considered, of whom 433 patients entered the study. Of the remaining 322 patients, 179 met one or more predefined exclusion criteria (previous myocardial infarction (n=102), diabetes mellitus type 1 (n=23), renal insufficiency defined as serum creatinine >200 μmol/1 (n=9), any chronic inflammatory disease (n=13), drug addiction (n=10), psychiatric disease (n=3) or unwillingness to enrol in the study (n=19)). Another 143 patients were not included for logistic and psychosocial reasons, resulting in a participation rate of eligible patients of 57%.

Three hundred eighty-seven patients completed the program. Of the 46 patients who did not complete the program, 19 withdraw their consent, whereas 19 were excluded due to occurrence of concomitant disease (n=12) or protocol violation (n=7), 3 died and 5 patients failed to complete the program. For each patient, a sex- and age-matched healthy control person was recruited from the general population of the same county.

Three months after the index cardiac event, patients and control subjects were interviewed about background facts, such as ethnical background, social situation, lifestyle characteristics, medical history and medication, and a medical examination was performed. Blood samples were drawn on the same occasion under fasting conditions.

The Ethics Committee of the Karolinska Hospital approved the study, and all subjects gave their informed consent to participation. Coronary angiography

All patients included at the Danderyd and Norrtalje hospitals (n=269) were offered routine coronary angiography, of whom 243 accepted to be included in the coronary angiography substudy. Coronary angiography was performed, if needed for clinical reason, during the initial hospital stay (n=35), or three months later (n=208). Angiograms were obtained, after intracoronary administration of glycerylnitrate (0.1 mg/ml, 2.5 ml), using the Philips Integris H3000 angiographic system. The first 200 examinations were stored on 36 mm cine film and the last 43 on CD-

ROM. Angiograms were then analysed by Quantitative Coronary Angiography (Medis QCA-CMS system, version 3.0). The coronary artery tree was divided into 15 segments according to AHA guidelines. In each segment, minimum lumen diameter (MLD), reference diameter, % diameter stenosis, mean segment diameter (MSD), segment length, plaque area, segment area and number of significant (>50%) stenoses were registered. Genotvping

DNA was prepared from peripheral blood cells using a genomic DNA isolation kit (Qiagen Inc, Valentia, CA) and stored in 96-well arrays at -20°C.

The PCR was performed in a 50 μL reaction volume containing: 50 ng of genomic DNA, IxPCR buffer, 10 nmol dNTP, 1 U AmpliTaq Gold, 0,1 μmol MgCl₂, 1 pmol of each forward and reverse primer and 29.8 μL distilled H₂O. The PCR was started by incubation at 95 °C for 9 minutes and then 35 repeated cycles of 94°C for 1 minute, 65⁰C for 50 seconds, 72°C for 2 minutes, finishing (after the cycles) with 10 minutes at 72⁰C. The primers used were the "forward" primers and the biotinylated "reverse" primers listed for each SNP in Table 1.

The nucleotide sequence was then identified by pyrosequencing™ as described by Ronaghi and Nyren (Science. 1998 JuI 17;281(5375):363- 365) using the PSQ™ 96 System with PSQ™ 96 SNP Reagent Kit according to the manufacturer's instructions (Pyrosequencing AB, Uppsala, Sweden). The primer used for pyrosequencing was the "sequencing" primers listed for each

SNP in Table 1. The dispensing order of the nucleotides for each SNP " is as in Table 1. The pattern of peaks appearing in the Pyrogram™ is unique for the genotype and the order in which one adds the dNTPs, as the height of the peak is proportional to the amount of incorporated nucleotide. Table 1.

