JP2006180876A - Method for detecting gene mutation associated with thrombotic disease by gene analysis of 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 mutation having possibility of causing the thrombotic disease (such as peripheral artery disease, coronary artery disease and cerebral accident) uses a means for detecting four kinds of single-base mutations present at the upstream from exon 2 in the genome sequence of a P2Y<SB>12</SB>receptor gene. The method for examining the morbid risk of the thrombotic disease utilizes the detection method. The gene having the single-base mutations, the polynucleotide usable for the method, and the reagent kit are also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention, by the analysis of single base mutations in human P2Y ₁₂ receptor gene, a method for detecting gene mutation with the potential to cause thrombotic disease. The present invention also relates to a method for examining the risk of thrombotic disease using the detection method. Furthermore, the present invention relates to a gene having the single nucleotide mutation, and 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 dark-stained granules in platelets, and is released to the outside of the cell upon platelet activation. 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 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 nucleotide 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 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.

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.

  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 thrombi, 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.

  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 reduced in risk by developing factors that increase the risk of these diseases, such as smoking, high blood pressure, high fat / high cholesterol diet habits, 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.

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).

An object of the present invention is to provide a means for enabling an early diagnosis and morbidity risk factor determination find useful single base mutations to identify of H2 haplotype of the human P2Y ₁₂ receptor gene, further thrombotic disease is there.

The present inventors have revealed that four one base mutation present upstream of exon 2 is H2 haplotype and linkage of the genes in the genome sequence of the human P2Y ₁₂ receptor gene. Then, by utilizing these single nucleotide mutations in a method for detecting a genetic mutation having a possibility of causing a thrombotic disease, and in utilizing the method for an early diagnosis of the disease and a determination method for risk of morbidity, the present invention. Was completed.

Detection of single base mutations in human _{P2Y 12} receptor in the gene, as a template of the human _{P2Y 12} receptor gene, a GenBank accession number NC_000003 gene sequence mutations and haplotypes are not taken into account (chromosome 3 nucleotide sequence, used including) the 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 of 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 of positions 15238300 to 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. It 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, and Table 2-3.

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, which uses the partial base sequence including the nucleotide of the one base mutation site as an index, is homologous to the partial base sequence. Is determined by identifying a single base 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 the 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, one to several bases may be different.

  Specifically, position 152381673 of the nucleotide sequence disclosed in GenBank accession number NC_000003.8 shows the position in the reference sequence when a nucleotide sequence different from the nucleotide sequence is used as the reference sequence: Can be specified. First, a base sequence homologous to the base sequence described in SEQ ID NO: 50 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 152381673 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: 50 in the Sequence Listing. Therefore, by comparing the base sequence set forth in SEQ ID NO: 50 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: 50 can be identified, and the position of the identified site in the reference sequence can be determined.

  In addition, position 15380188 of the base sequence disclosed in GenBank accession number NC_000003.8 is specified 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: 51 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 15238188 of the nucleotide sequence disclosed in GenBank accession number NC — 000003.8 corresponds to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 51 in the Sequence Listing. Therefore, by comparing the base sequence set forth in SEQ ID NO: 51 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: 51 can be identified, and the position of the identified site in the reference sequence can be determined.

  When the base sequence different from the base sequence is used as the reference sequence, the position of the base sequence disclosed in GenBank Accession No. NC_000003.8 is to be specified as shown below. Can do. First, using a base sequence described in SEQ ID NO: 52 of the Sequence Listing (partial base sequence of the 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. Position No. 152379350 of the nucleotide sequence disclosed in GenBank accession number NC — 000003.8 corresponds to the 51st nucleotide of the nucleotide sequence set forth in SEQ ID NO: 52 of the Sequence Listing. Therefore, by comparing the base sequence set forth in SEQ ID NO: 52 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: 52 can be identified, and the position of the identified site in the reference sequence can be determined.

  GenBank Accession No. NC — 000003.8, position 152378084 of the nucleotide sequence, if a nucleotide sequence different from the nucleotide sequence is used as the reference sequence, specify the position in the reference sequence as shown below Can do. First, a base sequence homologous to the base sequence described in SEQ ID NO: 55 of the sequence listing (partial base sequence of the base sequence disclosed in GenBank accession number NC_000003.8) is used as a reference sequence. Find out. Position 1523780884 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: 55 in the Sequence Listing. Therefore, by comparing the base sequence described in SEQ ID NO: 55 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: 55 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 nucleotide base 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 other than the base sequence as a reference sequence. When used, the position in the reference sequence can be specified as shown below. First, a base sequence homologous to the base sequence described in SEQ ID NO: 60 of the sequence listing (partial base sequence of base sequence disclosed in GenBank Accession No. NC — 000003.8) is used as a 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 described in SEQ ID NO: 60 in the sequence listing as an index.

