JP4666942B2 - Detection of gene mutations related to thrombotic diseases by gene analysis of von Willebrand factor cleaving enzyme - Google Patents

Description

  The present invention has the potential to cause thrombotic diseases such as thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, coronary artery disease or stroke by analyzing single nucleotide mutations in the human von Willebrand factor cleaving enzyme gene The present invention relates to a method for detecting a gene mutation, a method for examining a risk of developing a thrombotic disease using the detection method, a gene having the mutation, and a polynucleotide and a reagent kit used in the method.

  A von Willebrand factor cleaving enzyme (hereinafter sometimes abbreviated as vWF cleaving enzyme) is an enzyme having an action of cleaving a von Willebrand factor formed as a high polymer into a low polymer. The vWF-cleaving enzyme gene is a gene present in the 9th chromosome q34 locus (Non-patent Document 1), and is also referred to as ADAMTS13 [a disintegrin-like and metalloprotease (reactivesin type) with thrombospondin type 13]. The gene contains 29 exons, has a total length of about 37 kb, and encodes a protein consisting of 1427 amino acids deduced from the base sequence (Patent Document 1). The mutation and polymorphism of the gene have also been reported (Non-patent Document 2).

  Von Willebrand factor (hereinafter sometimes abbreviated as vWF) is a protein produced in vascular endothelial cells and bone marrow megakaryocytes, and is interposed between the platelet membrane protein GPIb and the subendothelial tissue. As an adhesion factor to be bound, it relates to platelet adhesion in the early stage of hemostasis. In circulating blood, it forms a complex with blood coagulation factor VIII and contributes to stabilization of factor VIII.

  The highly polymerized vWF having a strong ability to induce platelet aggregation released from vascular endothelial cells into blood by the vWF-cleaving enzyme is gradually cleaved to become a low polymer having a weak ability to induce platelet aggregation. It is known that when the vWF-cleaving enzyme activity is low, the amount of highly polymerized vWF is relatively increased. That is, platelet thrombi are easily formed when vascular endothelial cells such as microcirculation are damaged in a state where the vWF-cleaving enzyme activity is reduced and the plasma vWF polymerization degree is increased. In the vicinity of the formed platelet thrombus, a blood coagulation process further proceeds to form a fibrin clot, thereby forming a strong hemostatic thrombus. Thrombus is usually removed by fibrinolytic activity.

  In the living body, when a blood vessel is damaged, a physiological hemostasis mechanism works under close interaction such as blood vessel function, platelet function, and coagulation / fibrinolytic system. Abnormalities in the hemostasis mechanism cause various hemorrhagic diseases and thrombotic diseases. For example, thrombotic diseases are caused by damage and detachment of vascular endothelial cells due to active oxygen, change (decrease) in blood flow, and enhancement of blood coagulation ability.

  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.

  As a disease caused by occlusion of the coronary artery, a coronary artery disease (hereinafter abbreviated as CAD) is known. 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.

  Examples of diseases caused by cerebral artery occlusion include stroke. Stroke is a general term for ischemic and hemorrhagic cerebrovascular disorders. Hemorrhagic cerebrovascular disorders include intracerebral hemorrhage and subarachnoid hemorrhage, and ischemic cerebrovascular disorders include cerebral infarction and transient 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 becoming narrowed 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.

  In recent years, it has been reported that a decrease in the activity of vWF-cleaving enzyme is involved in thrombotic thrombocytopenic purpura (hereinafter sometimes abbreviated as TTP) (Non-patent Documents 3-5). In addition, the presence of inhibitors and antibodies against vWF-cleaving enzyme has been reported in the blood of TTP patients (Non-patent Document 6). Furthermore, mutations of vWF-cleaving enzyme have been identified in 7 families with TTP (Non-patent Document 1). It is considered that TTP develops when vascular endothelial cells such as microcirculation are damaged in a state where highly polymerized vWF is increased due to a decrease in vWF-cleaving enzyme activity. There are various causes of injury of vascular endothelial cells. For example, toxins produced by bacteria are known as one of the causes in infections. It is also known that vascular endothelial cells are damaged by drugs such as anticancer agents and immunosuppressants.

  TTP is primarily due to thrombus formation associated with a wide range of microvascular disorders, and is particularly caused by thrombus formation in small arteries such as the brain, kidney and coronary arteries. TTP is a systemic serious disease characterized by five signs of thrombocytopenic purpura, microvascular injury hemolysis, fluctuating neuropsychiatric symptoms, fever, and renal dysfunction. The disease type is classified into idiopathic and secondary, and idiopathic is further classified into acute, chronic and recurrent according to the course. Secondary may include pregnancy, autoimmune diseases (eg, systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, and polyarteritis), malignant tumor diseases (eg, malignant lymphoma, leukemia, gastric cancer) Etc.), infectious diseases (for example, viruses and bacteria, etc.), examples secondary to vaccination, examples of the use of various drugs (such as penicillin, sulfa drugs, and various anticancer agents) have been reported. As treatment methods for TTP, replacement therapy of vWF-cleaving enzyme or therapy for removing an inhibitor of vWF-cleaving enzyme activity, and therapy for introducing a normal vWF-cleaving enzyme gene into vascular endothelial cells are considered.

  Similar to TTP, hemolytic uremic syndrome (hereinafter abbreviated as HUS) is known as a disease having three signs of thrombocytopenia, hemolytic anemia, and renal dysfunction. TTP and HUS are sometimes collectively referred to as TTP / HUS or thrombotic microangiopathy (TMA) because of their similar symptoms. HUS is thought to develop due to platelet aggregation and platelet consumption at the local site, where the inner wall of the small arteries is damaged for some reason, reducing the antiplatelet function of vascular endothelial cells. Factors that cause frequent occurrence are not known and have been reported to be caused by various diseases and infections.

  In addition, as a disease caused by abnormal vWF, von Willebrand disease, which is mainly characterized by bleeding caused by genetic vWF deficiency, is known. Clinical findings of von Willebrand disease include prolonged bleeding time, decreased plasma vWF antigen, qualitative abnormalities in vWF, and decreased ristocetin-induced platelet aggregation. Von Willebrand disease is classified into three disease types, with a quantitative deficiency of vWF as type 1, a qualitative abnormality as type 2, and a complete deficiency as type 3. Furthermore, type 2 includes type 2A, which shows reduced function due to deficiency of vWF, and type 2B, which shows abnormally increased affinity with platelet membrane protein GPIb. A qualitative disorder in which the affinity with factor VIII is reduced is referred to as N-type. For the treatment, a method of increasing the plasma vWF concentration, for example, administration of a plasma-derived factor VIII / vWF complex preparation having a high vWF content or desmopressin acetate, which is a synthetic antidiuretic hormone, is performed.

The documents cited in this specification are listed below.
International Publication No. WO02 / 42441 pamphlet. Levy, GG, et al., “Nature”, 2001, Vol. 413, p. 488-494. Kokame, K, et al., "Proceeding of the National Academy of Science of the United States of America (PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF99, AMERICA 2nd year)". 11902-11907. Furlan, M. et al., “Blood”, 1997, 89, p. 3097-3103. Furlan, M. et al., “New England Journal of Medicine”, 1998, 339, p. 1578-1584. Bianchi, V. et al., “Blood”, 2002, 100, p. 710-713. Tsai, H.-M. et al., “New England Journal of Medicine”, 1998, 339, p. 1585-1594.

  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 factors by correcting factors that increase the risk of these diseases, such as smoking, high blood pressure, high fat / high cholesterol diet, obesity and stress. In addition, thrombotic thrombocytopenic purpura has various causes of its development, and it is necessary to select a treatment suitable for the cause. The development of various treatments has greatly improved the prognosis after onset. However, there are many cases of treatment resistance or recurrence. Therefore, early diagnosis of the disease is desired in order to avoid the risk of developing these diseases, to enable early treatment and to select treatment methods according to various etiologies.

  An object of the present invention is to provide a means that enables early diagnosis and risk determination of thrombotic diseases such as thrombotic thrombocytopenic purpura, coronary artery disease and stroke.

  The present inventors have found that a single base mutation found in the gene of vWF-cleaving enzyme related to platelet aggregation, which is the initial stage of thrombus formation, may cause thrombotic diseases such as TTP, HUS, CAD or Stroke The present invention has been completed by using the method for detecting mutations and the test method for early diagnosis of these diseases and determination of morbidity risk.