Forward primers

SNP rs677112 5'- ^■CTGCAGACCCCTCAG-CTCTG-3 ' SEQ ID NO:11 rs3900976 5'- CAACACCCTGGATCCCCA-3 ' SEQ IDNO:12 rsl69631 5'- TTTCCTTGTGGTTGCTGCTG-3 ' SEQ ID NO:13 rs208294 5'- ACAGGTCTTCTGGTTCCCTTCA-3 ' SEQ ID NO:14 rs208298 5'- AACACAAGACCCAGTGGCATT-3 ' SEQ IDNO:15 rsl718119 5'- AAATATGGGAGCGACAGCAGTT-S ' SEQ ID NO:16 rs891781 5'- CGGCACTCAGATTAAACAATTGAA-3 ' SEQ IDNO:17 rs3751143 5'- AGCTGCTTAGAAAGGAGGCGA-S ' SEQ IDNO:18 rsl718161 5'- TCAGGAGATGAAGGGCAGTCA-S ' SEQ IDNO:19

Reverse primers

SNP rs677112 5'-GGAACTGGGATTGTCACTATGCT-S ' SEQ ID NO:20 rs3900976 5'-GCCTGGTCCTATTCCTCCCA-S ' SEQ ID NO:21 rsl69631 5'-GCAGATGGAAATGGGACAGG-S' SEQ ID NO:22 rs208294 5'-CTCCGCAGTTCTTTCACATCTG-3 ' SEQ ID NO:23 rs208298 5'-AGTCCCAGATACGGAGCAAGTC-S ' SEQ ID NO:24 rsl718119 5'-AGTGACTAACGCAGCGCTTGT-S' SEQ ID NO:25 rs891781 5'-ATTGCAGGTGTGAGCCACC-S' SEQ ID NO:26 rs3751143 5 '-AGGAACTGCAGGACGTGTCTG-3 ' SEQ ID NO:27 rsl718161 5 '-CCTGATGTCAAAGCAGCACCT-3 ' SEQ ID NO:28

Sequencing primers

SNP rs677112 5'-GGAATGGAGATGGTGA-3 ' SEQ ID NO:29 rs3900976 5'-GGAGGAGGCGAGGAT-3 ' SEQ ID NO:30 rs 169631 5'-TTTATCCCATCAACTTCC-3 ' SEQ ID NO:31 rs208294 5'-GACCGGAAGGTGTGT-3 ' SEQ ID NO:32 rs208298 5'-TTTACTGTGGCACCTCT-S' SEQ ID NO:33 rsl718119 5'-CATTCTCCCCAGGC-3 ' SEQ ID NO:34 rs891781 5'-CCTTTTATGCCCCC-3 ' SEQ ID NO:35 rs3751143 5'-CCGGCAGCACAGC-3 ' SEQ ID NO:36 rsl718161 5'-CCCACCGTGCAGT-S' SEQ ID NO:37

Dispensing Order

SNP rs677112 GCTGATGAT SEQ ID NO:38 rs3900976 GCTGCAGCA SEQ ID NO:39 rs 169631 AGCATCTCT SEQ ID NO:40 rs208294 CAGTGCTGA SEQ ID NO:41 rs208298 GCTGACAGT SEQ ID NO:42 rsl718119 TCAGACTGT SEQ ID NO:43 rs891781 GCTGCATCA SEQ ID NO:44 rs3751143 TGACTGCTG SEQ ID NO:45 rsl718161 CGTGATGCT SEQ ID NO:46 Genomic organisation of P2X7 gene

P2X7 Gene (NCBI human genome build 35) Exon Chr Start End Length

1 12 120033398 120033618 221 bp

2 12 120055308 120055476 169 bp

3 12 120056602 120056670 69 bp

4 12 120061425 120061497 73 bp

5 12 120062947 120063043 97 bp

6 12 120065880 120065960 81 bp

7 12 120066581 120066710 130 bp

8 12 120068011 120068147 137 bp

9 12 120075911 120076001 91 bp

10 12 120077670 120077735 66 bp

11 12 120077820 120077969 150 bp

12 12 120080877 120080978 102 bp

13 12 120084828 120086576 1749 bp

Tested P2X7 SNP positions (NCBI human genomebuild 35)

SNP Chr rs677112 12 120020101 rs3900976 12 120033941 rs169631 12 120046471 rs208294 12 120062973 rs208298 12 120064958

IS1718119 12 120077823 rs891781 12 120081158 rs3751143 12 120085024 rsl718161 12 120090178

Reference sequence information:

