JP2007097569A - Method for detecting genotype relating to coronary artery disease by gene analysis of human p2y12 receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means enabling a thrombotic disease to be diagnosed at an early stage and the morbid risk thereof to be judged. <P>SOLUTION: The method for detecting the gene having possibility of causing the coronary artery disease comprises means identifying that the haplotype of human P2Y<SB>12</SB>receptor gene is a homozygote of H1 haplotype, and the genotype of the 152539314th position of the genomic base sequence of the gene is represented by C/C, (the position is defined as the position of the genomic base sequence described in Gen Bank accession number NC_000003.9, but the position corresponds to the 152377525th position of the genomic base sequence described in Gen Bank accession number NC_000003.8), and the invention relates to the method for detecting a gene having possibility of causing coronary artery disease, the method for judging morbid risk thereof by using the method, a method for identifying a compound inducing desensitization on thrombocyte and a polynucleotide and reagents kit to be used in the method for the diagnosis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention is a gene encoding human P2Y ₁₂ receptor by analysis of one base mutation site (hereinafter, human P2Y referred to as ₁₂ receptor gene), a method for detecting the genotype with the potential to cause coronary artery disease. Table More particularly, the present invention is to identify the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, and a 152 539 314 of the genotype of the genomic nucleotide sequence of said gene in C / C The present invention relates to a method for detecting a genotype having a possibility of causing coronary artery disease, wherein the position of the genome base sequence at position 15253314 is GenBank accession number NC — 000003.9. (This position corresponds to position 1523777525 in the genome base sequence described in GenBank Accession No. NC — 0000032.8). The present invention also relates to a test method for coronary artery disease risk using the detection method. The invention further relates to a method for identifying compounds that induce platelet desensitization. The present invention also relates to a polynucleotide and a reagent kit used in the detection method and the inspection method.

The P2Y ₁₂ receptor is a receptor that activates platelets by binding to adenosine 5 ′ diphosphate (hereinafter abbreviated as ADP) (Non-patent Documents 1 and 2). While platelet activation is an important physiological phenomenon related to hemostasis, it also involves thrombosis and causes vascular occlusion and ischemic damage.

ADP is contained in darkly-stained granules in platelets, and is released to the outside of cells as platelets are activated. The released ADP promotes platelet aggregation by binding to two ADP receptors present around the platelet surface (P2Y ₁ receptor and _{P2Y 12} receptor). The P2Y ₁ receptor is coupled to Gq, which is a trimeric G protein, and is involved in the initial reaction of platelet aggregation such as morphological change through an increase in intracellular calcium concentration. P2Y ₁₂ receptors are Gi and a conjugated a G protein is involved in growth and stabilization of the platelet clumps via activation of the Rap1b and serine-threonine kinase of the low molecular weight G protein Akt.

Human _{P2Y 12} receptor gene is a gene present in chromosome 3 q24-q25 locus. The gene contains two exons, the total length is about 3.1 kb, and encodes a protein consisting of 342 amino acids.

Polymorphisms in the human P2Y ₁₂ receptor gene, called as H1 haplotype and H2 haplotypes have been reported (Patent Document 1, Non-Patent Document 3 and Non-Patent Document 4). The H1 haplotype has a high allele frequency and is considered to be a normal genotype. On the other hand, it is reported that ADP-induced platelet aggregation ability is increased in individuals having H2 haplotype, and that H2 haplotype is a risk factor for developing peripheral arterial injury (Non-patent Document 3 and Non-patent Document 4). . P2Y ₁₂ platelet aggregation in individuals with of H2 haplotype receptor gene not clear the mechanism to enhance. Perhaps, P2Y ₁₂ receptor numbers on the cell surface are presumed that it would be increasing.

The H2 haplotype of the human _{P2Y 12} receptor gene, there are four kinds of single base mutations (Table 1). Hereinafter, these single base mutations are referred to as known single base mutations. It is disclosed that the H2 haplotype can be identified by identifying these known single-base mutations (Patent Document 1). Further, it is disclosed that there is a high correlation between H2 haplotype and peripheral arterial disease (hereinafter sometimes abbreviated as PAD) (Patent Document 1). Furthermore, the particular H2 haplotype, morbidity risk of atherothrombosis and thienopyridine antithrombotic agents (e.g., P2Y ₁₂ receptor clopidogrel or ticlopidine or the like to target) disclosed that is considered to be performing the detection of the low sensitivity to (Patent Document 1).

In Table 1, the position of one base mutation of the human P2Y ₁₂ receptor gene was expressed as a position in the genome sequence of the chromosome 3 there is a human P2Y ₁₂ receptor gene.

Chromosome 3 of the base sequence present human _{P2Y 12} receptor gene, as GenBank Accession No. NC_000003, disclosed in NCBI (National Center for Biotechnology Information) public databases. The base sequence disclosed in GenBank Accession No. NC — 000003 was revised on August 24, 2004. The version before August 24, 2004 is NC_000003.8 [gi: 42406220] (referred to as NC_000003.8), and the subsequent version is NC_000002.9 [gi: 51511463] (referred to as NC_000003.9). is there.

Thus, in Table 1, 1 a position at which base mutation is present, a third chromosome of the nucleotide sequence present human _{P2Y 12} receptor gene, that is, the position in the nucleotide sequence disclosed in GenBank accession number NC_000003.8, and Expressed as a position in the base sequence disclosed in GenBank Accession No. NC — 000003.9.

Others 1 base mutation of the human _{P2Y 12} receptor gene, the human _{P2Y 12} receptor gene genome sequence of the 152 539 314 (this position is defined as the position in the genome sequence described in GenBank accession number NC_000003.9 but In this position, a mutation from the base cytosine (C) to thymine (T) of the base of the genomic base sequence described in GenBank accession number NC_000003.8 (corresponding to position 1523777525) is known. This single nucleotide mutation has been reported to be associated with an increase in cerebrovascular events in peripheral arterial disease patients receiving clopidogrel treatment (Non-patent Document 5). Specifically, in patients with peripheral arterial disease receiving clopidogrel treatment, patients who show side effects of the nervous system, such as patients who develop ischemic cerebral infarction, patients who have undergone carotid stenting or arteriosclerotic incision, It has been reported that patients with this single nucleotide mutation are about 4 times higher than patients without this single nucleotide mutation. On the other hand, there is a report that the platelet aggregation ability of patients having this single nucleotide mutation is lower than that of patients not having this single nucleotide mutation, and the risk of myocardial infarction is low (Non-patent Document 6). . Further, the one base mutation, it is the H1 / H2 haplotype of the human P2Y ₁₂ receptor gene considered unlinked (Non-Patent Document 3 and Non-Patent Document 7), and the one base mutation and ADP-induced platelet It has been reported that there is no relation with aggregation (Non-patent Document 3).

  The hemostatic mechanism is a physiological mechanism that works under close interaction such as blood vessel function, platelet function, coagulation / fibrinolytic system, etc. when a blood vessel is damaged in a living body. Abnormalities in the hemostasis mechanism cause various bleeding diseases and thrombotic diseases. In pathophysiological conditions, for example, rupture of atherosclerotic plaques causes pathological platelet activation, resulting in the formation of arterial thrombus, which can lead to myocardial infarction, ischemic stroke, and peripheral arterial disease .

  Thrombotic diseases are diseases caused by stenosis or occlusion of blood vessels due to thrombus and can be classified into thrombosis and embolism. Thrombosis is a symptom caused by partial or complete blockage of the blood flow at the site where the thrombus is formed, and embolism is caused by removal of the thrombus from the formation site and movement by the blood flow. A symptom caused by partial or complete blockage of a flow. Coronary artery disease, peripheral arterial disease (PAD), cerebral infarction, and deep vein thrombosis can be exemplified as diseases accompanied by prothrombotic blood coagulation abnormalities.

  Coronary artery disease (hereinafter sometimes abbreviated as CAD) is known as a disease caused by occlusion of the coronary artery. CAD is also called ischemic heart disease, and blood supply to the myocardium is remarkably reduced, causing symptoms such as myocardial infarction, angina pectoris or arrhythmia.

  Ischemic stroke is an ischemic cerebrovascular disorder and includes cerebral infarction and transient cerebral ischemic attack. Cerebral infarction accounts for a high percentage of strokes. Cerebral infarction is roughly classified into two types, namely, those caused by cerebral thrombosis and those caused by cerebral embolism. Cerebral thrombosis is caused by cerebral blood vessels narrowing due to changes such as arteriosclerosis, resulting in poor blood flow and formation of thrombi. Cerebral embolism is caused by clogging of the cerebral blood vessels as a result of the thrombus formed in the heart or carotid artery being carried to the cerebral blood vessels by the blood flow. Cerebral infarction is classified as cardiogenic or non-cardiogenic. Anticoagulants have been shown to be particularly effective in cardiogenic cerebral infarction, and antiplatelet drugs in noncardiogenic cerebral infarction.

  Peripheral artery disease (PAD) is known as a disease caused by obstruction of the peripheral artery. PAD is based on atherosclerosis in most patients and is thought to be very close in pathophysiology to cerebral infarction excluding CAD and cardiogenicity. Acute ischemia is caused by rupture of the proximal atherosclerotic plaque, acute thrombosis due to preexisting atherosclerosis, emboli flying from the heart, aorta or other large vessels, and aneurysm dissection. Chronic ischemia is said to be caused by the gradual expansion of atherosclerotic plaque. In this condition, antiplatelet drugs are effective.

  Venous thrombosis can occur in any superficial or deep veins of the body, but deep vein thrombosis (Deep Vein Thrombosis, hereinafter referred to as DVT), such as the leg vein, femoral vein, and deep pelvic vein ) Is frequent and clinically important as it can cause fatal pulmonary embolism. In DVT, collagen is exposed due to injury of vascular endothelial cells, and tissue thromboblastin is released. It is considered that blood clotting is promoted and blood clots are formed when stasis is accompanied here. In this pathological condition, it is known that anticoagulants (such as heparin and warfarin) and antiplatelet drugs (such as aspirin) are effective in preventing thrombus formation and expansion.

International Patent Publication WO 2004/035826 Pamphlet. Dorsam, RT, et al., "The Journal of Clinical Investigation", 2004, Vol. 113, No. 3, p. 340-345. Kunapuli, SP, et al., “Biochemical Journal”, 1998, Vol. 336, p. 513-523. Fontana, P. et al., “Circlation”, 2003, 108, p. 989-995. Fontana, P. et al., “Circlation”, 2003, 108, p. 2971-2933. Ziegler, S. et al., “STROKE”, 2005, 36, p. 1394-1399. Von Veckerath N. et al., “Blood Coagulation Fibrinolysis” 2005, Vol. 16, p. 199-204.

The gene encoding the central role human P2Y ₁₂ receptor in platelet aggregation is the initial reaction in thrombus formation, genetic polymorphism called as H1 haplotype and H2 haplotypes have been reported. In individuals with the H2 haplotype, enhanced platelet aggregation ability has been observed (Non-Patent Document 3 and Non-Patent Document 4). Furthermore, the human _{P2Y 12} receptor gene genome sequence of the 152 539 314 (this position is defined as the position in the genome sequence described in GenBank accession number NC_000003.9, the present position is described NC_000003.8 genome It has been reported that a cytosine (C) to thymine (T) mutation in the base sequence (corresponding to position 1523777525 in the base sequence) is associated with an increase in cerebrovascular events in PAD patients receiving clopidogrel treatment. (Non-Patent Document 5).

  However, the relationship between complex elements such as haplotypes and single nucleotide mutations and platelet reactivity and the relationship between complex elements such as haplotypes and single nucleotide mutations and diseases caused by thrombus formation have been clarified. Not.

  Thrombotic diseases resulting from thrombus formation, such as coronary artery disease and stroke, are diseases that account for a large proportion of human deaths. Coronary artery disease and stroke can be prevented by correcting factors that increase the risk of developing these diseases, such as smoking, high blood pressure, high fat / high cholesterol diet, obesity, and stress. Early diagnosis of the disease is desired to enable avoidance of the risk of developing these diseases, early treatment, selection of treatment methods and dose setting of drugs.

An object of the present invention is to clarify the genotype of the human P2Y ₁₂ receptor gene associated with thrombotic disease, thereby to provide a means for enabling an early diagnosis and morbidity risk factor determination of the disease.

The present inventors have analyzed the association between genotype and the platelet aggregation ability and thrombotic disorders of the human P2Y ₁₂ receptor gene, the human P2Y ₁₂ receptor gene, which can be considered to have coronary artery disease and highly relevant As a result, the present invention was completed.

Genotype of the human P2Y ₁₂ receptor gene analysis was carried out by identifying the human P2Y ₁₂ receptor gene haplotype and 1 nucleotide mutation site nucleotides within the gene.

Identification of the human _{P2Y 12} receptor gene haplotype and 1 base mutation site within the gene nucleotide as a template for the human _{P2Y 12} receptor gene, genetic mutations and haplotypes are not considered GenBank Accession No. NC_000003 gene sequence ( a third chromosomal nucleotide sequence, using the included) genomic sequence of the human P2Y ₁₂ receptor gene was performed Japanese genomic sample as a population. This template sequence may be referred to as a reference sequence. In the present specification, the single base mutation site is defined as the position of the base in the reference sequence.

  The version of the nucleotide sequence disclosed in the NCBI public database as GenBank accession number NC — 000003 was revised on August 24, 2004. The version before August 24, 2004 is NC_000003.8 [gi: 42406220] (referred to as NC_000003.8), and the subsequent version is NC_000002.9 [gi: 51511463] (referred to as NC_000003.9). is there.

Position of the region containing the human _{P2Y 12} receptor gene is shifted to 161789bp downstream as compared to those disclosed in GenBank accession number NC_000003.8 the nucleotide sequence disclosed in GenBank accession number NC_000003.9 . Accordingly, the position of each base in the region containing the human _{P2Y 12} receptor gene in the nucleotide sequence disclosed in GenBank accession number NC_000003.9 are each such in the nucleotide sequence disclosed in GenBank accession number NC_000003.8 Compared to the position of the base, it is located 161789 bp downstream.

Region containing the human _{P2Y 12} receptor gene, corresponds from the 152,544,089 of the base sequence disclosed in GenBank accession number NC_000003.9 at position No. 152537783. On the other hand, this region corresponds to positions 152382300 to 15235994 in the base sequence disclosed in GenBank accession number NC_000003.8.

Region containing the human _{P2Y 12} receptor gene, namely a base sequence from the 152 544 089 largest position first 152,537,783 of the nucleotide sequence disclosed in GenBank accession number NC_000003.9, are disclosed in GenBank accession number NC_000003.8 The base sequences from position 15238300 to position 15235994 of the base sequence are completely identical. Thus, the revision before and after versions of GenBank Accession No. NC_000003, the base sequence of the human _{P2Y 12} receptor gene has not changed.

Thus, the region containing the human _{P2Y 12} receptor gene, since its position with GenBank accession number NC_000003.8 and GenBank accession number NC_000003.9 is only different, GenBank accession number NC_000003.8 The position of the single base mutation in the human P2Y12 receptor gene indicated as the position in the human chromosome 3 nucleotide sequence disclosed in the above is the human chromosome 3 nucleotide sequence disclosed in GenBank Accession No. NC — 000003.9 corresponding to the position Although the base number indicating the position is different when indicated as the position in, it is the same single base mutation that is specified by each base number.

In the present specification, unless otherwise defined, the base sequence disclosed in GenBank Accession No. NC — 000003.8 is used as a reference sequence, and the position of each base of the human P2Y ₁₂ receptor gene is GenBank Accession No. NC — 000003. This is shown as the position in the base sequence disclosed in FIG. In the base sequence disclosed in GenBank accession number NC_000003.9, each of these bases is located 161789 bp downstream of the position in the base sequence disclosed in GenBank accession number NC_000003.8. That is, the position of each base is the base number obtained by adding 161789 to the base number in the base sequence disclosed in GenBank Accession Number NC_000003.8 in the base sequence disclosed in GenBank Accession Number NC_000003.9. Can be expressed as

For the location of each base of the human _{P2Y 12} receptor gene, which will be described later, the position in the nucleotide sequence disclosed in GenBank accession number NC_000003.8, the position in the nucleotide sequence disclosed in GenBank accession number NC_000003.9 Are shown in Table 2-1, Table 2-2, Table 2-3, and Table 2-4.