That is, the present invention
1. In the base sequence of a human von Willebrand factor cleaving enzyme gene (hereinafter abbreviated as vWF-CP gene), a base sequence having one or more of the single base mutations shown below, or the complement of the base sequence Polynucleotide containing the base sequence:
(1) the base at position 3066539 is adenine;
(2) the base at position 1193 is thymine;
(3) the base at position 1460 is guanine;
(4) the base at position 1992 is adenine;
(5) the base at position 3050 is adenine;
(6) The base at position 3152 is thymine (where the position of the base at position 3066539 is defined as the position in the genomic base sequence of the human vWF-CP gene described in GenBank Accession No. NT — 03514.3, Other base positions are defined as positions in the cDNA base sequence of the gene described in GenBank Accession No. NM_139025).
2. In the genomic base sequence of the human vWF-CP gene, a cDNA derived from a genome having a single base mutation in which the base at position 3066539 is adenine, which is derived from the 3 ′ end of the base sequence derived from exon 3 of the gene and from exon 4 CDNA obtained by inserting the nucleotide sequence set forth in SEQ ID NO: 40 or the nucleotide sequence set forth in SEQ ID NO: 41 between the 5 'end of the nucleotide sequence (here, the position of the base at position 3066539 is the GenBank accession number) Defined as the position in the genomic base sequence of the human vWF-CP gene described in NT — 03514.3.
3. An allele-specific nucleic acid probe capable of detecting one or more of the single nucleotide mutations shown below in the genomic base sequence of the human vWF-CP gene:
(1) the base at position 3066539 is adenine; (2) the base at position 1193 is thymine;
(3) the base at position 1460 is guanine;
(4) the base at position 1786 is guanine;
(5) the base at position 1992 is adenine;
(6) the base at position 2160 is adenine;
(7) the base at position 2724 is cytosine;
(8) the base at position 3050 is adenine;
(9) The base at position 3152 is thymine (here, the position of the base at position 3066539 is defined as the position in the genomic base sequence of the human vWF-CP gene described in GenBank Accession No. NT — 03514.3, Other base positions are defined as positions in the cDNA base sequence of the gene described in GenBank Accession No. NM_139025).
4). In the genomic base sequence of the human vWF-CP gene, a base sequence having any one of the single base mutations shown below or a base at the position showing the single base mutation of a polynucleotide comprising a complementary base sequence of the base sequence A polynucleotide comprising 8 to 50 bases that hybridizes under stringent conditions to a gene fragment represented by the contained base sequence:
(1) the base at position 3066539 is adenine; (2) the base at position 1193 is thymine;
(3) the base at position 1460 is guanine;
(4) the base at position 1786 is guanine;
(5) the base at position 1992 is adenine;
(6) the base at position 2160 is adenine;
(7) the base at position 2724 is cytosine;
(8) the base at position 3050 is adenine;
(9) The base at position 3152 is thymine (here, the position of the base at position 3066539 is defined as the position in the genomic base sequence of the human vWF-CP gene described in GenBank Accession No. NT — 03514.3, Other base positions are defined as positions in the cDNA base sequence of the gene described in GenBank Accession No. NM_139025).
5. 2. A polynucleotide comprising 8 to 50 bases that hybridizes under stringent conditions to a polynucleotide comprising the base sequence inserted in the cDNA of or a polynucleotide fragment represented by a complementary base sequence of the base sequence,
6). A method for detecting a gene mutation having a possibility of causing a thrombotic disease, which comprises detecting one or more of the following single nucleotide mutations in the human vWF-CP gene:
(1) the base at position 3066539 is adenine; (2) the base at position 1193 is thymine;
(3) the base at position 1460 is guanine;
(4) the base at position 1786 is guanine;
(5) the base at position 1992 is adenine;
(6) the base at position 2160 is adenine;
(7) the base at position 2724 is cytosine;
(8) the base at position 3050 is adenine;
(9) The base at position 3152 is thymine (here, the position of the base at position 3066539 is defined as the position in the genomic base sequence of the human vWF-CP gene described in GenBank Accession No. NT — 03514.3, Other base positions are defined as positions in the cDNA base sequence of the gene described in GenBank Accession No. NM_139025).
7). A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single base mutation in which the base at position 3066539 in a human vWF-CP gene is adenine by a method comprising the following steps: (Here, the position of the base at position 3066539 is defined as the position in the genomic base sequence of the human vWF-CP gene described in GenBank accession number NT — 03514.3.):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers,
8). A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the base at position 1193 in a human vWF-CP gene is thymine by a method comprising the following steps: (Here, the position of the base at position 1193 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 as primers,
9. A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the 1460th base in a human vWF-CP gene is guanine by a method comprising the following steps: (Here, the position of the base at position 1460 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 42 and SEQ ID NO: 43 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 42 and SEQ ID NO: 43 as primers,
10. A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the 1786th base in a human vWF-CP gene is guanine by a method comprising the following steps: (Here, the position of the base at position 1786 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 11 and SEQ ID NO: 12 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers,
11. A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the base at position 1992 in the human vWF-CP gene is adenine by a method comprising the following steps: (Here, the position of the base at position 1992 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 15 and SEQ ID NO: 16 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 17 and SEQ ID NO: 18 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 17 and SEQ ID NO: 18 as primers,
12 A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the base at position 2160 in the human vWF-CP gene is adenine by a method comprising the following steps: (Here, the position of the 2160th base is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 19 and SEQ ID NO: 20 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 21 and SEQ ID NO: 22 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 21 and SEQ ID NO: 22 as primers,
13. Detection of a gene mutation having a possibility of causing a thrombotic disease, wherein the detection of a single nucleotide mutation in which the 2724th base in the human vWF-CP gene is cytosine is carried out by a method comprising the following steps: Method (Here, the position of the base at position 2724 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 24 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 25 and SEQ ID NO: 26 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 25 and SEQ ID NO: 26 as primers,
14 A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the base at position 3050 in the human vWF-CP gene is adenine by a method comprising the following steps: (Here, the position of the base at position 3050 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 27 and SEQ ID NO: 28 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 29 and SEQ ID NO: 30 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 29 and SEQ ID NO: 30 as primers,
15. A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single nucleotide mutation in which the base at position 3152 in a human vWF-CP gene is thymine by a method comprising the following steps: (Here, the position of the base at position 3152 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025):
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 31 and SEQ ID NO: 32 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 33 and SEQ ID NO: 34 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 33 and SEQ ID NO: 34 as primers,
16. A method for detecting a gene mutation having a possibility of causing a thrombotic disease, comprising detecting a single base mutation in which the base at position 3066539 in a human vWF-CP gene is adenine by a method comprising the following steps: (Here, the position of the 3066539 base is defined as the position in the genomic base sequence of the human vWF-CP gene described in GenBank Accession No. NT — 03514.3.):
(I) performing reverse transcription polymerase chain reaction (RT-PCR) using the polynucleotides of SEQ ID NO: 37 and SEQ ID NO: 39 as primers;
And (ii) determining the size of the PCR product obtained in the step (i),
17. 6. above. To 16. A method for examining the risk of developing a thrombotic disease using any of the detection methods of
18. 5. The thrombotic disease is thrombotic thrombocytopenic purpura (hereinafter abbreviated as TTP). To 16. Any detection method,
19. 16. The thrombotic disease is thrombotic thrombocytopenic purpura (hereinafter abbreviated as TTP). Inspection method,
20. A hemolytic uremic syndrome (hereinafter referred to as “a hemolytic uremic syndrome”) characterized by detecting a single nucleotide mutation in which the base at position 3066539 in the human vWF-CP gene is adenine and / or a single base mutation in which the base at position 1193 is thymine. A method for detecting a genetic mutation that has the potential to cause HUS) (here, the position of the base at position 3066539 is the position in the genomic base sequence of the human vWF-CP gene described in GenBank Accession No. NT_035014.3) And the position of the base at position 1193 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025).
21. 21. The method according to the above 20, wherein the detection of a single nucleotide mutation in which the base at position 3066539 in the human vWF-CP gene is adenine is performed by the method comprising the following steps. Detection method of
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers,
22. 21. The method according to the above 20, wherein the detection of a single nucleotide mutation in which the base at position 3066539 in the human vWF-CP gene is adenine is carried out by a method comprising the following steps. Detection method of
(I) performing reverse transcription polymerase chain reaction (RT-PCR) using the polynucleotides of SEQ ID NO: 37 and SEQ ID NO: 39 as primers;
And (ii) determining the size of the PCR product obtained in the step (i),
23. 21. The method according to the above 20, wherein the detection of a single nucleotide mutation in which the base at position 1193 in the human vWF-CP gene is thymine is performed by a method comprising the following steps. To 22. Any detection method of
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 as primers,
24. A method for detecting a gene mutation having a possibility of causing hemolytic uremic syndrome (hereinafter abbreviated as HUS), which comprises detecting all of the following single nucleotide mutations in the human vWF-CP gene:
(1) the base at position 1460 is guanine;
(2) the base at position 1786 is guanine;
(3) the base at position 2160 is adenine;
(4) The base at position 2296 is guanine (here, the base position is defined as the position in the cDNA base sequence of the gene described in GenBank accession number NM — 139025).
25. 24. The method further comprises detecting a single base mutation in which the position 2724 base in the human vWF-CP gene is cytosine in addition to the single base mutation. (Here, the base position is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025).
26. 24. The method according to 24, wherein the detection of a single nucleotide mutation in which the 1460th base in the human vWF-CP gene is guanine is performed by a method comprising the following steps. Detection method of
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 42 and SEQ ID NO: 43 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 42 and SEQ ID NO: 43 as primers,
27. 25. The detection method according to 24 above, wherein the detection of a single nucleotide mutation in which the base at position 1786 in the human vWF-CP gene is guanine is performed by a method comprising the following steps;
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 11 and SEQ ID NO: 12 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers,
28. 25. The detection method according to 24 above, wherein the detection of a single nucleotide mutation in which the base at position 2160 in the human vWF-CP gene is guanine is performed by a method comprising the following steps;
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 19 and SEQ ID NO: 20 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 21 and SEQ ID NO: 22 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 21 and SEQ ID NO: 22 as primers,
29. 25. The detection method according to 24, wherein the detection of a single nucleotide mutation in which the base at position 2296 in the human vWF-CP gene is guanine is performed by a method comprising the following steps;
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 44 and SEQ ID NO: 45 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 44 and SEQ ID NO: 45 as primers,
30. 26. The detection method according to 25 above, wherein the detection of a single nucleotide mutation in which the base at position 2724 in the human vWF-CP gene is guanine is performed by a method comprising the following steps;
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 23 and SEQ ID NO: 24 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 25 and SEQ ID NO: 26 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 25 and SEQ ID NO: 26 as primers,
31. 20 above. To 30. A method for examining the risk of developing HUS using any of the detection methods of
32. A method for detecting a gene mutation having a possibility of causing coronary artery disease (hereinafter abbreviated as CAD), characterized by detecting a single nucleotide mutation in which the base at position 1786 in the human vWF-CP gene is guanine (herein referred to as CAD) The position of the base at position 1786 is defined as the position in the cDNA base sequence of the gene described in GenBank Accession No. NM — 139025).
33. 32. The method according to 32, wherein the detection of a single nucleotide mutation in which the 1786th base in the human vWF-CP gene is guanine is carried out by a method comprising the following steps. Detection method of
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 11 and SEQ ID NO: 12 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers,
34. 32. Or 33. A method for detecting the risk of developing CAD using the detection method of
35. A method for detecting a genetic mutation having a possibility of causing a stroke, characterized by detecting a single base mutation in which the base at position 1786 in the human vWF-CP gene is guanine (wherein the base position at position 1786 is The position is defined as the position in the cDNA base sequence of the gene described in GenBank accession number NM — 139025).
36. 35. The method according to 35 above, wherein the detection of a single nucleotide mutation in which the position 1786 base in the human vWF-CP gene is guanine is performed by a method comprising the following steps. Detection method of
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 11 and SEQ ID NO: 12 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 as primers,
37. 35. Or 36. A method for examining the risk of stroke incidence using the detection method of
38. A polynucleotide comprising the base sequence set forth in any one of SEQ ID NO: 3 to SEQ ID NO: 39 and SEQ ID NO: 42 to SEQ ID NO: 43 in the Sequence Listing;
39. 3 above. Allele-specific nucleic acid probes of the above and 4. 5. And 38. A reagent kit comprising at least one of the polynucleotides of
Consists of.