Gene genomic location: 120033398 - 120086576 bp on chromosome 12 (NCBI build 35) Gene is located in sequences: AC079602.15.1.172008, Z98941.2.1.129192 & AC069209.38.1.157792 (these three, together with many others) overlap to form a contiguous sequence which is numbered as above)

RefSeq dna: NM_002562.4 (gene only)

SEQ ID NO: 1 provides the cDNA sequence of human P2X7 gene. SEQs 2-10 and 47-55 provide sequence around each of the 9 SNPs analysed. Description of haplotype

To ensure an exhaustive coverage of the P2X7 gene a total of 9 SNPs were used in the analysis. The SNPs selected were tested for association to myocardial infarction and to extent of atherosclerosis in a case control material (SCARF). The SNPs tested showed linkage to each other in two LD-blocks, the first block encompassing SNPs rs677112, rs3900976, rs 169631 and rs208294 and the second block encompassing SNPs rs208298, rsl718119, rs891781, rs3751143 and rsl718161. Within each block SNPs had D' values above 0.90 in the case population, with the exception of SNP rs208298 which only showed linkage with D'>0.90 to rs3751143, but which clearly belonged to the second block as it showed low linkage to the SNPs in the other LD-block (D' <0.4).

According to both the pair wise and triple-allele haplotype association data the best association was seen with haplotypes that include both of SNPs rs 169631 and rs3751143. Interestingly, these two SNPs are in different LD-blocks. To ensure that the linkage seen for SNP rsl 69631 was to P2X7 and not to a neighbouring gene, haplotypes were constructed that included SNPs from both LD blocks. The primary choice was to also include SNPs rs677112 and rs3751143 in addition to rsl 69631 as these showed a trend towards association with Myocardial Infarction (Table 2). SNP rs3751143 was also the SNP in strongest linkage to SNP rs208298 and thus using rs3751143 in the haplotype would ensure that genetic associations to SNP rs208298 would not be lost in the haplotype analysis. This haplotype showed high association to MI, p <0.0002 (Table 3). Haplotype 1 is associated with protection from disease (P<0.0000085 (uncorrected for multiple comparisons, see Table 5) or 0.0011 (corrected, see Table 6))

Table 2

SNP Alleles Minor Allele Frequency Association

Major/Minor Patients Controls P rs677112 G/A 0.23 0.19 0.13 rs3900976 C/T 0.05 0.04 0.36 rsl 69631 G/C 0.31 0.25 0.01 rs208294 C/T 0.45 0.46 0.82 rs208298 G/A 0.28 0.30 0.58 rsl718119 AJG 0.38 0.39 0.62 rs891781 C/T 0.38 0.38 0.96 rs3751143 A/C 0.17 0.14 0.15 rsl718161 A/G 0.18 0.18 0.46

The base for the major/minor allele is identified with reference to the nucleic strand disclosed in the sequence listings herein, in particular SEQ ID Nos: 2 - 10.

Table 3 Pair wise haplotypes

SNPl SNP2 Association to MI Cγ²-tesf)p rs677112 rs3900976 0.27 rs677112 rsl 69631 0.14 rs677112 rs208294 0.16 rs677112 rs208298 0.53 rs677112 rsl718119 0.44 rs677112 rs891781 0.55 rs677112 rs3751143 0.05 rs677112 rsl718161 0.06 rs3900976 rsl 69631 0.07 rs3900976 rs208294 0.69 rs3900976 rs208298 0.53 rs3900976 rsl718119 0.87 rs3900976 rs891781 0.62 rs3900976 rs3751143 0.28 rs3900976 rsl718161 0.47 rsl69631 rs208294 0.006 rsl69631 rs208298 0.21 rsl69631 rsl718119 0.14 rsl69631 rs891781 0.11 rsl69631 rs3751143 0.0001 rsl69631 »1718161 0.04 rs208294 rs208298 0.64 rs208294 »1718119 0.95 rs208294 rs891781 0.95 rs208294 rs3751143 0.02 rs208294 »1718161 0.89 rs208298 »1718119 0.28 rs208298 rs891781 0.41 rs208298 rs3751143 0.04 rs208298 »1718161 0.87 rsl718119 rs891781 0.41 rsl718119 rs3751143 0.23