As described above, the base number indicating the position of each base of the human P2Y ₁₂ receptor gene is determined according to the version of GenBank accession number NC — 000003 which discloses the human chromosome 3 base sequence, and the human P2Y ₁₂ receptor in that version. It may be different because the location of the region containing the gene may shift. Also, the basic number indicating the position of each base of the human _{P2Y 12} receptor gene, when used as a reference sequence to another nucleotide sequence is the nucleotide sequence disclosed in GenBank Accession No. NC_000003, the GenBank accession number NC_000003 It may be different from that in the disclosed base sequence.

When used as a reference sequence to another nucleotide sequence is the nucleotide sequence disclosed in GenBank accession number NC_000003.8 or NC_000003.9, 1 base mutation site of human _{P2Y 12} receptor gene in said reference sequence, then Can be specified as shown. First, the partial base sequence of the base sequence disclosed in GenBank accession number NC_000003.8 or NC_000003.9, using the partial base sequence including the nucleotide of the single base mutation site as an index, is homologous to the partial base sequence. Is determined by identifying the single nucleotide mutation site in the reference sequence by comparing the partial base sequence with a base sequence homologous to the partial base sequence in the reference sequence. be able to. Searching a reference sequence for a base sequence homologous to a partial base sequence of the base sequence disclosed in GenBank Accession No. NC_000003.8 or NC_000003.9 is a homology search method known per se, such as blast search (BLAST search). ). The partial base sequence of the base sequence disclosed in GenBank accession number NC_000003.8 or NC_000003.9 and the base sequence homologous to the partial base sequence in the reference sequence do not have to be completely homologous. As long as a plurality of base sequences homologous to the partial base sequence do not exist therein, one to several bases may be different.

  Specifically, position 1523777525 of the base sequence disclosed in GenBank accession number NC_000003.8 shows the position in the reference sequence when a base sequence different from the base sequence is used as the reference sequence. Can be specified. First, a base sequence homologous to the base sequence described in SEQ ID NO: 62 of the sequence listing (partial base sequence of the base sequence disclosed in GenBank Accession No. NC — 000003.8) is used as an index in the reference sequence Find out. Position No. 1523777525 of the base sequence disclosed in GenBank Accession No. NC — 000003.8 corresponds to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the Sequence Listing. Therefore, by comparing the base sequence set forth in SEQ ID NO: 62 of the sequence listing with the base sequence homologous to the base sequence in the reference sequence, the sequence listing in the base sequence homologous to the base sequence in the reference sequence The site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 can be identified, and the position of the identified site in the reference sequence can be determined.

  In addition, the position of the base sequence disclosed in GenBank accession number NC_000003.8 is identified as shown below when a base sequence different from the base sequence is used as a reference sequence. can do. First, a base sequence homologous to the base sequence described in SEQ ID NO: 58 of the sequence listing (partial base sequence of the base sequence disclosed in GenBank Accession No. NC — 000003.8) is used as a reference sequence. Find out. The position No. 152379350 of the nucleotide sequence disclosed in GenBank Accession No. NC — 000003.8 corresponds to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 58 in the Sequence Listing. Therefore, by comparing the base sequence set forth in SEQ ID NO: 58 of the sequence listing with the base sequence homologous to the base sequence in the reference sequence, the sequence listing in the base sequence homologous to the base sequence in the reference sequence A site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 58 can be identified, and the position of the identified site in the reference sequence can be determined.

  When using a base sequence different from the base sequence as the reference sequence, position No. 152377507 in the base sequence disclosed in GenBank accession number NC_000003.8 is to be specified as shown below. Can do. First, a base sequence homologous to the base sequence described in SEQ ID NO: 62 of the sequence listing (partial base sequence of the base sequence disclosed in GenBank Accession No. NC — 000003.8) is used as an index in the reference sequence Find out. No. 152377507 of the nucleotide sequence disclosed in GenBank Accession No. NC — 000003.8 corresponds to the 69th nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 in the Sequence Listing. Therefore, by comparing the base sequence set forth in SEQ ID NO: 62 of the sequence listing with the base sequence homologous to the base sequence in the reference sequence, the sequence listing in the base sequence homologous to the base sequence in the reference sequence A site corresponding to the 69th nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 can be identified, and the position of the identified site in the reference sequence can be determined.

Useful nucleotide sequence information to determine the single base mutation site of the human _{P2Y 12} receptor gene in a reference sequence, the partial base sequence information of the nucleotide sequence disclosed in NC_000003.8 or NC_000003.9, partial base sequence information Table 3 shows the 1 base mutation site of human _{P2Y 12} receptor gene in.

In Table 3, "*" Section 152,378,495 of the base sequence disclosed in GenBank accession number NC_000003.8 displaying at, as shown in Table 1, the genotype of the human _{P2Y 12} receptor gene H2 haplotype , The base of the nucleotide at that position is A, but if it is the H1 haplotype, the nucleotide at that position is missing. In such a case, the base sequence disclosed in GenBank Accession No. NC_000003.8 is positioned at position 15278495 (sometimes referred to as the target base position) using a base sequence different from the base sequence as a reference sequence. When used, the position in the reference sequence can be specified as shown below. First, using a base sequence described in SEQ ID NO: 60 (partial base sequence of base sequence disclosed in GenBank Accession No. NC — 000003.8) as an index, a base sequence homologous to the base sequence in the reference sequence Find out. Next, the position of the target base in the reference sequence can be identified using the site corresponding to the 107th to 108th nucleotides (AA) of the base sequence shown in SEQ ID NO: 60 of the sequence listing as an index.

The present inventors have found that was performed by the analysis of the genotype of the human P2Y ₁₂ receptor gene identifying one base mutation site at nucleotide haplotype and said the genes of the gene, the human genome P2Y ₁₂ receptor gene It was clarified that four types of single nucleotide mutations existing upstream of exon 2 in the nucleotide sequence were linked to the H2 haplotype of the gene.

As a result of analyzing the aggregation to low concentrations of ADP human platelets, homozygous haplotypes of the human _{P2Y 12} receptor gene H1 haplotype and second 152,539,314 of genomic nucleotide sequence of the gene (this position GenBank accession number NC_000003.9 is defined as a position in the genomic base sequence described in GenBank accession number NC_000003.8, and this position corresponds to the 1532777525th position in the genomic base sequence described in GenBank accession number NC_000003.8). It has been found that platelets derived from individuals having a genotype represented by C / C have a lower platelet aggregation ability than platelets derived from individuals not having these genotypes. On the other hand, almost no difference was observed in the aggregation ability for high concentration ADP between the former-derived platelets and the latter-derived platelets.

Haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype and second 152,539,314 of genomic nucleotide sequence of the gene (the present position is located in the genome nucleotide sequence described in GenBank accession number NC_000003.9 (This position is equivalent to position 1523777525 in the genomic base sequence described in GenBank Accession No. NC_000003.8). The platelet derived from an individual whose genotype is represented by C / C is As described above, the aggregating ability at the time of ADP stimulation is low, and therefore, it is difficult to activate and desensitize with a small amount of ADP constantly generated in a living blood vessel. Desensitization means a state in which platelets are not sufficiently reacted (aggregated) even if stimulated with a P2Y ₁₂ receptor agonist such as ADP and then stimulated again with the agonist. On the other hand, platelets derived from individuals that do not have the genotype as described above are highly reactive to ADP, and thus continuously react with trace amounts of ADP produced in living blood vessels and fall into a desensitized state. Cheap.

  Platelets are exposed to high concentrations of ADP at the onset of coronary artery disease. At this time, desensitized platelets cannot react sufficiently to ADP and form fragile and small thrombi, whereas platelets that are difficult to desensitize react sensitively to ADP and form strong and large thrombi. It is thought to contribute to the onset and worsening of coronary artery disease.

Haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype and second 152,539,314 of genomic nucleotide sequence of the gene (the present position is located in the genome nucleotide sequence described in GenBank accession number NC_000003.9 (This position is equivalent to position 1523777525 in the genomic base sequence described in GenBank Accession No. NC_000003.8). The platelet derived from an individual whose genotype is represented by C / C is Because of its low agglutination ability for low-concentration ADP, it is difficult to desensitize a small amount of ADP produced in a living blood vessel. As a result, it reacts sensitively to high-concentration ADP exposed at the onset of coronary artery disease, Of coronary artery disease and its worse This is thought to contribute to

In fact, in coronary artery disease patients, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and the genomic nucleotide sequence of the 152,539,314 of the gene (This position is described in GenBank accession number NC_000003.9 This position is a genotype represented by C / C. It has been found in the present invention that a certain frequency is significantly higher than that in healthy individuals.

The present inventors have found that these findings are homozygous haplotypes of the human _{P2Y 12} receptor gene H1 haplotype, and genome sequence of the 152,539,314 of the gene (present position GenBank accession number NC_000003.9 Is defined as a position in the genome base sequence described in the above, but this position corresponds to position 1523777525 in the genome base sequence described in GenBank Accession No. NC — 0000032.8). We believe that individuals with a genotype are more likely to cause coronary artery disease than individuals without such a genotype. The identification of H1 haplotype of the human P2Y ₁₂ receptor gene, and, utilizing the detection of the 152,539,314 of the genotype of the genomic nucleotide sequence of the gene, the method of detecting genotypes with the potential to cause coronary artery disease And the present invention was completed by using the method for early diagnosis of the disease and determination of morbidity risk.

That is, the present invention is to identify the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, and a 152 539 314 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C The present invention relates to a method for detecting a genotype having the possibility of causing coronary artery disease, comprising the following steps, wherein the means comprises the following steps (here, the 15325314th position of the genome base sequence is GenBank accession): (This position is defined as the position in the genome base sequence described in session number NC_000003.9, which corresponds to position 1523777525 in the genome base sequence described in GenBank accession number NC_000003.8):
(I) a DNA fragment containing the human _{P2Y 12} receptor gene haplotype 1 base mutation site at nucleotide that can determine, and polymerase chain reaction a DNA fragment containing the first 152 539 314 of the nucleotides of the genomic nucleotide sequence of the gene (PCR) Amplifying by,
And (ii) The sequence of the PCR product obtained in the step (i) is determined by a sequencing method, or the base at the above position is determined by a typing method for the PCR product obtained in the step (i). Process.

The present invention also the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and / Alternatively, it is identified by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 in the genomic base sequence of the gene is represented by C / C. The present invention relates to a method for detecting a genotype having the possibility of causing coronary artery disease, comprising detecting the genotype as a means, wherein the means comprises the following steps (here, positions 152541139 and 1525329296 of the genomic base sequence): No. and 15253314th place are described in GenBank accession number NC_000003.9 Are defined as positions in the genomic base sequence, which correspond to positions 152379350, 152377507, and 1523777525 of the genomic base sequence described in GenBank Accession No. NC — 0000032.8):
(I) the human _{P2Y 12} receptor gene DNA fragment comprising the first 152 541 139-position and / or the 152 539 296 of the nucleotides of the genomic nucleotide sequence and the gene of the genomic nucleotide sequence polymerase chain The DNA fragment containing the first 152 539 314 of the nucleotides of, Amplifying by reaction (PCR);
And (ii) The sequence of the PCR product obtained in the step (i) is determined by a sequencing method, or the base at the above position is determined by a typing method for the PCR product obtained in the step (i). Process.

The present invention further that haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and / Alternatively, it is identified by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 in the genomic base sequence of the gene is represented by C / C. a means to detect that a genotype, said means comprises first 152,541,139 of genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps A method for detecting a genotype having a possibility of causing coronary artery disease (where, Positions 41139, 15253296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are the genome base sequences described in GenBank Accession No. NC — 000003.8, respectively. Corresponds to positions 15379350, 1523777507 and 1523777525):
(I) amplified by a DNA fragment containing the first 152 541 139 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 17 and SEQ ID NO: 34 (PCR) The process of
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152541139 is determined by typing using the polynucleotide of SEQ ID NO: 41 or 43 as a primer Process.

The invention further also that haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and And / or by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is represented by C / C. that a means to detect that a genotype, detects that said means is a genotype which first 152,539,296 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is expressed in G / G by the following steps A method for detecting a genotype having a possibility of causing coronary artery disease (where the first of the genomic base sequence Positions 2541139, 1525392996, and 1525339314 are defined as positions in the genomic base sequence described in GenBank Accession No. NC — 000003.9. These positions are the genomic base sequences described in GenBank Accession No. NC — 0000032.8, respectively. Corresponds to positions 15379350, 1523777507 and 1523777525):
(I) amplified by a DNA fragment containing the first 152 539 296 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 23 and SEQ ID NO: 40 (PCR) The process of
And (ii) determining the base at the position corresponding to position 15253296 of the PCR product obtained in the step (i) by a typing method using the polynucleotide of SEQ ID NO: 42 as a primer.

The present invention also the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and / Alternatively, it is identified by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 in the genomic base sequence of the gene is represented by C / C. a means to detect that a genotype, said means comprises first 152,539,314 of genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps A method for detecting a genotype having a possibility of causing coronary artery disease, wherein The 1139th position, the 1525392996 position and the 1525339314 position are defined as the positions in the genome base sequence described in the GenBank accession number NC — 000003.9. These positions are the genomic base sequences described in the GenBank accession number NC — 000003.8, respectively. Corresponds to positions 15379350, 1523777507 and 1523777525):
(I) amplified by a DNA fragment containing the first 152 539 314 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 23 and SEQ ID NO: 40 (PCR) The process of
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 15253314 is determined by typing using the polynucleotide of SEQ ID NO: 50 or 55 as a primer Process.

The present invention further that haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and / Alternatively, it is identified by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 in the genomic base sequence of the gene is represented by C / C. a means to detect that a genotype, said means comprises first 152,541,139 of genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps A method for detecting a genotype having a possibility of causing coronary artery disease (where, Positions 41139, 15253296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are the genome base sequences described in GenBank Accession No. NC — 000003.8, respectively. Corresponds to positions 15379350, 1523777507 and 1523777525):
(I) amplified by a DNA fragment containing the first 152 541 139 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 5 and SEQ ID NO: 6 (PCR) The process of
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 18 and SEQ ID NO: 34 as primers.

The invention further also that haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and And / or by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is represented by C / C. that a means to detect that a genotype, detects that said means is a genotype which first 152,539,296 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is expressed in G / G by the following steps A method for detecting a genotype having a possibility of causing coronary artery disease (where the first of the genomic base sequence Positions 2541139, 1525392996, and 1525339314 are defined as positions in the genomic base sequence described in GenBank Accession No. NC — 000003.9. These positions are the genomic base sequences described in GenBank Accession No. NC — 0000032.8, respectively. Corresponds to positions 15379350, 1523777507 and 1523777525):
(I) a DNA fragment containing the first 152 539 296 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, according to SEQ ID NO: 7 or SEQ ID NO: 51 polynucleotide, and, according to SEQ ID NO: 8 or SEQ ID NO: 52 Amplifying by polymerase chain reaction (PCR) using the polynucleotide as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method.

The present invention also the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype, it first 152,541,139 of the genomic nucleotide sequence of the gene is a gene type represented by C / C and / Alternatively, it is identified by detecting that the genotype at position 1525392996 is a genotype represented by G / G, and the genotype at position 15253314 in the genomic base sequence of the gene is represented by C / C. a means to detect that a genotype, said means comprises first 152,539,314 of genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps A method for detecting a genotype having a possibility of causing coronary artery disease, wherein The 1139th position, the 1525392996 position and the 1525339314 position are defined as the positions in the genome base sequence described in the GenBank accession number NC — 000003.9. These positions are the genomic base sequences described in the GenBank accession number NC — 000003.8, respectively. Corresponds to positions 15379350, 1523777507 and 1523777525):
(I) a DNA fragment containing the first 152 539 314 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, according to SEQ ID NO: 7 or SEQ ID NO: 51 polynucleotide, and, according to SEQ ID NO: 8 or SEQ ID NO: 52 Amplifying by polymerase chain reaction (PCR) using the polynucleotide as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method.