According to the present invention, it is possible to determine the risk of morbidity such as thrombotic diseases such as TTP, HUS, CAD, or Stroke and to conduct early diagnosis due to abnormalities of vWF due to decreased activity of vWF cleaving enzyme. It becomes possible. Therefore, when it is determined that the risk of morbidity such as CAD or Stroke is high, the risk factors such as smoking, high blood pressure, high fat / high cholesterol diet, obesity and stress may be corrected before onset. it can. In addition, when it is determined that the risk of suffering from TTP is high, it is possible to select a treatment method that can avoid the induction of the disease. For example, if it is determined that the risk of developing TTP is high, select other treatment methods by avoiding vaccination or the use of various drugs (such as penicillin, sulfa drugs, and various anticancer drugs) that may induce the onset of TTP. be able to. Alternatively, when TTP develops, its etiology can be determined and an appropriate treatment method can be selected. For example, when a single nucleotide mutation in the vWF-cleaving enzyme gene is detected, abnormal expression of the gene and / or quantitative or qualitative abnormality of the enzyme due to the single nucleotide mutation is considered as the etiology. Allows selection of therapy. If a single base mutation is not detected, it will be possible to select the appropriate therapy considering that the onset of TTP is caused by other abnormalities. For example, if it is determined that the pathogenesis is due to an inhibitor of vWF-cleaving enzyme activity, removal therapy for the inhibitor can be selected.
Thus, the present invention is useful for diagnosis of thrombotic diseases, specifically, TTP, HUS, CAD, Stroke and the like, and tests for the prevention and treatment of the diseases.

  The present invention has been achieved by utilizing a single nucleotide mutation in the vWF-cleaving enzyme gene found in thrombotic thrombocytopenic purpura as a method for detecting a genetic mutation having a possibility of causing a thrombotic disease. 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. Examples of thrombotic diseases include thrombotic diseases caused by abnormal expression of the vWF-cleaving enzyme gene and / or quantitative or qualitative abnormality of the enzyme. Specifically, TTP, HUS, CAD, Stroke, etc. can be illustrated.

In the present invention, the cDNA base sequence (SEQ ID NO: 1) of the human vWF cleaving enzyme gene described in NM — 139025 was used as a reference sequence for judging single base mutations in exons. As a reference sequence for judging a single nucleotide mutation in an intron, the genomic nucleotide sequence (SEQ ID NO: 46) of the human vWF cleaving enzyme gene described in the chromosome 9 contig sequence of GenBank accession number NT — 03514.3 was used. These standard sequences are sometimes referred to as reference sequences. The position of the base where the single base mutation exists is defined as the position of the base in these reference sequences.

  A single base mutation in the vWF cleaving enzyme gene consists of a number indicating the position of the single base mutation in the genomic base sequence or the cDNA base sequence (SEQ ID NO: 1) and a symbol indicating the base at the position in the gene having the single base mutation. May be shown in combination. For example, 1193T means that there is a single base mutation at position 1193 of the cDNA base sequence (SEQ ID NO: 1) of the vWF cleaving enzyme gene, and the base at that position is thymine (T).

  The amino acid encoded by the codon containing a single base mutation is a number indicating the position of the amino acid in the amino acid sequence (SEQ ID NO: 2) encoded by the cDNA (SEQ ID NO: 1) of the vWF cleaving enzyme gene described in GenBank Accession No. NM — 139025 May be shown in combination with a symbol representing an amino acid. In this case, the amino acid notation is represented by one letter. For example, Q448E indicates that the 448th amino acid is glutamine (Q) in the vWF-cleaving enzyme without a single base mutation, but is mutated to glutamic acid (E) by a single base mutation.

  The single nucleotide mutation found in the vWF-cleaving enzyme gene derived from a TTP patient includes four single nucleotide mutations with amino acid mutations [1193T (A250V), 1786G (Q448E), 3050A (G869S) and 3152T (S903L)], splicing variants. One type that occurs (intron 3 G / A), and three single-base mutations that do not have amino acid mutations [1992A (T506T), 2160A (T572T), and 2724C (G760G)] (see Tables 1 and 2). Intron 3 G / A means a mutation from guanine (G) to adenine (A) at the base at the 5 ′ end of intron 3 (position 3066539 in the genomic base sequence).

In the genomic base sequence as the reference sequence, the base corresponding to position 3066539 is G. In the cDNA base sequence (SEQ ID NO: 1) as a reference sequence, the bases corresponding to the 1193rd position, the 1786th position, the 1992 position, the 2160th position, the 2724th position, the 3050th position, and the 3152st position are respectively , C, C, C, A, C, G and C (see Tables 1 and 2).
However, according to the knowledge of the present inventors, in the Japanese, the base at position 2160 is that G is dominant, and mutating to A is considered to be strongly related to the onset of TTP. In addition, the base at position 2724 has many alleles in which the base at this position is T in Japanese, and mutating to C is considered to be strongly related to the onset of TTP.

  Intron 3 G / A, 1193T (A250V), 1992A (T506T), 3050A (G869S) and 3152T (S903L) were searched for gene databases such as GenBank. It was a mutation. Meanwhile, 1786G (Q448E), 2160A (T572T), and 2724C (G760G) have already been reported [Kokame, K. et al., “Proceeding of the National Academy of Science of the United States of America (PROCEEDINGS OF THE National Academy of SCIENCES OF THE THE UNITED STATES OF AMERICA) ”, 2002, Vol. 99, p. 11902-11907]. However, the relationship between the reported single nucleotide mutation and TTP is not known.

  In addition to the above base mutations, single nucleotide mutations at positions 1460 and 2296 were found in the WF-cleaving enzyme gene derived from TTP / HUS patients. Specifically, in the vWF-cleaving enzyme gene derived from one TTP / HUS patient, all base mutations corresponding to positions 1460, 1786, 2160 and 2296 were found. Furthermore, in each vWF cleaving enzyme gene of 2 patients with TTP / HUS, mutations of all bases corresponding to positions 1460, 1786, 2160, 2296, and 2724 were found. Among these mutations, the mutation at position 1460 is a single base mutation [1460G (T339R)] accompanied by an amino acid mutation. As described above, the mutations at positions 1786, 2160, and 2724 were guanine (G), adenine (A), and cytosine (C), respectively. The mutation at position 2296 was a single nucleotide mutation [2296G (P618A)] accompanied by an amino acid mutation. In the cDNA base sequence (SEQ ID NO: 1) as the reference sequence, the bases corresponding to the 1460th position and the 2296th position are C and C, respectively (see Table 5).

  1460G (T339R) was a novel single nucleotide mutation that has not been reported so far when gene database searches such as GenBank were conducted. On the other hand, 2296G (P618A) and the possibility of an association between this mutation and TTP have already been reported [Antonie G. et al., “British Journal of Hematology”, 2003, vol. 120. , P.821-824].

  Intron 3 G / A is a mutation in the base at the 5 ′ end of intron 3. For this reason, it was found that the 5′-splice site of intron 3 was changed downstream of the splice site in the gene without mutation by the mutation, resulting in a splicing variant. That is, from the vWF cleaving enzyme gene having intron 3 G / A, the intron 3 of the gene is located between the 3 ′ end of the base sequence derived from exon 3 of the gene and the 5 ′ end of the base sequence derived from exon 4. A splicing variant in which the base sequence derived therefrom is inserted is generated. Specific examples of the base sequence derived from intron 3 include the base sequence consisting of 27 bases described in SEQ ID NO: 40 or the base sequence consisting of 105 bases described in SEQ ID NO: 41. Such splicing variants cause mutations in the amino acid sequence of the vWF cleaving enzyme.

  Proteins with amino acid mutations are expected to reduce or eliminate the function of normal vWF cleaving enzymes with structural changes. For example, since the metalloprotease domain is present in the region containing exon 3, intron 3 G / A is considered to change the function of the domain and thus attenuate or eliminate the function of normal vWF-cleaving enzyme. Furthermore, the RNA stability of splicing variants is also considered to be low, and there is a disadvantage in terms of biological control from gene to gene product expression and function compared to normal.

1193T (A250V) is located next to a highly conserved methionine residue in the amino acid sequence of the active site of the ADAMTS family enzyme to which the vWF cleaving enzyme belongs [Cal, S. et al., “Gene ( Gene) ", 2002, 283, p. 49-62]. The methionine turn (Met-turn) structure containing the methionine residue is a sequence characteristic of zinc peptidases such as metalloproteases, and is essential for binding of Zn ²⁺ together with three histidine residues [Boud, W ) Et al., “FEBS LETTERS”, 1993, Vol. 331, p. 134-140]. 1193T (A250V) in the vWF-cleaving enzyme is considered to have a high possibility of reducing the protease activity by imparting a Zn ²⁺ binding change or non-binding state by affecting this methionine turn structure.

