JP4129910B2 - Coronary artery spasm risk diagnosis method - Google Patents

Description

【図面の簡単な説明】
【図１】図１は実施例におけるスクリーニング関連解析において検討した112遺伝子多型をまとめた表である。
【図２】図２は同じく実施例におけるスクリーニング関連解析において検討した112遺伝子多型をまとめた表である。
【図３】図３は実施例において遺伝子型を決定するために使用されるプライマー（上から順に配列番号１９、２０、２１、１６、１７、１８、２５、２６、２７、３１、３２、３３、３４、３５、３６、２８、２９、３０、１３、１４、１５、２２、２３、２４、１０、１１、１２）、プローブ（上から順に配列番号３７、３８、３９、４０）及びその他の条件をまとめた表である。図中、FITCはフルオレセインイソチオシアネートを、TxRはテキサスレッドを、Biotinはビオチンをそれぞれ表す。
【図４】図４は実施例の関連解析において検討した一塩基多型をまとめた表である。
【図５】図５は実施例における関連解析の対象とした男性1215例及び女性973例の背景データをまとめた表である。年齢及びBody mass indexの各データは平均±標準偏差で表される。表中、*1はP<0.0001を、*2はP<0.05を、*3はP<0.001を、*4はP<0.01をそれぞれ表す。
【図６】図６は関連解析の対象とした遺伝子多型と多項ロジスティック回帰分析の結果をまとめた表である。各SNPにおいて低い方のP値は太字で表される。
【図７】図７は冠動脈攣縮と関連のある遺伝子多型について遺伝子型の分布を示した図である。
【図８】図８は冠動脈攣縮と関連のある遺伝子多型における多項ロジスティック回帰分析のstep forward selection methodを行った結果を示す表である。
【図９】図９は男性における4個の組合せ遺伝子多型を用いた冠動脈攣縮の遺伝的リスク診断を行った結果を示す表である。
【図１０】図１０は女性における4個の組合せ遺伝子多型を用いた冠動脈攣縮の遺伝的リスク診断を行った結果を示す表である。
【図１１】図１１は冠動脈攣縮のリスク診断における累積オッズ比と一塩基多型の数の関連を表したグラフである。（A）は男性、(B)は女性における関連を示す。(A)における各SNPは、SNP1: TNFα(-863C→A)多型、SNP2:p22フォックス(242C→T)多型、SNP3:ApoC-III(-482C→T)多型、SNP4:ApoE(-219G→T)多型である。（B）における各SNPは、SNP1:ストロメライシン1(-1171/5A→6A)多型、SNP2:IL-6(-634C→G)多型、SNP3:FABP2 (2445G→A)多型、SNP4:ApoE(4070C→T)多型、SNP5:ENOS(-786T→C)多型である。[0001]
[Industrial application fields]
The present invention relates to a detection method using a gene associated with coronary artery spasm. More specifically, the present invention relates to a detection method using polymorphisms of a plurality of genes related to coronary artery spasm and a kit used in the method. The present invention can be used to determine the risk of developing coronary spasm.
[0002]
[Prior art]
Coronary spasm has an important role in the pathogenesis of variant angina, myocardial infarction and sudden cardiac death. Although calcium antagonists (diltiazem) and nitrites are known to be effective for coronary spasm, the molecular mechanism of coronary spasm is still unclear. One way to prevent coronary spasm is to identify coronary spasm susceptibility genes. Until now, by genome epidemiological studies, endothelial nitric oxide synthase (Non-patent Documents 1 and 2), angiotensin converting enzyme (Non-patent document 3), angiotensin II type 1 receptor (Non-patent document 4), paraoxonase (Non-patent document 5), the association between genetic polymorphisms such as phospholipase C-δ1 (non-patent document 6) and the onset of coronary artery spasm has been reported, but coronary artery spasm susceptibility genes have not yet been sufficiently identified. The incidence of coronary artery spasm is reported to be higher in Japanese than in Caucasians, and the diversity of genetic factors by race may be related to the pathology of coronary artery spasm (Non-Patent Document 1). Therefore, it is important to construct a database on the association between polymorphism and coronary spasm in each race.
[0003]
[Non-Patent Document 1]
Nakayama M, Yasue H, Yoshimura M, et al. T ^-786 → C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999; 99: 2864-2870.
[Non-Patent Document 2]
Yoshimura M, Yasue H, Nakamura M, et al. Genetic risk factors for coronary artery spasm: significance of endothelial nitric oxide synthase gene T-786 → C and missense Glu298Asp. J Invest Med. 2000; 48: 367-374.
[Non-Patent Document 3]
Oike Y, Hata A, Ogata Y, et al. Angiotensin converting enzyme as a genetic risk factor for coronary artery spasm: implication in the pathogenesis of myocardial infarction. J Clin Invest. 1995; 96: 2975-2979.
[Non-Patent Document 4]
Amant C, Hamon M, Bauters C, et al. The angiotensin II type 1 receptor gene polymorphism is associated with coronary artery vasoconstriction.J Am Coll Cardiol. 1997; 29: 486-490.
[Non-Patent Document 5]
Ito T, Yasue H, Yoshimura M, et al. Paraoxonase gene Gln192Arg (Q192R) polymorphism is associated with coronary artery spasm.Hum Genet. 2002; 110: 89-94.
[Non-Patent Document 6]
Nakano T, Osanai T, Tomita H, et al. Enhanced activity of variant phospholipase C-d1 protein (R257H) detected in patients with coronary artery spasm. Circulation. 2002; 105: 2024-2029.
[0004]
[Problems to be solved by the invention]
As described above, association analysis between several gene polymorphisms and coronary artery spasm has been performed so far. However, coronary spasm susceptibility genes have not yet been fully identified. The main reason is that in many studies, the target population is not large enough, and not only genetic polymorphism but also environmental factors such as smoking differ among races.
The present invention has been made in view of the above background, and an object thereof is to provide a means for diagnosing a genetic risk of coronary spasm with high accuracy and a high probability of prediction. To contribute to the primary prevention of infarction and sudden cardiac death.
[0005]
[Means for Solving the Problems]
In order to achieve the above object, the present inventors extracted 71 genes estimated to be associated with coronary atherosclerosis, coronary artery spasm, hypertension, diabetes, hyperlipidemia, etc. using several types of public databases, 112 polymorphisms were selected, mainly those that are expected to be associated with changes in gene function. Subsequently, the association analysis of myocardial infarction with respect to this 71 gene 112 polymorphism was performed for 445 myocardial infarctions and 464 control cases. It was found to be related to the onset of myocardial infarction (Yamada Y, Izawa H, Ichihara S, et al. Prediction of risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med. In press.) The group also included a candidate gene for coronary artery spasm. Therefore, a large-scale association analysis was performed on the relationship between these SNPs and coronary spasm. As a result, we succeeded in identifying 4 SNPs and 5 women in association with coronary artery spasm. In addition, the multi-factor logistic regression analysis stepwise forward selection method, which analyzes these polymorphisms in combination, showed a maximum odds ratio of coronary artery spasm of 5.08 for males and 53.37 for females. The highest odds ratio was shown. From these results, we have obtained knowledge that if multiple SNPs are selected from these SNPs and the results of analysis of each SNP are used in combination, the risk diagnosis of coronary artery spasm with high reliability and high predictive probability can be obtained. It was. The present invention is based on the above knowledge and provides the following configuration.
[1] A method for detecting the genotype of a nucleic acid sample, comprising the following step (a):
(A) a step of analyzing two or more polymorphisms selected from the group consisting of the following (1) to (4) in a nucleic acid sample;
(1) Polymorphism at base number -863 of the tumor necrosis factor α gene,
(2) a polymorphism at nucleotide position 242 of the NADH / NADPH oxidase p22 fox gene,
(3) a polymorphism at base position -219 of the apolipoprotein E gene, and
(4) A polymorphism at position -482 of the apolipoprotein C-III gene.
[2] A method for detecting the genotype of a nucleic acid sample, comprising the following step (b):
(B) a step of analyzing two or more polymorphisms selected from the group consisting of the following (5) to (9) in a nucleic acid sample;
(5) a polymorphism at base position -1171 of the stromelysin 1 gene,
(6) a polymorphism at position 4070 of the apolipoprotein E gene,
(7) a polymorphism at base position -634 of the interleukin 6 gene,
(8) a polymorphism at base number -786 of the endothelial nitric oxide synthase gene, and
(9) A polymorphism at nucleotide position 2445 of the fatty acid binding protein 2 gene.
[3] A risk diagnosis method for coronary artery spasm, comprising the following steps (i) to (iii):
(i) a step of analyzing two or more polymorphisms selected from the group consisting of the following (1) to (4) in a nucleic acid sample;
(1) Polymorphism at base number -863 of the tumor necrosis factor α gene,
(2) a polymorphism at nucleotide position 242 of the NADH / NADPH oxidase p22 fox gene,
(3) a polymorphism at base position -219 of the apolipoprotein E gene, and
(4) a polymorphism at base number -482 of the apolipoprotein C-III gene,
(ii) determining the genotype of the nucleic acid sample from the polymorphism information obtained by the step, and
(iii) A step of determining a genetic risk of coronary artery spasm from the determined genotype.
[4] Coronary artery spasm risk diagnosis method comprising the following steps (iv) to (vi):
(iv) analyzing two or more polymorphisms selected from the group consisting of the following (5) to (9) in a nucleic acid sample;
(5) a polymorphism at base position -1171 of the stromelysin 1 gene,
(6) a polymorphism at position 4070 of the apolipoprotein E gene,
(7) a polymorphism at base position -634 of the interleukin 6 gene,
(8) a polymorphism at base number -786 of the endothelial nitric oxide synthase gene, and
(9) Polymorphism at nucleotide position 2445 of the fatty acid binding protein 2 gene,
(v) determining the genotype of the nucleic acid sample from the polymorphism information obtained by the step, and
(vi) A step of determining a genetic risk of coronary artery spasm from the determined genotype.