  The present invention further relates to a method for examining the risk of coronary artery disease, characterized by using a method for detecting a genotype having the possibility of causing any of the above-mentioned coronary artery diseases.

The invention further genes are homozygous for the haplotype H1 haplotype a human P2Y ₁₂ receptor gene, and a 152 539 314 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C and platelets to hold the protein shown by the amino acid sequence encoded by the P2Y ₁₂ receptor gene is a type (platelet a), control platelets (platelet B, where platelets B is any one selected from the following group 1 is platelets for holding a protein shown by the amino acid sequence encoded by the P2Y ₁₂ receptor gene: (i) a human P2Y ₁₂ receptor gene homozygous in its haplotype H1 haplotype and, The genotype of the gene at position 15253314 of the genomic base sequence is C / T or T / T; (ii) human P2 A Y ₁₂ receptor gene that haplotype is H1 / H2 haplotype heterozygotes, and genome sequence No. 152539314 of the genotype of the gene is C / C, C / T or T / T, ; and (iii) homozygous for the haplotype H2 haplotype a human _{P2Y 12} receptor gene, and the genomic nucleotide sequence No. 152539314 of genotypes C / C in the gene, C / T or, T / T) with a human P2Y ₁₂ receptor agonist in the presence of a compound, respectively, to induce platelet aggregation, followed by a dose-dependent curve of platelet aggregation by platelet A and platelet B and comparing the dose-dependent curve of platelet aggregation agglutination, induces desensitization of platelets to human P2Y ₁₂ receptor agonists Here, position 15253314 of the genomic base sequence is defined as a position in the genomic base sequence described in GenBank accession number NC_000003.9, but this position is defined in GenBank accession number NC_000003.8. Corresponding to position 15237775 in the described genomic nucleotide sequence).

The present invention also relates to a method for identifying said compound, wherein the human P2Y ₁₂ receptor agonist is adenosine 5 ′ diphosphate.

  The present invention further relates to a polynucleotide represented by the base sequence described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing.

  The present invention further relates to a reagent kit comprising the polynucleotide described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing.

  The present invention also provides a reagent kit comprising the polynucleotide described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing and the polynucleotide described in any one of SEQ ID NO: 1 to SEQ ID NO: 49 in the sequence listing. About.

The present invention further provides that the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genome sequence 58 The genotype of the site corresponding to the 51st nucleotide of the base sequence described in is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, and SEQ ID NO: in the sequence listing 62 by identifying that the genotype of the site corresponding to the 69th nucleotide of the base sequence described in 62 is a genotype represented by G / G, and in the genomic base sequence of the gene, The genotype of the site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genome base sequence is C / The present invention relates to a method for detecting a genotype having the possibility of causing coronary artery disease, the method comprising detecting the genotype represented by C as a means, wherein the means comprises the following steps:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, site corresponds to a 51 th nucleotide of the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence and / or A DNA fragment comprising a partial base sequence of the genomic base sequence and comprising a nucleotide corresponding to the 69th nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing, and the genomic base sequence of the gene, A step of amplifying a DNA fragment containing a nucleotide at a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genomic base sequence by the polymerase chain reaction (PCR);
And (ii) The sequence of the PCR product obtained in the step (i) is determined by a sequencing method, or the base at the above position is determined by a typing method for the PCR product obtained in the step (i). Process.

The invention further also that haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence 58. The genotype of the site corresponding to the 51st nucleotide of the base sequence described in 58 is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, the sequence of the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the nucleotide sequence of number 62 is a genotype represented by G / G, and in the genomic nucleotide sequence of the gene A genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the sequence listing, which is a partial base sequence of the genomic base sequence A means to detect that the genotype represented by C / C, distribution said means at genomic nucleotide sequence of the human P2Y ₁₂ receptor gene by the following step, a partial base sequence of the genomic nucleotide sequence The possibility of causing coronary artery disease comprising detecting that the genotype of the site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 58 of the sequence table is a genotype represented by C / C A method for detecting genotypes having:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 17 and SEQ ID NO: 34 as primers;
And (ii) the process for the PCR product obtained in the (i), in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, according to SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of determining a base at a site corresponding to the 51st nucleotide of the base sequence by a typing method using the polynucleotide of SEQ ID NO: 41 or 43 as a primer.

The present invention also provides that the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genome sequence 58 The genotype of the site corresponding to the 51st nucleotide of the base sequence described in is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, and SEQ ID NO: in the sequence listing 62 by identifying that the genotype of the site corresponding to the 69th nucleotide of the base sequence described in 62 is a genotype represented by G / G, and in the genomic base sequence of the gene, The genotype of the site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genomic base sequence is C / C A means to detect that in a genotype represented in the means genome sequence of the human P2Y ₁₂ receptor gene by the following process, the sequence of the sequence listing a partial nucleotide sequence of the genomic nucleotide sequence A gene having the possibility of causing coronary artery disease, comprising detecting that the genotype of the site corresponding to the 69th nucleotide of the nucleotide sequence of number 62 is a genotype represented by G / G Regarding type detection:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the first 69-th site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 40 as primers;
And (ii) the process for the PCR product obtained in the (i), in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, according to SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of determining a base at a site corresponding to the 69th nucleotide of the base sequence by a typing method using the polynucleotide of SEQ ID NO: 42 as a primer.

The present invention further provides that the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genome sequence 58 The genotype of the site corresponding to the 51st nucleotide of the base sequence described in is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, and SEQ ID NO: in the sequence listing 62 by identifying that the genotype of the site corresponding to the 69th nucleotide of the base sequence described in 62 is a genotype represented by G / G, and in the genomic base sequence of the gene, The genotype of the site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genome base sequence is C / A means to detect that a genotype represented in C, and the means in the genome sequence of the human P2Y ₁₂ receptor gene by the following steps in the sequence listing a partial nucleotide sequence of the genomic nucleotide sequence It has the possibility of causing coronary artery disease, including detecting that the genotype of the site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 is the genotype represented by C / C Regarding genotype detection methods:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 40 as primers;
And (ii) the process for the PCR product obtained in the (i), in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, according to SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of determining the base of the site corresponding to the 51st nucleotide of the base sequence by a typing method using the polynucleotide of SEQ ID NO: 50 or 55 as a primer.

The invention further also that haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence 58. The genotype of the site corresponding to the 51st nucleotide of the base sequence described in 58 is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, the sequence of the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the nucleotide sequence of number 62 is a genotype represented by G / G, and in the genomic nucleotide sequence of the gene A genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the sequence listing, which is a partial base sequence of the genomic base sequence A means to detect that the genotype represented by C / C, distribution said means at genomic nucleotide sequence of the human P2Y ₁₂ receptor gene by the following step, a partial base sequence of the genomic nucleotide sequence The possibility of causing coronary artery disease comprising detecting that the genotype of the site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 58 of the sequence table is a genotype represented by C / C A method for detecting genotypes having:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 18 and SEQ ID NO: 34 as primers.

The present invention also provides that the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genome sequence 58 The genotype of the site corresponding to the 51st nucleotide of the base sequence described in is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, and SEQ ID NO: in the sequence listing 62 by identifying that the genotype of the site corresponding to the 69th nucleotide of the base sequence described in 62 is a genotype represented by G / G, and in the genomic base sequence of the gene, The genotype of the site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genomic base sequence is C / C A means to detect that in a genotype represented in the means genome sequence of the human P2Y ₁₂ receptor gene by the following process, the sequence of the sequence listing a partial nucleotide sequence of the genomic nucleotide sequence A gene having the possibility of causing coronary artery disease, comprising detecting that the genotype of the site corresponding to the 69th nucleotide of the nucleotide sequence of number 62 is a genotype represented by G / G Regarding type detection:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the first 69-th site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence Amplifying a DNA fragment containing the polynucleotide according to SEQ ID NO: 7 or 51 and polymerase chain reaction (PCR) using the polynucleotide according to SEQ ID NO: 8 or 52 as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method.

The present invention further provides that the haplotypes of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, SEQ ID NO: of the Sequence Listing a partial nucleotide sequence of the genome sequence 58 The genotype of the site corresponding to the 51st nucleotide of the base sequence described in is a genotype represented by C / C, and / or a partial base sequence of the genomic base sequence, and SEQ ID NO: in the sequence listing 62 by identifying that the genotype of the site corresponding to the 69th nucleotide of the base sequence described in 62 is a genotype represented by G / G, and in the genomic base sequence of the gene, The genotype of the site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genome base sequence is C / A means to detect that a genotype represented in C, and the means in the genome sequence of the human P2Y ₁₂ receptor gene by the following steps in the sequence listing a partial nucleotide sequence of the genomic nucleotide sequence It has the possibility of causing coronary artery disease, including detecting that the genotype of the site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 is the genotype represented by C / C Regarding genotype detection methods:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence Amplifying a DNA fragment containing the polynucleotide according to SEQ ID NO: 7 or 51 and polymerase chain reaction (PCR) using the polynucleotide according to SEQ ID NO: 8 or 52 as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method.

  The present invention further relates to a method for examining the risk of coronary artery disease, characterized by using the method for detecting a genotype having the possibility of causing any of the above-mentioned coronary artery diseases.

The present invention also are homozygous for the haplotype H1 haplotype a human P2Y ₁₂ receptor gene, and, in the genome sequence of the gene, the sequence listing and a partial nucleotide sequence of the genomic nucleotide sequence the protein 51st genotypes site corresponding to nucleotide base sequence represented by the amino acid sequence encoded by the P2Y ₁₂ receptor gene, a genotype represented by C / C according to SEQ ID NO: 62 and platelets which holds (platelets a), control platelets (platelet B, where platelets B holds protein shown by the amino acid sequence encoded by the P2Y ₁₂ receptor gene of any one selected from the following group there platelets: (i) homozygous for the haplotype H1 haplotype a human P2Y ₁₂ receptor gene, and,該遺In the genome base sequence of a gene, the genotype of the site corresponding to the 51st nucleotide of the base sequence shown in SEQ ID NO: 62 in the sequence listing is a partial base sequence of the genomic base sequence is C / T or T / It is T; (ii) is its haplotype a human P2Y ₁₂ receptor gene H1 / H2 haplotype heterozygotes, and, in the genome sequence of the gene, a partial nucleotide sequence of the genomic nucleotide sequence genotype of a portion corresponding to the 51 th nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 of the sequence Listing is a C / C, C / T or T / T,; and (iii) the human _{P2Y 12} receptor gene The haplotype is a homozygote of the H2 haplotype and is a partial base sequence of the genomic base sequence in the genomic base sequence of the gene. And the genotype of the site corresponding to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 62 in the Sequence Listing is C / C, C / T, or T / T) in the presence of a certain compound. In contact with a human P2Y ₁₂ receptor agonist to induce platelet aggregation and then comparing the dose-dependent curve of platelet aggregation by platelet A with the dose-dependent curve of platelet aggregation by platelet B And a method for identifying compounds that induce platelet desensitization to human P2Y ₁₂ receptor agonists.

In the present invention, to identify the haplotypes of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, and a 152 539 314 of the genomic nucleotide sequence of the gene (This position is described in GenBank accession number NC_000003.9 Defined as the position in the genomic base sequence, which corresponds to position 15237725 in the genomic base sequence described in GenBank Accession No. NC — 0000032.8). Therefore, it is possible to provide a method for detecting a genotype having a possibility of causing coronary artery disease.

In coronary artery disease patients, homozygous haplotypes of the human _{P2Y 12} receptor gene H1 haplotype and second 152,539,314 of genomic nucleotide sequence of the gene (This position is described in GenBank accession number NC_000003.9 This position is a genotype in which the genotype of the genome base sequence described in GenBank accession number NC_000003.8 is equivalent to position 1523777525) in C / C. It was clarified in the present invention that a certain frequency is significantly higher than that of healthy subjects.

Therefore, by the detection method, homozygous haplotypes of the human _{P2Y 12} receptor gene H1 haplotype and second 152,539,314 of genomic nucleotide sequence of the gene (This position is described in GenBank accession number NC_000003.9 Defined as a position in the genomic base sequence, which corresponds to position 1523777525 in the genomic base sequence described in GenBank Accession No. NC — 0000032.8). In the case, it can be determined that the coronary artery disease has a possibility of causing.

  In the present invention, it is possible to provide a method for examining the risk of coronary artery disease using the detection method. Furthermore, a polynucleotide and a reagent kit used in the detection method and the inspection method can be provided.

  When it is determined that the risk of coronary artery disease is high according to the present invention, the risk factors such as smoking, high blood pressure, high fat / high cholesterol diet, obesity and stress may be corrected before onset. it can.

In the present invention, homozygous for the haplotype H1 haplotype a human _{P2Y 12} receptor gene, and a 152 539 314 of the genomic nucleotide sequence of the gene (present position GenBank accession number NC_000003.9 Is defined as a position in the genome base sequence described in the above, but this position corresponds to position 1523777525 in the genome base sequence described in GenBank Accession No. NC — 0000032.8). Identification of a compound that induces desensitization of platelets to P2Y ₁₂ receptor agonists, for example, ADP, using platelets that retain the protein represented by the amino acid sequence encoded by the genotype human P2Y ₁₂ receptor gene Can provide a method. Since the compound identified by the present identification method can induce desensitization of platelets to ADP, it can reduce the aggregation reaction of platelets to high concentration ADP seen at the onset of thrombotic diseases such as coronary artery disease, As a result, thrombus formation can be inhibited or reduced. Such compounds can be used as active ingredients in platelet desensitization inducers, thrombus formation inhibitors, and preventive and therapeutic agents for thrombotic diseases such as coronary artery disease. Moreover, prevention and treatment of thrombotic diseases can be carried out using these drugs.

In the present invention, to analyze the association between genotype and the platelet aggregation ability and thrombotic disorders of the human P2Y ₁₂ receptor gene, as a result, the human P2Y ₁₂ receptor, which can be considered to have coronary artery disease and highly relevant The genotype of the body gene was found.

Genotype of the human P2Y ₁₂ receptor gene analysis was carried out by identifying a nucleotide mutation site at nucleotide haplotype and said the genes of the gene.

But when indicating the position of each base of the human _{P2Y 12} receptor gene in the following, unless otherwise defined, indicated as positions in the base sequence disclosed in GenBank accession number NC_000003.8.

The P2Y ₁₂ receptor gene, hereinafter, means a human _{P2Y 12} receptor gene.

  “Genotype” means a combination of two alleles present in one individual for one genetic locus.

  “Allele” means two or more genes having a difference based on a difference in DNA base sequence occurring at the same locus.

  “Haplotype” means a combination type of multiple loci on a single chromosome (haploid; haploid) or a combination of multiple single nucleotide mutation sites on the same gene. .

The "haplotype of P2Y ₁₂ receptor gene" means a type of combination of a plurality of single base mutation site of P2Y ₁₂ receptor on gene. Polymorphisms in P2Y ₁₂ receptor gene, called as H1 haplotype and H2 haplotypes have been reported (Patent Document 1, Non-Patent Document 3 and Non-Patent Document 4). The H1 haplotype has a high allele frequency and is considered to be a normal genotype. On the other hand, it is reported that ADP-induced platelet aggregation ability is increased in individuals having H2 haplotype, and that H2 haplotype is a risk factor for developing peripheral arterial injury (Non-patent Document 3 and Non-patent Document 4). .

  “Single base mutation site” means a position where a single base mutation has occurred in the base sequence of a gene. A single base mutation means a change from a single base to another base due to a mutation such as substitution, deletion and insertion.