That is, the present invention
1. And means to detect one or more single base mutation selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, with the potential to cause thrombotic disease Gene mutation detection method:
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine;
(4) a 152 378 084 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.8),
2.1 Detection of base mutation, amplified by (i) the human _{P2Y 12} receptor No. 152381673 of intragenic, IN 152 380 188, the polymerase chain reaction a DNA fragment containing the first 152,379,350 position or first 152,378,084 of nucleotide (PCR) And (ii) determining the sequence of the obtained PCR product by a sequencing method, or determining the base at the above position for the obtained PCR product by a typing method, A method for detecting genetic mutations in
3. Comprising the steps, in the detection method (here a 152 381 673-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,381,673 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 381 673 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 1 and SEQ ID NO: 2,
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: 10 and SEQ ID NO: 28 as primers,
4). Comprising the steps, in the detection method (here a 152 380 188-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,380,188 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 380 188 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 3 and SEQ ID NO: 4,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide of SEQ ID NO: 32 as a primer,
5. Comprising the steps, in the detection method (here a 152 379 350-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,379,350 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 379 350 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 5 and SEQ ID NO: 6,
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,
6). Comprising the steps, in the detection method (here a 152 378 084-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,378,084 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 378 084 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 7 and SEQ ID NO: 8,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide of SEQ ID NO: 39 as a primer,
7). Comprising the steps, in the detection method (here a 152 379 350-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,379,350 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 379 350 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 17 and SEQ ID NO: 34,
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152379350 is determined by a typing method using the polynucleotide of SEQ ID NO: 41 or SEQ ID NO: 43 as a primer. The process of
8). Comprising the steps, in the detection method (here a 152 381 673-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,381,673 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 381 673 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152381673 is determined by typing using the polynucleotide of SEQ ID NO: 44 or 45 as a primer The process of
9. Comprising the steps, in the detection method (here a 152 380 188-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,380,188 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 380 188 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 15238188 is determined by typing using the polynucleotide of SEQ ID NO: 46 or 47 as a primer. The process of
10. Comprising the steps, in the detection method (here a 152 378 084-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,378,084 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 378 084 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 1523778084 is determined by typing using the polynucleotide of SEQ ID NO: 48 or 49 as a primer. The process of
11. 3 above. To 10. 1. The method for detecting a single nucleotide mutation according to any one of 1) above. A method for detecting a genetic mutation having the possibility of causing thrombotic disease of
12 Polynucleotides in nucleotide sequence of the human P2Y ₁₂ receptor gene comprising a complementary base sequence of the base sequence or the base sequence having one or more single base mutation selected from the following (1) (4):
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) a 152 379 350 of the base is a thymine; and (4) a 152 378 084 of the base is cytosine (where the position of each base human _{P2Y 12} receptor according to GenBank accession number NC_000003.8 Defined as a position in the genome sequence of a body gene),
13. One or more single base mutation capable of detecting allele-specific nucleic acid probes selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene:
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine;
(4) a 152 378 084 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.8),
14 Of complementary base sequence consisting of a polynucleotide of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene from the following (1) (4) nucleotide sequence or base sequence having from any one of single nucleotide mutations selected, the 1 A polynucleotide comprising 8 to 50 bases that hybridizes under stringent conditions to a gene fragment represented by a base sequence containing a base at a position showing a base mutation:
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine;
(4) a 152 378 084 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.8),
15. The polynucleotide according to any one of SEQ ID NO: 1 to SEQ ID NO: 49 in the sequence listing;
16. 1 above. 2. Or 11. A thrombotic disease morbidity risk test method, characterized by using a method for detecting a genetic mutation having a possibility of causing thrombotic disease
17. 16. The above-mentioned 16. wherein the thrombotic disease is atherothrombosis. Thrombotic disease morbidity risk testing method,
18. 16. The thrombotic disease is a peripheral arterial disease. Thrombotic disease morbidity risk testing method,
19. 1 above. 2. Or 11. A method for selecting a prophylactic and / or therapeutic agent for a thrombotic disease, characterized by using a method for detecting a genetic mutation having a possibility of causing a thrombotic disease of
20. 1 above. 2. Or 11. A method for determining the dose of a prophylactic and / or therapeutic agent for thrombotic disease, comprising using a method for detecting a genetic mutation having the possibility of causing thrombotic disease of
21. 13. Allele-specific nucleic acid probes of the above and 14. Or 15. A reagent kit comprising at least one of the polynucleotides of
22. And means to detect one or more single base mutation selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, with the potential to cause thrombotic disease Gene mutation detection method:
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine;
(4) The 152,539,873 of base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.9),
Detection of 23.1 base mutation, amplified by (i) the human _{P2Y 12} receptor No. 152,543,462 of intragenic, IN 152 541 977, the polymerase chain reaction a DNA fragment containing the first 152,541,139 position or first 152,539,873 of nucleotide (PCR) And (ii) determining the sequence of the obtained PCR product by a sequencing method, or determining the base at the position of the obtained PCR product by a typing method, 22. A method for detecting genetic mutations in
24. Comprising the steps, in the detection method (here a 152 543 462-positional base in the human _{P2Y 12} receptor gene 1 base mutation is cytosine, position of the 152,543,462 positional base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 543 462 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 1 and SEQ ID NO: 2,
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: 10 and SEQ ID NO: 28 as primers,
25. Comprising the steps, in the detection method (here a 152 541 977-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,541,977 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 541 977 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 3 and SEQ ID NO: 4,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide of SEQ ID NO: 32 as a primer,
26. Comprising the steps, in the detection method (here a 152 541 139-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,541,139 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 541 139 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 5 and SEQ ID NO: 6,
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,
27. Comprising the steps, in the detection method (here a 152 539 873-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,539,873 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 539 873 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 7 and SEQ ID NO: 8,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide of SEQ ID NO: 39 as a primer,
28. Comprising the steps, in the detection method (here a 152 541 139-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,541,139 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 541 139 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 17 and SEQ ID NO: 34,
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 SEQ ID NO: 43 as a primer. The process of
29. Comprising the steps, in the detection method (here a 152 543 462-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,543,462 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 543 462 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 15543462 is determined by typing using the polynucleotide of SEQ ID NO: 44 or 45 as a primer The process of
30. Comprising the steps, in the detection method (here a 152 541 977-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,541,977 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 541 977 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152541977 is determined by typing using the polynucleotide of SEQ ID NO: 46 or 47 as a primer The process of
31. Comprising the steps, in the detection method (here a 152 539 873-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,539,873 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 539 873 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152539873 is determined by typing using the polynucleotide of SEQ ID NO: 48 or 49 as a primer The process of
32. 24. To 31. 21. The method for detecting a single base mutation according to any one of the above. A method for detecting a genetic mutation having the possibility of causing thrombotic disease of
33. Polynucleotides in nucleotide sequence of the human P2Y ₁₂ receptor gene comprising a complementary base sequence of the base sequence or the base sequence having one or more single base mutation selected from the following (1) (4):
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) a 152 541 139 of the base is a thymine; and (4) a 152 539 873 of the base is cytosine (where the position of each base human _{P2Y 12} receptor according to GenBank accession number NC_000003.9 Defined as a position in the genome sequence of a body gene),
34. One or more single base mutation capable of detecting allele-specific nucleic acid probes selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene:
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine;
(4) a 152 539 873 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.9),
35. Of complementary base sequence consisting of a polynucleotide of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene from the following (1) (4) nucleotide sequence or base sequence having from any one of single nucleotide mutations selected, the 1 A polynucleotide comprising 8 to 50 bases that hybridizes under stringent conditions to a gene fragment represented by a base sequence containing a base at a position showing a base mutation:
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine;
(4) a 152 539 873 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.9),
36. 22. 23. Or 32. A thrombotic disease morbidity risk test method, characterized by using a method for detecting a genetic mutation having a possibility of causing thrombotic disease
37. 36. The thrombotic disease is atherothrombosis. Thrombotic disease morbidity risk testing method,
38. 36. The thrombotic disease is a peripheral artery disease. Thrombotic disease morbidity risk testing method,
39. 22. 23. Or 32. A method for selecting a prophylactic and / or therapeutic agent for a thrombotic disease, characterized by using a method for detecting a genetic mutation having a possibility of causing a thrombotic disease of
40. 22. 23. Or 32. A method for determining the dose of a prophylactic and / or therapeutic agent for thrombotic disease, comprising using a method for detecting a genetic mutation having the possibility of causing thrombotic disease of
41. 34. 15. Allele-specific nucleic acid probes of the above and 15. Or 35. A reagent kit comprising at least one of the polynucleotides of
Consists of.