[5] A genotype detection kit comprising two or more nucleic acids selected from the group consisting of the following (1) to (4):
(1) a nucleic acid for analyzing a polymorphism at base position -863 of the tumor necrosis factor α gene,
(2) a nucleic acid for analyzing the polymorphism at position 242 of the NADH / NADPH oxidase p22 fox gene,
(3) a nucleic acid for analyzing the polymorphism at base number -219 of the apolipoprotein E gene, and
(4) A nucleic acid for analyzing a polymorphism at base position -482 of the apolipoprotein C-III gene.
[6] A genotype detection kit comprising two or more nucleic acids selected from the group consisting of the following (5) to (9):
(5) a nucleic acid for analyzing the polymorphism at base number -1171 of the stromelysin 1 gene,
(6) a nucleic acid for analyzing a polymorphism at base position 4070 of the tumor necrosis factor apolipoprotein E gene,
(7) a nucleic acid for analyzing the polymorphism at base number -634 in the interleukin 6 gene,
(8) a nucleic acid for analyzing the polymorphism at base number -786 of the endothelial nitric oxide synthase gene, and
(9) A nucleic acid for analyzing a polymorphism at nucleotide position 2445 of the fatty acid binding protein 2 gene.
[7] An immobilized nucleic acid in which two or more nucleic acids selected from the group consisting of the following (1) to (4) are immobilized on an insoluble support,
(1) a nucleic acid for analyzing a polymorphism at base position -863 of the tumor necrosis factor α gene,
(2) a nucleic acid for analyzing the polymorphism at position 242 of the NADH / NADPH oxidase p22 fox gene,
(3) a nucleic acid for analyzing the polymorphism at base number -219 of the apolipoprotein E gene, and
(4) A nucleic acid for analyzing a polymorphism at base position -482 of the apolipoprotein C-III gene.
[8] An immobilized nucleic acid in which two or more nucleic acids selected from the group consisting of the following (5) to (9) are immobilized on an insoluble support,
(5) a nucleic acid for analyzing the polymorphism at base number -1171 of the stromelysin 1 gene,
(6) a nucleic acid for analyzing the polymorphism at base number 4070 of the apolipoprotein E gene,
(7) a nucleic acid for analyzing the polymorphism at base number -634 in the interleukin 6 gene,
(8) a nucleic acid for analyzing the polymorphism at base number -786 of the endothelial nitric oxide synthase gene, and
(9) A nucleic acid for analyzing a polymorphism at nucleotide position 2445 of the fatty acid binding protein 2 gene.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
1st aspect of this invention is related with the method of detecting the genotype of a nucleic acid sample, The one aspect | mode has the process of analyzing two or more polymorphisms selected from the group which consists of the following (1)-(4). It is characterized by including. Another aspect is characterized by including a step of analyzing two or more polymorphisms selected from the group consisting of the following (5) to (9). The genetic risk of coronary artery spasm can be determined by determining the genotype of the nucleic acid sample from the polymorphism information obtained as a result of the above steps.
(1) Tumor necrosis factor-α gene base number -863 polymorphism: -863C → A (hereinafter also referred to as “TNFα (-863C → A) polymorphism)”
(2) Polymorphism at position 242 of the NADH / NADPH oxidase p22 phox gene: 242C → T (hereinafter also referred to as the “p22 fox (242C → T) polymorphism”)
(3) Polymorphism at position -219 of the apolipoprotein E gene: -219G → T (hereinafter also referred to as “ApoE (-219G → T) polymorphism”)
(4) Polymorphism at base number -482 of the Apolipoprotein C-III gene: -482C → T (hereinafter also referred to as “ApoC-III (-482C → T) polymorphism)”
(5) Polymorphism at base position -1171 of the Stromelysin-1 gene: -1171 / 5A → 6A (hereinafter also referred to as “Stromelysin-1 (-1171 / 5A → 6A) polymorphism”) )
(6) Apolipoprotein E (Apolipoprotein E) gene base number 4070 polymorphism: 4070C → T (hereinafter also referred to as “ApoE (4070C → T) polymorphism”)
(7) Interleukin-6 (Interleukin-6) gene base number -634 polymorphism: -634C → G (hereinafter also referred to as “IL-6 (-634C → G) polymorphism”)
(8) Polymorphism at base number -786 in the endothelium nitric oxide synthase gene: -786T → C (hereinafter also referred to as “ENOS (-786T → C) polymorphism”)
(9) Polymorphism at nucleotide position 2445 of the Fatty acid-binding protein 2 gene: 2445G → A (hereinafter also referred to as “FABP2 (2445G → A) polymorphism”)
[0007]
In the above, the notation such as −863C → A means that the polymorphism at the base number position consists of two genotypes that are bases before or after the arrow. However, −1171 / 5A → 6A means a polymorphism consisting of a genotype in which 5 A (adenine) are present in succession in the 3 ′ direction from the base number position −1711, and a genotype in which 6 are present.
[0008]
The base number in each gene is represented on the basis of a known sequence registered in GenBank (NCBI) which is a public database. In the nucleotide sequence of SEQ ID NO: 1 (Accession No. L11698: Homo sapiens tumor necrosis factor alpha gene, promoter region), the 197th base corresponds to the -863 position base of the tumor necrosis factor α gene. Similarly, in the nucleotide sequence of SEQ ID NO: 2 (Accession No. M61107: Homo sapiens cytochrome b light chain (CYBA) gene, exons 3 and 4), the 684th base corresponds to the 242nd base of the NADH / NADPH oxidase p22 fox gene. In the base sequence of SEQ ID NO: 3 (Accession No. AF055343: Homo sapiens apolipoprotein E (APOE) gene, 5 'regulatory region, partial sequence), the base 801 corresponds to the -219th base of the apolipoprotein E gene. In the base sequence of No. 4 (Accession No. X13367: Human DNA for apolipoprotein C-III 5'-flank), the 936th base corresponds to the -482 position base of the apolipoprotein C-III gene, and the base sequence of SEQ ID NO: 5 (Accession No. U43511: Homo sapiens stromelysin-1 gene, promoter region), the 698th base corresponds to the -1171 base of the stromelysin 1 gene, and the nucleotide sequence of SEQ ID NO: 6 (Accession No. M10065 J03053 J03054: Human apolipoprotein E (epsilon-4 allele) gene, complete cds), the 4070th base corresponds to the 4070th base of the apolipoprotein E gene, and the nucleotide sequence of SEQ ID NO: 7 (Accession No. AF372214: Homo sapiens interleukin 6 (IL6) gene, complete cds), the 1111th base corresponds to the -634th base of the interleukin 6 gene and 815 in the base sequence of SEQ ID NO: 8 (Accession No. L10693: Human constitutive endothelial nitric oxide synthase gene, exon 1 and promoter region). The second base corresponds to the -786th base of the endothelial nitric oxide synthase gene, and the nucleotide sequence of SEQ ID NO: 9 (Accession No. M18079 J03465: Human, intestinal fatty acid binding protein gene, complete cds, and an Alu repetitive element), the 2445th base corresponds to the 2445th base of the fatty acid binding protein 2 gene.
[0009]
In the present invention, “analyzing a polymorphism” means examining what genotype the nucleic acid sample has for the gene polymorphism to be analyzed, and the base (base sequence) at the position where the polymorphism exists. Is synonymous with Typically, taking the analysis of TNFα (-863C → A) polymorphism as an example, the genotype of tumor necrosis factor α in the nucleic acid sample is CC (the -863 base is a homozygote where both alleles are C), Means to check whether CA (base 863 is a heterozygote of C allele and A allele) or AA (base 863 is a homozygote for both alleles A) To do.
[0010]
The polymorphisms (1) to (4) above are recognized to be particularly effective for use in the determination of the genetic risk of coronary artery spasm in the analysis of Japanese men, as shown in the examples below. Polymorphism. Therefore, using these polymorphisms as analysis targets enables a diagnosis with higher accuracy and a higher probability of prediction when a male (particularly a Japanese male) is employed as a subject.
[0011]
Similarly, the polymorphisms (5) to (9) described above are particularly used for determination of the genetic risk of coronary artery spasm in the analysis of Japanese women, as shown in the examples described later. It is a polymorphism recognized as effective. Therefore, the analysis of these polymorphisms enables a diagnosis with higher accuracy and a higher prediction probability when a female (particularly a Japanese female) is employed as a subject.
[0012]
Here, in principle, the genotype of the nucleic acid sample is more finely classified in proportion to the increase in the number of polymorphisms to be analyzed, which makes it possible to diagnose the genetic risk of coronary artery spasm with a higher probability of prediction. . From this viewpoint, it is preferable to detect a genotype by analyzing more polymorphisms among the polymorphisms (1) to (4) described above. Therefore, it is most preferable to analyze all the polymorphisms (1) to (4). When genotype detection is performed by combining three or less polymorphisms, it is preferable to preferentially select and use polymorphisms having a high odds ratio shown in the examples described later. For example, if three polymorphisms are used in combination, it is preferable to select three polymorphisms having the highest odds ratio, that is, (2), (3), and (4). Similarly, for example, when two polymorphisms are used in combination, it is preferable to select (3) and (4).