  3152T (S903L) is present in the thrombospondin 1 (TSP1) domain. TSP1 is known to bind to other proteins such as heparin, fibrinogen, fibronectin, plasminogen, type V collagen and sulfatide. From this, it is considered that this single base mutation affects the specific binding ability and binding ability with the substrate of vWF-cleaving enzyme.

  On the other hand, even a single base mutation without amino acid mutation may cause an abnormality such as a decrease in the stability of the gene transcript. The vWF cleaving enzyme gene is a gene in which single nucleotide mutations are frequently present, and several single nucleotide mutations have already been reported. However, single nucleotide mutations reported so far are silent mutations that do not affect gene expression or gene product properties, or mutations between amino acids with similar properties, even if they involve amino acid mutations in the gene product. Therefore, the single nucleotide mutation according to the present invention has a significant influence on the transcription / translation of the vWF-cleaving enzyme gene and the change in the activity of the gene product as compared with the conventionally reported single nucleotide mutation. Yes, we believe that it is deeply related to diseases caused by abnormalities in the gene product.

  The gene mutation detection method according to the present invention has been achieved on the basis of these findings. The base at the position where a single base mutation is present in the human vWF cleaving enzyme gene is determined, and the type of the base is determined as a normal gene. It is characterized by detecting a genetic mutation having a possibility of causing a thrombotic disease by comparing with those of the above.

  The base is determined for the base corresponding to the next position of the base sequence of the human vWF cleaving enzyme gene: preferably position 3066539 in the genomic base sequence, and position 1193 in the cDNA base sequence (SEQ ID NO: 1). No. 1460, No. 1786, No. 1992, No. 2160, No. 2724, No. 3050 and No. 3152, more preferably No. 3066539, No. 1193, No. 1786, No. 1992, 30th and 3152nd. Base determinations are made for one or more of these positions.

  The determination method of a base can be implemented using a method known per se. For example, a partial sequence of a vWF cleaving enzyme gene containing a base at a desired position is amplified by polymerase chain reaction (hereinafter abbreviated as PCR), and the base sequence of the obtained amplification product is sequenced, 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 analysis method, and the like can be used. Analysis of single nucleotide mutations according to the present invention is not limited to these methods, and can be carried out by applying a method known per se. As a method for analyzing single nucleotide mutations, a method using fluorescence, for example, Invader assay [Lyamichev, V. et al., “Nature Biotechnology”, 1999, Vol. 17, p. 292-296] and the Luminex method [Chen, J. et al., “Genome Research”, 2000, Vol. 10, p. 549-557]. Further, a method using a mass spectrum, for example, a method using a primer extension method can be used. Such methods include the pinpoint assay [Ross, P. et al., “Nature Biotechnology”, 1998, Vol. 16, p. 1347-1351] and the probe method [Braun, A. et al., "Clinical Chemistry", 1997, Vol. 43, p. 1151-1158]. Furthermore, Dole Assay [DOL assay, Chen, X. et al., "Proceeding of the National Academy of Science of the United States of America", 1997, Vol. 94, p. 10756-10661], Sniper Assay [Piatek, AS, et al., “Nature Biotechnology”, 1998, Vol. 16, p. 359-363] and the tag array method [Fan, JB, et al., “Genome Research”, 2000, Vol. 10, p. 853-860] can also be used. These methods can be easily carried out by referring to the cited references in which they are reported.

  As the test sample, a nucleic acid sample prepared from human blood, cerebrospinal fluid, bronchoalveolar lavage fluid, sputum, other body fluid, tissue, or the like is used. The nucleic acid sample can be prepared by a nucleic acid preparation method known per se. For detection of a single base mutation present in an exon among single base mutations according to the present invention, any of genomic DNA, cDNA or RNA can be used as a nucleic acid sample. Further, since intron 3 G / A is a mutation that causes a splicing variant, any of genomic DNA, cDNA, and RNA can be used as a nucleic acid sample for detection. The prepared nucleic acid may be used directly for detection or may be amplified enzymatically by using the polymerase chain reaction (hereinafter abbreviated as PCR) or other amplification method before analysis. . When a single base mutation is detected using cDNA or RNA, the base at the position in cDNA or RNA corresponding to the position where the single base mutation is present in the genomic base sequence is determined.

  As an example of the method for detecting a genetic mutation according to the present invention, the detection of a single nucleotide mutation at position 3066539 is performed by, for example, using the polynucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 as primers for the genomic DNA of the vWF cleaving enzyme gene. PCR was performed using the polynucleotides described in SEQ ID NO: 5 and SEQ ID NO: 6 as primers, and then the primers used for the second PCR were used. It can be carried out by determining the base by the sequencing method. Similarly, the detection of the single nucleotide mutation at position 1193 uses the polynucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as the first PCR primer, and SEQ ID NO: 9 and SEQ ID NO: 10 as the second PCR primer and the sequencing primer. It can carry out using the polynucleotide as described in above. For the detection of the single nucleotide mutation at position 1786, the polynucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 12 were used as the first PCR primers, and the SEQ ID NO: 13 and SEQ ID NO: 14 were used as the second PCR primers and sequencing primers. It can be carried out using a polynucleotide. The detection of the single-base mutation at position 1992 uses the polynucleotides shown in SEQ ID NO: 15 and SEQ ID NO: 16 as the first PCR primers, and the SEQ ID NO: 17 and SEQ ID NO: 18 as the second PCR primers and sequencing primers. It can be carried out using a polynucleotide. For the detection of the single nucleotide mutation at position 2160, the polynucleotides shown in SEQ ID NO: 19 and SEQ ID NO: 20 were used as the first PCR primers, and the SEQ ID NO: 21 and SEQ ID NO: 22 were used as the second PCR primers and sequencing primers. It can be carried out using a polynucleotide. For the detection of the single nucleotide mutation at position 2724, the polynucleotides shown in SEQ ID NO: 23 and SEQ ID NO: 24 were used as the first PCR primers, and the SEQ ID NO: 25 and SEQ ID NO: 26 were used as the second PCR primers and sequencing primers. It can be carried out using a polynucleotide. The detection of the single nucleotide mutation at position 3050 uses the polynucleotides shown in SEQ ID NO: 27 and SEQ ID NO: 28 as the first PCR primers, and the SEQ ID NO: 29 and SEQ ID NO: 30 as the second PCR primers and sequencing primers. It can be carried out using a polynucleotide. For the detection of the single nucleotide mutation at position 3152, the polynucleotides shown in SEQ ID NO: 31 and SEQ ID NO: 32 were used as the first PCR primers, and the SEQ ID NO: 33 and SEQ ID NO: 34 were used as the second PCR primers and sequencing primers. It can be carried out using a polynucleotide. Alternatively, these single nucleotide mutations can be detected by performing PCR using each of the first PCR primers and determining the base by a sequencing method using the primer as a sequencing primer. For example, detection of the single nucleotide mutation at position 1460 is performed by performing PCR using the polynucleotides of SEQ ID NO: 42 and SEQ ID NO: 43 as primers on the genomic DNA of the vWF cleaving enzyme gene, and the sequence of the amplification product obtained. Can be carried out by determining the base by a sequencing method using the primer.

  The detection of the single base mutation at position 3066539 can also be carried out by detecting the splicing variant by utilizing the splicing variant caused by the mutation. That is, RNA is extracted from a test sample, the RNA sample is subjected to reverse transcription polymerase reaction (RT-PCR) using the polynucleotides of SEQ ID NO: 37 and SEQ ID NO: 39 as primers, and the obtained amplification product The mutation can be detected by determining the base sequence or measuring the size. When the amplification product is larger in size than the amplification product obtained by performing RT-PCR in the same manner using the gene having no single nucleotide mutation as a template, a splicing variant is present in the test sample, that is, It can be determined that there is a single base mutation at position 3066539. The size of the amplification product can be determined by a known method such as agarose gel electrophoresis.

  Single base mutations at positions 3066539, 1193, 1460, 1786, 1992, 2160, 2724, 3050 or 3152 also use allele-specific nucleic acid probes. Thus, it can also be detected by a hybridization method. An allele refers to two or more genes having a difference based on a difference in DNA base sequence occurring at the same locus.

  As the nucleic acid probe, a polynucleotide comprising a nucleotide sequence comprising a single nucleotide mutation represented by 3066539A, 1193G, 1460G, 1786G, 1992A, 2160A, 2724C, 3050A or 3152T in the nucleotide sequence of the vWF cleaving enzyme gene or a complementary nucleotide sequence thereof The polynucleotide which has a base sequence substantially complementary to the base sequence containing the base of the position which shows the said 1 base mutation can be illustrated. “Having a substantially complementary base sequence” means that when used as a probe, a base sequence containing a single base mutation in a test sample or a complementary base sequence thereof, for example, under stringent hybridization conditions It means that it can be detected. The conditions for hybridization 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].

  The mutation at position 3066539 results in a splicing variant in which a base sequence derived from intron 3 is inserted between exon 3 and exon 4, so that the mutation can be detected by detecting the splicing variant. Therefore, the mutation can be detected by a hybridization method using a nucleic acid probe capable of specifically detecting the inserted base sequence. An example of such a probe is a polynucleotide having a base sequence derived from intron 3 such as a base sequence substantially complementary to the base sequence described in SEQ ID NO: 40 or SEQ ID NO: 41.