According to the present invention, by analyzing single nucleotide mutations in the genome sequence of the human P2Y ₁₂ receptor gene, it can be identified of H2 haplotype of the gene. In individuals with of H2 haplotype P2Y ₁₂ receptor gene, it is observed increased platelet aggregation (Non-patent Document 3). Moreover, H2 haplotype of the human P2Y ₁₂ receptor gene, be a risk factor for the development peripheral artery disorders (Non-Patent Document 4), and high that the correlation exists between the peripheral arterial disease (PAD) (Patent Reference 1) has been reported. Furthermore, the particular H2 haplotype of the human P2Y ₁₂ receptor gene, performing the detection of the low sensitivity morbidity risk of atherothrombosis and thienopyridine antithrombotic agents against (e.g. clopidogrel or ticlopidine or the like to target P2Y ₁₂ receptor) It is thought that it can be done (patent document 1). Accordingly, by the present invention to identify of H2 haplotype of the human P2Y ₁₂ receptor gene, it allows detection of genetic variations with disease according to enhancement of platelet aggregation, the potential to cause, for example, thrombotic diseases. When it is determined that the risk of thrombotic 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. Further, the present invention by identifying of H2 haplotype of the human P2Y ₁₂ receptor gene, thienopyridines and antithrombotic drugs, it is possible to determine a low sensitivity to antithrombotic agents which competitively inhibit P2Y ₁₂ receptor. When it is determined with respect to antithrombotic agents which competitively inhibits the thienopyridine antithrombotic drugs and P2Y ₁₂ receptor by the present invention as a low sensitivity, and selected such that increasing the dose of these agents, with these agents It is possible to select a treatment method other than the treatment method.

  Thus, the present invention is useful for diagnosis of thrombotic diseases and tests for prevention and treatment of the diseases.

The present invention is a single base mutation in genomic sequence of the gene encoding the P2Y ₁₂ receptor present on human platelet surface, diseases related to increased platelet aggregation, e.g. detection of gene mutation which has the potential to cause a thrombotic disease This is achieved by using the method. Furthermore, in the present invention, it is possible to provide a method for examining the morbidity rate of a thrombotic disease characterized by using the detection method.

In the present invention, it revealed that the four types of one base mutation present upstream of exon 2 is H2 haplotype and linkage of the genes in the genome sequence of the human P2Y ₁₂ receptor gene. Hereinafter, the _{P2Y 12} receptor gene, means a human _{P2Y 12} receptor gene.

In individuals with of H2 haplotype P2Y ₁₂ receptor gene, it has been reported that increased platelet aggregation is observed (Non-Patent Document 3). Moreover, H2 haplotype P2Y ₁₂ receptor gene, higher that the correlation exists (Patent Document between be a risk factor for the development peripheral artery disorders (Non-Patent Document 4), and peripheral arterial disease (PAD) 1) has been reported. Similarly stroke or coronary artery disease and PAD is considered because it is a disease pathogenesis of arterial thrombosis, and there is a high correlation between the H2 haplotype of stroke and coronary artery disease and P2Y ₁₂ receptor gene. On the other hand, deep vein thrombosis is a disease caused by venous thrombus, unlike stroke, coronary artery disease and PAD. In deep vein thrombosis, since antiplatelet drugs are effective in preventing thrombus formation and expansion, increased platelet aggregation may be involved in the development of deep vein thrombosis. These, considered one base mutation found in H2 haplotype P2Y ₁₂ receptor gene is a gene mutation with the potential to cause thrombotic disease.

By and means for detecting a single base mutation in P2Y ₁₂ receptor gene of the present invention, it is possible to identify of H2 haplotype of the gene, further diseases related to increased platelet aggregation, possible causes for example thrombotic disease It is possible to detect genetic mutations having sex.

But when indicating the position of each base of _{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.

1 base mutation of _{P2Y 12} receptor gene of the present invention, IN 152 381 673, IN 152 380 188, one base mutation in the 152,379,350 position and positions the 152,378,084. These 1 in _{P2Y 12} receptor gene base mutation was observed, each base at each position, cytosine (C), cytosine (C), thymine (T) and was cytosine (C) (Examples and Table 7 reference). Bases of each of these positions, in the _{P2Y 12} receptor gene H1 haplotype, respectively guanine (G), thymine (T), cytosine (C) and thymine (T). Reports suggesting the chain of the H2 haplotype of one base mutation and P2Y ₁₂ receptor gene is not.