[0013]
Similarly, when analyzing two or more polymorphisms selected from the polymorphisms (5) to (9), it is most preferable to analyze all of these polymorphisms, that is, five polymorphisms. When genotype detection is performed by combining four or less polymorphisms, it is preferable to preferentially select and use polymorphisms having a high odds ratio shown in the examples described later. For example, if four polymorphisms are used in combination, it is preferable to select four polymorphisms having the highest odds ratio, that is, (5), (6), (7), and (8). Similarly, for example, when three polymorphisms are used in combination, it is preferable to select (5), (6), and (7). Similarly, for example, if two polymorphs are used in combination, it is preferable to select (5) and (7).
[0014]
The method of analyzing each gene polymorphism is not particularly limited, for example, using an allele-specific primer (and probe), amplification by PCR method, and analysis of polymorphism of amplification product by fluorescence or luminescence, PCR-RFLP (restriction fragment length polymorphism) method using PCR (polymerase chain reaction) method, PCR-SSCP (single strand conformation polymorphism) method (Orita, M et al., Proc. Natl. Acad. Sci., USA, 86, 2766-2770 (1989), etc.), PCR-SSO (specific sequence oligonucleotide) method, PCR-SSO method and dot high Hybridization method combined with ASO (allele specific oligonucleotide) hybridization method (Saiki, Nature, 324, 163-166 (1986) etc.) or TaqMan-PCR method (Livak, KJ, Genet Anal, 14, 143) (1999), Morris, T. et al., J. Clin. Microbiol., 34, 2933 (1996)), Invader method (Lyamichev V et al., Nat Biotechnol, 17, 292 (1999)), MALDI-TOF / MS (matrix) method using primer extension method (Haff LA, Smirnov IP, Genome Res 7,378 (1997)), RCA ( rolling cycle amplification) method (Lizardi PM et al., Nat Genet 19,225 (1998)), DNA chip or microarray method (Wang DG et al., Science 280,1077 (1998), etc.), primer extension method, Southern Known methods such as blot hybridization and dot hybridization (Southern, E., J. Mol. Biol. 98, 503-517 (1975)) can be employed. Furthermore, the analysis may be performed by directly sequencing the polymorphic portion to be analyzed. Note that polymorphism analysis may be performed by arbitrarily combining these methods.
[0015]
When the amount of nucleic acid sample is small, it is preferable to analyze by a method using PCR method (for example, PCR-RFLP method) from the viewpoint of detection sensitivity or accuracy. In addition, any of the above analysis methods can be applied after a nucleic acid sample has been previously amplified (including amplification of a partial region of the nucleic acid sample) by a PCR method or a gene amplification method such as a method applying the PCR method. On the other hand, when analyzing a large number of nucleic acid samples, allele-specific PCR method, allele-specific hybridization method, TaqMan-PCR method, Invader method, MALDI-TOF / MS (matrix) method using primer extension method, It is particularly preferable to use an analysis method capable of analyzing a large number of specimens in a relatively short time, such as an RCA (rolling cycle amplification) method or a method using a DNA chip or a microarray.
[0016]
In the above method, a nucleic acid such as a primer or a probe corresponding to each method (also referred to as “polymorphic analysis nucleic acid” in the present invention) is used. As an example of the nucleic acid for polymorphism analysis, a nucleic acid having a sequence complementary to a certain region (partial DNA region) containing the polymorphic site in a gene containing the polymorphism to be analyzed can be mentioned. In addition, the gene containing the polymorphism to be analyzed has a sequence complementary to a certain region (partial DNA region) containing the polymorphic site, and is designed so that the DNA fragment containing the polymorphic portion can be specifically amplified. Can be mentioned. As such a nucleic acid, for example, when the polymorphism at position −863 of tumor necrosis factor α gene is to be analyzed, the base at position −863 of tumor necrosis factor α gene whose base at position −863 is C (cytosine) is used. Nucleic acid having a sequence complementary to the partial DNA region containing, or nucleic acid having a sequence complementary to the partial DNA region containing the 3932th base of the tumor necrosis factor α gene whose base at -863 is A (adenine) To do.
[0017]
As another specific example of the nucleic acid for polymorphism analysis, it was designed to specifically amplify a partial DNA region containing the polymorphic site only when the polymorphic site to be analyzed is any genotype. A nucleic acid set can be mentioned. More specifically, it is a nucleic acid set designed to specifically amplify a partial DNA region containing a polymorphic site to be analyzed, and the polymorphic site of the antisense strand in which any genotype is present. Examples of the nucleic acid set include a sense primer that specifically hybridizes to a partial DNA region including a type site and an antisense primer that specifically hybridizes to a partial region of the sense strand. In such a nucleic acid set, when the polymorphism at position -863 of tumor necrosis factor α gene is the target of analysis, a partial DNA region containing the base at position −863 of tumor necrosis factor α gene is specifically amplified. Sense primer which is a designed nucleic acid set and specifically hybridizes to a partial DNA region containing the -863 base in the antisense strand of the tumor necrosis factor α gene whose -863 base is C (cytosine) -863 in a nucleic acid set consisting of an antisense primer that specifically hybridizes to a partial region of the sense strand, or an antisense strand of a tumor necrosis factor α gene in which the base at -863 is A (adenine) A sense primer that specifically hybridizes to a partial DNA region containing a base and an antisense primer that specifically hybridizes to a partial region of the sense strand. This corresponds to the nucleic acid set. Here, the length of the amplified partial DNA region is appropriately set within a range suitable for the detection, and is, for example, 50 bp to 200 bp, preferably 80 bp to 150 bp. More specifically, for example, a nucleic acid primer for TNFα (−863C → A) polymorphism analysis having the following sequence can be exemplified. The underlined portion in the following array represents a portion corresponding to the polymorphism. N in the sequence means any one of A, T, C, and G.
[0018]
Antisense primer
GGCCCTGTCTTCGTTAAN G G: SEQ ID NO: 8, or
ATGGCCCTGTCTTCGTTAAN T G: SEQ ID NO: 9
Sense primer
CCAGGGCTATGGAAGTCGAGTATC: SEQ ID NO: 10
[0019]
Similarly, nucleic acid primers for p22 fox (242C → T) polymorphism analysis include those having the following sequences.
Antisense primer
ACCACGGCGGTCATGN G C: SEQ ID NO: 11, or
ACCACGGCGGTCATGN A C: SEQ ID NO: 12
Sense primer
GCAGCAAAGGAGTCCCGAGT: SEQ ID NO: 15
[0020]
Similarly, nucleic acid primers for ApoE (-219G → T) polymorphism analysis can be exemplified by those having the following sequences.
Sense primer
GAATGGAGGAGGGTGTCTN G A: SEQ ID NO: 16, or
AGAATGGAGGAGGGTGTCTN T A: SEQ ID NO: 17
Antisense primer
CCAGGAAGGGAGGACACCTC: SEQ ID NO: 18
[0021]
Similarly, nucleic acid primers for ApoC-III (-482C → T) polymorphism analysis can be exemplified by those having the following sequences.
Sense primer
CGGAGCCACTGATGCN C G: SEQ ID NO: 19, or
CGGAGCCACTGATGCN T G: SEQ ID NO: 20
Antisense primer
TGTTTGGAGTAAAGGCACAGAA: SEQ ID NO: 21
[0022]
Similarly, nucleic acid primers for stromelysin 1 (-1171 / 5A → 6A) polymorphism analysis include those having the following sequences.
Sense primer
TTTGATGGGGGG AAAANA C: SEQ ID NO: 22, or
TTGATGGGGGG AAAAN CC: SEQ ID NO: 23
Antisense primer
CCTCATATCAATGTGGCCAA: SEQ ID NO: 24
[0023]
Similarly, nucleic acid primers for ApoE (4070C → T) polymorphism analysis include those having the following sequences.
Sense primer
CCGATGACCTGCAGAAN C G: SEQ ID NO: 25, or
GCCGATGACCTGCAGAAN T G: SEQ ID NO: 26
Antisense primer
CGGCCTGGTACACTGCCAG: SEQ ID NO: 27
[0024]
Similarly, those having the following sequences can be exemplified as nucleic acid primers for IL-6 (-634C → G) polymorphism analysis.
Sense primer
GGCAGTTCTACAACAGCN C C: SEQ ID NO: 28, or
GCAGTTCTACAACAGCN G C: SEQ ID NO: 29
Antisense primer
CTGTGTTCTGGCTCTCCCTG: SEQ ID NO: 30
[0025]
Similarly, nucleic acid primers for ENOS (-786T → C) polymorphism analysis can be exemplified by those having the following sequences.
Sense primer
ATCAAGCTCTTCCCTGGN C G: SEQ ID NO: 31, or
ATCAAGCTCTTCCCTGGN T G: SEQ ID NO: 32
Antisense primer
TCAGCAGAGAGACTAGGGCTGA: SEQ ID NO: 33
[0026]
Similarly, nucleic acid primers for FABP2 (2445G → A) polymorphism analysis include those having the following sequences.
Sense primer
TCACAGTCAAAGAATCAAGN G C: SEQ ID NO: 34, or
ATTCACAGTCAAAGAATCAAGN A C: SEQ ID NO: 35
Antisense primer
CAAAAACAACTTCAATGTTTCGA: SEQ ID NO: 36
[0027]
On the other hand, the following can be mentioned as a specific example of a probe.
As a probe for ApoC-III (-482C → T) polymorphism analysis
AGCCACTGATGCN C GGTCT: SEQ ID NO: 37, or
AGCCACTGATGCN T GGTCT: SEQ ID NO: 38.
[0028]
FABP2 (2445G → A) as a probe for polymorphism analysis
GAATCAAGN G CTTTTCGAAACATT: SEQ ID NO: 39, or
GAATCAAGN A CTTTTCGAAACATT: SEQ ID NO: 40.