  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. The probe is designed based on the base sequence of the vWF cleaving enzyme gene having a single base mutation represented by 3066539A, 1193G, 1460G, 1786G, 1992A, 2160A, 2724C, 3050A or 3152T or a complementary base sequence thereof, and is a conventional method. For example, it can be produced by chemical synthesis. A string from the obtained polynucleotide to a gene fragment represented by a base sequence containing a base at the position showing the single base mutation of a polynucleotide comprising the base sequence having the single base mutation or a complementary base sequence of the base sequence. The target probe can be obtained by selecting one that hybridizes under a gentle condition. A probe for detecting intron 3 G / A is designed based on the base sequence of intron 3, for example, the base sequence described in SEQ ID NO: 40 or SEQ ID NO: 41, and can be obtained in the same manner. These probes may be labeled with a marker for detection, 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 single nucleotide mutation according to the present invention is a mutation found in TTP. Therefore, by detecting one or two or more of the above-mentioned single nucleotide mutations, it is possible to detect a genetic mutation that can cause TTP. .

  Among the single-base mutations according to the present invention, a mutation at position 3066539 to adenine and a mutation at position 1193 to thymine were found in genomic DNA samples from HUS patients. From this, it is possible to detect a gene mutation that has the possibility of causing HUS by detecting a single base mutation at positions 3066539 and / or 1193. The vWF-cleaving enzyme activity in patients with these single nucleotide mutations was 3% or less (the normal human enzyme activity was taken as 100%). Although the vWF-cleaving enzyme activity of the patient's parents was 46% father and 46% mother, no TTP / HUS-like symptoms were observed. Genetic analysis revealed that the patient's vWF-cleaving enzyme gene has these mutations in heterozygos, and that the mother has a mutation at position 3066539 in heterogeneity but no mutation at position 1193. It was. The patient is thought to have developed a condition due to both of these mutations.

  It was clarified by a statistical method that the mutation of the 1786th base to guanine among the single base mutations according to the present invention is associated with the onset of CAD or Stroke. That is, the single base mutation is considered to be one of the risk factors for CAD or Stroke. Therefore, by detecting a single base mutation at position 1786, a genetic mutation that can cause CAD or Stroke can be detected.

  The method for detecting a genetic mutation according to the present invention can be used as a test method for determining the risk of thrombotic disease. The disease risk rate can be determined by comparing the base sequence of the vWF-cleaving enzyme gene in the sample with the control. As a control, preferably the genomic base sequence of the human vWF cleaving enzyme gene described in GenBank accession number NT — 03514.3 or the cDNA base sequence of the human vWF cleaving enzyme gene described in GenBank accession number NM — 139025 (SEQ ID NO: 1) Can be used. Alternatively, it is more preferable to use the base sequence of the vWF cleaving enzyme gene contained in a nucleic acid sample derived from a healthy person or a patient with a disease having no abnormality in the vWF cleaving enzyme gene. That is, the genome base sequence No. 3066539 of the vWF-cleaving enzyme gene in the test sample and the cDNA base sequence (SEQ ID NO: 1) of the gene No. 1193, No. 1786, No. 1992, No. 2160, No. 2724 1 or two or more bases selected from the bases at positions corresponding to positions 3050 and 3152, and comparing the base type at each position with the control, a base different from the control, For example, when it is recognized that they are A, T, G, A, A, C, A, and T, respectively, it can be determined that the disease prevalence is high. These mutations may be detected on both alleles or only one.

  Diseases whose risk of morbidity can be determined by this test method are diseases caused by abnormal expression of the gene and / or quantitative or qualitative abnormality of the enzyme due to a single nucleotide mutation in the vWF-cleaving enzyme gene. For example, it is preferably used to detect the risk of morbidity of thrombotic diseases associated with these abnormalities, specifically TTP, HUS, CAD or Stroke. Alternatively, it is also possible to detect a risk of morbidity of von Willebrand disease caused by quantitative and / or qualitative abnormalities of vWF, particularly type 2 von Willebrand disease due to qualitative abnormalities of vWF polymer.

  In the genomic DNA sample derived from the TTP / HUS patient, mutations were found in all of the nucleotides at positions 1460, 1786, 2160, and 2296 of the vWF-cleaving enzyme gene. By detecting these mutations, Genetic mutations that can cause HUS can be detected. Furthermore, in each genomic DNA sample of 2 patients with TTP / HUS, mutations in all of the nucleotides at positions 1460, 1786, 2160, 2296 and 2724 were found. By detecting, it is possible to detect genetic mutations that have the potential to cause thrombotic diseases such as HUS. For example, a gene mutation that can cause a thrombotic disease by detecting a mutation of at least one of the bases at positions 1460, 1786, 2160, 2296, and 2724 Can be detected. Preferably, genetic mutations that have the potential to cause HUS can be detected by detecting mutations in all of the nucleotides at positions 1460, 1786, 2160, and 2296. In addition, by detecting mutations in all of the bases at positions 1460, 1786, 2160, 2296, and 2724, a genetic mutation that can cause HUS can be detected.

  The detection of the single nucleotide mutation at position 2296 is performed by, for example, performing genomic PCR of the vWF cleaving enzyme gene using the polynucleotides of SEQ ID NO: 44 and SEQ ID NO: 45 as primers, and the sequence of the amplification product obtained. Can be performed by sequencing using the polynucleotides set forth in SEQ ID NO: 44 and SEQ ID NO: 45.

  Since these mutations were found in a genomic DNA sample derived from a TTP / HUS patient, the mutation detection method can be used as a test method for determining the risk of HUS disease. Specifically, bases at positions corresponding to positions 1460, 1786, 2160, and 2296 of the cDNA base sequence of the vWF-cleaving enzyme gene (SEQ ID NO: 1) in the test sample are determined, As a result of comparing the base type at each position with the control, when the bases at all these positions are different from the control, such as G, G, A and G, respectively, the HUS prevalence is high. Can be determined. In addition, the base sequence at positions corresponding to the 1460th, 1786th, 2160th, 2296th, and 2724th positions of the cDNA base sequence of the vWF-cleaving enzyme gene (SEQ ID NO: 1) in the test sample was determined. It can be determined that the prevalence of HUS is high when the base at position is found to be a base different from the control, eg, G, G, A, G and C, respectively.

  The present invention also provides a polynucleotide having a base sequence of a vWF cleaving enzyme gene having at least one single base mutation according to the present invention or a base sequence complementary to the base sequence. It is also possible to provide a polynucleotide generated by splicing abnormality due to a single base mutation according to the present invention. In addition, the allele-specific probes and primers can also be provided. In the present invention, the polynucleotide includes polydeoxyribonucleotide (DNA) composed of deoxyribonucleotide and polyribonucleotide (RNA) composed of ribonucleotide. The ribonucleotide may be a modified ribonucleotide.

  The polynucleotide according to the present invention is a known genetic engineering technique [edited by Sambrook et al., “Molecular Cloning, Laboratory Manual No. 1” based on information relating to the specific nucleotide sequence disclosed by the present invention. 2nd edition ", 1989, Cold Spring Harbor Laboratory; and Muramatsu Masami, edited by" Lab Manual Genetic Engineering ", 1988, Maruzen Co., Ltd.]. Specifically, a cDNA library is prepared according to a conventional method from an appropriate source in which the expression of the polynucleotide according to the present invention has been confirmed, and the desired clone is selected from the cDNA library. It can be acquired. Examples of the origin of cDNA include various cells and tissues in which the expression of this polynucleotide has been confirmed, or cultured cells derived therefrom. Isolation of total RNA from these sources, isolation and purification of mRNA, acquisition of cDNA and its cloning, etc. can all be performed according to conventional methods. A method for selecting a desired clone from the cDNA library is not particularly limited, and a conventional method can be used. For example, a plaque hybridization method, a colony hybridization method, etc., a method combining these, and the like can be exemplified. In addition, polymerase chain reaction [hereinafter abbreviated as PCR, Ulmer, KM, “Science”, 1983, Vol. 219, p. 666-671; edited by Ehrlich, HA, “PCR Technology, Principles and Applications of DNA Amplification”, 1989, Stockton Press; and Saiki, RK, et al., “Science. ), 1985, Vol. 230, p. 1350-1354] can be suitably used. Also, the RACE method (“Experimental Medicine”, 1994, Vol. 12, No. 6, p. 35-) and the 5′-RACE method [Frohman, MA, et al., “Proceedings of the National Academy of Sciences of the United States of America (Proceedings of The National of Sciences of The United States of America), 1988, Vol. 23, p. 8998-9002] or the like can also be used. The determination of the nucleotide sequence of a polynucleotide obtained by such a method can be performed by a conventional method, for example, the dideoxy method ["Proceedings of the Nationals of Science of the United States of America". of America) ", 1977, 74, p. 5463-5467] and the Maxam-Gilbert method [“Methods in Enzymology”, 1980, vol. 65, p. 499-560] and the like, or simply using a commercially available sequence kit or 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 base 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 reagents and reagent kits can be used as test agents and test kits 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.
In addition, the experiments described in the examples were performed by obtaining informed consent of a patient who was a sample provider.

(Analysis of each exon base sequence of vWF-cleaving enzyme gene)
For 29 exons of the vWF cleaving enzyme gene, mutations in the base sequence of each exon were detected using a genomic DNA sample. Genomic DNA samples were donated from 7 TTP patients. Detection was carried out by amplifying the target region from the genomic DNA of the vWF-cleaving enzyme gene in the sample and determining the base sequence of the obtained amplification product using the direct sequencing method.

  Amplification of the target region was performed by PCR. PCR primers were designed and prepared based on the base sequences upstream and downstream of each exon of the genomic base sequence of the vWF cleaving enzyme gene (GenBank accession number NT — 03514.3) (see below).

48 μl of the PCR reaction solution, 1 μl of the first PCR primer (10 μM) and 1 μl of the DNA sample (50 μl in total) were mixed, and the first PCR was performed under the following conditions.
PCR conditions 1) 95 ° C for 1 minute 2) 95 ° C for 30 seconds 3) 48 ° C for 30 seconds 4) 72 ° C for 2.5 minutes
Steps 2) to 4) were performed 30 cycles.
5) 72 ° C for 10 minutes

  Next, 48 μl of the PCR reaction solution, 1 μl of the second PCR primer (10 μM) and 1 μl of the first PCR amplification product (total 50 μl) were mixed, and the second PCR was performed under the same conditions as the first PCR.