P2Y ₁₂ receptor gene genotype and platelet aggregation ability and the results of analysis of the association with thrombotic diseases, in particular, the haplotype of P2Y ₁₂ receptor gene is homozygous for H1 haplotype, and the gene It has been revealed that the genotype at position 1523777525 of the genomic base sequence of is a genotype represented by C / C, which is associated with coronary artery disease.

More specifically, using platelets from healthy individuals homozygous for haplotype H1 haplotype of P2Y ₁₂ receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of said gene C / C As a result of comparing the platelet aggregation ability to ADP for platelets derived from individuals of the genotype represented by and those derived from individuals not having these genotypes, the former platelets were compared with the platelets derived from the latter It was found that the aggregating ability for low concentration ADP was low. On the other hand, almost no difference was observed in the aggregation ability for high concentration ADP between the former-derived platelets and the latter-derived platelets.

  “Homozygote”, also called homozygote, refers to a zygote that has the same or experimentally indistinguishable allele at a locus. In contrast, a conjugate having another allele at a certain locus is called a heterozygote.

The "haplotype of _{P2Y 12} receptor gene is homozygous for H1 haplotype" means the allele of the _{P2Y 12} receptor gene in both a _{P2Y 12} receptor gene H1 haplotype.

Thing is "a is genotypes No. 152377525 of genotype is expressed in C / C", in both alleles of the _{P2Y 12} receptor gene, bases of the 152,377,525 of the nucleotides are cytosine (C) Means.

As used herein, reference to "H1 homo & 152377525 C / C" is a homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene Is a genotype represented by C / C.

The phrase "Others" is genotype No. 152377525 of the genotype and / or the gene in a genomic base sequence haplotype _{P2Y 12} receptor gene is not a homozygote of the H1 haplotype is represented by C / C Means not. The "Others" includes the following examples: (1) _{P2Y 12} homozygous haplotypes receptor gene H1 haplotype, and a 152 377 525 of the genotype of the genomic nucleotide sequence of said gene C (2) H1 / H2 haplotype heterozygote and the genotype at position 15237775 of the genome base sequence of the gene is C / C, C / T, or T / T And (3) a homozygote of H2 haplotype, and the genotype at position 15237775 of the genomic base sequence of the gene is C / C, C / T, or T / T.

On the other hand, the individual derived platelet is a genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the P2Y ₁₂ receptor gene is expressed in C / C, the site of genotypes genotype other than C / C As a result of comparing the platelet aggregation ability with respect to ADP for platelets derived from an individual, the former platelets were found to have a lower aggregation ability regardless of the concentration of ADP than the latter.

As used herein, reference to "152377525 C / C" is regardless of haplotype _{P2Y 12} receptor is the genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C Means that.

The phrase "152377525 T Carriers" are irrespective of the haplotypes of _{P2Y 12} receptor gene, the 152 377 525 of the genotype of the genomic nucleotide sequence of the gene it is meant that genotype other than C / C. To "152377525 T Carriers" includes examples No. 152377525 of the genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene is an example or T / T is a C / T.

In contrast, the individual derived platelet homozygous haplotypes H1 haplotype of P2Y ₁₂ receptor gene, Platelet derived from an individual a haplotype haplotype other than homozygous for H1 haplotype of the gene, ADP As a result of comparison of the platelet aggregation ability, almost no difference was observed in the aggregation ability between the two regardless of the concentration of ADP.

As used herein, reference to "H1 homo" means regardless of the 152,377,525 of the genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene, haplotype of the gene is homozygous for H1 haplotype .

The phrase "H2 Carriers" are irrespective of the 152,377,525 of the genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene, haplotype of the gene means that it is not a homozygote of the H1 haplotype. “H2 carriers” includes examples in which the genotype of the P2Y ₁₂ receptor gene is an H1 / H2 haplotype heterozygote or an H2 haplotype homozygote.

P2Y ₁₂ homozygous haplotypes of H1 haplotype receptor gene, and platelets from an individual is a genotype which first 152,377,525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C is As described above, the aggregation ability at the time of ADP stimulation is low, and therefore, it is difficult to activate and desensitize with a small amount of ADP constantly generated in a living blood vessel. Desensitization means a state in which platelets are not sufficiently reacted (aggregated) even if stimulated with a P2Y ₁₂ receptor agonist such as ADP and then stimulated again with the agonist. On the other hand, platelets derived from individuals that do not have the genotype as described above are highly reactive to ADP, and thus continuously react with trace amounts of ADP produced in living blood vessels and fall into a desensitized state. Cheap.

  Platelets are exposed to high concentrations of ADP at the onset of coronary artery disease. At this time, desensitized platelets cannot react sufficiently to ADP and form fragile and small thrombi, whereas platelets that are difficult to desensitize react sensitively to ADP and form strong and large thrombi. It is thought to contribute to the onset and worsening of coronary artery disease.

P2Y ₁₂ homozygous haplotypes of H1 haplotype receptor gene, and platelets from an individual is a genotype which first 152,377,525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C is Because of its low agglutination ability for low-concentration ADP, a small amount of ADP produced in a living blood vessel is difficult to desensitize. As a result, it reacts sensitively to high-concentration ADP exposed at the onset of coronary artery disease, A large thrombus is thought to contribute to the onset and worsening of coronary artery disease.

The association between genotype and disease P2Y ₁₂ receptor gene, coronary artery disease patients and healthy individuals genomic samples from each (hereinafter sometimes referred to as the CAD and control groups, respectively) using a Case - Control results of the analysis studies, homozygous haplotypes H1 haplotype of P2Y ₁₂ receptor gene, and is the genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C The frequency was found to be significantly higher in patients with coronary artery disease.

  Analysis was performed by univariate analysis and multivariate analysis. Univariate analysis was performed using the single nucleotide polymorphism of the gene as an independent variable. Multivariate analysis uses factors such as single nucleotide polymorphisms of genes, hyperlipidemia, hypertension, presence / absence of diabetes, history of smoking, and body mass index (BMI value) as independent variables, and significant differences are detected by univariate analysis. Carried out when it was.

  Specifically, as a result of univariate analysis and multivariate analysis based on the number of cases of “H1 homo & 15237725 C / C” and “Others” in the CAD group and the control group, univariate analysis and multivariate analysis were performed. A high odds ratio (OR) was obtained in both analyzes of the variable analysis, and the obtained OR was determined to be reliable from the value of the 95% confidence interval (95% CI). This result reliably demonstrated that “H1 homo & 15237725 C / C” is associated with CAD.

  On the other hand, when univariate analysis and multivariate analysis were performed based on the number of examples of “152377575 C / C” and “1523777525 T carriers” in the CAD group and the control group, respectively, univariate analysis and multivariate analysis The OR obtained in both analyzes was low, and the obtained OR was found to be unreliable from the 95% CI value. From this result, it is considered that “152377525 C / C” is unlikely to be associated with CAD.

  In addition, when univariate analysis and multivariate analysis were performed based on the number of cases of “H1 homo” and “H2 carriers” in the CAD group and the control group, both univariate analysis and multivariate analysis were performed. A high OR was obtained, but the obtained OR was found to be unreliable from a value of 95% CI. From this result, it is considered that “H1 homo” is unlikely to be associated with CAD.

Thus, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and the genotype of the 152,377,525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C is the frequency Are significantly higher in coronary artery disease patients than in healthy individuals, the present inventors believe that it is strongly associated with the onset of coronary artery disease. It has been confirmed in many case-control studies that hypertension, high cholesterol, diabetes, and smoking used as independent variables in the multivariate analysis are independent risk factors for coronary artery disease. In the multivariate analysis in the present invention, it was confirmed by all the above analyzes that hypertension, high cholesterol, diabetes, and smoking are independent risk factors. This means that the specimen sampling and analysis is reasonably made in the analysis, therefore, homozygous for haplotype H1 haplotype of P2Y ₁₂ receptor gene, and the genomic nucleotide sequence of the gene It is strongly supported that the genotype at position 15237775 of the above is a genotype represented by C / C, which is a risk factor for coronary artery disease.

1 embodiment of the present invention is to identify the haplotype of P2Y ₁₂ receptor gene is homozygous for H1 haplotype, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C The present invention relates to a method for detecting a genotype having a possibility of causing coronary artery disease, which comprises detecting the genotype.

It haplotypes P2Y ₁₂ receptor gene is homozygous for H1 haplotype can be carried out by performing the determination of the H1 haplotype and H2 haplotype for _{P2Y 12} receptor gene.

In genomic sample from a human, H1 _{P2Y 12} receptor gene haplotype is detected, and if _{the P2Y 12} receptor gene of H2 haplotype is not detected, _{P2Y 12} receptor gene in said sample is homozygous for H1 haplotype It can be determined that there is. In contrast, from a human in the genome samples, H2 _{P2Y 12} receptor gene haplotype is detected, and if _{the P2Y 12} receptor gene H1 haplotype is not detected, _{P2Y 12} receptor gene in said sample is the H2 haplotype It can be determined that it is a homozygote. Further, in the genomic sample from a human, if _{the P2Y 12} receptor gene of _{P2Y 12} receptor gene and H2 haplotype H1 haplotype was detected neither, _{P2Y 12} receptor gene in said sample is the H1 / H2 haplotype heterodimer It can be determined that it is a joined body.

P2Y ₁₂ determine the H1 haplotype and H2 haplotype of the receptor gene may be carried out by identifying the type of the nucleotide of one base mutation site which can identify these haplotypes.

  Four types of single nucleotide mutations existing in the H2 haplotype have already been reported (Table 1), and it has been disclosed that the H2 haplotype can be identified by identifying these known single nucleotide mutations (Patent Document 1). These known single nucleotide mutations have been found in genomic samples derived from healthy white people. Further, it has been reported that the allele frequencies of H1 haplotype and H2 haplotype are 86.2% and 13.8%, respectively, in Westerners (Non-patent Document 3).

In the present invention, the genomic sequence of the P2Y ₁₂ receptor gene, a new one base mutation in four that H2 haplotype linkage was found to be present upstream of exon 2 in the genome sequence of the gene ( See Example 1 and Table 7).

In the present invention, among the known single nucleotide mutations shown in Table 1, 5 cases having an H2 haplotype in which no single nucleotide mutation (mutation from G to T at position 152377507) present in exon 2 is observed. It was found in a genome sample derived from a healthy human. In these examples, P2Y ₁₂ receptor gene was found in the present invention the four 1 base mutation (see Table 7) was observed, further exons of the four known one base mutation shown in Table 1 2 Three types of single-base mutations were observed except for the single-base mutation present in. Thus, one base mutation present in exon 2 of the P2Y ₁₂ receptor gene (to T mutation No. 152377507 of the G) is revealed that the example shown different genotypes and conventional H2 haplotype is present (See Table 8). The genotype of _{P2Y 12} receptor gene found here referred to as H2' haplotype (eg number = 5). The presence of the H2 ′ haplotype was found by analysis using a Japanese genome sample. However, since the reported haplotype exists in the same position in the Japanese, it is assumed that the H2 ′ haplotype also exists in Westerners. H2' haplotype, of the known single-base mutations shown in Table 1, present in exon 2 1 base mutation (mutation to T from the 152,377,507 position G) but is not observed, _{P2Y 12} receptor according to the present invention All four types of single-base mutations in body genes were observed. Since the H2 ′ haplotype is different from the H2 haplotype only in the presence or absence of a single base mutation at one site, it is considered that the H2 ′ haplotype is related to the enhancement of platelet aggregation ability like the H2 haplotype.

Specific H1 haplotype P2Y ₁₂ receptor gene, specifically, can be carried out by detection of one or more of the following single base mutation site nucleotides of P2Y ₁₂ receptor gene:
(1) The base at position 152381673 is guanine (G),
(2) the base at position 15238188 is thymine (T),
(3) the base at position 152379350 is cytosine (C);
(4) the base at position 15379156 is cytosine (C),
(5) the base at position 152388551 is thymine (T),
(6) There is no insertion mutation in the base at position 15278495,
(7) the base at position 152378084 is thymine (T),
(8) The base at position 152377507 is guanine (G).

P2Y ₁₂ of H1 haplotype receptor gene identification is preferably that the 152,379,350 of the base is cytosine (C), and / or the position first 152,377,507 bases detects that a guanine (G) Can be implemented. More preferably, the identification can be carried out by detecting that the base at position 15379350 is cytosine (C) and that the base at position 152377507 is guanine (G).

In genomic sample from a human, it is a genotype which one of the one base mutation sites capable of identifying the H1 haplotype and H2 haplotype of P2Y ₁₂ receptor gene or more is represented only by nucleotides present in H1 haplotype If, P2Y ₁₂ receptor gene in said sample can be determined to be homozygous for H1 haplotype.

The "single base mutation site one or more of a genotype represented only by nucleotides present in the H1 haplotype" in both alleles of the P2Y ₁₂ receptor gene, of the single base mutation site It means that the nucleotide is a nucleotide present in the H1 haplotype of the gene.

In genomic sample from a human, P2Y ₁₂ receptor gene one or more of the single base mutation site nucleotides may specify H1 haplotype and H2 haplotype is a nucleotide present in the H1 haplotype is detected, and the If one base mutation site nucleotide P2Y ₁₂ receptor gene was not detected is a nucleotide present in H2 haplotype, P2Y ₁₂ receptor gene in said sample can be determined to be homozygous for H1 haplotype. In contrast, the single base mutation site nucleotides is detected P2Y ₁₂ receptor gene is a nucleotide present in H2 haplotype, P2Y ₁₂ receptor gene the single base mutation site nucleotides are nucleotides present in H1 haplotype If but not detected, P2Y ₁₂ receptor gene in said sample can be determined to be homozygous H2 haplotype. Further, both the P2Y ₁₂ receptor gene is a nucleotide in which the 1 base mutation site at nucleotide is a nucleotide that exists H1 haplotype P2Y ₁₂ receptor gene and the single nucleotide mutation site of the nucleotide is present in the H2 haplotype is detected If, _{P2Y 12} receptor gene in said sample can be determined to be H1 / H2 haplotype heterozygotes.

Table example, in the genomic sample from a human, a genotype No. 152379350 of the genotype of _{P2Y 12} receptor gene is expressed in C / C, and / or the genotype at position No. 152377507 is at G / G If a genotype that is, P2Y ₁₂ receptor gene in said sample can be determined to be homozygous for H1 haplotype.

The "a is genotypes genotype of the 152,379,350 is represented by C / C", in both alleles of the _{P2Y 12} receptor gene, means that the 152,379,350 of the base is cytosine (C) . The "a is genotypes genotype of the 152,377,507 is represented by G / G", in both alleles of the _{P2Y 12} receptor gene, means that the 152,377,507 of the base is guanine (G) .

In genomic sample from a human, _{P2Y 12} receptor No. 152379350 of bases is detected _{P2Y 12} receptor gene is a cytosine (C), and a base of said one base mutation site is a base other than cytosine (C) If the gene is not detected, the genotype of the P2Y ₁₂ receptor gene in said sample can be determined to be genotype genotypes of the 152 379 350 is represented by C / C. Further, the 152377507 of bases is detected _{P2Y 12} receptor gene is guanine (G), and a base of said one base mutation sites not detected _{P2Y 12} receptor gene is a base other than guanine (G) If the genotype of the _{P2Y 12} receptor gene in said sample can be determined to be genotype genotypes of the 152 377 507 it is represented by G / G.

On the other hand, if the genotype at position 15237775 of the P2Y ₁₂ receptor gene is a genotype represented by C / C, the nucleotide at position 15237775 of the P2Y ₁₂ receptor gene is identified, and the base of the nucleotide is It can be determined by detecting that it is cytosine (C).

In genomic sample from a human, _{P2Y 12} receptor No. 152377525 of bases is detected _{P2Y 12} receptor gene is a cytosine (C), and a base of said one base mutation site is a base other than cytosine (C) If the gene is not detected, the genotype of the P2Y ₁₂ receptor gene in said sample can be determined to be genotype genotypes of the 152 377 525 is represented by C / C.