[0029]
The above nucleic acid primers and nucleic acid probes are merely examples, and some nucleic acid primers can be used to perform the target amplification reaction without hindrance, and other nucleic acid probes can be used to perform the target hybridization reaction without hindrance. The base sequence may be modified. Here, “partial modification” means that a part of the base is deleted, substituted, inserted and / or added. The number of bases involved in the modification is, for example, 1 to 7, preferably 1 to 5, and more preferably 1 to 3. Such modification is generally performed at a portion other than the base corresponding to the polymorphic site. However, when the polymorphism to be analyzed is the stromelysin 1 (-1171 / 5A → 6A) polymorphism, a nucleic acid obtained by modifying a part of the base corresponding to the polymorphic site is used as a primer or probe. It is also possible.
[0030]
As the polymorphism analysis nucleic acid (probe, primer), a DNA fragment or an RNA fragment is appropriately used depending on the analysis method. The base length of the nucleic acid for polymorphism analysis is not particularly limited as long as each function is exhibited. Examples of the base length when used as a primer are about 10 to 50 bp, preferably about 15 to 40 bp, more preferably It is about 15-30bp.
When used as a primer, there may be some mismatch to the template sequence as long as it can specifically hybridize to the amplification target and amplify the target DNA fragment. Similarly, in the case of the probe, there may be some mismatch with the sequence to be detected as long as specific hybridization with the sequence to be detected can be performed. The degree of mismatch is 1 to several, preferably 1 to 5, and more preferably 1 to 3.
The polymorphism analysis nucleic acid (primer, probe) can be synthesized by a known method such as a phosphodiester method. Regarding the design, synthesis, etc. of the nucleic acid for polymorphism analysis, reference can be made to a written book (for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York).
[0031]
The nucleic acid for polymorphism analysis in the present invention can be labeled with a labeling substance in advance. By using such a labeled nucleic acid, for example, polymorphism can be analyzed using the labeling amount of the amplification product as an index. In addition, if two types of primers designed to specifically amplify partial DNA regions in each genotype gene constituting the polymorphism are labeled with different labeling substances, they can be detected from the amplification product. The genotype of the nucleic acid sample can be discriminated by the labeling substance and the labeling amount. As a specific example of the detection method using such a labeled primer, two kinds of nucleic acid primers (allele-specific sense primers) that specifically hybridize to the sense strand of each genotype constituting the polymorphism are used as fluorescein. In each of the amplification products obtained by labeling with isothiocyanate and Texas Red, amplifying a partial DNA region containing a polymorphic site using these labeled primers and an antisense primer that specifically hybridizes to the antisense strand. A method for detecting the polymorphism by measuring the labeling amount of each fluorescent substance can be mentioned. If the antisense primer here is labeled with, for example, biotin, amplification products can be separated using specific binding between biotin and avidin.
[0032]
As labeling substances used for labeling nucleic acids for polymorphism analysis ³² Fluorescent substances such as radioisotopes such as P, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Texas Red, etc. And 3 'end labeling method using Klenow fragment, nick translation method, random primer method (Molecular Cloning, Third Edition, Chapter 9, Cold Spring Harbor Laboratory Press, New York).
[0033]
The nucleic acid for polymorphism analysis described above can also be used in a state immobilized on an insoluble support. If the insoluble support used for immobilization is processed into a chip shape, a bead shape, or the like, polymorphism analysis can be performed more easily using these immobilized nucleic acids.
[0034]
The nucleic acid sample can be prepared from a subject's blood, skin cells, mucosal cells, hair, and the like using a known extraction method and purification method. As long as it contains a gene to be analyzed for polymorphism, genomic DNA of any length can be used as a nucleic acid sample. In addition, it is not always necessary to use a nucleic acid sample in which all the genes to be analyzed are present on one nucleic acid. That is, as the nucleic acid sample of the present invention, any of those in which all the genes to be analyzed are present on one nucleic acid and those in which the genes to be analyzed are separately present on two or more nucleic acids are used. be able to. Even if the gene to be analyzed is not in a complete state (that is, a state in which the full length of the gene exists) in the nucleic acid sample, at least as long as the polymorphic site to be analyzed exists, a fragmented or partial state It may be.
[0035]
Analysis of each gene polymorphism is performed for each gene polymorphism, or a plurality or all of them simultaneously. In the former case, for example, a nucleic acid sample obtained from a subject is dispensed according to the number of polymorphisms to be analyzed, and each polymorphism is analyzed individually. In the latter case, for example, a DNA chip or a microarray can be used. The term "simultaneous" here does not mean that all operations in the analysis process are performed simultaneously, but some operations (for example, nucleic acid amplification operation, probe hybridization, or detection) are performed simultaneously. Including.
[0036]
It is also possible to analyze polymorphisms of each gene using mRNA which is a transcription product of the gene to be analyzed. For example, after extracting and purifying mRNA of a gene to be analyzed from blood, urine, etc. of a subject, Northern blotting (Molecular Cloning, Third Edition, 7.42, Cold Spring Harbor Laboratory Press, New York), dot blotting (Molecular Cloning) , Third Edition, 7.46, Cold Spring Harbor Laboratory Press, New York), RT-PCR (Molecular Cloning, Third Edition, 8.46, Cold Spring Harbor Laboratory Press, New York), methods using DNA chips (DNA arrays), etc. By executing the above, polymorphism analysis can be performed using mRNA as a starting material.
[0037]
Furthermore, among the polymorphisms described above, those with amino acid changes can be subjected to polymorphism analysis using the expression product of the gene to be analyzed. In this case, as long as the amino acid corresponding to the polymorphic site is included, even a partial protein or partial peptide can be used as a sample for analysis.
[0038]
Examples of the analysis method using such gene expression products include a method of directly analyzing amino acids at polymorphic sites, a method of immunological analysis using a change in the three-dimensional structure, and the like. As the former, a well-known amino acid sequence analysis method (method using Edman method) can be used. Examples of the latter include ELISA (enzyme-linked immunosorbent assay), radioimmunoassay using a monoclonal antibody or polyclonal antibody having specific binding activity to the expression product of a gene having any of the genotypes constituting the polymorphism. , Immunoprecipitation method, immunodiffusion method and the like can be used.
[0039]
The polymorphism information obtained by executing the detection method of the present invention described above can be used for diagnosis of the genetic risk of coronary artery spasm. That is, the present invention comprises a step of determining the genotype of a nucleic acid sample from the polymorphism information obtained by the above detection method, and a step of obtaining a genetic risk from the determined genotype of the nucleic acid sample. A risk diagnosis method is also provided. The determination of the genotype here is typically to determine which genotype each of the alleles of the nucleic acid sample has with respect to the polymorphism to be detected. For example, if the TNFα (-863C → A) polymorphism is the target of detection, the genotype of tumor necrosis factor α in the nucleic acid sample is typically CC (the base at -863 is homozygous for both alleles C). ), CA (the -863 base is a heterozygote between the C allele and the A allele), and AA (the -863 base is both a homozygote for both alleles). It is to be.
[0040]
Considering the results obtained in the examples described later, in order to enable diagnosis of the genetic risk of coronary artery spasm with high accuracy and high predictive probability, for example, if the TNFα (-863C → A) polymorphism, the nucleic acid It is determined whether the genotype of the sample is AA, or it is either CA or CC. Similarly, p22 Fox (242C → T) polymorphism is either TT or CT, or CC, or ApoE (-219G → T) polymorphism is TT, or GT or GG or ApoC-III (-482C → T) polymorphism, either TT or CT, or CC, or stromelysin 1 (-1171 / 5A → 6A) If it is a polymorphism, it is either 6A / 6A or 5A / 6A, or 5A / 5A, if it is an ApoE (4070C → T) polymorphism, it is either TT or CT, or CC Or IL-6 (-634C → G) polymorphism, GG, or CG or CC, or ENOS (-786T → C) polymorphism, CC or TC If it is either, TT, or FABP2 (2445G → A) polymorphism, it is determined whether it is AA, or GA or GG.
[0041]
By diagnosing the genetic risk of coronary artery spasm, the degree of risk of developing coronary artery spasm (ease of onset) in the future, that is, the risk of onset (vulnerability of onset) is predicted. Based on this, it is possible to recognize coronary artery spasm and to grasp the medical condition. In other words, the diagnosis method of the present invention can be used to evaluate the risk of developing coronary artery spasm, to recognize that it is suffering from coronary artery spasm, or to grasp the symptoms. Above all, the evaluation of the onset risk is extremely significant clinically. This is because knowing the risk of onset in advance contributes to the primary prevention of coronary artery spasm and makes it possible to take appropriate preventive measures.
Information obtained by the diagnostic method of the present invention can be used to select an appropriate treatment method, improve prognosis, improve the quality of life (QOL) of a patient, or reduce the risk of onset.
[0042]
By executing the diagnostic method of the present invention, the risk of developing coronary artery spasm or the like can be monitored. As a result of such monitoring, if there is a correlation between an external factor (environmental factor, administration of drugs, etc.) and an increase in onset risk, the external factor is recognized as a risk factor, and this information Based on this, it is considered possible to reduce the risk of onset.
[0043]
The genetic information related to the onset of coronary artery spasm obtained in the present invention can be used for the treatment (including prophylactic treatment) of coronary artery spasm. For example, as a result of performing the diagnostic method of the present invention, when the polymorphism to be analyzed is a genotype that increases the risk of developing coronary artery spasm, a gene having a genotype with a low risk of onset is introduced into the living body and expressed. Therefore, expression of the gene can be expected to reduce symptoms, suppress onset, reduce risk of onset, and the like. A similar therapeutic effect can be expected by introducing an antisense strand against mRNA of a gene having a genotype with a high risk of onset to suppress the expression of the mRNA.