IS1718119 »1718161 0.73 rs891781 rs3751143 0.13 rs891781 »1718161 0.76 rs3751143 »1718161 0.43

Table4

SNPl SNP2 Association to MI (γ²-tesf) Global p-value* rsl69631 rs3751143 0.0129 * p-value corrected for in a conservative fashion for multiple comparisons by multiplying the p-value obtained in the chi² analysis by the total number of combinations possible (single SNPs = 9, Pairwise= 36 & three-way combinations = 84 , total n=129). Table 5 Three-way haplotypes

SNPl SNP2 SNP3 Association to MI (χ²-test)p rs677112 rs3900976 rsl 69631 0.079 rs677112 rs3900976 rs208294 0.035 rs677112 rs3900976 rs208298 0.16 rs677112 rs3900976 rsl718119 0.32 rs677112 rs3900976 rs891781 0.26 rs677112 rs3900976 rs3751143 0.00089 rs677112 rs3900976 rsl718161 0.30 rs677112 rsl69631 rs208294 0.0054 rs677112 rsl69631 rs208298 0.11 rs677112 rsl 69631 rsl718119 0.12 rs677112 rs 169631 rs891781 0.11 rs677112 rsl 69631 rs3751143 0.0000089 rs677112 rsl 69631 rsl718161 0.081 rs677112 rs208294 rs208298 0.0019 rs677112 rs208294 rsl718119 0.068 rs677112 rs208294 rs891781 0.062 rs677112 rs208294 rs3751143 0.000033 rs677112 rs208294 rsl718161 0.10 rs677112 rs208298 rsl718119 0.0093 rs677112 rs208298 rs891781 0.067 rs677112 rs208298 rs3751143 0.0000048 rs677112 rs208298 rsl718161 0.51 rs677112 IS1718119 rs891781 0.29 rs677112 rsl718119 rs3751143 0.000012 rs677112 rsl718119 rsl718161 0.27 rs677112 rs891781 rs3751143 0.0000014 rs677112 rs891781 rsl718161 0.30 rs677112 rs3751143 rsl718161 0.0030 rs3900976 rsl69631 rs208294 0.0025 rs3900976 rsl 69631 rs208298 0.048 rs3900976 rsl 69631 rsl718119 0.08 rs3900976 rsl 69631 rs891781 0.046 rs3900976 rsl 69631 rs3751143 0.0000017 rs3900976 rsl 69631 rsl718161 0.046 rs3900976 rs208294 rs208298 0.021 rs3900976 rs208294 rsl718119 0.37 rs3900976 rs208294 rs891781 0.30 rs3900976 rs208294 rs3751143 0.000094 rs3900976 rs208294 rsl718161 0.61 rs3900976 rs208298 rsl718119 0.12 rs3900976 rs208298 rs891781 0.08 rs3900976 rs208298 rs3751143 0.00015 rs3900976 rs208298 rsl718161 0.44 rs3900976 rsl718119 rs891781 0.64 rs3900976 rsl718119 rs3751143 0.00026 rs3900976 rsl718119 rsl718161 0.65 rs3900976 rs891781 rs3751143 0.000033 rs3900976 rs891781 rsl718161 0.63 rs3900976 rs3751143 rsl718161 0.14 rsl 69631 rs208294 rs208298 0.0054 rs 169631 rs208294 rsl718119 0.00078 rsl 69631 rs208294 rs891781 0.00060 rsl 69631 rs208294 rs3751143 0.00000029 rsl 69631 rs208294 rsl718161 0.0079 sl 69631 rs208298 rsl718119 0.050 sl 69631 rs208298 rs891781 0.039 sl 69631 rs208298 rs3751143 0.000000073 sl 69631 rs208298 rsl718161 0.13 sl 69631 rsl718119 rs891781 0.06 sl 69631 rsl718119 rs3751143 0.000000027 sl 69631 rsl718119 rsl718161 0.039 rs169631 rs891781 rs3751143 0.000000015 rsl69631 rs891781 «1718161 0.037 rsl69631 rs3751143 «1718161 0.0000030 rs208294 rs208298 «1718119 0.012 rs208294 rs208298 rs891781 0.013 rs208294 rs208298 rs3751143 0.0000010 rs208294 rs208298 «1718161 0.063 rs208294 «1718119 rs891781 0.67 rs208294 «1718119 rs3751143 0.0000030 rs208294 «1718119 «1718161 0.79 rs208294 rs891781 rs3751143 0.0000024 rs208294 rs891781 «1718161 0.78 rs208294 rs3751143 «1718161 0.0022 rs208298 «1718119 rs891781 0.21 rs208298 «1718119 rs3751143 0.000011 rs208298 «1718119 «1718161 0.28 rs208298 rs891781 rs3751143 0.0000079 rs208298 rs891781 «1718161 0.22 rs208298 rs3751143 «1718161 0.0027 rsl718119 rs891781 rs3751143 0.00022 rsl718119 rs891781 «1718161 0.80