As described above, the detection method of the present invention is the genomic nucleotide sequence of the P2Y ₁₂ receptor gene, single nucleotide mutation site may determine the H1 haplotype and H2 haplotype of the gene, and a 152 377 525-position of the genomic nucleotide sequence It is a means to detect the nucleotide and identify its type.

Detection of nucleotides and identification of their types can be performed using methods known per se. For example, the polymerase chain reaction (hereinafter, abbreviated as PCR) by, amplifying the partial sequence of the P2Y ₁₂ receptor gene comprising a nucleotide at a desired position, sequencing the nucleotide sequence of the amplification products obtained, for example, direct sequencing A method of sequencing by the method can be used. As a sequencing method, in addition to the sequencing method, a hybridization method, a restriction enzyme fragment length polymorphism (RFLP) analysis method and the like can be used.

Specifically, detection of nucleotides and identification of their types can be carried out as follows. The detection of the nucleotide at position 152379350 and the identification of the type thereof are carried out, for example, by performing PCR on genomic DNA using the polynucleotides described in SEQ ID NO: 5 and SEQ ID NO: 6 as primers, and the base sequence of the obtained amplification product Can be carried out by determining by the sequencing method. Examples of the primers used in the sequencing method (hereinafter referred to as sequencing primers) include the polynucleotides described in SEQ ID NO: 18 and SEQ ID NO: 34. The detection of the nucleotide at position 152377507 and the identification of the type thereof use, for example, each polynucleotide described in SEQ ID NO: 7 or SEQ ID NO: 51 and each polynucleotide described in SEQ ID NO: 8 or SEQ ID NO: 52 as a PCR primer. The same method as described above can be performed using each polynucleotide described in SEQ ID NO: 24 or 53 and each polynucleotide described in SEQ ID NO: 40 or 54 as sequencing primers. The detection of the nucleotide at position 15237775 and the identification of the type thereof use, for example, each polynucleotide described in SEQ ID NO: 7 or 51 and each polynucleotide described in SEQ ID NO: 8 or 52 as a PCR primer. The same method as described above can be performed using each polynucleotide described in SEQ ID NO: 24 or 53 and each polynucleotide described in SEQ ID NO: 40 or 54 as sequencing primers. Detection of the nucleotide at position 152381673 and identification of the type thereof are carried out by using, for example, the polynucleotides described in SEQ ID NO: 1 and SEQ ID NO: 2 as PCR primers and SEQ ID NO: 10 and SEQ ID NO: 28 as sequencing primers. It can carry out by the same method as described above using each polynucleotide. The detection of the nucleotide at position 15238188 and the identification of the type thereof are carried out using, for example, the polynucleotides shown in SEQ ID NO: 3 and SEQ ID NO: 4 as PCR primers and the polynucleotide shown in SEQ ID NO: 32 as sequencing primers. Thus, the same method as described above can be used. For example, the detection of the nucleotide at position 152378084 and the identification of the type thereof use the polynucleotides shown in SEQ ID NO: 7 and SEQ ID NO: 8 as PCR primers and the polynucleotide shown in SEQ ID NO: 39 as a sequencing primer. Thus, the same method as described above can be used. Ranked No. 152379156, IN 152378551, detection and the type of identification of the 152378495 of nucleotide also acquired designed based appropriate PCR primers and sequencing primers in the nucleotide sequence of the _{P2Y 12} receptor gene, these It can be carried out in the same manner as described above using a primer. P2Y ₁₂ primers used in PCR to amplify the desired region including the respective positions of the receptor gene is not limited to the above exemplified polynucleotides, but any primer capable of amplifying a region of said desired can be used . Further, a primer for sequencing is not limited to the above exemplified polynucleotides, but any primer capable of determining the nucleotide sequence of the desired region including the respective positions of the P2Y ₁₂ receptor gene can be used. PCR and sequencing methods can be performed according to generally known methods.

Identification of the type of nucleotide can be performed using other known single-base mutation detection methods. Examples of single nucleotide mutation detection methods include genotyping methods such as SNuPe method. Specifically, the identification of the type of nucleotide by the SNuPe method is as follows. Taking the nucleotide at position 152379350 as an example, a DNA fragment containing the base is used as a primer for each polynucleotide described in SEQ ID NO: 17 and SEQ ID NO: 34. It can be carried out by amplifying genomic DNA by PCR and typing the type of base at the target position of the obtained amplification product by SNuPe method using the polynucleotide described in SEQ ID NO: 41 or SEQ ID NO: 43 as a typing primer ( See Examples). Identification of the type of nucleotide at position 152377507 is the same as described above, for example, using the polynucleotides set forth in SEQ ID NO: 23 and SEQ ID NO: 40 as PCR primers and the polynucleotide set forth in SEQ ID NO: 42 as a typing primer. It can carry out by the method of. The identification of the type of the nucleotide at position 1523777525 is, for example, using the polynucleotides shown in SEQ ID NO: 23 and SEQ ID NO: 40 as PCR primers and the polynucleotide shown in SEQ ID NO: 50 or 55 as typing primers. The same method as described above can be used. The determination of the bases at positions 152381673, 15238188 and 1523778084 can be similarly performed. The typing primer used for typing the bases at these positions can be appropriately designed and obtained from the upstream or downstream base sequence adjacent to the position. Specifically, the polynucleotide described in SEQ ID NO: 44 or SEQ ID NO: 45 can be exemplified as a primer for typing the base at position 152381673. Examples of the primer for typing the base at position 15238188 include the polynucleotide described in SEQ ID NO: 46 or SEQ ID NO: 47. Examples of the nucleotide typing primer for position 1523778084 include the polynucleotide described in SEQ ID NO: 48 or SEQ ID NO: 49. Ranked No. 152379156, IN 152378551, identification of the type of the first 152378495 of nucleotide also acquired designed based appropriate PCR primers and the typing primer on the nucleotide sequence of _{P2Y 12} receptor gene, using these primers The same method as described above can be used. P2Y ₁₂ primers used in PCR to amplify the desired region including the respective positions of the receptor gene is not limited to the above exemplified polynucleotides, but any primer capable of amplifying a region of said desired can be used . Furthermore, typing primer is not limited to the above exemplified polynucleotides, but any primers capable of identifying the type of nucleotides of the position of the P2Y ₁₂ receptor gene can be used. PCR and genotyping methods can be performed according to generally known methods.

  The detection of nucleotides and identification of their types can be carried out by applying other known single-base mutation analysis methods. As a single base mutation analysis method, a method using fluorescence, for example, Invader assay (Lyamichev, V. et al., “Nature Biotechnology”, 1999, Vol. 17, p. 292-296) or Luminex (Chen, J. et al., “Genome Research”, 2000, Vol. 10, pp. 549-557). Further, a method using a mass spectrum, for example, a method using a primer extension method can be used. As such a method, a pinpoint assay (Ross, P. et al., “Nature Biotechnology”, 1998, Vol. 16, pp. 1347-1351) or a probe method (Brown, Braun, A.) et al., “Clinical Chemistry”, 1997, Vol. 43, p. 1151-1158). In addition, DOL assay, Chen, X. et al., “Proceedings of the National of Science of the United States of America, United States of Science. 94, 10756-10761), Sniper Assay (Piatek, AS, et al., “Nature Biotechnology”, 1998, 16, 359-363). And the tag array method (Fan, J-B. Et al., “Genome Research ch) ", 2000, Vol. 10, p. 853-860). These methods can be easily carried out by referring to the cited references in which they are reported. In addition, a well-known method for detecting a single base mutation, such as fluorescence resonance energy transfer (FRET) or Taqman method, can be used.

  Nucleotide detection and type identification can also be performed by hybridization methods, using allele-specific nucleic acid probes, respectively.

The nucleic acid probes, substantially nucleotide sequence comprising the genomic nucleotide sequence of the P2Y ₁₂ receptor gene of a polynucleotide represented by the nucleotide sequence, or its complementary nucleotide sequence comprises the single base mutation site, the one base mutation site And a polynucleotide having a complementary base sequence. “Having a substantially complementary base sequence” means a base sequence containing a single base mutation site in a sample to be examined (hereinafter referred to as a test sample) or its complementary when used as a probe. This means that the base sequence can be detected, for example, under stringent hybridization conditions. The hybridization conditions can be in accordance with, for example, the method described in the book (Sambrook et al., “Molecular Cloning, Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory). Specifically, stringent hybridization conditions include heating in a solution of 6 × SSC, 0.5% SDS and 50% formamide at 42 ° C., then 0.1 × SSC, 0.5% SDS. Examples of conditions in which a positive hybridization signal is still observed even under the condition of washing at 68 ° C. in the above solution.

The polynucleotide used as the probe is preferably composed of 8 to 50 nucleotides, more preferably 17 to 35 nucleotides, and further preferably 17 to 30 nucleotides. Probe was designed based on the nucleotide sequence, or its complementary nucleotide sequence of the P2Y ₁₂ receptor gene, conventional methods, for example, be prepared by chemical synthesis or the like. A string from the obtained polynucleotide to a gene fragment represented by the base sequence containing the single base mutation site of the polynucleotide represented by the base sequence containing the single base mutation site or a complementary base sequence of the base sequence. The target probe can be obtained by selecting one that hybridizes under a gentle condition. Specifically as probes, it can be exemplified a polynucleotide consisting of the nucleotide sequence, or its complementary nucleotide sequence having any one of 1 base mutation according to the present invention there is provided a partial nucleotide sequence of the P2Y ₁₂ receptor gene. These probes may be labeled with a marker for detection, for example, a label such as a fluorescent substance or a radioisotope. The label is not limited to these, and any label can be used as long as hybridization between the probe and the corresponding polynucleotide is not inhibited. The detection method of the said 1 base mutation is only an illustration, and the detection method which can be used in this invention is not limited to these.

  The test sample may be any of human-derived blood, spinal fluid, bronchoalveolar lavage fluid, sputum, other body fluids, or nucleic acid samples prepared from tissues and the like. The nucleic acid sample can be prepared by a nucleic acid preparation method known per se. As the nucleic acid sample, preferably genomic DNA is used. The prepared nucleic acid may be used for direct detection or the desired region may be amplified enzymatically by using PCR or other amplification methods prior to analysis.

  One aspect of the present invention relates to a method for examining the risk of coronary artery disease, characterized by using the method for detecting a genotype having a possibility of causing coronary artery disease according to the present invention. An early diagnosis of coronary artery disease can be performed by using the genotype detection method having the possibility of causing coronary artery disease according to the present invention.

Inspection method of morbidity risk of coronary artery disease, specifically, the P2Y ₁₂ receptor gene in a test sample, performs the identification of haplotypes, and the first 152,377,525 of the genotype of the genomic nucleotide sequence of the gene It can be implemented by detecting. That haplotypes P2Y ₁₂ receptor gene in the test sample is homozygous for H1 haplotype, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is a gene type represented by C / C When detected, it can be determined that the risk of coronary artery disease is high.

1 embodiment of the present invention are homozygous for the haplotype H1 haplotype a P2Y ₁₂ receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C gene A method for identifying a compound that induces platelet desensitization to a P2Y ₁₂ receptor agonist, comprising using a platelet having a protein represented by an amino acid sequence encoded by a P2Y ₁₂ receptor gene, About.

P2Y ₁₂ that haplotype A receptor gene is homozygous for H1 haplotype, and _{P2Y 12} receptor No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is a gene type represented by C / C Platelet retaining a protein represented by the amino acid sequence encoded by the gene (hereinafter sometimes referred to as platelet A) has a lower concentration of ADP than control platelets (hereinafter sometimes referred to as platelet B). It became clear that the aggregation ability with respect to was low. On the other hand, almost no difference was observed between platelet A and platelet B in the aggregation ability for high concentration ADP. Platelet A has a low aggregation ability when stimulated with ADP, and therefore is hardly activated or desensitized by a small amount of ADP constantly generated in a living blood vessel. On the other hand, since platelet B is highly reactive to ADP, it reacts constantly with a small amount of ADP produced in a living blood vessel and tends to fall into a desensitized state. Here, the control platelets (platelet B), means platelets to hold the protein shown by the amino acid sequence encoded from the P2Y ₁₂ receptor gene with different genotypes and P2Y ₁₂ receptor gene with platelet A To do. Specifically, platelets B is a platelet for holding a protein shown by the amino acid sequence encoded by the P2Y ₁₂ receptor gene of any one selected from the following group: (i) P2Y ₁₂ receptor gene a is homozygous for the haplotype H1 haplotype, and a 152 377 525 of the genotype of the genomic nucleotide sequence of said gene is C / T or T / T; (ii) at _{P2Y 12} receptor gene The haplotype is a H1 / H2 haplotype heterozygote and the genotype at position 1523777525 of the genomic base sequence of the gene is C / C, C / T, or T / T; and (iii) a P2Y ₁₂ receptor a gene homozygous for the haplotype H2 haplotype and the genomic nucleotide sequence of the gene 52377525 position genotype is C / C, C / T or T / T,.

  Platelets are exposed to high concentrations of ADP at the onset of coronary artery disease. At this time, desensitized platelets such as platelet B cannot react sufficiently with ADP and form fragile and small thrombi, whereas platelets that are difficult to desensitize such as platelet A react sensitively with ADP. However, it is thought that it forms a strong and large thrombus and contributes to the onset and worsening of coronary artery disease.

A compound that induces desensitization of platelets that are difficult to desensitize, such as the above-mentioned platelet A, can bring the platelets into a desensitized state, and as a result, to a high concentration ADP at the onset of coronary artery disease. It can reduce the response of the platelets to exposure and reduce thrombus formation.
Such a compound can be used as an active ingredient of a preventive and / or therapeutic agent for coronary artery disease. Moreover, the prevention and / or treatment method of a coronary artery disease can be implemented using such a compound.

The following identification method can be exemplified as a method for identifying a platelet that is difficult to desensitize, for example, a compound that induces desensitization of the above-mentioned platelet A. First, the platelet A and the platelet B are each brought into contact with a P2Y ₁₂ receptor agonist in the presence of a certain compound (hereinafter referred to as a test compound) to induce a platelet aggregation reaction. The platelet aggregation reaction can be performed in vitro or in vivo. Preferably, the platelet aggregation reaction is performed in vitro. Next, a dose-dependent curve of the platelet aggregation reaction by platelet A and a dose-dependency curve of the platelet aggregation reaction by platelet B are prepared and compared. Compounds in which there is no difference between the dose-dependent curve of platelet aggregation reaction by platelet A and that of platelet B can be determined to induce desensitization of platelet A. Preparation of the Platelet A is, for example, homozygous for the haplotype H1 haplotype a P2Y ₁₂ receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C It can be carried out by preparing platelet-rich plasma or washed platelets from blood derived from an individual having a genotype. Blood can be obtained by collecting blood from an individual in the presence of an anticoagulant. Preparation of platelet-rich plasma or washed platelets from blood can be performed by a general method. Preparation of the Platelet B is, for example, homozygous for the haplotype H1 haplotype a P2Y ₁₂ receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C It can be carried out by the same method as described above using blood derived from an individual having no genotype.

1 embodiment of the present invention relates to primers and allele-specific probes that can be used in the detection and the type identification of one base mutation site nucleotides capable of determining the H1 haplotype and H2 haplotypes P2Y ₁₂ receptor gene. Examples of such a primer and probe include the polynucleotide described in any one of SEQ ID NO: 1 to SEQ ID NO: 55. More preferably, the polynucleotide described in any one of SEQ ID NOs: 50 to 55 can be exemplified.

For example, a combination of the polynucleotides described in SEQ ID NO: 17 and SEQ ID NO: 34 can be provided as a primer for amplifying a region containing position 152379350 in the P2Y ₁₂ receptor gene. Moreover, the combination of the polynucleotide of sequence number 23 and sequence number 40 can be provided as a primer used for determination of the base sequence of this area | region. Moreover, the polynucleotide of SEQ ID NO: 41 or 43 can be provided as a primer that can be used for typing the base at position 152379350.