[0044]
The gene or antisense strand can be introduced, for example, by a method using a gene introduction plasmid or a viral vector, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984 )), Ultrasonic microbubbles (Lawrie, A., et al. Gene Therapy 7, 2023-2027 (2000)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84,7413- 7417 (1984)), microinjection (Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci. USA 73, 366-370 (1976)). Using these methods, a desired gene or the like can be directly introduced into a living body (in vivo method) or indirectly introduced (ex vivo method).
[0045]
The second aspect of the present invention provides a kit (genotype detection kit or coronary artery spasm diagnosis kit) used in the detection method or diagnosis method of the present invention described above. Such a kit includes a nucleic acid for analyzing two or more polymorphisms selected from the group consisting of the polymorphisms (1) to (4) (nucleic acid for polymorphism analysis). In another embodiment, a kit is constructed that includes a nucleic acid (nucleic acid for polymorphism analysis) for analyzing two or more polymorphisms selected from the group consisting of the polymorphisms of (5) to (9) above. The
Nucleic acids for polymorphism analysis are applied to analysis methods (method using PCR method using allyl-specific nucleic acid mentioned above, PCR-RFLP method, PCR-SSCP, TaqMan-PCR method, Invader method, etc.) Are designed as those that can specifically amplify (primer) or specifically detect (probe) a DNA region containing a polymorphic part to be analyzed or mRNA corresponding thereto. Specific examples of kits provided in the present invention are shown below.
[0046]
A genotype detection kit comprising two or more nucleic acids selected from the group consisting of the following (1) to (4):
(1) Nucleic acid having a sequence complementary to the partial DNA region containing the -863 base of the tumor necrosis factor α gene whose base at -863 is C, or the tumor necrosis factor α gene whose base at -863 is A A nucleic acid having a sequence complementary to a partial DNA region containing the base at position -863,
(2) a nucleic acid having a sequence complementary to a partial DNA region containing the 242nd base of the NADH / NADPH oxidase p22 fox gene whose base 242 is C, or the NADH / NADPH oxidase p22 fox gene whose base 242 is T A nucleic acid having a sequence complementary to a partial DNA region containing the 242 base of
(3) a nucleic acid having a sequence complementary to a partial DNA region containing the -219 base of the apolipoprotein E gene whose base is -219, or -219 of the apolipoprotein E gene whose T is -T A nucleic acid having a sequence complementary to a partial DNA region containing a coordinate base, and
(4) a nucleic acid having a complementary sequence to a partial DNA region containing the -482 base of the apolipoprotein C-III gene whose base at -482 is C, or apolipoprotein C-III whose -482 base is T A nucleic acid having a sequence complementary to a partial DNA region containing the -482 base of the gene.
In the above, a kit is constructed by selecting two or more nucleic acids from the group consisting of (1) to (4), but two or more from (1) to (4) can be arbitrarily selected. A kit may be constructed by selecting a group and selecting two or more nucleic acids from the group. For example, it is possible to construct a kit by selecting two or more nucleic acids from the group consisting of (2) to (4) (nucleic acids for analyzing polymorphisms with odds ratios of the top three in the examples described later). it can.
[0047]
A genotype detection kit comprising two or more nucleic acids selected from the group consisting of the following (5) to (9):
(5) a nucleic acid having a sequence complementary to a partial DNA region containing the sequence portion of the stromelysin 1 gene in which 5 A's are continuously present in the 3 'direction from position -1171, or 3 from position -1171 A nucleic acid having a sequence complementary to a partial DNA region containing the sequence portion of the stromelysin 1 gene, in which six A's are continuously present in the direction;
(6) Nucleic acid having a complementary sequence to the partial DNA region containing the 4070th base of the apolipoprotein E gene whose 4070th base is C, or the 4070th base of the apolipoprotein E gene whose 4070th base is T A nucleic acid having a sequence complementary to a partial DNA region,
(7) a nucleic acid having a sequence complementary to a partial DNA region containing the -634 base in the interleukin 6 gene whose base at -634 is C, or -634 of the interleukin 6 gene whose base in the -634 position is G A nucleic acid having a sequence complementary to a partial DNA region containing a base,
(8) Nucleic acid having a complementary sequence to the partial DNA region containing the -786th base of the endothelial nitric oxide synthase gene whose base at -786 is T, or the endothelium type 1 whose base at -786 is C A nucleic acid having a sequence complementary to a partial DNA region containing the -786 base of the nitric oxide synthase gene, and
(9) Nucleic acid having a complementary sequence to the partial DNA region containing the 2445th base of the fatty acid binding protein 2 gene whose base at 2445 is G, or the 2445th base of the fatty acid binding protein 2 gene whose 2445th base is A A nucleic acid having a sequence complementary to a partial DNA region containing
In the above, a kit is constructed by selecting two or more nucleic acids from the group consisting of (5) to (9), but two or more from (5) to (9) can be arbitrarily selected. A kit may be constructed by selecting a group and selecting two or more nucleic acids from the group. For example, a kit can be constructed by selecting two or more nucleic acids from the group consisting of (5) to (8) (nucleic acids for analyzing polymorphisms with odds ratios of the top four in the examples described later), A kit can be constructed by selecting two or more nucleic acids from the group consisting of (5) to (7) (nucleic acids for analyzing polymorphisms with odds ratios up to the top three in the examples described later) .
[0048]
A genotype detection kit comprising two or more nucleic acid sets selected from the group consisting of the following (1) to (4):
(1) Designed to specifically amplify a partial DNA region containing the −863 base of the tumor necrosis factor α gene only when the −863 base of the tumor necrosis factor α gene in the nucleic acid sample is C The partial DNA region containing the -863 base of the tumor necrosis factor α gene is specifically amplified only when the -863 base of the tumor necrosis factor α gene in the prepared nucleic acid set or nucleic acid sample is A Nucleic acid set, designed to
(2) Only when the 242nd base of the NADH / NADPH oxidase p22 fox gene in the nucleic acid sample is C, the partial DNA region containing the 242nd base of the NADH / NADPH oxidase p22 fox gene is specifically amplified. Only when the 242nd base of the NADH / NADPH oxidase p22 fox gene in the nucleic acid set or nucleic acid sample is T, the partial DNA region containing the 242nd base of the NADH / NADPH oxidase p22 fox gene is specified. A nucleic acid set designed to amplify automatically,
(3) Designed to specifically amplify a partial DNA region containing the -219 base of the apolipoprotein E gene only when the -219 base of the apolipoprotein E gene in the nucleic acid sample is G Designed to specifically amplify a partial DNA region containing the -219 base of the apolipoprotein E gene only when the -219 base of the apolipoprotein E gene in the nucleic acid set or nucleic acid sample is T. Nucleic acid set, and
(4) Only when the -482 base of the apolipoprotein C-III gene in the nucleic acid sample is C, the partial DNA region containing the -482 base of the apolipoprotein C-III gene is specifically amplified. Only when the -482 position base of the apolipoprotein C-III gene in the nucleic acid set or nucleic acid sample in the nucleic acid sample is T, the partial DNA region containing the -482 base position of the apolipoprotein C-III gene is specified Nucleic acid set designed to amplify automatically.
In the above, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (1) to (4), but any two or more from (1) to (4) can be arbitrarily selected. The kit may be configured by selecting two or more nucleic acid sets from the group. For example, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (2) to (4) (in the examples described later, a nucleic acid set for analyzing polymorphisms with an odds ratio of the top three). be able to.
[0049]
A genotype detection kit comprising two or more nucleic acid sets selected from the group consisting of the following (5) to (9):
(5) The partial DNA region containing the sequence portion of the stromelysin 1 gene is specified only when five A's are continuously present in the 3 'direction from position -1171 in the stromelysin 1 gene in the nucleic acid sample. The stromelysin 1 gene only when six consecutive A's are present in the 3 'direction from the -1171 position in the stromelysin 1 gene in the nucleic acid set or nucleic acid sample designed to be amplified A nucleic acid set designed to specifically amplify a partial DNA region containing the sequence portion of
(6) A nucleic acid set designed to specifically amplify a partial DNA region containing the 4070th base of the apolipoprotein E gene only when the 4070th base of the apolipoprotein E gene in the nucleic acid sample is C Or a nucleic acid set designed to specifically amplify a partial DNA region containing the 4070th base of the apolipoprotein E gene only when the 4070th base of the apolipoprotein E gene in the nucleic acid sample is T,
(7) Designed to specifically amplify a partial DNA region containing the -634 base of the interleukin 6 gene only when the -634 base of the interleukin 6 gene in the nucleic acid sample is C Designed to specifically amplify a partial DNA region containing the -634 base of the interleukin 6 gene only when the -634 base of the interleukin 6 gene in the nucleic acid set or nucleic acid sample is G. Nucleic acid set,
(8) A partial DNA region containing the -786th base of the endothelial nitric oxide synthase gene is specific only when the -786th base of the endothelial nitric oxide synthase gene in the nucleic acid sample is T A nucleic acid set designed to amplify or a -786 position of the endothelial nitric oxide synthase gene only when the -786 position base of the endothelial nitric oxide synthase gene in the nucleic acid sample is C A nucleic acid set designed to specifically amplify a partial DNA region containing a base; and
(9) Designed to specifically amplify a partial DNA region containing the 2445th base of the fatty acid binding protein 2 gene only when the 2445th base of the fatty acid binding protein 2 gene in the nucleic acid sample is G Designed to specifically amplify a partial DNA region containing the 2445th base of the fatty acid binding protein 2 gene only when the 2445th base of the fatty acid binding protein 2 gene in the nucleic acid set or nucleic acid sample is A. Nucleic acid set.