«1718119 rs3751143 «1718161 0.0011 rs891781 rs3751143 «1718161 0.0003

Table 6

SNPl SNP2 SNP3 A Assssoocciiaattiioon to MI (y -test) Global p-value* rs677112 rsl 69631 rs3751143 0.0011 rs3900976 rsl 69631 rs3751143 0.00016 rsl69631 rs208294 rs3751143 0.000038 rs 169631 rs208298 rs3751143 0.0000094 rsl69631 IS1718119 rs3751143 0.0000081 rsl 69631 rs891781 rs3751143 0.0000019 rs 169631 rs3751143 rsl718161 0.00033

* p-value corrected for in a conservative fashion for multiple comparisons by multiplying the p-value obtained in the cm^'2 analysis by the total number of combinations possible (single SNPs = 9, Pairwise= 36 & three-way combinations = 84 , total n=129)

The global p-value is a sum of contributions from all haplotypes, so one may find (as in Table 7) that some haplotypes are strongly associated with protection against disease, while others are weakly associated with risk or protection and some not showing any association at all.

Table 7

SNP Frequency Association to MI (Yates corrected χ -test)

Haplotype rs677112 rsl69631 rs3751143 Controls Cases P

1 C G A 0.62 0.52 0.000034 Protective

2 T C A 0.18 0.23 0.014 Risk

3 C G C 0.13 0.17 0.024 Risk

4 C C A 0.05 0.08 0.063 Neutral

5 T C C 0.01 O.001 0.025 Protective (rare)

6 C C C 0.01 <0.001 0.026 Protective (rare)

10 7 T G A 0.001 O.001 0.99 Neutral (rare)

SNP Frequency Association to MI (Yates corrected χ²-test)

Haplotype rsl 69631 rs891781 rs3751143 Controls Cases P

8 G C A 0.42 0.35 0.0018 Protective

15 9 G T A 0.20 0.17 0.19 Neutral

10 C T A 0.17 0.20 0.056 Neutral u>

11 G C C 0.11 0.17 0.0014 Risk

12 C C A 0.07 0.11 0.012 Risk

13 G T C 0.02 O.001 0.0016 Protective (rare)

20 14 C C C 0.01 <0.001 0.014 Protective (rare)

15 C T C 0.01 O.001 0.14 Neutral (rare)

25

Table 8

The most protective haplotypes for all three-marker combinations containing marker 3 (rs 169631) and 8 (rs3751143) are:

1-3-8 2-3-8 3-4-8 3-5-8 3-6-8 3-7-8 3-8-9

C-G-A C-G-A G-C-A G-C-A G-G-A G-C-A G-A-A

Cont freq 0.62 0.62 0.28 0.46 0.42 0.42 0.58 Case freq 0.52 0.52 0.22 0.35 0.34 0.35 0.48 p-value 0.000034 0.000034 0.0064 0.000043 0.0017 0.0018 0.000085

10

Key: marker 1 = rs677112; marker 2 = rs3900976; marker 3 = rsl69631; marker 4 = rs208294; marker 5 = rs208298; marker 6 = rsl718119; marker 7 = rs891781; marker 8 = rs3751143

Such "protective" haplotypes can also be written as: 3-8-9/G-A-A, 1-3-8/C-G-A, 2-3-8/C- G-A, 3-4-8/G-C-A, 3-5-8/G-C-A, 3-6-8/G-Q-A and 3-7-8/G-C-A, wherein the three numerals (e.g. 1-3-8) represent the three markers that are present in the haplotype and the letters (e.g. C-G-A) represent the corresponding nucleotide present on at least one chromosomal copy (e.g. with 1-3-8/C-G-A there is a C at rs677112, a G at rsl69631 and an A at rs3751143).