As primers for amplifying a region including the first 152,377,507 of the P2Y ₁₂ receptor in the gene, a polynucleotide according to SEQ ID NO: 7 or SEQ ID NO: 51, a set of the polynucleotide according to SEQ ID NO: 8 or SEQ ID NO: 52 Can provide tailoring. Moreover, a combination of the polynucleotide described in SEQ ID NO: 24 or 53 and the polynucleotide described in SEQ ID NO: 40 or 54 can be provided as a primer for use in determining the base sequence of the region. . Moreover, the polynucleotide of SEQ ID NO: 42 can be provided as a primer that can be used for typing the base at position 152379507.

As primers for amplifying a region including the first 152,377,525 of the P2Y ₁₂ receptor in the gene, a polynucleotide according to SEQ ID NO: 7 or SEQ ID NO: 51, a set of the polynucleotide according to SEQ ID NO: 8 or SEQ ID NO: 52 Can provide tailoring. Moreover, a combination of the polynucleotide described in SEQ ID NO: 24 or 53 and the polynucleotide described in SEQ ID NO: 40 or 54 can be provided as a primer for use in determining the base sequence of the region. . Moreover, the polynucleotide of SEQ ID NO: 50 or SEQ ID NO: 55 can be provided as a primer that can be used for typing the base at position 1523777525.

As primers for amplifying a region including the first 152,381,673 of the P2Y ₁₂ receptor in the gene, we can provide a combination of the polynucleotide set forth in SEQ ID NO: 1 and SEQ ID NO: 2. Moreover, the combination of the polynucleotide of sequence number 10 and sequence number 28 can be provided as a primer used for determination of the base sequence of this area | region. Moreover, the polynucleotide of SEQ ID NO: 44 or SEQ ID NO: 45 can be provided as a primer that can be used for typing the base at position 152381673.

As primers for amplifying a region including the first 152,380,188 of the P2Y ₁₂ receptor in the gene, we can provide a combination of the polynucleotide set forth in SEQ ID NO: 3 and SEQ ID NO: 4. Moreover, the polynucleotide of SEQ ID NO: 32 can be provided as a primer for use in determining the base sequence of the region. Moreover, the polynucleotide of SEQ ID NO: 46 or SEQ ID NO: 47 can be provided as a primer that can be used for typing the base at position 15238188.

As primers for amplifying a region including the first 152,378,084 of the P2Y ₁₂ receptor in the gene, we can provide a combination of the polynucleotide set forth in SEQ ID NO: 7 and SEQ ID NO: 8. Moreover, the polynucleotide of SEQ ID NO: 39 can be provided as a primer for use in determining the base sequence of the region. Moreover, the polynucleotide of SEQ ID NO: 48 or 49 can be provided as a primer that can be used for typing the base at position 152378084.

  The polynucleotide according to the present invention can be obtained from known genetic engineering techniques (eg, Sambrook et al., “Molecular Cloning, Laboratory,” based on information related to the specific nucleotide sequence disclosed by the present invention. Manual 2nd Edition ”, 1989, Cold Spring Harbor Laboratory; and Muramatsu, Masami,“ Lab Manual Genetic Engineering ”, 1988, Maruzen Co., Ltd.).

Probes and primers designed based on the nucleotide sequence, or its complementary nucleotide sequence of the P2Y ₁₂ receptor gene, conventional methods, for example, be prepared by chemical synthesis or the like.

  One aspect of the present invention relates to a reagent kit containing the polynucleotide. The scope of the reagent kit according to the present invention also includes a reagent kit comprising one or more containers filled with one or more of the above polynucleotides. Preferred examples of the reagent kit according to the present invention include a reagent kit containing the polynucleotide described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the Sequence Listing. Moreover, as a reagent kit according to the present invention, in addition to the polynucleotide described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing, the polynucleotide described in any one of SEQ ID NO: 1 to SEQ ID NO: 49 is used. The reagent kit containing can be illustrated. This reagent kit can contain substances required for carrying out the detection method, the test method, and the compound identification method according to the present invention, such as a labeling substance, a buffer solution, and a salt. Furthermore, substances such as stabilizers and / or preservatives may be included. In the formulation, a known formulation means may be introduced according to each substance to be used, for example, a polynucleotide. Such a reagent and reagent kit can be used as a test agent and a test kit in the detection method and test method according to the present invention.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to the following Example. When in this embodiment indicates the position of each base _{P2Y 12} receptor gene, unless otherwise defined, it indicated as positions in the base sequence disclosed in GenBank accession number NC_000003.8.

(Analysis of the genomic sequence of the _{P2Y 12} receptor gene)
P2Y ₁₂ for 5'-side about 150bp from the point of exon 1 upstream 3kb of exon 2 of the receptor gene (total length of about 5 kb), were detected in the mutant by analysis of the genomic sequence. The sample used was genomic DNA provided by 141 healthy Japanese individuals.

First, using 20 cases of genomic DNA, from genomic DNA to amplify the target region of P2Y ₁₂ receptor gene was performed to detect the mutation by determining the nucleotide sequence of the amplification products obtained using the direct sequencing method .

Amplification of the target region was performed by PCR. Target region as a reference sequence to genomic nucleotide sequence disclosed in GenBank accession number NC_000003.8, set at four locations of the genomic sequence of the _{P2Y 12} receptor gene contained in the genome sequence, PCR primers are Designed and prepared based on the nucleotide sequence (Table 4). PCR was performed in a 20 μl reaction system using Ex Taq (manufactured by Takara Bio Inc.) or LA Taq (manufactured by Takara Bio Inc.) using 2 μl of the genomic DNA sample as a template. PCR reaction conditions used were different for each target region (Table 5).

PCR reaction conditions A
1) 94 ° C for 4 minutes 2) 94 ° C for 30 seconds 3) 62 ° C for 30 seconds 4) 72 ° C for 2 minutes
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 10 minutes

PCR reaction condition B
1) 94 ° C for 4 minutes 2) 94 ° C for 30 seconds 3) 58 ° C for 30 seconds 4) 72 ° C for 2 minutes
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 10 minutes

  The PCR amplification product was purified using exosap (Exo SAP, manufactured by Amersham Biosciences) and used as a template for sequencing reaction. The sequencing reaction was carried out using the primers shown in Table 6 and a dynamic ET dye terminator kit (DyDynamic ET dye terminator kit, manufactured by Amersham Biosciences). The reaction product was purified by gel filtration using Sephadex-50. As the sequencer, Megabase 1000 (MegaBACE1000, manufactured by Amersham Biosciences) was used.

  Using the obtained sequence data, the accuracy (quality value) of each base was calculated, the assembly, and the mutation were detected by Fred / Frap / Polyfred software. As the reference base sequence and base position, the base sequence and base position disclosed in GenBank Accession No. NC — 000003.8 were used.

  As a result of nucleotide sequence analysis, it was revealed that the four types of single nucleotide mutations shown in Table 7 were completely linked to the H2 haplotype. In the genomic DNA in which four types of single nucleotide mutations shown in Table 7 were observed, all three types of single nucleotide mutations existing in intron 1 among the known single nucleotide mutations (Table 1) were observed.

As a result of nucleotide sequence analysis known single base mutation has been reported to be H2 haplotype linkage of P2Y ₁₂ receptor gene of (Table 1, Patent Document 1, Non-Patent Documents 3 and 4), An example was found having an H2 haplotype in which a single base mutation (a mutation from G to T at position 152377507) present in exon 2 is not observed. That is, this single base mutation present in exon 2 is considered not completely linked to the H2 haplotype. In this example, the P2Y ₁₂ receptor gene, single nucleotide mutation of four types found in the present invention (Table 7) was observed, further present in exon 2 of the four known one base mutation shown in Table 1 Three types of single base mutations were observed except for the single base mutation.

Therefore, revealed that the example shown a genotype is different from the conventional H2 haplotype (mutation G to T at position No. 152377507) 1 nucleotide mutation present in exon 2 of the P2Y ₁₂ receptor gene is present It was. The genotype of _{P2Y 12} receptor gene found here referred to as H2' haplotype. Since the H2 ′ haplotype is different from the H2 haplotype only in the presence or absence of a single base mutation at one site, it is considered that the H2 ′ haplotype is related to the enhancement of platelet aggregation ability like the H2 haplotype.

1 base mutation of H2 haplotype and H2' haplotypes and linkage to _{P2Y 12} receptor gene are summarized in Table 8.

  In Table 8, single base mutations shown in # 1, # 2, # 3, and # 7 are single base mutations that have been shown to be completely linked to the H2 haplotype in the present invention. The single base mutations shown in # 4, # 5, and # 6 are known single base mutations (Table 1, Patent Document 1, Non-Patent Document 3, and Patent Document 4). The single base mutation shown in # 8 is a single base mutation reported to be linked to the H2 haplotype (Patent Document 1, Non-Patent Document 3 and Patent Document 4), but this single base mutation was recognized in Japanese. I found several examples that could not be done. Since the reported haplotype exists in the same position in Japanese, it is assumed that the H2 'haplotype also exists in Westerners.

(Analysis of the base at position 15379350 and the base at position 152377507)
Next, the healthy person 121 cases using genomic DNA, H2 haplotype and completely chain to have one base mutation in _{P2Y 12} receptor gene, and H2 linkage with haplotypes of the 152,377,507 of which turned out not completely One base mutation was analyzed. As a fully chain to have one base mutation and H2 haplotypes of P2Y ₁₂ receptor gene was analyzed one base mutation at position No. 152379350. Since _{P2Y 12} receptor gene having a single base mutation at position No. 152379350 is H2 haplotype in the genomic DNA one base mutation at position No. 152 379 350 was observed, completely chain and H2 haplotype _{P2Y 12} receptor gene There are other single-base mutations (such as single-base mutations at positions 152381673, 15238188, and 1523788084).

Detection of these two types of single-base mutations, the detection of the _{P2Y 12} receptor gene was amplified by PCR from the genomic DNA using a DNA fragment containing a base in the target position was performed by SNuPe reaction. PCR was performed using PCR primers using each genomic DNA as a template. As the PCR primer, each polynucleotide described in SEQ ID NO: 17 and SEQ ID NO: 34 was used for amplification of a DNA fragment containing the base at position 152379350. In addition, each polynucleotide described in SEQ ID NO: 23 and SEQ ID NO: 40 was used for amplification of a DNA fragment containing the base at position 152377507. Specifically, 2 μl of each template was used, and PCR was performed in a 10 μl reaction system using Ex Taq (Takara Bio). For positive control, human genomic DNA (Clontech) prepared at 10 ng / μl was used, and for negative control, PCR was performed without adding a template.

PCR reaction conditions are shown below.
1) 94 ° C 4 minutes 2) 94 ° C 30 seconds 3) 54 ° C 30 seconds 4) 72 ° C 45 seconds
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 4 minutes

  The PCR amplification product was cooled at 4 ° C., purified by gel filtration using Sephadex G50 Fine (manufactured by Amersham Biosciences), and used for SNuPe reaction.

The SNuPe reaction was performed using an SNuPe primer designed immediately before the position where each single nucleotide mutation exists, a purified PCR amplification product as a template, and a megabase SNuPe genotyping kit (MegaBACE SNuPe Genotyping kit, manufactured by Amersham Biosciences). It was carried out according to the method described in the attachment. As the SNuPe primers for typing the bases at positions 152379350 and 152377507, the respective polynucleotides described in Table 9 were used. Note that the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 42 is a polynucleotide designed based on the nucleotide sequence consisting of 25 nucleotides from the first 152,377,532 of the _{P2Y 12} receptor gene genome sequence to place the 152,377,508 It is a polynucleotide consisting of 24 nucleotides that does not include the nucleotide at position 15237775.

  Typing analysis was performed using a capillary sequencer Megabase 1000 (manufactured by Amersham Biosciences) in a 96-well plate format, and typing was performed using a SNP profiler which is data analysis software.

  In 141 cases examined (including 20 cases analyzed by the direct sequencing method), there were 94 H1 haplotype homozygotes. The H1 / H2 haplotype heterozygote, the H1 / H2 ′ haplotype heterozygote, and the H2 / H2 ′ haplotype heterozygote were 42 cases, 4 cases, and 1 example, respectively.

(Association analysis of genotype and platelet reactivity phenotype of P2Y ₁₂ receptor gene)
P2Y ₁₂ genotype measurement typing and platelet reactivity phenotype of the receptor gene was performed on genomic DNA by the recipient from the Japanese healthy subjects of 217 cases.

Genotype typing was performed on 1 base mutation site at 152,377,525 of the H1 haplotype and 1 nucleotide mutation site may determine and H2 haplotype, and the genes of the genomic nucleotide sequence of the _{P2Y 12} receptor gene.

Determination of H1 haplotype and H2 haplotype of P2Y ₁₂ receptor gene was performed on two 1 base mutation site at 152,379,350 position and positions the 152,377,507 genomic nucleotide sequence of the _{P2Y 12} receptor gene. Haplotype P2Y ₁₂ receptor gene is H1 haplotype when first 152,379,350 position and positions the 152,377,507 is C and G, respectively, which is determined to be H2 haplotype when a T and T, respectively. In genomic DNA, _{P2Y 12} receptor gene H1 haplotype is detected, and if _{the P2Y 12} receptor gene of H2 haplotype is not detected, _{P2Y 12} receptor gene of said genomic DNA is When it is homozygous for H1 haplotype Judged. In contrast, in the genomic DNA, H2 _{P2Y 12} receptor gene haplotype is detected, and if _{the P2Y 12} receptor gene H1 haplotype is not detected, _{P2Y 12} receptor gene of said genomic DNA is homozygous H2 haplotype It was determined to be a body. Further, in the genomic DNA, _{if P2Y 12} receptor gene of _{P2Y 12} receptor gene and H2 haplotype H1 haplotype was detected neither, _{P2Y 12} receptor gene heterozygous H1 / H2 haplotype of the genomic DNA It was determined that

The 152,377,525 of the genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene in genomic DNA, in the genomic DNA, _{P2Y 12} receptor gene of the 152,377,525 base is cytosine (C) is detected and the If one base mutation site bases P2Y ₁₂ receptor gene was not detected is a base other than cytosine (C), is determined that the genotype represented by C / C. In genomic DNA, when the first 152,377,525 of the base _{P2Y 12} receptor gene was detected cytosine (C) at a _{P2Y 12} receptor gene and the single nucleotide mutation site of the base is thymine (T), said genomic DNA the 152377525 of genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene was determined that the genotype represented by C / T. In genomic DNA, when the first 152,377,525 of the base _{P2Y 12} receptor gene was detected is thymine (T) at a _{P2Y 12} receptor gene and the single nucleotide mutation site of the base cytosine (C), said genomic DNA the 152377525 of genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene was determined that the genotype represented by T / T.

P2Y ₁₂ receptor No. 152379350 of the genomic nucleotide sequence of a gene, IN 152 377 507, and determination of the 152,377,525 of genotypes by SNuPe method was performed as follows. First, PCR was performed using genomic DNA as a template and PCR primers to amplify region 1 containing a single nucleotide mutation site at position 152379350 and region 2 containing single nucleotide mutation sites at positions 152377507 and 1523777525. . As a primer for amplification of region 1, a combination of polynucleotides represented by the nucleotide sequences described in SEQ ID NO: 17 and SEQ ID NO: 34 (Table 10) was used. As the amplification primer for region 2, a combination of polynucleotides represented by the nucleotide sequences described in SEQ ID NO: 23 and SEQ ID NO: 40 (Table 10) was used. PCR was performed in a reaction system of 20 μl using 0.5 to 1 μl of each template and using ThermoStart, buffer, and deoxynucleotide triphosphates (dNTPs) (all manufactured by AB gene). PCR was performed without adding a template to the negative control. PCR reaction conditions are shown below.