  The base sequence of the amplification product of the second PCR was determined by performing a sequence reaction using an ABI 3100 sequencer according to the ABI measurement manual. Sequencing primers were the same as those used for the second PCR. The results are shown in Table 1.

^* : The position of the base is shown as the position in the genomic base sequence of the human vWF cleaving enzyme gene described in GenBank Accession No. NT — 03514.3.
^** : The position of the base is shown as the position in the cDNA base sequence (SEQ ID NO: 1) of the human vWF cleaving enzyme gene described in GenBank Accession No. NM — 139025.
^# : The base corresponding to position 2160 of the cDNA base sequence shown in SEQ ID NO: 1 is A. However, in Japanese, G is a dominant base at this position.
^## : The base corresponding to position 2724 of the cDNA base sequence shown in SEQ ID NO: 1 is C. However, in Japanese, there are many alleles whose base at this position is T.

  As shown in Table 1, three single nucleotide mutations with amino acid mutations (Q448E, G869S and S903L), one expected to produce splicing variants (intron 3 G / A), and one without amino acid mutation Three base mutations (T506T, T572T, and G760G) were found. Regarding exon 3, no mutation was detected in the base sequence of exon 3 itself, and a nucleotide mutation corresponding to the 5 ′ end of the intron following the 3 ′ end of exon 3 was detected in one example. It was expected that this mutation would change the splice site and result in a splicing variant. Among the detected mutations, intron 3 G / A, 1992A (T506T), 3050A (G869S) and 3152T (S903L) were novel single nucleotide mutations that have not been reported so far. On the other hand, 1786G (Q448E), 2160A (T572T) and 2724C (G760G) have already been reported, but the relationship between these single nucleotide mutations and thrombotic diseases is not known.

The primers used for detecting the single nucleotide mutations shown in Table 1 are shown below.
Exon 3 first PCR primer forward primer; SEQ ID NO: 3
Reverse primer; SEQ ID NO: 4
Exon 3 second PCR primer Forward primer; SEQ ID NO: 5
Reverse primer; SEQ ID NO: 6
First PCR primer for exon 12 Forward primer; SEQ ID NO: 11
Reverse primer; SEQ ID NO: 12
Exon 12 second PCR primer Forward primer; SEQ ID NO: 13
Reverse primer; SEQ ID NO: 14
First PCR primer for exon 13 Forward primer; SEQ ID NO: 15
Reverse primer; SEQ ID NO: 16
Exon 13 second PCR primer Forward primer; SEQ ID NO: 17
Reverse primer; SEQ ID NO: 18
First PCR primer of exon 15 Forward primer; SEQ ID NO: 19
Reverse primer; SEQ ID NO: 20
Exon 15 second PCR primer Forward primer; SEQ ID NO: 21
Reverse primer; SEQ ID NO: 22
Exon 19 first PCR primer Forward primer; SEQ ID NO: 23
Reverse primer; SEQ ID NO: 24
Exon 19 second PCR primer Forward primer; SEQ ID NO: 25
Reverse primer; SEQ ID NO: 26
First PCR primer of exon 20 Forward primer; SEQ ID NO: 27
Reverse primer; SEQ ID NO: 28
Second PCR primer of exon 20 Forward primer; SEQ ID NO: 29
Reverse primer; SEQ ID NO: 30
First PCR primer of exon 21 Forward primer; SEQ ID NO: 31
Reverse primer; SEQ ID NO: 32
Second PCR primer of exon 21 Forward primer; SEQ ID NO: 33
Reverse primer; SEQ ID NO: 34

  For detection of mutations in the vWF cleaving enzyme gene exon 7, 2 μl each of the same DNA sample as in Example 1 was used to amplify the target region from the genomic DNA of the vWF cleaving enzyme gene in the sample, and the base of the obtained amplification product The sequence was performed by determining using the direct sequencing method.

Amplification of the target region was performed by PCR. The PCR primers were designed based on the upstream and downstream base sequences of exon 7 of the genomic base sequence of the vWF cleaving enzyme gene (GenBank accession number NT — 03514.3). The PCR reaction was carried out under the following conditions using KOD-plus as a PCR enzyme and adding DMSO to a final concentration of 5%.
PCR conditions 1) 94 ° C for 4 minutes 2) 94 ° C for 30 seconds 3) 64 ° C for 30 seconds 4) 72 ° C for 1 minute
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 3 minutes

  A part of the reaction solution was subjected to agarose gel electrophoresis, and after confirming the band of the estimated product length, the PCR amplification product was purified using Exo SAP (manufactured by Amersham Biosciences).

  Using the purified amplification product as a template, the nucleotide sequence was analyzed by the dye terminator method. The sequence reaction was carried out using the following sequence primer, MegaBACE ET Terminator (DYEmetic ET dye terminator kit, manufactured by Amersham Biosciences), or purified by gel filtration using CleanSEQ (Agencourt) or Sephadex G-50. MegaBACE1000 (Amersham Biosciences) was used as a sequencer. The results are shown in Table 2.

^* : The position of the base is shown as the position in the cDNA base sequence (SEQ ID NO: 1) of the human vWF cleaving enzyme gene described in GenBank accession number NM — 139025.

  As shown in Table 2, a single base mutation (A250V) with an amino acid mutation was found in exon 7. This mutation was a novel single nucleotide mutation that has not been reported so far.

The primers used to detect a single base mutation in exon 7 are shown below.
PCR primer for exon 7 Forward primer; SEQ ID NO: 7
Reverse primer; SEQ ID NO: 8
Sequence primer for exon 7 Forward primer; SEQ ID NO: 9
Reverse primer; SEQ ID NO: 10

  The influence of intron 3 G / A on the vWF-cleaving enzyme phenotype was examined by constructing a mini-gene expression system.

The minigene expression system was constructed as follows. The region from exon 3 to exon 4 of the vWF cleaving enzyme gene (hereinafter referred to as exon 3-exon 4 fragment) was cloned from human genomic DNA by PCR. PCR was performed by mixing 98 μl of PCR reaction solution, 0.5 μl of each first PCR primer (final concentration 0.5 μM), and 1 μl of DNA sample (100 μl in total), and the first PCR was performed under the following conditions. The enzyme used was KOD.
First PCR primer Forward primer; SEQ ID NO: 3
Reverse primer; SEQ ID NO: 35
PCR conditions 1) 98 ° C for 5 minutes 2) 98 ° C for 1 minute 3) 50 ° C for 1 minute 4) 72 ° C for 1 minute
Steps 2) to 4) were performed 30 cycles.
5) 72 ° C for 5 minutes

Next, 98 μl of the PCR reaction solution, 0.5 μl each of the second PCR primer (final concentration 0.5 μM), and 1 μl of the amplification product of the first PCR (100 μl in total) are mixed, and the second PCR is performed under the same conditions as the first PCR reaction conditions. It was. However, Ex-Taq was used as the enzyme.
Second PCR primer Forward primer; SEQ ID NO: 5
Reverse primer; SEQ ID NO: 36

  Intron 3 G / A mutation was introduced into the obtained exon 3 to exon 4 fragment using QuikChange Site-Directed Mutagenesis Kit (manufactured by STRATAGENE). Each expression vector was prepared by introducing a wild type exon 3-exon 4 fragment and an intron 3 G / A mutant exon 3-exon 4 fragment into pcDNA3.1 (+), respectively. RNA was extracted from HEK293 cells introduced with these vectors using RNeasy mini kit (QIAGEN), and RT-PCR was performed using ImProm-II Reverse Transfer System (Promega). RT reaction was performed using oligo dT primer. PCR was performed using primer set 1 to detect mRNA from exon 3, and primer set 2 to detect mRNA from exon 3 to exon 4.

Primer set 1
Forward primer; SEQ ID NO: 37
Reverse primer; SEQ ID NO: 38
Primer set 2
Forward primer; SEQ ID NO: 37
Reverse primer; SEQ ID NO: 39

  Although the expression of mRNA of the expected size was confirmed in the cells into which the wild type exon 3-exon 4 fragment was introduced, the cells into which the intron 3 G / A mutant exon 3-exon 4 fragment was introduced were larger than the wild type. Size mRNA was expressed (FIG. 1).

  The amplification product obtained by RT-PCR using primer set 2 using RNA extracted from HEK293 cells introduced with the above vectors as a template was subjected to TA cloning using QIAGEN PCR Cloning Kit, and then the nucleotide sequence was analyzed. .

  As a result, splicing occurred as expected in the mRNA obtained from the cells into which the wild-type exon 3-exon 4 fragment was introduced (see FIG. 2-A). In clones (56) obtained from mRNA obtained from cells into which intron 3 G / A mutant exon 3-exon 4 fragment was introduced, 27 clones inserted into wild-type cDNA (11); 105 base insertions (38) were observed (see FIGS. 2-B and 2-C). The other 7 clones were the self ligation products of the TA cloning vector. The sequences of 27 bases and 105 bases inserted in cDNA obtained from the cells into which the mutant exon 3-exon 4 fragment was introduced are shown in SEQ ID NOs: 40 and 41, respectively.

  From these, it became clear that the intron 3 G / A mutation causes a splicing variant, suggesting that it may cause pathogenesis caused by abnormalities in the vWF-cleaving enzyme gene.

  Among the mutations of the vWF-cleaving enzyme gene detected in Examples 1 and 2, four types of single-base mutations with amino acid mutations [1193T (A250V), 1786G (Q448E), 3050A (G869S) and 3152T (S903L)] and splicing One type of variant (Intron 3 G / A) was examined for its association with disease. Mutation was detected in the same manner as in Examples 1 and 2.