1. Statistics

Associations were calculated by the nonparametric ANOVA Kruskal Wallis test for the association between the haplotype 1 (homozygous carriers, heterozygous carriers and non- carriers), and atherosclerosis.

2. Haplotype 1 in patients with previous MI (n=382) versus atherosclerosis. A significant association was seen between the presence of haplotype 1 and mean lumen diameter (MSD) in coronary arteries, as assessed by quantitative coronary angiography (Table 9). No significant association was seen with percentage stenosis, number of stenoses or minimum lumen diameter in coronary arteries.

Table 9 Haplotype 1 versus atherosclerosis in the whole study population. Atherosclerosis values are presented as mean ± SE.

Table 10 SNPs that have a D' >Q.9 with rsl69631 or rs3751143; (see http://www.ncbi.nlm.nih.gov/SNP/ (dbSNP))

rs!69631 rs3751143 rsl 1065441 rsl0849849 rsl2301635 rsl7525809 rsl 1065443 rsl0849850 rsl718161 rs2051551 rsl 1065450 rslO849851 rs2686384 rs2Q8282 rsl 183296 rsl1065464 rsl1065464 rs208288 rsl 2297280 rsl2813980 rsl1065468 rs208290 rsl2310654 rsl2821688 rsl186055 rs208291 rsl 2312695 rsl653583 rsl2297280 rs208293 rsl2312758 rsl653585 rsl2304035 rs208298 rsl2314721 rsl718124 rsl2310654 rs208302 rsl2830607 rsl718125 rsl2312695 rs208305 rsl7434640 rsl718162 rsl2312758 rs208306 rsl7434647 rsl7434731 rsl2314721 rs208307 rsl7512249 rsl7434809 rsl2321707 rs208310 rs208280 rsl7525767 rsl2813980 rs208311 rs208282 rsl7525809 rsl2815078 rs2230912 rs3861798 rs2051551 rsl2821688 rs2567988 rs487417 rs208288 rsl2829218 rs2567989 rs535557 rs208290 rsl2830607 rs2567991 rs560422 rs208292 rsl653583 rs2686369 rs568531 rs208293 rsl653585 rs2686371 rs591874 rs208294 rsl653598 rs2857589 rs594423 rs2230912 rsl653601 rs2857590 rs656612 rs2686369 rsl653602 rs2857592 rs669547 rs2686371 rsl653609 rs487417 rs7310821 rs2857589 rsl653610 rs503720 rs7959194 rs3751143 rsl653614 rs504677 rs9805004 rs6489794 rsl653618 rs507085 rs7973360 rsl69631 rs535557

IS1718119 rs568531 rsl718124 rs594423 rsl718133 rs656612 rsl718134 rs669547 rsl718135 rs7137837 rsl718136 rs7958311 rsl718162 rs891781

Claims

Claims:

1. A method for haplotyping P2X7 gene in a human individual comprising the steps of: a) treating nucleic acid from a sample that has been removed from the individual so as to identify the nucleotides present at each allelic position of an "atherosclerosis- susceptibility haplotype", which haplotype includes one or both of the P2X7 gene SNPs rsl69631, or a polymorphism in linkage disequilibrium above D' 0-9 therewith, and rs3751143 , or a polymorphism in linkage disequilibrium above D'

0.9 therewith, and b) assigning the individual a particular haplotype according to the nucleotides detected in step a).

2. The method as claimed in claim 1, wherein the haplotype comprises 2 or 3 alleles.

3. The method as claimed in 1 or 2, wherein at least two of the SNPs making up the haplotype are located within the P2X7 gene.