PCR reaction conditions for region 1 amplification 1) 94 ° C. for 4 minutes 2) 94 ° C. for 30 seconds 3) 55 ° C. for 30 seconds 4) 72 ° C. for 2 minutes
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 4 minutes

PCR reaction conditions for region 2 amplification 1) 94 ° C. for 4 minutes 2) 94 ° C. for 30 seconds 3) 65 ° C. for 30 seconds 4) 72 ° C. for 2 minutes
Steps 2) to 4) were performed for 45 cycles.
5) 72 ° C for 4 minutes

  The PCR amplification product was purified by gel filtration using ExoSAP-IT (manufactured by Amersham Biosciences), and genotype typing was performed by SNuPe method. The SNuPe reaction was carried out using the SNuPe primer designed immediately before each single-base mutation site, the purified PCR amplification product as a template, and a megabase SNuPe genotyping kit (Amersham Biosciences) according to the method described in the kit attachment. did. Polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 41 and the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 42 as SNuPe primers for typing the single nucleotide mutation sites at positions 15379350, 152737507 and 1523777525, respectively The nucleotide and the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 50 were used (Table 11). The product of the SNuPe reaction was purified by gel filtration using Sephadex G50 Fine (Amersham Biosciences). Typing analysis was performed using a capillary sequencer Megabase 1000 (manufactured by Amersham Biosciences) in a 96-well plate format, and typing was performed using a SNP profiler which is data analysis software.

When performing genotype typing only 1 base mutation site at 152,377,525 of the genomic nucleotide sequence of the P2Y ₁₂ receptor gene, using the direct sequencing or SNuPe method.

The 152377525 of genotype typing of genomic nucleotide sequence of the _{P2Y 12} receptor gene by direct sequencing was carried out as follows. First, PCR was performed using genomic DNA as a template and two polynucleotides (Table 12) represented by the nucleotide sequences of SEQ ID NO: 51 and SEQ ID NO: 52 as PCR primers, and a single nucleotide mutation at position 1523777525 The region containing the site (1816 bp) was amplified. PCR was performed using 2 μl of each template and 20 μl of reaction system using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) under the following reaction conditions.

PCR reaction conditions 1) 94 ° C for 4 minutes 2) 94 ° C for 30 seconds 3) 56 ° C for 30 seconds 4) 72 ° C for 2 minutes
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 4 minutes

  The PCR amplification product was purified using Sephadex G50 Fine (manufactured by Amersham Biosciences) and used as a template for sequencing reaction. The sequencing reaction was performed using a dynamic ET dye terminator kit (manufactured by Amersham Biosciences) using two polynucleotides (Table 13) represented by the respective nucleotide sequences described in SEQ ID NO: 53 and SEQ ID NO: 54 as primers. . The product of the sequence reaction was purified using Sephadex G50 Fine (manufactured by Amersham Biosciences). As the sequencer, Megabase 1000 (manufactured by Amersham Biosciences) was used.

  Using the obtained sequence data, the accuracy (quality value) of each base was calculated, the assembly and the mutation were detected by Fred / Flap / Polyfred software. As the reference base sequence and base position, the base sequence and base position disclosed in GenBank Accession No. NC — 000003.8 were used.

The 152377525 of genotype typing of genomic nucleotide sequence of the _{P2Y 12} receptor gene by SNuPe method was carried out as follows. First, PCR was performed using genomic DNA as a template and two polynucleotides (Table 10) represented by the nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 40 as PCR primers, and a single nucleotide mutation at position 1523777525 A region containing the site (699 bp) was amplified. PCR was performed using 2 μl of each template in a 20 μl reaction system using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) under the following reaction conditions.

PCR reaction conditions 1) 94 ° C 4 minutes 2) 94 ° C 30 seconds 3) 54 ° C 30 seconds 4) 72 ° C 45 seconds
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 4 minutes

  The PCR amplification product was purified by gel filtration using ExoSAP-IT (manufactured by Amersham Biosciences), and genotype typing was performed by SNuPe method. The SNuPe reaction was performed in the same manner as the SNuPe reaction except that the polynucleotide (Table 14) represented by the nucleotide sequence shown in SEQ ID NO: 55 was used as the SNuPe primer. The product of the SNuPe reaction was purified by gel filtration using Sephadex G50 Fine (Amersham Biosciences). Typing analysis was performed as described above.

  Platelet reactivity phenotype was evaluated by optical agglutination assay using platelet rich plasma (PRP). Aggregation was induced with 2 or 5 μmol / L ADP. Analyzed parameters are maximum aggregation (%) and area of the aggregation curve from 0 to 6 minutes (AUC).

A P2Y ₁₂ receptor homozygous haplotypes H1 haplotype genes, and the individuals from platelet is a genotype which first 152,377,525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C, As a result of comparison with platelets derived from individuals not having such genotypes, the maximum aggregation and AUC induced by 2 μmol / L ADP were statistically significantly lower in the former platelets than in the latter. (FIG. 1). However, maximal aggregation and AUC induced by 5 μmol / L ADP showed almost no difference between the former-derived platelets and the latter-derived platelets (FIG. 1). In FIG. 1, the former is expressed as “H1 homo & 15237725 C / C”, and the latter is expressed as “Others”.

On the other hand, from individuals and from individuals platelet 152377525 of genotype of the genomic nucleotide sequence of the P2Y ₁₂ receptor gene is C / C, genotype of the single nucleotide mutation site is a genotype other than C / C As a result of comparison of the platelets of the former regardless of the haplotype of the gene, the maximum aggregation and AUC induced by 2 or 5 μmol / L ADP were statistically significantly higher in the former platelets than in the latter. It was low (Figure 2). In FIG. 2, the former is expressed as “1523777525 C / C”, and the latter is expressed as “1523777525 T carriers”.

In contrast, the haplotype H1 haplotype of P2Y ₁₂ receptor gene from the individual platelets are homozygous, and platelets from individuals with P2Y ₁₂ receptor gene haplotype is H2 haplotype of the gene As a result of comparison regardless of the genotype at position 15237775 of the genomic base sequence, there was almost no difference in maximum aggregation and AUC regardless of the concentration of ADP (FIG. 3). In FIG. 2, the former genotype is represented as “H1 homo”, and the latter genotype is represented as “H2 carriers”.

(- control study case on the genotype of the _{P2Y 12} receptor gene)
The genotype of typing of P2Y ₁₂ receptor gene, performed on genomic DNA was provided from CAD patients and healthy persons, respectively, thereby to analyze the association between genotype and disease of the gene.

Genotyping was performed on 194 CAD patients (CAD group) and 325 healthy individuals (control group). Genotype typing, _{P2Y 12} H1 haplotype and H2 haplotype and may determine a base mutation site (position No. 152379350 and of the 152,377,507) of the receptor gene, and the second 152 377 525 of the genomic nucleotide sequence of the gene, carried Performed in a manner similar to that described in Example 2.

  In order to prevent differences in the age and gender composition between the control group and the CAD group, data from the CAD group data on women, data on teenage CAD patients, and data on patients over 57 years old Was excluded. In addition, data on normal cases of cardiac catheters were excluded. Data on patients under 42 years of age were excluded from the control group data. As a result, the average age of the control group and the CAD group used in the case-control study was 48.6 years and 48.2 years, respectively.

Analyze relevance between genotype and disease P2Y ₁₂ receptor gene, it performs univariate analysis First, when a significant difference was detected in the univariate analysis were carried out more multivariate analysis. Univariate analysis was performed using single nucleotide polymorphisms of genes as independent variables. Multivariate analysis was performed using factors such as single nucleotide polymorphism of the gene, hyperlipidemia, hypertension, presence / absence of diabetes, history of smoking, and body mass index (BMI value) as independent variables. When a significant difference is detected in the odds ratio (OR) obtained by both the univariate analysis and the multivariate analysis, it can be determined that there is an association between the genotype of this gene and the disease. Significant difference was determined by P value, and when P <0.05, it was determined that there was significant difference. In both analyses, a 95% confidence interval (95% CI) was established and the reliability of the results of univariate analysis and multivariate analysis was determined.

As shown in Table 15, between the CAD group and the control group, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of said gene C As a result of analyzing an example having a genotype represented by / C and an example having no such genotype, the OR value in univariate analysis was 1.9, and at this time, the 95% CI value was 1.2. -3.1. The OR value in the multivariate analysis was 2.0, and at this time, the 95% CI value was 1.1-3.3. Significant differences were detected in the results of both analyses. Moreover, since both 95% CI values were 1 or more in both analyses, it was determined that the OR value obtained in both analyzes was highly reliable. That is, in the CAD group, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and is the genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C It was proved reliably that the frequency was significantly higher.

On the other hand, between the CAD group and the control group, and examples are genotypes No. 152377525 of the genotype of the genomic nucleotide sequence of the _{P2Y 12} receptor gene is expressed in C / C, genotype of the single nucleotide mutation site As a result of analyzing the case where genotypes other than C / C (C / T or T / T) are analyzed regardless of the haplotype of the gene, the OR value in univariate analysis is 1.4. The 95% CI value was 0.8-2.2 (Table 16). The OR value in the multivariate analysis was 1.2, and at this time, the 95% CI value was 0.7-2.2. No significant difference was detected in the results of both analyses. In addition, since both 95% CI values in both analyzes are values sandwiching 1, the OR value obtained in both analyzes was determined to be low in reliability (Table 16).

Further, between the CAD group and the control group, _{P2Y 12} and example haplotype receptor gene is homozygous for H1 haplotype, example having _{P2Y 12} receptor gene H2 haplotype (H1 / H2 heterozygotes or H2 / H2 homozygote), the OR value in univariate analysis was 1.6 as a result of the analysis regardless of the genotype at position 15237775 of the genomic base sequence of the gene. At this time, the 95% CI value Was 1.0-2.8 (Table 17). The OR value in the multivariate analysis was 1.7, and at this time, the 95% CI value was 0.9-3.1. No significant difference was detected in the results of both analyses. In addition, since both 95% CI values in both analyzes are values sandwiching 1, the OR value obtained in both analyzes was determined to be low in reliability (Table 17).

From the above results, and haplotypes P2Y ₁₂ receptor gene is homozygous for H1 haplotype and a genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C , It became clear that there is an association with CAD.

(Association analysis between genotype and CAD severity of _{P2Y 12} receptor gene)
Using donor genomic DNA from CAD patients, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C The relationship between the genotype and the severity of CAD was analyzed.

  The data accumulated in Example 3 was used as genotyping data for genomic DNA provided by CAD patients. The severity of CAD was classified by the number of coronary artery branches. That is, the greater the number of coronary branches, the more severe it is, and when the number of coronary branches is 3, the severity is 1 or 2, and the severity is 2 or 1. Classified as degree 3.

As a result, as shown in Table 18, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and a 152 377 525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C In the case of genotype, it was found that the number of cases with a severity of 1 was significantly higher than in the case of no genotype.

From this result, homozygous haplotypes H1 haplotype of _{P2Y 12} receptor gene, and a genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C, I think that the symptoms of CAD may be severe.

According to the present invention, by identifying the genotype of the human P2Y ₁₂ receptor gene, it can be detected genotypes with the potential to cause coronary artery disease. As a result, it becomes possible to determine the risk of coronary artery disease and to conduct early diagnosis. The present invention is useful for diagnosis of coronary artery disease and examination for prevention and treatment of the disease, and greatly contributes to the pharmaceutical field.

Haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype and a genotype No. 152377525 of the genotype of the genomic nucleotide sequence of the gene is represented by C / C (in the figure, H1 The maximum aggregation and AUC induced by ADP for platelets derived from individuals (denoted as homo & 15237725 C / C) and those derived from individuals not having such a genotype (denoted as Others in the figure) It is a figure which shows the result compared. The platelets derived from the former had statistically significantly lower maximum aggregation and AUC induced by 2 μmol / L ADP than the platelets derived from the latter. However, the maximum aggregation and AUC induced by 5 μmol / L ADP showed little difference between the former and the latter platelets. In the figure, the position of the base of the human _{P2Y 12} receptor gene, indicated as position in the nucleotide sequence disclosed in GenBank accession number NC_000003.8. (Example 2) The 152377525 of genotype of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene is a gene type represented by C / C (in the figure, referred to as 152377525 C / C) individuals and from platelets, said one base Comparison of ADP-induced maximum aggregation and AUC for platelets derived from individuals whose mutation site genotype is a genotype other than the genotype represented by C / C (denoted 1523777525 T carriers in the figure) It is a figure which shows a result. Maximum aggregation and AUC induced by 2 or 5 μmol / L ADP were statistically significantly lower in the former derived platelets compared to the latter derived platelets. In the figure, the position of the base of the human _{P2Y 12} receptor gene, indicated as position in the nucleotide sequence disclosed in GenBank accession number NC_000003.8. (Example 2) Haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype (in the figure, denoted as H1 homo) and platelets from an individual haplotype has a _{P2Y 12} receptor gene is H2 haplotype (in the figure, It is a figure which shows the result of having compared the maximum aggregation and AUC induced | guided | derived with ADP about the platelet derived from an individual | organism | solid (it describes with H2 carriers). Regardless of the concentration of ADP, there was little difference in maximum aggregation and AUC. (Example 2)

SEQ ID NO: 1 designed polynucleotide for primer.
SEQ ID NO: 2: designed polynucleotide for primer.
SEQ ID NO 3: Designed polynucleotide for primer.
SEQ ID NO: 4: Designed polynucleotide for primer.
SEQ ID NO: 5: designed polynucleotide for primers.
SEQ ID NO: 6: designed polynucleotide for primer.
SEQ ID NO: 7: designed polynucleotide for primer.
SEQ ID NO: 8: designed polynucleotide for primer.
SEQ ID NO: 9: designed polynucleotide for primer.
SEQ ID NO: 10: designed polynucleotide for primer.
SEQ ID NO: 11: designed polynucleotide for primer.
SEQ ID NO: 12: designed polynucleotide for primer.
SEQ ID NO: 13: designed polynucleotide for primer.
SEQ ID NO: 14: designed polynucleotide for primer.
SEQ ID NO: 15: designed polynucleotide for primer.
SEQ ID NO: 16: designed polynucleotide for primer.
SEQ ID NO: 17: designed polynucleotide for primer.
SEQ ID NO: 18: designed polynucleotide for primer.
SEQ ID NO: 19: Designed polynucleotide for primer.
SEQ ID NO: 20: designed polynucleotide for primer.
SEQ ID NO: 21: designed polynucleotide for primer.
SEQ ID NO: 22: designed polynucleotide for primer.
SEQ ID NO: 23: designed polynucleotide for primer.
SEQ ID NO: 24: designed polynucleotide for primer.
SEQ ID NO: 25: designed polynucleotide for primer.
SEQ ID NO: 26: designed polynucleotide for primer.
SEQ ID NO: 27: designed polynucleotide for primer.
SEQ ID NO: 28: designed polynucleotide for primer.
SEQ ID NO: 29: designed polynucleotide for primer.
SEQ ID NO: 30: designed polynucleotide for primer.
SEQ ID NO: 31: designed polynucleotide for primer.
SEQ ID NO: 32: designed polynucleotide for primer.
SEQ ID NO: 33: designed polynucleotide for primer.
SEQ ID NO: 34: designed polynucleotide for primer.
SEQ ID NO: 35: designed polynucleotide for primer.
SEQ ID NO: 36: designed polynucleotide for primer.
SEQ ID NO: 37: designed polynucleotide for primer.
SEQ ID NO: 38: designed polynucleotide for primer.
SEQ ID NO: 39: designed polynucleotide for primer.
SEQ ID NO: 40: designed polynucleotide for primer.
SEQ ID NO: 41: designed polynucleotide for primer.
SEQ ID NO: 42: designed polynucleotide for primer.
SEQ ID NO: 43: designed polynucleotide for primer.
SEQ ID NO: 44: designed polynucleotide for primer.
SEQ ID NO: 45: designed polynucleotide for primer.
SEQ ID NO: 46: designed polynucleotide for primer.
SEQ ID NO: 47: designed polynucleotide for primer.
SEQ ID NO: 48: designed polynucleotide for primer.
SEQ ID NO: 49: designed polynucleotide for primer.
SEQ ID NO: 50: designed polynucleotide for primer.
SEQ ID NO: 51: designed polynucleotide for primer.
SEQ ID NO: 52: designed polynucleotide for primer.
SEQ ID NO: 53: designed polynucleotide for primer.
SEQ ID NO: 54: designed polynucleotide for primer.
SEQ ID NO: 55: designed polynucleotide for primer.
SEQ ID NO: 56: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 57: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 58: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 59: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO 60: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 60: (30): (30) N may be A, C, G or T.
SEQ ID NO: 60: (68): (68) N may be A, C, G or T.
SEQ ID NO 61: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 61: (1): (6) N may be A, C, G or T.
SEQ ID NO: 62: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.