  As a result, both intron 3 G / A and 1193T (A250V) mutations were detected in one patient (corresponding to sample number 3 in Tables 1 and 2). The patient was a 51-year-old male who frequently showed HUS symptoms, and the vWF-cleaving enzyme activity was 3% or less (the enzyme activity of a healthy person is 100%). The vWF-cleaving enzyme activity of the patient's parents was 46% father and 46% mother, and no TTP / HUS-like symptoms were observed. Genetic analysis revealed that the patient had intron 3 G / A and 1193T (A250V) in heterozygos. It has been shown that the mother's vWF-cleaving enzyme gene has intron 3 G / A in heterogeneity but no 1193T (A250V). The father has not obtained a genetic sample and has not analyzed it. From this, intron 3 G / A of the patient is considered to be a maternal mutation, and 1193T (A250V) is considered to be a paternal mutation.

  It is considered that the HUS patient to be analyzed this time has developed a disease state by having both intron 3 G / A and 1193T (A250V) mutations.

  Among the mutations of the vWF-cleaving enzyme gene detected in Examples 1 and 2, three kinds of single nucleotide mutations [1786G (Q448E), 3050A (G869S) and 3152T (S903L)] accompanied by amino acid mutations and one kind that produces splicing variants Regarding (Intron 3 G / A), the relationship between CAD and cerebrovascular disease (hereinafter abbreviated as CVD) was examined. Mutation was detected in the same manner as in Examples 1 and 2.

  A typing study was conducted using genomic DNA of 342 healthy individuals, 191 CAD patients, and 233 CVD patients. As a result, the expression frequency of each single nucleotide mutation was as follows. Expression of 3050A (G869S) and intron 3 G / A was not observed in the samples examined this time.

  Among the mutations of the vWF-cleaving enzyme gene detected in Examples 1 and 2, three kinds of single nucleotide mutations [1786G (Q448E), 3050A (G869S) and 3152T (S903L)] accompanied by amino acid mutations and one kind that produces a splicing variant Regarding (Intron 3 G / A), the relationship with CAD was examined. Mutation was detected in the same manner as in Examples 1 and 2.

  A typing study was conducted using genomic DNA from 140 healthy individuals and CAD patients. Healthy individuals and patients were prepared to have the same sex ratio and age (± 2 years). That is, the sex ratio in healthy individuals and patients was 93.6% for men and 6.4% for women. The average age was 55.8 ± 6.3 years for healthy individuals and 55.4 ± 6.5 years for patients.

  As a result, it was statistically revealed that a mutation in exon 12 [1786G (Q448E)] is associated with CAD. Wild-type CC, heterozygous CG and homozygous GG expression was observed in CAD in 88 (62.9%), 45 (32.1%) and 7 (5.0%), respectively. . On the other hand, in healthy individuals, 98 cases (70.0%), 36 cases (25.7%) and 6 cases (4.3%) were observed, respectively. The McNemar's Test was used as a statistical method, and the expression frequency of mutations in healthy individuals and patients was analyzed. As a result, in CAD, an association was observed between wild-type CC and heterozygous CG with an odds ratio of 1.667 and a 90% confidence interval (1.026-1.748).

  This suggests that the 1786G (Q448E) mutation may be one of the risk factors for developing CAD.

  Among the mutations of the vWF-cleaving enzyme gene detected in Examples 1 and 2, three kinds of single nucleotide mutations [1786G (Q448E), 3050A (G869S) and 3152T (S903L)] accompanied by amino acid mutations and one kind that produces a splicing variant Regarding (Intron 3 G / A), the relationship with Stroke was examined.

  Typing studies were performed using 195 genomic DNA each of healthy and Stroke patients. Healthy individuals and patients were prepared to have the same sex ratio and age (± 2 years). That is, the sex ratio in healthy individuals and patients was 81.0% for men and 19.0% for women. The average age was 57.7 ± 5.7 years for healthy individuals and 57.8 ± 6.1 years for patients.

  As a result, data suggesting that the mutation in exon 12 [1786G (Q448E)] is associated with Stroke was obtained. Expression of wild type CC, heterozygous CG and homozygous GG was observed in 130 cases (66.7%), 53 cases (27.2%) and 12 cases (6.2%) in Stroke, respectively. . On the other hand, in healthy individuals, 145 cases (74.4%), 42 cases (21.5%) and 8 cases (4.1%) were recognized, respectively. The McNemar method was used as a statistical method, and the expression frequency of mutations in healthy individuals and patients was analyzed. As a result, an odds ratio of 1.034 was obtained for the heterozygous CG and an odds ratio of 4.000 for the homozygous GG in Stroke, and the genotype relative risk increased.

  This suggests that the 1786G (Q448E) mutation may be one of the risk factors for developing Stroke.

  For a genomic DNA sample derived from a TTP / HUS patient, a mutation in the vWF cleaving enzyme gene was detected. Detection was carried out by amplifying the target region from the genomic DNA of the vWF-cleaving enzyme gene in the sample and determining the base sequence of the obtained amplification product by the dye terminator method.

  Amplification of the target region was performed by PCR. PCR primers were designed and prepared based on the base sequences upstream and downstream of each exon of the genomic base sequence of the vWF cleaving enzyme gene (GenBank accession number NT — 03514.3) (see below).

A PCR reaction solution and a PCR primer were added to 2 μl of a DNA sample, and PCR was performed under the following conditions. Ex-taq (manufactured by Takara Bio Inc.) was used as the polymerase for PCR. The solution obtained by PCR was stored at 4 ° C. A part of the solution obtained by PCR was developed on an agarose gel, and it was confirmed that the amplification product had an estimated length. Thereafter, the amplification product was purified from the solution obtained by PCR using ExoSAP (manufactured by Amersham Biosciences).
PCR conditions 1) 94 ° C for 4 minutes 2) 94 ° C for 30 seconds 3) 60 ° C for 30 seconds 4) 72 ° C for 1 minute
Steps 2) to 4) were performed 40 cycles.
5) 72 ° C for 3 minutes

  The base sequence of the PCR amplification product was determined by the dye terminator method using the PCR product as a template. Sequence was performed using sequencing primers and MegaBACE ET Terminator (Amersham Biosciences), and the resulting reaction product was purified with CleanSEQ (Agencourt) or Sephadex G50. Sequencing primers used were the same as those used for PCR. MegaBACE1000 (Amersham Biosciences) was used as the sequencer. The results are shown in Table 5.

^** : The position of the base is shown as the position in the cDNA base sequence (SEQ ID NO: 1) of the human vWF cleaving enzyme gene described in GenBank Accession No. NM — 139025.
^# : The base corresponding to position 2160 of the cDNA base sequence shown in SEQ ID NO: 1 is A. However, in Japanese, G is a dominant base at this position.
^## : The base corresponding to position 2724 of the cDNA base sequence shown in SEQ ID NO: 1 is C. However, in Japanese, there are many alleles whose base at this position is T.

  As shown in Table 5, in the vWF cleaving enzyme gene of TTP / HUS patients, 4 single nucleotide mutations with amino acid mutations (T339R, Q448E and P618A) and 1 single nucleotide mutation without amino acid mutations (T572T and G760G). ) Was detected. Of the mutations detected, 1460G (T339R) was a novel single nucleotide mutation that has not been reported so far. On the other hand, the possibility of association between 2296G (P618A) and this mutation with TTP has already been reported. 1786G (Q448E), 2160A (T572T) and 2724C (G760G) are mutations detected in the vWF cleaving enzyme gene of TTP patients in Example 1.

The primers used for detecting the single nucleotide mutations shown in Table 1 are shown below.
Exon 9 primer Forward primer; SEQ ID NO: 42
Reverse primer; SEQ ID NO: 43
Exon 12 primer Forward primer; SEQ ID NO: 11
Reverse primer; SEQ ID NO: 12
PCR primer for exon 15 Forward primer; SEQ ID NO: 19
Reverse primer; SEQ ID NO: 20
Exon 16 primer Forward primer; SEQ ID NO: 44
Reverse primer; SEQ ID NO: 45
Exon 19 primer Forward primer; SEQ ID NO: 23
Reverse primer; SEQ ID NO: 24

  According to the present invention, it is possible to determine the risk of morbidity such as thrombotic diseases such as TTP, HUS, CAD, or Stroke and to conduct early diagnosis due to abnormalities of vWF due to decreased activity of vWF cleaving enzyme. It becomes possible. 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.