In the above, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (5) to (9), but two or more of (5) to (9) are arbitrarily selected. The kit may be configured by selecting two or more nucleic acid sets from the group. For example, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (5) to (8) (nucleic acid sets for analyzing polymorphisms with odds ratios of the top four in the examples described later). Or two or more nucleic acid sets are selected from the group consisting of (5) to (7) (nucleic acid sets for analyzing polymorphisms with odds ratios up to the top three in the examples described later). can do.
[0050]
A genotype detection kit comprising two or more nucleic acid sets selected from the group consisting of the following (1) to (4):
(1) A set of nucleic acids designed to specifically amplify a partial DNA region containing the −863 base of the tumor necrosis factor α gene, wherein the −863 base of the tumor necrosis factor α gene is − Antisense primer that specifically hybridizes to the partial DNA region containing the 863th base, and / or the partial DNA region containing the -863th base in the tumor necrosis factor α gene whose -863th base is A A nucleic acid set consisting of an antisense primer that specifically hybridizes and a sense primer that specifically hybridizes to a partial region of the tumor necrosis factor α gene,
(2) NADH / NADPH oxidase p22 fox gene, a nucleic acid set designed to specifically amplify a partial DNA region containing the 242nd base of the NADH / NADPH oxidase p22 fox gene, wherein the 242nd base is C A partial DNA region containing the 242 base in the NADH / NADPH oxidase p22 fox gene wherein the antisense primer specifically hybridizes to the partial DNA region containing the 242 base in FIG. A nucleic acid set consisting of an antisense primer that specifically hybridizes to and a sense primer that specifically hybridizes to a partial region of the NADH / NADPH oxidase p22 fox gene,
(3) A nucleic acid set designed to specifically amplify a partial DNA region containing the -219th base of the apolipoprotein E gene, the -219th position in the apolipoprotein E gene where the -219th base is G Sense primer that specifically hybridizes to a partial DNA region containing a base and / or a specific DNA region containing a -219 base in the apolipoprotein E gene where the -219 base is T A nucleic acid set comprising a hybridizing sense primer and an antisense primer that specifically hybridizes to a partial region of the apolipoprotein E gene; and
(4) Apolipoprotein C-III gene, which is a nucleic acid set designed to specifically amplify a partial DNA region containing the -482 base of the apolipoprotein C-III gene, wherein the -482 base is C A partial primer region containing the -482 base in the apolipoprotein C-III gene wherein the sense primer that specifically hybridizes to the partial DNA region containing the -482 base in FIG. A nucleic acid set comprising a sense primer that specifically hybridizes to and a antisense primer that specifically hybridizes to a partial region of the apolipoprotein C-III gene.
In the above, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (1) to (4), but any two or more from (1) to (4) can be arbitrarily selected. The kit may be configured by selecting two or more nucleic acid sets from the group. For example, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (2) to (4) (in the examples described later, a nucleic acid set for analyzing polymorphisms with an odds ratio of the top three). be able to.
[0051]
A genotype detection kit comprising two or more nucleic acid sets selected from the group consisting of the following (5) to (9):
(5) A nucleic acid set designed to specifically amplify a partial DNA region containing the -1171 position base of the stromelysin 1 gene, comprising 5 consecutive A's in the 3 'direction from the -1171 position. A sense primer that specifically hybridizes to the partial DNA region containing the sequence portion in the existing stromelysin 1 gene and / or stromelyi in which six As are present continuously in the 3 ′ direction from position −1171. A sense primer that specifically hybridizes to a partial DNA region containing the sequence portion in the syn-1 gene, and an antisense primer that specifically hybridizes to a partial region of the stromelysin 1 gene. A nucleic acid set,
(6) A nucleic acid set designed to specifically amplify a partial DNA region containing the 4070th base of the apolipoprotein E gene, which contains the 4070th base in the apolipoprotein E gene whose 4070th base is C A sense primer that specifically hybridizes to a partial DNA region and / or a sense primer that specifically hybridizes to a partial DNA region containing 4070 bases in the apolipoprotein E gene whose base is 4070 A nucleic acid set comprising an antisense primer that specifically hybridizes to a partial region of the apolipoprotein E gene,
(7) A nucleic acid set designed to specifically amplify a partial DNA region containing the base at position −634 of interleukin 6 gene, and position −634 in the interleukin 6 gene whose base at position −634 is C. Sense primer that specifically hybridizes to a partial DNA region containing a base and / or a partial DNA region containing a base at -634 in the interleukin 6 gene where the base at -634 is G A nucleic acid set comprising a hybridizing sense primer and an antisense primer that specifically hybridizes to a partial region of the interleukin-6 gene, (8) -786th base of the endothelial nitric oxide synthase gene Is a nucleic acid set designed to specifically amplify a partial DNA region containing γ--, and in the endothelial nitric oxide synthase gene whose base at −786 is T, Specific primer that specifically hybridizes to the partial DNA region, and / or the partial DNA region containing the -786 base in the endothelial nitric oxide synthase gene whose C-786 base is C A nucleic acid set consisting of a sense primer that hybridizes specifically, and an antisense primer that specifically hybridizes to a partial region of the endothelial nitric oxide synthase gene, and
(9) A nucleic acid set designed to specifically amplify a partial DNA region containing nucleotide 2445 in the fatty acid binding protein 2 gene, and nucleotide 2445 in the fatty acid binding protein 2 gene in which the nucleotide 2445 is G Sense primer that specifically hybridizes to the partial DNA region that contains, and / or specifically hybridizes to the partial DNA region that contains the 2445th base in the fatty acid binding protein 2 gene whose base is the 2445th position. A nucleic acid set comprising: a sense primer that specifically hybridizes to a partial region of the fatty acid-binding protein 2 gene.
In the above, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (5) to (9), but two or more of (5) to (9) are arbitrarily selected. The kit may be configured by selecting two or more nucleic acid sets from the group. For example, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (5) to (8) (nucleic acid sets for analyzing polymorphisms with odds ratios of the top four in the examples described later). Or two or more nucleic acid sets are selected from the group consisting of (5) to (7) (nucleic acid sets for analyzing polymorphisms with odds ratios up to the top three in the examples described later). can do.
[0052]
A genotype detection kit comprising two or more nucleic acid sets selected from the group consisting of the following (1) to (4):
(1) a first hybrid which specifically hybridizes to a partial region containing a base at position −863 and is labeled with a first labeling substance in the sense strand of a tumor necrosis factor α gene whose base at position −863 is C A second hybrid that specifically hybridizes to the nucleic acid and a partial region containing the base at position −863 in the sense strand of the tumor necrosis factor α gene whose base at −863 is A, and is labeled with a second labeling substance -863 base of the tumor necrosis factor α gene that hybridizes specifically with the nucleic acid and a partial region of the antisense strand of the tumor necrosis factor α gene and is used together with the first nucleic acid or the second nucleic acid A nucleic acid set comprising a third nucleic acid capable of specifically amplifying a partial DNA region containing
(2) a first hybrid that specifically hybridizes to a partial region containing the base at position 242 in the sense strand of the NADH / NADPH oxidase p22 fox gene whose base at position 242 is C and is labeled with a first labeling substance A second hybrid that specifically hybridizes to a nucleic acid and a partial region containing the base at position 242 in the sense strand of the NADH / NADPH oxidase p22 fox gene whose base at position 242 is T and is labeled with a second labeling substance. Specifically hybridized to a nucleic acid and to a partial region of the antisense strand of the NADH / NADPH oxidase p22 fox gene and used together with the first nucleic acid or the second nucleic acid of the NADH / NADPH oxidase p22 fox gene A nucleic acid set comprising a third nucleic acid capable of specifically amplifying a partial DNA region containing the 242nd base,
(3) a first hybrid that specifically hybridizes to a partial region containing the -219 base in the antisense strand of the apolipoprotein E gene whose base -219 is G and is labeled with a first labeling substance A second hybrid that specifically hybridizes to a nucleic acid and a partial region containing the -219 base in the antisense strand of the apolipoprotein E gene whose T-219 base is T and is labeled with a second labeling substance A portion that specifically hybridizes to a nucleic acid and to a partial region of the sense strand of the apolipoprotein E gene and includes the -219th base of the apolipoprotein E gene when used together with the first nucleic acid or the second nucleic acid A nucleic acid set comprising a third nucleic acid capable of specifically amplifying a DNA region,
(4) a first nucleic acid that specifically hybridizes to a partial region containing the base at -482 in the antisense strand of the apolipoprotein C-III gene in which -482 is C, and -482 is T A second nucleic acid that specifically hybridizes to the partial region containing the -482 base in the antisense strand of a certain apolipoprotein C-III gene, and a partial region of the sense strand of the apolipoprotein C-III gene A third nucleic acid capable of specifically amplifying a partial DNA region containing the -482 base of the apolipoprotein C-III gene by being specifically hybridized and used together with the first nucleic acid or the second nucleic acid And a first labeling substance that specifically hybridizes to the nucleic acid amplified using the first nucleic acid and the third nucleic acid using the apolipoprotein C-III gene at position -482 C as a template. 4th labeled A second label that specifically hybridizes to a nucleic acid and to a nucleic acid amplified using the second and third nucleic acids using the apolipoprotein C-III gene having a T at -482 as a template A nucleic acid set comprising a fifth nucleic acid labeled with a substance.
In the above, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (1) to (4), but any two or more from (1) to (4) can be arbitrarily selected. The kit may be configured by selecting two or more nucleic acid sets from the group. For example, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (2) to (4) (in the examples described later, a nucleic acid set for analyzing polymorphisms with an odds ratio of the top three). be able to.