H2 haplotype P2Y ₁₂ receptor gene is disclosed that can be identified by known single nucleotide mutation 4 types of detection as shown in Table 1 (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).

However, among the known single nucleotide mutations shown in Table 1, five cases with H2 haplotypes that do not have a single nucleotide mutation (mutation from G to T at position 152377507) present in exon 2 are derived from healthy Japanese individuals Of genomic samples. In these examples, the P2Y ₁₂ receptor gene, single nucleotide mutation of four types found in the present invention (see Examples and Table 7) were observed, four of known single-base mutations further shown in Table 1 Among them, three types of single-base mutations except for the single-base mutation present in exon 2 were observed.

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 Examples and 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 genomic DNA sample, but since the reported haplotype exists in the same position in the Japanese, it is assumed that the H2' haplotype also exists in Westerners. . Among the known single nucleotide mutations shown in Table 1, the H2 ′ haplotype does not have a single nucleotide mutation present in exon 2 (a mutation from G to T at position 152377507), but P2Y _{12 according to the} present invention. All four types of single-base mutations in the receptor gene 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.

In DNA141 example genome was examined 1 base mutation of P2Y ₁₂ receptor gene, homozygous for H1 haplotype was 94 cases. 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.

The _{P2Y 12} receptor gene is identified as H2 haplotype by detection of single base mutations in _{P2Y 12} receptor gene of the present invention include the _{P2Y 12} receptor gene H2' haplotype. As described above, since the H2 ′ haplotype is different from the H2 haplotype only in the presence or absence of single nucleotide mutation at one site, it is considered that the H2 ′ haplotype is related to the enhancement of platelet aggregation ability like the H2 haplotype. Therefore, the detection of single base mutations in P2Y ₁₂ receptor gene of the present invention, a disease caused by enhancement of platelet aggregation, for example, a method of detecting a gene mutation having the potential to cause a thrombotic diseases can be carried out.

P2Y ₁₂ specific H2 haplotype receptor gene can be performed by detecting a single base mutation in _{P2Y 12} receptor gene of the present invention. Specific H2 haplotype of the gene, can be performed by known 1 base mutation 4 types of detection shown in Table 1, by further performing the detection of single base mutations in P2Y ₁₂ receptor gene of the present invention The certain accuracy will be higher. Of the four known single-base mutations (Table 1), all single-base mutations in intron 1 are completely linked to H2 haplotype, whereas single-base mutations in exon 2 and H2 haplotype are completely linked. Is not allowed. Therefore, it is difficult to identify the H2 haplotype only by detecting a single base mutation present in exon 2. In addition, it is known that detection of single-base mutations involving single-base insertions in intron 1 is generally low in detection accuracy of mutations due to insertion of bases. . In contrast, single nucleotide mutation of P2Y ₁₂ receptor gene of the present invention are any substitution mutation, it is completely linked with H2 haplotype. Therefore, by and means for detecting a single base mutation in P2Y ₁₂ receptor gene of the present invention, as compared with the detection of single base mutations previously reported (Table 1), of H2 haplotype with higher precision and accuracy Can be detected.

Moreover, it turned out that the example which shows a genotype (H2 'haplotype) different from the conventional H2 haplotype exists. 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. Therefore, in the method for detecting all four types of known single-base mutations shown in Table 1, the H2 ′ haplotype is not detected. By and means for detecting a single base mutation in P2Y ₁₂ receptor gene of the present invention, it is detected in both the H2 haplotype and H2' haplotypes.

Detection method of gene mutation according to the present invention is the genomic nucleotide sequence of the P2Y ₁₂ receptor gene, and means to detect single nucleotide mutation that H2 haplotype and linkage of the gene. More specifically, the present detection method comprises (i) determining a base at a position where a single base mutation is present in the P2Y ₁₂ receptor gene, and (ii) the type of the base and the H2 haplotype of the P2Y ₁₂ receptor gene. And (iii) detecting a gene mutation that can cause a disease related to the enhancement of platelet aggregation ability, for example, a thrombotic disease.

Determination of the base is, _{P2Y 12} performs the bases corresponding to the next position in the nucleotide sequence of the receptor gene: preferably of the 152,381,673, IN 152 380 188, a position No. 152379350 and of the 152,378,084. The base is determined at at least one of these positions, preferably two or more, more preferably three or more, and still more preferably four. In addition to the single base mutation in these positions, it is also possible to further detect known single base mutation has been reported that linked to H2 haplotype P2Y ₁₂ receptor gene.

Particular of the presence or absence of linkage with H2 haplotype of the determined base type and P2Y ₁₂ receptor gene, it can be carried out by comparing the 1 base mutation of P2Y ₁₂ receptor gene of the present invention the type of the base. 1 base mutation of _{P2Y 12} receptor gene of the present invention are the following four kinds of single base mutation; (1) No. 152381673 of the base is cytosine (C); (2) a 152 380 188 of the base cytosine (3) the base at position 152379350 is thymine (T); (4) the base at position 152378084 is cytosine (C). When the determined base type is compared with these four types of single-base mutations, if it is found that at least 1, preferably 2 or more, more preferably 3 or more, and even more preferably 4 matches, the mutation is P2Y _12. It can be determined that it is linked to the H2 haplotype of the receptor gene. In addition to the comparison of the 1 base mutation of P2Y ₁₂ receptor gene of the present invention, identical with the known single nucleotide mutations compared to known single base mutation it has been reported that linked to H2 haplotype P2Y ₁₂ receptor gene by determining the presence or absence of one base mutation, it is possible to identify the presence or absence of linkage with H2 haplotype P2Y ₁₂ receptor gene.