The expected size of mRNA (exon 3 + exon 4) was detected in cells expressing wild-type exon 3-exon 4 fragment derived from the genome of vWF-cleaving enzyme gene, but intron 3 G / A mutant exon 3- It is a figure explaining that mRNA of the size larger than a wild type was detected in the cell which expressed the exon 4 fragment. Lanes 3 and 4 show exon 3-derived portions in mRNA using RNA extracted from cells expressing wild-type exon 3-exon 4 fragment and intron 3 G / A mutant exon 3-exon 4 fragment, respectively, as a template. The PCR product obtained using the primer set 1 that can be detected is shown. Lanes 6 and 7 show PCR products obtained using Primer Set 2 that can detect exon 3 to exon 4 derived portions in mRNA using RNA extracted from each cell as a template. Lanes 2 and 5 show amplification products obtained by PCR using primer set 1 and primer set 2, respectively, using RNA extracted from cells into which none of the above fragments have been introduced as a template. Lane 1 is a 100 bp marker. Example 3 It is a figure explaining that normal splicing was performed in the cell in which the wild type exon 3-exon 4 fragment of the vWF cleaving enzyme gene was expressed. Example 3 In the genome of the vWF cleaving enzyme gene, the 5 ′ end of intron 3 (position 3066539 in the genomic DNA sequence) following exon 3 is mutated from G to A, so that the 5′-splice site of intron 3 is 27. It is a figure explaining that the splicing variant of mRNA produced by it changing to the base downstream. The examination was performed in a minigene expression system using intron 3 G / A mutant exon 3 to exon 4 fragment. Example 3 In the vWF cleaving enzyme gene, the base at the 5 ′ end of intron 3 (position 3066539 in the genomic DNA sequence) following exon 3 is mutated from G to A, so that the 5′-splice site of intron 3 is 105 bases downstream. It is a figure explaining that it changed and the splicing variant of mRNA produced by it. The examination was performed in a minigene expression system using intron 3 G / A mutant exon 3 to exon 4 fragment. Example 3

SEQ ID NO: 3: A polynucleotide designed based on the base sequence upstream of exon 3 of genomic DNA of vWF cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 4: A polynucleotide designed based on the base sequence downstream of exon 3 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 5: A polynucleotide designed based on the nucleotide sequence upstream of exon 3 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 6: Polynucleotide designed based on the nucleotide sequence downstream of exon 3 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 7: A polynucleotide designed based on the nucleotide sequence upstream of exon 7 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 8: A polynucleotide designed based on the nucleotide sequence downstream of exon 7 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 9: A polynucleotide designed based on the nucleotide sequence upstream of exon 7 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 10: a polynucleotide designed based on the nucleotide sequence downstream of exon 7 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 11: Polynucleotide designed based on the nucleotide sequence upstream of exon 12 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 12: A polynucleotide designed based on the nucleotide sequence downstream of exon 12 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 13: a polynucleotide designed based on the nucleotide sequence upstream of exon 12 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 14: Polynucleotide designed based on the nucleotide sequence downstream of exon 12 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 15: A polynucleotide designed based on the nucleotide sequence upstream of exon 13 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 16: Polynucleotide designed based on the nucleotide sequence downstream of exon 13 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 17: Polynucleotide designed based on the nucleotide sequence upstream of exon 13 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 18: Polynucleotide designed based on the nucleotide sequence downstream of exon 13 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 19: A polynucleotide designed based on the nucleotide sequence upstream of exon 15 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 20: Polynucleotide designed based on the nucleotide sequence downstream of exon 15 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 21: a designed polynucleotide based on the nucleotide sequence upstream of exon 15 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 22: A polynucleotide designed based on the nucleotide sequence downstream of exon 15 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 23: A polynucleotide designed based on the nucleotide sequence upstream of exon 19 in the genomic DNA of the vWF cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 24: Polynucleotide designed based on the nucleotide sequence downstream of exon 19 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 25: A polynucleotide designed based on the base sequence upstream of exon 19 in the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 26: Polynucleotide designed based on the nucleotide sequence downstream of exon 19 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 27: a polynucleotide designed based on the nucleotide sequence upstream of exon 20 in the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 28: Polynucleotide designed based on the nucleotide sequence downstream of exon 20 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 29: A polynucleotide designed based on the nucleotide sequence upstream of exon 20 of the genomic DNA of the vWF cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 30: Polynucleotide designed based on the nucleotide sequence downstream of exon 20 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 31: a designed polynucleotide based on the upstream nucleotide sequence of exon 21 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 32: a polynucleotide designed based on the nucleotide sequence downstream of exon 21 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 33: Polynucleotide designed based on the nucleotide sequence upstream of exon 21 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 34: A polynucleotide designed based on the nucleotide sequence downstream of exon 21 of the genomic DNA of the vWF cleaving enzyme gene (described in GenBank Accession No. NT — 03514.3).
SEQ ID NO: 35: Polynucleotide designed based on the nucleotide sequence downstream of exon 4 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 36: Polynucleotide designed based on the nucleotide sequence downstream of exon 4 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 37: a polynucleotide designed based on the nucleotide sequence of exon 3 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 38: Polynucleotide designed based on the nucleotide sequence of exon 3 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 39: a polynucleotide designed based on the nucleotide sequence of exon 4 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 40: Base sequence derived from intron 3 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 41: base sequence derived from intron 3 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 42: a polynucleotide designed based on the nucleotide sequence upstream of exon 9 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 43: Polynucleotide designed based on the nucleotide sequence downstream of exon 9 of genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 44: a polynucleotide designed based on the upstream nucleotide sequence of exon 16 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 45: a polynucleotide designed based on the nucleotide sequence downstream of exon 16 of the genomic DNA of vWF-cleaving enzyme gene (described in GenBank accession number NT — 03514.3).
SEQ ID NO: 46: Genomic DNA of the vWF cleaving enzyme gene shown in GenBank Accession No. NT — 03514.3.

Claims (11)

In the genomic base sequence of the human von Willebrand factor cleaving enzyme gene (hereinafter abbreviated as vWF-CP gene), it is defined as the 3066539th base as the position in the genomic base sequence described in SEQ ID NO: 46 of the Sequence Listing. 1 nucleotide bases bases is defined as the vWF-CP gene cDNA nucleotide No. 1193 of the nucleotide as a position in the sequence shown in SEQ ID NO: 1 of 1 base mutation and / or the sequence listing is an adenine is is thymine A polynucleotide comprising a nucleotide sequence having a mutation or a complementary nucleotide sequence of the nucleotide sequence. In the genomic base sequence of the human vWF-CP gene, it is derived from a genome having a single base mutation in which the base defined as the 3066539th base as a position in the genomic base sequence shown in SEQ ID NO: 46 of the Sequence Listing is adenine. a base that is defined as the base at position 3066538 as the position in the genomic base sequence described in SEQ ID NO: 46 of the sequence listing and the 3 ′ end of the base sequence derived from exon 3 A base sequence described in SEQ ID NO: 40 between the 5 ′ end of the base sequence and a base defined as the 3075589th position as a position in the genomic base sequence described in SEQ ID NO: 46 of the Sequence Listing; CDNA obtained by inserting the nucleotide sequence set forth in SEQ ID NO: 41. An allele-specific nucleic acid probe for detecting a genetic mutation having a possibility of causing hemolytic uremic syndrome (hereinafter abbreviated as HUS), which is a sequence of the sequence listing in the genomic base sequence of human vWF-CP gene A single nucleotide mutation in which the base defined as the 3066539th base is adenine as a position in the genome base sequence described in No. 46 or a position in the vWF-CP gene cDNA base sequence shown in SEQ ID NO: 1 in the Sequence Listing An allele-specific nucleic acid probe comprising 17 to 30 bases capable of detecting a single base mutation in which the base defined at position 1193 is thymine. A nucleic acid probe for detecting a genetic mutation having a possibility of causing hemolytic uremic syndrome (hereinafter abbreviated as HUS), wherein the nucleotide sequence inserted in the cDNA according to claim 2 can be specifically detected. A nucleic acid probe comprising 17 to 30 bases. A base that is a single base mutation in the genomic base sequence of the human vWF-CP gene and is defined as the 3066539th base as a position in the genomic base sequence described in SEQ ID NO: 46 of the Sequence Listing is 1 It is characterized by detecting a base mutation and / or a single base mutation in which the base defined as the base at position 1193 is thymine as the position in the cDNA base sequence of the vWF-CP gene shown in SEQ ID NO: 1 in the sequence listing A method for detecting a genetic mutation having a possibility of causing hemolytic uremic syndrome (hereinafter abbreviated as HUS). A base that is a single base mutation in the genomic base sequence of the human vWF-CP gene and is defined as the 3066539th base as a position in the genomic base sequence described in SEQ ID NO: 46 of the Sequence Listing is 1 The detection method according to claim 5, wherein the detection of the base mutation is performed by a method comprising the following steps;
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 as primers;
(Ii) performing PCR using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers for the PCR product obtained in the step (i),
And (iii) determining the sequence of the PCR product obtained in the step (ii) by a sequencing method using the polynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 as primers. A base that is a single base mutation in the genomic base sequence of the human vWF-CP gene and is defined as the 3066539th base as a position in the genomic base sequence described in SEQ ID NO: 46 of the Sequence Listing is 1 The detection method according to claim 5, wherein the detection of the base mutation is performed by a method comprising the following steps;
(I) performing reverse transcription polymerase chain reaction (RT-PCR) using the polynucleotides of SEQ ID NO: 37 and SEQ ID NO: 39 as primers;
(Ii) determining the size of the PCR product obtained in the step (i),
(Iii) comparing the size of the resulting PCR product with the size of the wild-type derived PCR product; and
(Iv) A step of determining that the single base mutation is present when the size of the obtained PCR product is 27 bases or 105 bases longer than the size of the PCR product derived from the wild type . A base defined as a 1-base mutation in the genomic base sequence of the human vWF-CP gene and defined as the base at position 1193 as the position in the vWF-CP gene cDNA base sequence described in SEQ ID NO: 1 in the Sequence Listing is thymine The detection method according to any one of claims 5 to 7, wherein detection of a single nucleotide mutation is performed by a method comprising the following steps;
(I) performing a polymerase chain reaction (PCR) using the polynucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers;
And (ii) determining the sequence of the PCR product obtained in the step (i) by a sequencing method using the polynucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 as primers. A base that is a single base mutation in the genomic base sequence of the human vWF-CP gene and is defined as the 3066539th base as a position in the genomic base sequence described in SEQ ID NO: 46 of the Sequence Listing is 1 A polynucleotide for detecting a base mutation, comprising a polynucleotide comprising any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 37, and SEQ ID NO: 39 in the sequence listing nucleotide. A base defined as a 1-base mutation in the genomic base sequence of the human vWF-CP gene and defined as the base at position 1193 as the position in the vWF-CP gene cDNA base sequence described in SEQ ID NO: 1 in the Sequence Listing is thymine A polynucleotide for detecting a single-base mutation, comprising the nucleotide sequence set forth in any one of SEQ ID NO: 7 to SEQ ID NO: 10 in the sequence listing. A reagent kit comprising the allele-specific nucleic acid probe according to claim 3 and 4 and at least one of the polynucleotides according to claims 9 and 10.