[0053]
A genotype detection kit comprising two or more nucleic acid sets selected from the group consisting of the following (5) to (9):
(5) specifically hybridizing to the partial region containing the base moiety in the antisense strand of the stromelysin 1 gene, in which five A's are continuously present in the 3 'direction from position -1171 and the first To the first nucleic acid labeled with the labeling substance and a partial region containing the base portion in the antisense strand of the stromelysin 1 gene in which 6 A's are continuously present in the 3 'direction from the base at position -1171 And specifically hybridizing to the second nucleic acid labeled with the second labeling substance and the partial region of the sense strand of the stromelysin 1 gene, and the first nucleic acid or A nucleic acid set comprising: a third nucleic acid that can be used together with the second nucleic acid to specifically amplify a partial DNA region containing the base part -1171 of the stromelysin 1 gene;
(6) a first nucleic acid that specifically hybridizes to the partial region containing the 4070th base in the antisense strand of the apolipoprotein E gene whose 4070th base is C and is labeled with a first labeling substance; A second nucleic acid that specifically hybridizes to the partial region containing the 4070th base in the antisense strand of the apolipoprotein E gene whose 4070th base is T and labeled with a second labeling substance; and Specifically hybridizes to a partial region of the sense strand of the apolipoprotein E gene and is used together with the first nucleic acid or the second nucleic acid to specifically specify a partial DNA region containing the 4070th base of the apolipoprotein E gene A nucleic acid set comprising a third nucleic acid capable of being amplified
(7) a first hybrid that specifically hybridizes to the partial region containing the base at position −634 and is labeled with a first labeling substance in the antisense strand of the interleukin 6 gene in which the base at position −634 is C; A second hybrid that specifically hybridizes to a nucleic acid and a partial region containing a base at position -634 in the antisense strand of the interleukin 6 gene having a base at position -634 as G and is labeled with a second labeling substance. A portion that specifically hybridizes to a nucleic acid and a partial region of the sense strand of the interleukin 6 gene and is used together with the first nucleic acid or the second nucleic acid and includes the -634th base of the interleukin 6 gene A nucleic acid set comprising a third nucleic acid capable of specifically amplifying a DNA region,
(8) It specifically hybridizes to the partial region containing the -786 base in the antisense strand of the endothelial nitric oxide synthase gene whose base is -786 and is labeled with the first labeling substance The second nucleic acid specifically hybridized to the partial region containing the -786 base in the antisense strand of the endothelial nitric oxide synthase gene whose C-786 base is C; It specifically hybridizes to a second nucleic acid labeled with a labeling substance and a partial region of the sense strand of the endothelial nitric oxide synthase gene and is used together with the first nucleic acid or the second nucleic acid A nucleic acid set comprising: a third nucleic acid capable of specifically amplifying a partial DNA region containing the -786 base of the endothelial nitric oxide synthase gene; and
(9) The first nucleic acid that specifically hybridizes to the partial region containing the nucleotide at position 2445 in the antisense strand of the fatty acid-binding protein 2 gene whose position is 2445 G, and the fatty acid-binding protein whose position 2445 is A It specifically hybridizes to the second nucleic acid that specifically hybridizes to the partial region containing the base at position 2445 in the antisense strand of the two genes and to the partial region of the sense strand of the fatty acid binding protein 2 gene. And a third nucleic acid which can be used together with the first nucleic acid or the second nucleic acid to specifically amplify a partial DNA region containing the nucleotide position 2445 of the fatty acid binding protein 2 gene, and a fatty acid whose position 2445 is G. A fourth nucleus that specifically hybridizes to a nucleic acid amplified using the first nucleic acid and the third nucleic acid using a binding protein 2 gene as a template and is labeled with a first labeling substance And specifically hybridizing to the nucleic acid amplified using the second nucleic acid and the third nucleic acid using the fatty acid binding protein 2 gene having A at position 2445 as a template, and labeling with the second labeling substance And a fifth nucleic acid set.
In the above, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (5) to (9), but two or more of (5) to (9) are arbitrarily selected. The kit may be configured by selecting two or more nucleic acid sets from the group. For example, a kit is constructed by selecting two or more nucleic acid sets from the group consisting of (5) to (8) (nucleic acid sets for analyzing polymorphisms with odds ratios of the top four in the examples described later). Or two or more nucleic acid sets are selected from the group consisting of (5) to (7) (nucleic acid sets for analyzing polymorphisms with odds ratios up to the top three in the examples described later). can do.
[0054]
In each of the above kits, one or more reagents (buffer, reaction reagent, detection reagent, etc.) according to the method of using the kit may be combined.
Hereinafter, the present invention will be described in more detail with reference to examples.
[0055]
【Example】
<Example 1> Selection of gene polymorphism
PubMed [National Center for Biological Information (NCBI)], Online Mendelian inheritance in Men (NCBI), Single Nucleotide Polymorphism (NCBI) 71 genes predicted to be associated with coronary atherosclerosis, coronary artery spasm, hypertension, diabetes, hyperlipidemia, etc. from comprehensive aspects such as biology, platelet / leukocyte biology, coagulation / fibrinolysis system, lipid / sugar / other metabolic factors Excerpt. Furthermore, among the polymorphisms present in these genes, there are 112 polymorphisms, mainly those present in promoter regions and exons, or those that are located in splice donor sites or acceptor sites and are expected to be associated with functional changes in gene products. The mold was selected (FIGS. 1 and 2).
[0056]
<Example 2> Determination of gene polymorphism
The subjects were 2188 Japanese (1215 males, 973 females) who visited (or hospitalized) 15 participating facilities between July 1994 and December 2001. All 593 coronary spasms (453 males, 140 females) have coronary angiography and left ventricular angiography, and coronary artery spasm with ST change of the electrocardiogram occurs spontaneously, or ergonobin or acetylcholine intracoronary artery It was confirmed by angiography that it was induced by injection.
Controls were 1595 (762 males, 833 females) who visited the participating facilities and had traditional risk factors for coronary artery disease: smoking (more than 10 per day), obesity (body mass index> 26 kg / m2), High blood pressure (systolic blood pressure> 140 mmHg or diastolic blood pressure> 90 mmHg or both), diabetes (fasting blood glucose> 126 mg / dL or hemoglobin A1c> 6.5% or both), hyperlipidemia (total serum) Cholesterol> 220 mg / dL), hyperuricemia (serum uric acid> 7.7 mg / dL in men, serum uric acid> 5.5 mg / dL in women) but not coronary artery disease or coronary spasm is there. These controls had normal resting electrocardiograms, and no myocardial ischemic changes were observed in exercise tests.
Cardiac catheterization was performed as follows. All medications were stopped 24-48 hours before coronary angiography. Induction of coronary artery spasm has been reported (Nakayama M, Yasue H, Yoshimura M, et al. T ^-786 → C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm.Cirulation. 1999; 99: 2864-2870., Yoshimura M, Yasue H, Nakamura M, et al. Genetic risk factors for coronary artery spasm: significance of endothelial nitric oxide synthase gene T ^-786 → C and missense Glu298Asp. J Invest Med. 2000; 48: 367-374.) In summary, after left and right coronary angiography, acetylcholine was injected into the left coronary artery in two doses of 50 mg and 100 mg, and coronary angiography was performed 1 minute after each injection. Next, 50 mg of acetylcholine was injected into the right coronary artery, and coronary angiography was performed again. Coronary spasm was defined as complete or partial occlusion of the coronary artery with chest pain and electrocardiogram ST change, or severe vasoconstriction. Finally, 1 mg isosorbide nitrate was injected into the coronary artery, and left and right coronary angiograms were performed from multiple directions. In all cases, the stenosis of the coronary artery was less than 25%.
[0057]
From each subject, 7 mL of venous blood was collected into a tube containing 50 mmol / L EDTA-2Na, and genomic DNA was extracted using a DNA extraction kit (Qiagen, Chatsworth, CA). The genotype of the single nucleotide polymorphism was determined using an allele-specific primer / probe measurement system (Toyobo Gene Analysis, Tsuruga, Japan) by fluorescence / luminescence method (FIG. 3). The DNA fragment containing the polymorphic site is composed of two allele-specific sense (or antisense) primers labeled with fluorescein isothiocyanate (FITC) or Texas red (TxR) at the 5 'end and antisense labeled with biotin at the 5' end. Amplification was performed by polymerase chain reaction (PCR) using (or sense) primers. Alternatively, the DNA fragment containing the polymorphic site can be obtained using two allele-specific sense (or antisense) primers and an antisense (or sense) primer labeled with biotin at the 5 'end, or Amplification was performed by PCR using an antisense primer labeled with biotin at the 5 ′ end. The reaction solution (25 mL) contains 20 ng of DNA, 5 pmol of each primer, 0.2 mmol / L of each deoxynucleoside triphosphate, 1-4 mmol / L of magnesium chloride, 1 U of DNA polymerase (rTaq or KODplus ; Toyobo, Osaka, Japan), and each DNA polymerase buffer was used. Amplification protocol includes initial denaturation at 95 ° C for 5 minutes, 35-45 cycles with denaturation at 95 ° C for 30 seconds, annealing at 55-67.5 ° C for 30 seconds, extension at 72 ° C for 30 seconds, and final extension at 72 ° C. 2 minutes.
For genotyping by fluorescence, the amplified DNA was incubated at room temperature in a solution containing streptavidin-conjugated magnetic beads in each well of a 96-well plate. Place this plate on a magnetic stand, collect the supernatant from each well, transfer it to each well of a 96-well plate containing 0.01 M NaOH, and then FITC is excited and fluorescence wavelength is 485 and 538 nm by a microplate reader. TxR measured fluorescence at excitation and fluorescence wavelengths of 584 and 612 nm. For genotyping by the luminescence method, the amplified DNA was denatured with 0.3 M NaOH and hybridized with one of the allele-specific complementary probes immobilized on the bottom of each well of a 96-well plate and 35-40% formamide. Hybridization was performed with buffer at 37 ° C. for 30 minutes. After thoroughly washing the wells, alkaline phosphatase-conjugated streptavidin was added to each well and the plate was shaken at 37 ° C. for 15 minutes. The wells were washed again and 0.8 mM 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium (monosodium salt) and 0.4 mM 5-bromo-4- After adding a solution containing chloro-3-indolyl phosphate p-toluidine salt, the absorbance at 450 nm was measured.