The base can be determined using a method 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, the base can be determined as follows. Determination of the base at position 152381673 is performed by, for example, performing genomic PCR using the polynucleotides described in SEQ ID NO: 1 and SEQ ID NO: 2 as primers, and determining the base sequence of the obtained amplification product by a sequencing method. Can be implemented. Examples of the primer used in the sequencing method include the polynucleotides described in SEQ ID NO: 10 and SEQ ID NO: 28. For example, the base at position 15238188 is determined by performing PCR on genomic DNA using the polynucleotides described in SEQ ID NO: 3 and SEQ ID NO: 4 as primers, and determining the base sequence of the obtained amplification product by a sequencing method. Can be implemented. An example of the primer used in the sequencing method is the polynucleotide described in SEQ ID NO: 32. The determination of the base at position 152379350 is, for example, PCR for genomic DNA using each polynucleotide described in SEQ ID NO: 5 and SEQ ID NO: 6 as a primer, and the base sequence of the obtained amplification product is determined by a sequencing method Can be implemented. Examples of the primers used in the sequencing method include the polynucleotides described in SEQ ID NO: 18 and SEQ ID NO: 34. For example, the base at position 152378084 is determined by performing PCR on genomic DNA using the polynucleotides described in SEQ ID NO: 7 and SEQ ID NO: 8 as primers, and determining the base sequence of the obtained amplification product by a sequencing method. Can be implemented. As a primer used for the sequencing method, the polynucleotide described in SEQ ID NO: 39 can be exemplified. 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 it can be used . Further, the primer used 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.

  In addition, the determination of the base can be performed using a known single-base mutation detection method. Examples of single nucleotide mutation detection methods include genotyping methods such as SNuPe method. Specifically, the determination of the base by the SNuPe method is based on the determination of the base at position 152379350, and the DNA fragment containing the base is used as a primer for each of the polynucleotides shown in SEQ ID NO: 17 and SEQ ID NO: 34. Amplification of the genomic DNA by PCR, and the type of base at the target position of the obtained amplification product can be carried out by typing using the polynucleotide of SEQ ID NO: 41 as a typing primer with the SNuPe method (see Examples). ). In addition, the type of base at position 152379350 can be similarly determined using the polynucleotide set forth in SEQ ID NO: 43 as a typing primer. The determination of the bases at positions 152381673, 15238188 and 1523778084 can be similarly performed. Typing primers used for typing the bases at these positions can be appropriately designed and obtained from upstream or downstream base sequences adjacent to the positions. 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. As a primer for typing the base at position 15238188, the polynucleotide described in SEQ ID NO: 46 or SEQ ID NO: 47 can be exemplified. Examples of the nucleotide typing primer for position 1523778084 include the polynucleotide set forth in SEQ ID NO: 48 or 49.

Further, to detect single base mutations in _{P2Y 12} receptor gene of the present invention, furthermore, by or means first 152,377,507 of the base is detected that the guanine (G), it is certain H2' haplotypes. Specifically, the determination of the base at position 152377507 is performed by, for example, performing genomic PCR on each of the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 40, and obtaining the base position at the target position of the obtained amplification product. This can be done by determining the type by genotyping (see Examples). The SNuPe method is an example of the genotyping method. An example of the typing primer is the polynucleotide set forth in SEQ ID NO: 42.

Detection and means of one base mutation of P2Y ₁₂ receptor gene of the present invention, other can be carried out by applying the per se known single nucleotide 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 Academy of Sciences, United States of America, The United States of Science of the United States of America. 94, 10756-10761), Sniper Assay (Piatek, AS, et al., “Nature Biotechnology”, 1998, 16, 359-363). And the tag array method (Fan, JB, et al., “Genome Research”, 2000 , Vol. 10, P.853-860), etc. can be used. 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 a fluorescence resonance energy transfer (FRET) or a Taqman method, can be used.

Detection and means of one base mutation of P2Y ₁₂ receptor gene of the present invention is also using allele-specific nucleic acid probes, it can be carried out by hybridization method, respectively. An allele refers to two or more genes having a difference based on a difference in DNA base sequence occurring at the same locus.

The nucleic acid probe, a polynucleotide represented by the nucleotide sequence, or its complementary nucleotide sequence comprises a single base mutation according to the present invention in the genome sequence of the P2Y ₁₂ receptor gene, the nucleotide positions representing the 1 base mutation A polynucleotide having a base sequence substantially complementary to the base sequence to be included can be exemplified. “Having a substantially complementary base sequence” means a base sequence containing a single base mutation in a sample to be examined (hereinafter referred to as a test sample) or its complementary base when used as a probe. It means that the 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 with single base mutation according to the present invention, conventional methods, for example, be prepared by chemical synthesis or the like. From the obtained polynucleotide, a gene represented by a base sequence comprising the base at the position showing the single base mutation of the polynucleotide represented by the base sequence having the single base mutation or a complementary base sequence of the base sequence By selecting one that hybridizes to the fragment under stringent conditions, the target probe can be obtained. 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.

  By using the method for detecting a gene mutation having a possibility of causing a disease related to enhancement of platelet aggregation ability according to the present invention, a method for examining a disease risk rate of a disease relating to enhancement of platelet aggregation ability can be carried out. In addition, by using this gene mutation detection method, an early diagnosis of a disease associated with enhanced platelet aggregation ability can be performed.

A thrombotic disease is mentioned as a disease related to enhancement of platelet aggregation ability. Preferably, atherothrombosis is mentioned. More specifically, examples include coronary artery disease, stroke, and peripheral artery disease (PAD) caused by occlusion of the artery, and venous thrombosis caused by occlusion of the vein. Since the P2Y ₁₂ high correlation between PAD and in individuals with of H2 haplotype of the receptor gene has been reported to be present (Patent Document 1), even more preferably include PAD.