Claims (23)

The haplotype of the human P2Y ₁₂ receptor gene is identified to be a homozygote of the H1 haplotype, and a 152 539 314 of the genotype of the genomic nucleotide sequence of the gene is a gene type represented by C / C A method of detecting a genotype having a possibility of causing coronary artery disease, wherein the means comprises the following steps (where the genome base sequence at position 15253314 is described in GenBank Accession No. NC — 000003.9) (This position corresponds to position 15237725 in the genome sequence described in GenBank Accession No. NC — 0000032.8):
(I) a DNA fragment containing the human _{P2Y 12} receptor gene haplotype 1 base mutation site at nucleotide that can determine, and polymerase chain reaction a DNA fragment containing the first 152 539 314 of the nucleotides of the genomic nucleotide sequence of the gene (PCR) Amplifying by,
And (ii) The sequence of the PCR product obtained in the step (i) is determined by a sequencing method, or the base at the above position is determined by a typing method for the PCR product obtained in the step (i). Process. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C And a method for detecting a genotype having the possibility of causing coronary artery disease, wherein the means comprises the following steps (where, the genome base sequence at positions 152541139, 15253296, and 15253314 are GenBank Accession No. NC_000003.9 The positions are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.8, respectively, corresponding to positions 15379350, 1523777507, and 1523777525):
(I) the human _{P2Y 12} receptor gene DNA fragment comprising the first 152 541 139-position and / or the 152 539 296 of the nucleotides of the genomic nucleotide sequence and the gene of the genomic nucleotide sequence polymerase chain The DNA fragment containing the first 152 539 314 of the nucleotides of, Amplifying by reaction (PCR);
And (ii) The sequence of the PCR product obtained in the step (i) is determined by a sequencing method, or the base at the above position is determined by a typing method for the PCR product obtained in the step (i). Process. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C a means to detect, said means comprises first 152,541,139 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps, coronary Method for detecting genotype having possibility of causing arterial disease (here, positions 15251139, 152 of the genome base sequence) Positions 39296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are positions 152379350 in the genome base sequence described in GenBank Accession No. NC — 0000033.8, respectively. , Corresponding to positions 15237507 and 15237575):
(I) amplified by a DNA fragment containing the first 152 541 139 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 17 and SEQ ID NO: 34 (PCR) The process of
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152541139 is determined by typing using the polynucleotide of SEQ ID NO: 41 or 43 as a primer Process. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C a means to detect, said means comprises first 152,539,296 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the G / G by the following steps, coronary Method for detecting genotype having possibility of causing arterial disease (here, positions 15251139, 152 of the genome base sequence) Positions 39296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are positions 152379350 in the genome base sequence described in GenBank Accession No. NC — 0000033.8, respectively. , Corresponding to positions 15237507 and 15237575):
(I) amplified by a DNA fragment containing the first 152 539 296 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 23 and SEQ ID NO: 40 (PCR) The process of
And (ii) determining the base at the position corresponding to position 15253296 of the PCR product obtained in the step (i) by a typing method using the polynucleotide of SEQ ID NO: 42 as a primer. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C a means to detect, said means comprises first 152,539,314 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps, coronary Method for detecting genotype having the possibility of causing arterial disease (here, positions 15541139 and 1515 of genomic base sequence Positions 539296 and 15259314 are defined as positions in the genome base sequence described in GenBank accession number NC — 000003.9. These positions are positions 152379350 in the genome base sequence described in GenBank accession number NC — 000003.8, respectively. , Corresponding to positions 15237507 and 15237575):
(I) amplified by a DNA fragment containing the first 152 539 314 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 23 and SEQ ID NO: 40 (PCR) The process of
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 15253314 is determined by typing using the polynucleotide of SEQ ID NO: 50 or 55 as a primer Process. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C a means to detect, said means comprises first 152,541,139 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps, coronary Method for detecting genotype having possibility of causing arterial disease (here, positions 15251139, 152 of the genome base sequence) Positions 39296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are positions 152379350 in the genome base sequence described in GenBank Accession No. NC — 0000033.8, respectively. , Corresponding to positions 15237507 and 15237575):
(I) amplified by a DNA fragment containing the first 152 541 139 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, the polymerase chain reaction using as primers the polynucleotide of SEQ ID NO: 5 and SEQ ID NO: 6 (PCR) The process of
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 18 and SEQ ID NO: 34 as primers. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C a means to detect, said means comprises first 152,539,296 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the G / G by the following steps, coronary Method for detecting genotype having possibility of causing arterial disease (here, positions 15251139, 152 of the genome base sequence) Positions 39296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are positions 152379350 in the genome base sequence described in GenBank Accession No. NC — 0000033.8, respectively. , Corresponding to positions 15237507 and 15237575):
(I) a DNA fragment containing the first 152 539 296 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, according to SEQ ID NO: 7 or SEQ ID NO: 51 polynucleotide, and, according to SEQ ID NO: 8 or SEQ ID NO: 52 Amplifying by polymerase chain reaction (PCR) using the polynucleotide as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method. The haplotype of the human _{P2Y 12} receptor gene is homozygous for H1 haplotype, the 152,541,139 of genomic nucleotide sequence of the gene of and / or positions the 152,539,296 a genotype expressed in C / C It is identified by detecting that the genotype is a genotype represented by G / G, and the genotype at position 15253314 of the genomic base sequence of the gene is a genotype represented by C / C a means to detect, said means comprises first 152,539,314 of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene is detected that the genotype represented by the C / C according to the following steps, coronary Method for detecting genotype having possibility of causing arterial disease (here, positions 15251139, 152 of the genome base sequence) Positions 39296 and 15253314 are defined as positions in the genome base sequence described in GenBank Accession No. NC — 000003.9. These positions are positions 152379350 in the genome base sequence described in GenBank Accession No. NC — 0000033.8, respectively. , Corresponding to positions 15237507 and 15237575):
(I) a DNA fragment containing the first 152 539 314 of the nucleotides of the genomic nucleotide sequence of the human _{P2Y 12} receptor gene, according to SEQ ID NO: 7 or SEQ ID NO: 51 polynucleotide, and, according to SEQ ID NO: 8 or SEQ ID NO: 52 Amplifying by polymerase chain reaction (PCR) using the polynucleotide as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method. A method for examining the risk of coronary artery disease, comprising using the method for detecting a genotype having a possibility of causing coronary artery disease according to any one of claims 1 to 8. Homozygous for the haplotype H1 haplotype a human _{P2Y 12} receptor gene, and _{P2Y 12} receptor No. 152539314 of the genotype of the genomic nucleotide sequence of the gene is a gene type represented by C / C and platelets to hold the protein shown by the amino acid sequence encoded by the body gene (platelets a), control platelets (platelet B, where platelets B is any one of the P2Y ₁₂ receptor gene selected from the following group is platelets for holding a protein shown by the amino acid sequence encoded by: (i) homozygous for the haplotype H1 haplotype a human P2Y ₁₂ receptor gene, and the genomic nucleotide sequence of the gene genotype at position No. 152539314 is a C / T or T / T; (ii) the human _{P2Y 12} receptor gene The haplotype is an H1 / H2 haplotype heterozygote and the genotype of the gene at position 15253314 is C / C, C / T, or T / T; and (iii) human a P2Y ₁₂ receptor gene in a by homozygous for the haplotype H2 haplotype and, genome sequence No. 152539314 of the genotype of the gene is C / C, C / T or T / T,) And a human P2Y ₁₂ receptor agonist in the presence of each compound to induce platelet aggregation, followed by a dose-dependent curve of platelet aggregation by platelet A and the dose of platelet aggregation by platelet B and comparing the dependence curves, methods for identifying compounds that induce desensitization of platelets to human P2Y ₁₂ receptor agonists Here, the 15253314th position of the genome base sequence is defined as the position in the genome base sequence described in GenBank accession number NC_000003.9, but this position is in the genome base sequence described in GenBank accession number NC_000003.8. No. 1523777525). The method for identifying a compound according to claim 10, wherein the human P2Y ₁₂ receptor agonist is adenosine 5 'diphosphate. A polynucleotide represented by the base sequence described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing. A reagent kit comprising the polynucleotide according to any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing. A reagent kit comprising the polynucleotide described in any one of SEQ ID NO: 50 to SEQ ID NO: 55 in the sequence listing and the polynucleotide described in any one of SEQ ID NO: 1 to SEQ ID NO: 49 in the sequence listing. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A method for detecting a genotype having the possibility of causing coronary artery disease, the method comprising detecting a subtype as a means, wherein the means comprises the following steps:
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, site corresponds to a 51 th nucleotide of the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence and / or A DNA fragment comprising a partial base sequence of the genomic base sequence and comprising a nucleotide corresponding to the 69th nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing, and the genomic base sequence of the gene, A step of amplifying a DNA fragment containing a nucleotide at a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 in the partial sequence of the genomic base sequence by the polymerase chain reaction (PCR);
And (ii) The sequence of the PCR product obtained in the step (i) is determined by a sequencing method, or the base at the above position is determined by a typing method for the PCR product obtained in the step (i). Process. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A means to detect that a child type, in said means genome sequence of the human P2Y ₁₂ receptor gene by the following steps, described in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A method for detecting a genotype having a possibility of causing coronary artery disease, comprising detecting that a genotype of a site corresponding to the 51st nucleotide of the nucleotide sequence of the nucleotide sequence is a genotype represented by C / C :
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 17 and SEQ ID NO: 34 as primers;
And (ii) the process for the PCR product obtained in the (i), in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, according to SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of determining a base at a site corresponding to the 51st nucleotide of the base sequence by a typing method using the polynucleotide of SEQ ID NO: 41 or 43 as a primer. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A means to detect that a child type, in said means genome sequence of the human P2Y ₁₂ receptor gene by the following steps, described in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A method for detecting a genotype having a possibility of causing coronary artery disease, comprising detecting that the genotype of a site corresponding to the 69th nucleotide of the nucleotide sequence of GnG is a genotype represented by G / G :
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the first 69-th site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 40 as primers;
And (ii) the process for the PCR product obtained in the (i), in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, according to SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of determining a base at a site corresponding to the 69th nucleotide of the base sequence by a typing method using the polynucleotide of SEQ ID NO: 42 as a primer. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A means to detect that a child type, in said means genome sequence of the human P2Y ₁₂ receptor gene by the following steps, described in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A method for detecting a genotype having a possibility of causing coronary artery disease, comprising detecting that a genotype of a site corresponding to the 51st nucleotide of the nucleotide sequence of the nucleotide sequence is a genotype represented by C / C :
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 40 as primers;
And (ii) the process for the PCR product obtained in the (i), in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, according to SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of determining the base of the site corresponding to the 51st nucleotide of the base sequence by a typing method using the polynucleotide of SEQ ID NO: 50 or 55 as a primer. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A means to detect that a child type, in said means genome sequence of the human P2Y ₁₂ receptor gene by the following steps, described in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A method for detecting a genotype having a possibility of causing coronary artery disease, comprising detecting that a genotype of a site corresponding to the 51st nucleotide of the nucleotide sequence of the nucleotide sequence is a genotype represented by C / C :
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A step of amplifying the contained DNA fragment by polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 18 and SEQ ID NO: 34 as primers. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A means to detect that a child type, in said means genome sequence of the human P2Y ₁₂ receptor gene by the following steps, described in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A method for detecting a genotype having a possibility of causing coronary artery disease, comprising detecting that the genotype of a site corresponding to the 69th nucleotide of the nucleotide sequence of GnG is a genotype represented by G / G :
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the first 69-th site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence Amplifying a DNA fragment containing the polynucleotide according to SEQ ID NO: 7 or 51 and polymerase chain reaction (PCR) using the polynucleotide according to SEQ ID NO: 8 or 52 as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method. The haplotype of the human P2Y ₁₂ receptor gene is homozygous for H1 haplotype in the genome nucleotide sequence of the gene, the nucleotide sequence set forth in SEQ ID NO: 58 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence The genotype of the site corresponding to the 51st nucleotide of the gene is a genotype represented by C / C and / or a partial base sequence of the genomic base sequence and the base set forth in SEQ ID NO: 62 in the sequence listing By detecting that the genotype of the site corresponding to the 69th nucleotide of the sequence is a genotype represented by G / G, and in the genomic base sequence of the gene, Genetics having a partial base sequence and a genotype of a site corresponding to the 51st nucleotide of the base sequence set forth in SEQ ID NO: 62 of the Sequence Listing as C / C A means to detect that a child type, in said means genome sequence of the human P2Y ₁₂ receptor gene by the following steps, described in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence A method for detecting a genotype having a possibility of causing coronary artery disease, comprising detecting that a genotype of a site corresponding to the 51st nucleotide of the nucleotide sequence of the nucleotide sequence is a genotype represented by C / C :
(I) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, the 51st site corresponding to nucleotides nucleotide base sequence set forth in SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence Amplifying a DNA fragment containing the polynucleotide according to SEQ ID NO: 7 or 51 and polymerase chain reaction (PCR) using the polynucleotide according to SEQ ID NO: 8 or 52 as a primer;
And (ii) using the sequence of the PCR product obtained in the step (i) as a primer with the polynucleotide of SEQ ID NO: 24 or SEQ ID NO: 53 and the polynucleotide of SEQ ID NO: 40 or SEQ ID NO: 54 The process of using and determining by a sequencing method. A method for examining the risk of coronary artery disease according to any one of claims 15 to 21, wherein the method for detecting a genotype having a possibility of causing coronary artery disease is used. A human P2Y ₁₂ receptor gene homozygous in its haplotype H1 haplotype, and in the genome nucleotide sequence of the gene, according to SEQ ID NO: 62 of the Sequence Listing a partial nucleotide sequence of the genomic nucleotide sequence platelets (platelets 51st genotypes site corresponding to nucleotides holds the protein shown by the amino acid sequence encoded by the P2Y ₁₂ receptor gene, a genotype represented by C / C in nucleotide sequence in a), but the control platelets (platelet B, where platelets B is a platelet for holding a protein shown by the amino acid sequence encoded by the P2Y ₁₂ receptor gene of any one selected from the following group :( i) homozygous for the haplotype H1 haplotype a human P2Y ₁₂ receptor gene, and the genome salts of the gene In the base sequence, the genotype of the site corresponding to the 51st nucleotide of the base sequence described in SEQ ID NO: 62 of the sequence listing in the partial base sequence of the genomic base sequence is C / T or T / T; (ii) is its haplotype a human P2Y ₁₂ receptor gene H1 / H2 haplotype heterozygotes, and, in the genome sequence of the gene, the sequence of the sequence listing a partial nucleotide sequence of the genomic nucleotide sequence As a and (iii) the human _{P2Y 12} receptor gene; the 51-th genotype sites corresponding to nucleotide is C / C, C / T or T / T, the nucleotide sequence set forth in ID NO: 62 The haplotype is a homozygote of the H2 haplotype, and the genomic base sequence of the gene is a partial base sequence of the genomic base sequence and the sequence listing 51st genotypes site corresponding to nucleotide base sequence C / C according to the numbers 62, a is a) a C / T or T / T,, human P2Y ₁₂ receptor in the presence of a compound in each Human P2Y ₁₂ receptor comprising contacting a bodily agonist to induce platelet aggregation and then comparing a dose-dependent curve of platelet aggregation by platelet A with a dose-dependent curve of platelet aggregation by platelet B A method of identifying a compound that induces platelet desensitization to a body agonist.

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