4. The method as claimed in any of claims 1 - 3, wherein all of the SNPs making up the haplotype are located within the P2X7 gene.

5. The method as claimed in any of claims 1 to 4, wherein the haplotype includes the SNPs rsl69631 and rs3751143.

6. The method as claimed in claim, wherein the haplotype is selected from the group consisting of: haplotypes 1 to 15.

7. The method as claimed in claim 1, further comprising determining the individual's potential risk to develop atherosclerosis or an atherosclerosis-mediated disease according to the particular haplotype that the individual possesses.

8. The method as claimed in claim 7, wherein the particular haplotype present predicts a lower predisposition to develop atherosclerosis and atherosclerosis mediated disease compared to a control.

9. The method as claimed in claim 8, wherein the haplotype is one selected from the group consisting of: 3-8/G-A, 3-8-9/G-A-A, 1-3-8/C-G-A, 2-3-8/C-G-A, 3-4-8/G-C-A, 3- 5-8/G-C-A, 3-6-8/G-G-A and 3-7-8/G-C-A.

10. The method as claimed in claim 7, wherein the particular haplotype present predicts a higher predisposition to develop atherosclerosis and atherosclerosis mediated disease compared to a control.

11. The method as claimed in claim 10, wherein the haplotype is one selected from the group consisting of: 3-7-8/G-C-C, 3-7-8/C-C-A, 1-3-8/T-C-A and 1-3-8/C-G-C.

12. The method as claimed in any of claims claim 7 to 11, wherein atherosclerosis- mediated disease is selected from the group consisting of: myocardial infarction (MI), stroke or peripheral vascular disease (PVD).

13. The method as claimed in any of the preceding claims, wherein the individual has suffered a previous myocardial infarction event.

14. The method as claimed in any of the preceding claims, wherein the individual is male.

15. The method according to claim 1 , wherein the nucleotide present at an allelic position is identified by a method that comprises enzymatic amplification of nucleic acid from said individual.

16. The method as claimed in claim 15, wherein said amplification is polymerase chain reaction.

17. The method as claimed in claim 15, wherein said amplification is allele-specifϊc amplification.

18. The method as claimed in claim 1 , wherein the nucleotides present at the allelic positions are determined by sequence analysis.

19. A method for selecting individuals for participation in a clinical trial to assess the efficacy of a drug for treating atherosclerosis or an atherosclerosis-mediated disease, comprising: grouping the individuals according to the particular P2X7 gene haplotype that each individual belongs to and selecting members from one or more haplotype grouped individuals for inclusion in the clinical trial.

20. The method as claimed in claim 19, wherein the particular haplotype group that an individual belongs to is determined by identifying the nucleotide present at each polymorphic position in an atherosclerosis-mediated haplotype on one or both copies of the relevant chromosome in a sample that has previously been removed from the individual, which haplotype includes the SNP rs 169631 or the SNP rs3751143 and at least one other SNPs

21. A method for selecting individuals for participation in a clinical trial to assess the efficacy of a drug for treating atherosclerosis or an atherosclerosis-mediated disease, comprising:

(a) individually haplotyping the P2X7 gene at two or more allele positions from a nucleic acid containing sample already isolated from each individual, wherein at least one of the two or more allele positions is selected from the group consisting of: is rsl69631 and rs3751143,

(b) grouping the individuals according to the particular P2X7 gene haplotype that each individual belongs to, and

(c) selecting individuals from one or more haplotype groups for inclusion in the clinical trial.

22. Use of a diagnostic kit capable of haplotyping P2X7 gene at at least three allele positions, at least one of which is rs 169631 or rs3751143, to stratify individuals into particular haplotype groups.

23. Use as claimed in claim 22, which further comprises selecting members from one or more of the stratified groups for inclusion in a clinical trial to determine the efficacy of a drug.

24. Use as claimed in claim 23, wherein the clinical trial is measuring the efficacy of the drug at treating atherosclerosis or an atherosclerosis mediated disease.

25. An in vitro diagnostic kit for determining the identity of SNPs rs 169631 and rs3751143 in the human P2X7 gene, said kit comprising components for the determination of said SNPs.