Inspection method of morbidity risk of disease, specifically, the P2Y ₁₂ receptor gene in a test sample, the position corresponding to position 1 base mutation according to the present invention was recognized at least one of the base base was determined, the type of base at the position can be performed by comparing the nucleotide sequence of the H2 haplotype P2Y ₁₂ receptor gene. 1 base mutation of _{P2Y 12} receptor gene of the present invention are the following four kinds of single base mutation; (1) No. 152381673 of the base is cytosine (C); (2) a 152 380 188 of the base cytosine (3) the base at position 152379350 is thymine (T); (4) the base at position 152378084 is cytosine (C). When the determined base type is compared with these four types of single-base mutations, when it is recognized that at least 1, preferably 2 or more, more preferably 3 or more, and even more preferably 4 matches, P2Y ₁₂ genotypes of the receptor gene may be determined that the H2 haplotype, thereby determined to have a high morbidity risk of diseases related to increased platelet aggregation. Even if both genotypes of the alleles are H2 haplotypes, even if only one of the genotypes is H2 haplotypes, it can be determined that there is a high morbidity risk for diseases associated with enhanced platelet aggregation ability.

A method for predicting responsiveness to a prophylactic and / or therapeutic agent for a disease associated with increased platelet aggregation ability by using the method for detecting a genetic mutation having a possibility of causing a disease associated with enhanced platelet aggregation ability according to the present invention. Can be implemented. The specific H2 haplotype (clopidogrel targeting _{P2Y 12} receptor, ticlopidine and CS-747 etc.) thienopyridine antithrombotic drugs and other _{P2Y 12} receptor competitively inhibit antithrombotic agents (hereinafter, _{P2Y 12} It is thought that low sensitivity to a receptor-competitive antithrombotic drug can be detected (Patent Document 1). Furthermore, in individuals with of H2 haplotype has been reported that platelet aggregation is increased (Non-Patent Document 3), an increase of P2Y ₁₂ receptor numbers on the cell surface are involved in enhancement of the platelet aggregation It is speculated that. Increase in P2Y ₁₂ receptor number is considered to give cause low sensitivity to anti-thrombotic agents that target P2Y ₁₂ receptor. Therefore, by identifying of H2 haplotype by the present invention allows the determination of the low sensitivity to thienopyridine antithrombotic agent and P2Y ₁₂ receptor competitive antithrombotics.

Method of predicting responsiveness to anti-thrombotic agents according to the present invention, specifically, the P2Y ₁₂ receptor gene in a test sample, the position corresponding to position 1 base mutation according to the present invention was observed bases at least one base to determine the type of base at the position can be performed by comparing the nucleotide sequence of the H2 haplotype P2Y ₁₂ receptor gene of. 1 base mutation of _{P2Y 12} receptor gene of the present invention are the following four kinds of single base mutation; (1) No. 152381673 of the base is cytosine (C); (2) a 152 380 188 of the base cytosine (3) the base at position 152379350 is thymine (T); (4) the base at position 152378084 is cytosine (C). When the determined base type is compared with these four types of single-base mutations, when it is recognized that at least 1, preferably 2 or more, more preferably 3 or more, and even more preferably 4 matches, P2Y ₁₂ genotypes of the receptor gene may be determined that the H2 haplotype can be determined thereby to be less reactive to anti-thrombotic agents. Even if both genotypes of the allele are H2 haplotypes, even if only one genotype is H2 haplotype, it can be determined that the reactivity to the antithrombotic drug is low.

Method of predicting responsiveness to anti-thrombotic agents according to the present invention are preferably (clopidogrel targeting P2Y ₁₂ receptor, ticlopidine and CS-747 etc.) thienopyridine antithrombotic drugs and other P2Y ₁₂ receptor competitive anti Can be used to predict reactivity to thrombotic drugs.

By using the method for detecting a gene mutation having a possibility of causing a disease related to enhancement of platelet aggregation ability according to the present invention, a method for selecting a preventive and / or therapeutic agent for a disease related to enhancement of platelet aggregation ability, and A method of determining the dose of the prophylactic and / or therapeutic agent can be performed. The detection method of the present mutations, if the genotype of the P2Y ₁₂ receptor gene is determined to be H2 haplotype, since it can be determined that a low sensitivity to antithrombotic agents, etc. to increase the dose of antithrombotic agents Adjustments can be made. Alternatively, other antithrombotic drugs can be selected without selecting an antithrombotic drug that is considered to be insensitive.

The present invention, genome sequence of the 152381673 provides a polynucleotide having a complementary base sequence to the base sequence or the base sequence of the P2Y ₁₂ receptor gene with single base mutation is a cytosine (C). Moreover, the said allele specific probe and primer can also be provided. For example, as a primer for amplifying a region including the first 152,381,673 position in _{P2Y 12} receptor gene, it 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.

The present invention also provides genomic nucleotide sequence of the 152380188 provides a polynucleotide having a complementary base sequence to the base sequence or the base sequence of the P2Y ₁₂ receptor gene with single base mutation is a cytosine (C). Moreover, the said allele specific probe and primer can also be provided. For example, a combination of the polynucleotides set forth in SEQ ID NO: 3 and SEQ ID NO: 4 can be provided as a primer for amplifying a region containing position 15238188 within the P2Y ₁₂ receptor gene. 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.

The present invention also provides a polynucleotide genome sequence of the 152 379 350 has a complementary base sequence to the base sequence or the base sequence of the P2Y ₁₂ receptor gene with single base mutation is thymine (T). Moreover, the said allele specific probe and primer can also be provided. For example, as a primer for amplifying a region including the first 152,379,350 position in P2Y ₁₂ receptor gene, according to the combination of the polynucleotide according to SEQ ID NO: 5 and SEQ ID NO: 6 or SEQ ID NO: 17 and SEQ ID NO: 34, poly Nucleotide combinations can be provided. Moreover, the combination of the polynucleotide of sequence number 18 and sequence number 34 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 can be provided as a typing primer used for typing of the base at the target position contained in the region. In addition, the polynucleotide of SEQ ID NO: 41 or SEQ ID NO: 43 can be provided as a primer that can be used for typing the base at position 152379350.

The present invention also provides genomic nucleotide sequence of the 152378084 provides a polynucleotide having a complementary base sequence to the base sequence or the base sequence of the P2Y ₁₂ receptor gene with single base mutation is a cytosine (C). Moreover, the said allele specific probe and primer can also be provided. For example, a combination of the polynucleotides shown in SEQ ID NO: 7 and SEQ ID NO: 8 can be provided as a primer for amplifying a region containing position 1523778084 in the P2Y ₁₂ receptor gene. 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 is based on the information relating to the specific base sequence disclosed by the present invention, based on known genetic engineering techniques (eg, Sambrook et al., “Molecular Cloning, Laboratory Manual 2nd Edition ”, 1989, Cold Spring Harbor Laboratory; and Muramatsu, Masami,“ Lab Manual Genetic Engineering ”, 1988, Maruzen Co., Ltd.).

Probes and primers may be produced by designed based on the nucleotide sequence, or its complementary nucleotide sequence of the P2Y ₁₂ receptor gene with single base mutation according to the present invention, conventional methods, such as chemical synthesis and the like.

  Any of the polynucleotide, the probe and the primer can be used as reagents. Further, a reagent kit comprising one or more containers filled with one or more of these is also included in the scope of the present invention. This reagent kit can contain substances required for carrying out the measurement, such as a labeling substance for detecting a single nucleotide mutation, 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, 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 standard 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 (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. Each of the polynucleotides shown in Table 9 was used as SNuPe primers for typing the bases at positions 152379350 and 152377507. 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.

According to the present invention, P2Y ₁₂ can identify the H2 haplotype and H2' haplotypes receptor gene, as a result, it is possible to detect easily thrombotic associated with H2 haplotype of the gene. Furthermore, according to the present invention, it is possible to determine the risk of thrombotic diseases such as atherothrombosis, specifically myocardial infarction, stroke and peripheral arterial disease, and to conduct early diagnosis. The present invention is useful for diagnosis of thrombotic diseases and tests for the prevention and treatment of the diseases, and greatly contributes to the pharmaceutical field.

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: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 51: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 52: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 53: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO 54: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 54: (30): (30) N may be A, C, G or T.
SEQ ID NO: 54: (68): (68) N may be A, C, G or T.
SEQ ID NO: 55: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.
SEQ ID NO: 55: (1): (6) N may be A, C, G or T.
SEQ ID NO: 56: sequence information for specifying the position of one base mutation of the human P2Y ₁₂ receptor gene.

Claims (41)

And means to detect one or more single base mutation selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, with the potential to cause thrombotic disease Gene mutation detection method:
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine;
(4) a 152 378 084 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.8). The detection of one base mutation, were amplified by: (i) 152 381 673 of the human _{P2Y 12} receptor in the gene, the 152,380,188 position, first 152,379,350 position or polymerase chain reaction (PCR) the DNA fragment containing the first 152 378 084 nucleotide positions, (Ii) The detection of a gene mutation according to claim 1, wherein the sequence of the obtained PCR product is determined by a sequencing method, or the base at the position of the obtained PCR product is determined by a typing method. Method. Comprising the steps, in the detection method (here a 152 381 673-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,381,673 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 381 673 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 1 and SEQ ID NO: 2,
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: 10 and SEQ ID NO: 28 as primers. Comprising the steps, in the detection method (here a 152 380 188-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,380,188 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 380 188 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 3 and SEQ ID NO: 4,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide described in SEQ ID NO: 32 as a primer. Comprising the steps, in the detection method (here a 152 379 350-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,379,350 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 379 350 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 5 and SEQ ID NO: 6,
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. Comprising the steps, in the detection method (here a 152 378 084-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,378,084 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 378 084 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 7 and SEQ ID NO: 8,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide described in SEQ ID NO: 39 as a primer. Comprising the steps, in the detection method (here a 152 379 350-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,379,350 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 379 350 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 17 and SEQ ID NO: 34,
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152379350 is determined by a typing method using the polynucleotide of SEQ ID NO: 41 or SEQ ID NO: 43 as a primer. Process. Comprising the steps, in the detection method (here a 152 381 673-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,381,673 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 381 673 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152381673 is determined by typing using the polynucleotide of SEQ ID NO: 44 or 45 as a primer Process. Comprising the steps, in the detection method (here a 152 380 188-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,380,188 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 380 188 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 15238188 is determined by typing using the polynucleotide of SEQ ID NO: 46 or 47 as a primer. Process. Comprising the steps, in the detection method (here a 152 378 084-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,378,084 of base is described in GenBank accession number NC_000003.8 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 378 084 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 1523778084 is determined by typing using the polynucleotide of SEQ ID NO: 48 or 49 as a primer. Process. The method for detecting a gene mutation having the possibility of causing a thrombotic disease according to claim 1, wherein the method for detecting a single nucleotide mutation according to any one of claims 3 to 10 is used. Polynucleotides in nucleotide sequence of the human P2Y ₁₂ receptor gene comprising a complementary base sequence of the base sequence or the base sequence having one or more single base mutation selected from the following (1) (4):
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine; and (4) the base at position 152377844 is cytosine (where the position of each base is the human P2Y ₁₂ receptor described in GenBank Accession No. NC — 000003.8) Defined as the position in the genome sequence of the body gene). One or more single base mutation capable of detecting allele-specific nucleic acid probes selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene:
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine;
(4) a 152 378 084 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.8). Of complementary base sequence consisting of a polynucleotide of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene from the following (1) (4) nucleotide sequence or base sequence having from any one of single nucleotide mutations selected, the 1 A polynucleotide comprising 8 to 50 bases that hybridizes under stringent conditions to a gene fragment represented by a base sequence containing a base at a position showing a base mutation:
(1) the base at position 152381673 is cytosine;
(2) the base at position 15238188 is cytosine;
(3) the base at position 152379350 is thymine;
(4) a 152 378 084 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.8). The polynucleotide according to any one of SEQ ID NO: 1 to SEQ ID NO: 49 in the sequence listing. A method for examining a thrombotic disease morbidity risk, characterized in that the method for detecting a gene mutation having the possibility of causing a thrombotic disease according to claim 1, 2 or 11 is used. The thrombotic disease morbidity test method according to claim 16, wherein the thrombotic disease is atherothrombosis. The thrombotic disease morbidity risk test method according to claim 16, wherein the thrombotic disease is a peripheral artery disease. A method for selecting a prophylactic and / or therapeutic agent for a thrombotic disease, comprising using the method for detecting a gene mutation having the possibility of causing a thrombotic disease according to claim 1, 2 or 11. A method for determining the dose of a prophylactic and / or therapeutic agent for a thrombotic disease, characterized by using the method for detecting a gene mutation having the possibility of causing a thrombotic disease according to claim 1, 2 or 11. A reagent kit comprising the allele-specific nucleic acid probe according to claim 13 and at least one of the polynucleotides according to claim 14 or 15. And means to detect one or more single base mutation selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene, with the potential to cause thrombotic disease Gene mutation detection method:
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine;
(4) a 152 539 873 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.9). The detection of one base mutation, were amplified by: (i) 152 543 462 of the human _{P2Y 12} receptor in the gene, the 152,541,977 position, first 152,541,139 position or polymerase chain reaction (PCR) the DNA fragment containing the first 152 539 873 nucleotide positions, The detection of a gene mutation according to claim 22, wherein (ii) the sequence of the obtained PCR product is determined by a sequencing method, or the base at the position of the obtained PCR product is determined by a typing method. Method. Comprising the steps, in the detection method (here a 152 543 462-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,543,462 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 543 462 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 1 and SEQ ID NO: 2,
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: 10 and SEQ ID NO: 28 as primers. Comprising the steps, in the detection method (here a 152 541 977-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,541,977 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 541 977 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 3 and SEQ ID NO: 4,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide described in SEQ ID NO: 32 as a primer. Comprising the steps, in the detection method (here a 152 541 139-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,541,139 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 541 139 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 5 and SEQ ID NO: 6,
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. Comprising the steps, in the detection method (here a 152 539 873-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,539,873 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 539 873 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 7 and SEQ ID NO: 8,
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotide described in SEQ ID NO: 39 as a primer. Comprising the steps, in the detection method (here a 152 541 139-positional base in the human _{P2Y 12} receptor gene 1 base mutation is thymine, the position of the 152,541,139 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) a step of amplifying by polymerase chain reaction (PCR) using a DNA fragment containing the first 152 541 139 of the nucleotides of the human _{P2Y 12} receptor in the gene, as a primer a polynucleotide according to SEQ ID NO: 17 and SEQ ID NO: 34,
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. Comprising the steps, in the detection method (here a 152 543 462-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,543,462 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 543 462 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 15543462 is determined by typing using the polynucleotide of SEQ ID NO: 44 or 45 as a primer Process. Comprising the steps, in the detection method (here a 152 541 977-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,541,977 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 541 977 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152541977 is determined by typing using the polynucleotide of SEQ ID NO: 46 or 47 as a primer Process. Comprising the steps, in the detection method (here a 152 539 873-positional base in the human _{P2Y 12} receptor gene single nucleotide mutation is a cytosine, the position of the 152,539,873 of base is described in GenBank accession number NC_000003.9 human P2Y ₁₂ is defined as the position in the genome sequence of the receptor gene):
(I) step of the DNA fragment containing the first 152 539 873 of the nucleotides of the human _{P2Y 12} receptor in the gene amplified by polymerase chain reaction (PCR),
And (ii) For the PCR product obtained in the step (i), the base at the position corresponding to position 152539873 is determined by typing using the polynucleotide of SEQ ID NO: 48 or 49 as a primer Process. The method for detecting a gene mutation having a possibility of causing a thrombotic disease according to claim 22, wherein the method for detecting a single nucleotide mutation according to any one of claims 24 to 31 is used. Polynucleotides in nucleotide sequence of the human P2Y ₁₂ receptor gene comprising a complementary base sequence of the base sequence or the base sequence having one or more single base mutation selected from the following (1) (4):
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine; and (4) the base at position 152539873 is cytosine (where the position of each base is the human P2Y ₁₂ receptor described in GenBank Accession No. NC — 000003.9) Defined as the position in the genome sequence of the body gene). One or more single base mutation capable of detecting allele-specific nucleic acid probes selected from the following (1) (4) in the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene:
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine;
(4) a 152 539 873 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.9). Of complementary base sequence consisting of a polynucleotide of the genomic nucleotide sequence of the human P2Y ₁₂ receptor gene from the following (1) (4) nucleotide sequence or base sequence having from any one of single nucleotide mutations selected, the 1 A polynucleotide comprising 8 to 50 bases that hybridizes under stringent conditions to a gene fragment represented by a base sequence containing a base at a position showing a base mutation:
(1) the base at position 15543462 is cytosine;
(2) the base at position 152541977 is cytosine;
(3) the base at position 152541139 is thymine;
(4) a 152 539 873 of the base is cytosine (where the position of each base is defined as the position in the genome sequence of the human _{P2Y 12} receptor gene as described in GenBank accession number NC_000003.9). A method for examining a thrombotic disease morbidity risk, characterized in that the method for detecting a gene mutation having a possibility of causing a thrombotic disease according to claim 22, 23 or 32 is used. The thrombotic disease morbidity risk test method according to claim 36, wherein the thrombotic disease is atherothrombosis. The thrombotic disease morbidity risk test method according to claim 36, wherein the thrombotic disease is a peripheral artery disease. A method for selecting a prophylactic and / or therapeutic agent for a thrombotic disease, comprising using the method for detecting a gene mutation having the possibility of causing a thrombotic disease according to claim 22, 23 or 32. A method for determining the dose of a prophylactic and / or therapeutic agent for a thrombotic disease, comprising using the method for detecting a gene mutation having the possibility of causing a thrombotic disease according to claim 22, 23 or 32. A reagent kit comprising an allele-specific nucleic acid probe according to claim 34 and at least one of the polynucleotides according to claim 15 or 35.