[0058]
In order to confirm the accuracy of genotyping by this method, 50 DNA samples were randomly selected and subjected to PCR-restriction fragment length polymorphism or direct PCR sequencing. In all samples, the genotype determined by the allele-specific primer / probe measurement system was the same as that determined by the PCR-restriction fragment length polymorphism method or the DNA sequencing method.
[0059]
Statistical analysis in the following related analysis was performed as follows. Clinical data were compared between patients with coronary artery spasm and controls using unpaired Student's t test or Mann-Whitney U test. Qualitative data was tested by chi-square test. The allele frequency was estimated by gene counting method, and whether or not it deviated from Hardy-Weinberg equilibrium was tested by chi-square test. The inventors performed a multinomial logistic regression analysis with risk factors corrected. With coronary spasm as a dependent factor, age, body mass index (BMI), smoking status (0 = non-smoking, 1 = smoking), metabolic factors (0 = no history of hypertension / diabetes / hypercholesterolemia / hyperuricemia) 1 = with a history), and the genotype of each polymorphism was regarded as an independent factor. Each genotype was analyzed using dominant, recessive, and additive genetic models, and P values, odds ratios, and 95% confidence intervals were calculated. In the combined genotype analysis, the odds ratio for each genotype was calculated by the stepwise forward selection method of logistic regression analysis.
[0060]
<Example 3> Selection of polymorphism related to coronary artery spasm and development of coronary artery spasm risk diagnosis method
In the previous report, we analyzed the association between 71 candidate gene 112 polymorphisms and myocardial infarction in 451 men (219 myocardial infarctions, 232 controls) and 458 women (226 myocardial infarctions, 232 controls). (Yamada Y, Izawa H, Ichihara S, et al. Prediction of risk of myocardial infarction from polymorphisms in candidatre genes. N Engl J Med. In press.) The study found that 19 single nucleotide polymorphisms in men and 18 in women were associated with the onset of myocardial infarction, but those polymorphism groups also included candidate genes for coronary artery spasm ( (See FIGS. 1, 2 and 4). In this example, 2188 large-scale association analyzes were performed on the relationship between these single nucleotide polymorphisms and coronary artery spasm.
[0061]
The background data of all 2188 subjects studied (1215 males and 973 females) are shown in FIG. In men, the frequency of smoking and hypercholesterolemia was significantly higher in patients with coronary artery spasm than in controls, and age was significantly lower in patients with coronary artery spasms compared with controls. In women, the frequency of smoking, hypertension and hyperuricemia was significantly higher in patients with coronary artery spasm compared with controls, and age was significantly lower in patients with coronary artery spasms compared with controls.
[0062]
In the association analysis between male 19 polymorphism, female 18 polymorphism and coronary spasm, multiple logistic regression analysis corrected for age, BMI, and frequency of smoking, hypertension, diabetes, hypercholesterolemia, and hyperuricemia in males 4 polymorphisms in women and 5 showed significant association with the onset of coronary artery spasm (either dominant or recessive genetic model was P <0.05) (Figure 6). The genotype distribution of these polymorphisms is shown in FIG.
[0063]
The present inventors performed a stepwise forward selection method of multinomial logistic regression analysis (FIG. 8). In this method, a dominant or recessive genetic model was adopted based on the P value (lower P value) in relation to coronary spasm of each polymorphism shown in FIG. The locus of these genes on the chromosome is shown in FIG. The odds ratio of the onset of coronary artery spasm by the combined genotype calculated by the Stepwise forward selection method is shown in FIGS. 9 and 11A for males and in FIGS. 10 and 11B for females. In males, the four polymorphic combination genotypes (TNFα (-863C → A) polymorphism, p22 Fox (242C → T) polymorphism, ApoC-III (-482C → T) polymorphism, and ApoE (-219G) → T) polymorphism), the maximum odds ratio was 5.08 (FIG. 9, FIG. 11 (A)). In women, the combined genotype of 5 polymorphisms (Stromelysin 1 (-1171 / 5A → 6A) polymorphism, IL-6 (-634C → G) polymorphism, FABP2 (2445G → A) polymorphism, ApoE (4070C → T) polymorphism and ENOS (−786T → C) polymorphism) resulted in a maximum odds ratio of 53.37 (FIGS. 10 and 11B).
[0064]
As described above, 4 single-nucleotide polymorphisms in men and 5 in women were associated with coronary artery spasm by multinomial logistic regression analysis. That is, the present inventors conducted a large-scale association analysis of 2188 cases of 19 single nucleotide polymorphisms in men and 18 single nucleotide polymorphisms in women and coronary spasm, and 4 polymorphisms related to the onset of coronary artery spasm in men. Individuals and 5 females were identified. Furthermore, we developed a genetic risk diagnostic method for coronary artery spasm using a combined genotype with a maximum odds ratio of 5.08 for men and a maximum odds ratio of 53.37 for women using the stepwise forward selection method of multinomial logistic regression analysis.
[0065]
In the results of this example, the smoking frequency was significantly higher in patients with coronary artery spasm than in the control, which is consistent with the reported results (Sugiishi M, Takatsu F. Cigarette smoking is a major risk factor for coronary spasm. Circulation. 1993; 87: 76-79.). Coronary artery spasm is mainly caused by vascular endothelial cell dysfunction (Yasue H, Horio Y, Nakamura N, et al. Induction of coronary artery spasm by acetylcholine in patients with variant angina: possible role of the parasympathetic nervous system in the pathogenesis of coronary artery spasm. Circulation. 1986; 74: 955-963., Vanhoutte PM, Shimokawa H. Endotheliun-derived relaxing factor and coronary vasospasm. Circulation. 1989; 80: 1-9.) and excessive contraction of vascular smooth muscle (Egashira K, Inou T, Yamada A, et al. Preserved endothelium-dependent vasodilation at the vasospastic site in patients with variant angina. J Clin Invest. 1992; 89: 1047-1052.) It causes failure of regulation of contraction and relaxation. Furthermore, vasoactive substances are released from activated platelets (Vanhoutte PM, Shimokawa H. Endotheliun-derived relaxing factor and coronary vasospasm. Circulation. 1989; 80: 1-9.). It is also important that arteriosclerotic changes exist as the underlying lesion (Shimokawa H, Tomoike H, Nabeyama S, et al. Coronary artery spasm induced in atherosclerotic miniature swine. Science. 1983; 221: 560-562.). Based on comprehensive viewpoints such as vascular biology, platelet / leukocyte biology, fibrinolytic system, lipid / sugar / other metabolic factors, we have 19 male and 18 single nucleotide polymorphisms and coronary spasm. The relationship was examined. In fact, genes related to coronary artery spasm have various roles in the pathology. Vascular biology (NADH / NADPH oxidase p22 fox and endothelial nitric oxide synthase), vascular inflammation (tumor necrosis factor α and interleukin 6), lipid metabolism (apolipoprotein C-III, apolipoprotein E) And fatty acid binding protein 2), substrate metabolism (stromelysin 1) and the like. Of the 9 polymorphisms associated with coronary artery spasm, no other polymorphisms other than the -786T → C polymorphism of endothelial nitric oxide synthase have been reported so far. . Among these polymorphisms, the -1171 / 5A → 6A polymorphism of the stromelysin 1 gene exhibited the highest odds ratio. This polymorphism has been reported in coronary artery disease (Ye S, Watts GF, Mandalia S, et al. Preliminary report: genetic variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis. Br Heart J. 1995; 73: 209 -15.) And restenosis after balloon dilatation (Humphries S, Bauters C, Meirhaeghe A, et al. The 5A6A polymorphism in the promoter of the stromelysin-1 (MMP3) gene as a risk Eur Heart J. 2002; 23: 721-725.), especially in the latter pathology, it is known that chronic vascular remodeling (vasoconstriction) is important. Our genetic risk diagnostic system shows that the maximum odds ratio of coronary spasm onset is 5.08 for men and 53.37 for women, especially among women, the largest odds ratio among the large-scale association analyzes reported so far. showed that.
[0066]
Some of the polymorphisms examined in this example may be in linkage disequilibrium with polymorphisms of genes that are truly associated with coronary artery spasm in the vicinity. However, our results showed that 4 genes for men and 5 genes for women are Japanese coronary artery spasm susceptibility loci. Furthermore, the combined genotype of these gene polymorphisms is considered useful for the diagnosis of genetic risk of coronary artery spasm.
[0067]
The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
[0068]
【The invention's effect】
According to the present invention, genetic polymorphism associated with coronary artery spasm is analyzed, and the genotype of the nucleic acid sample is detected. By using polymorphism information obtained by detecting this genotype, coronary artery spasm risk diagnosis with high accuracy and high probability of prediction can be performed. Therefore, the present invention is an effective means for knowing in advance the onset risk of coronary artery spasm, and can be expected to contribute to primary prevention of atypical angina, myocardial infarction and sudden cardiac death due to coronary artery spasm. In addition, according to the present invention, auxiliary information useful for diagnosis of coronary artery spasm can be obtained, and more appropriate treatment can be performed to improve the prognosis. Furthermore, since the present invention provides effective information for elucidating the onset mechanism of coronary artery spasm, it is an extremely important means for establishing a method for preventing coronary artery spasm.
[0069]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a table summarizing 112 gene polymorphisms examined in screening-related analysis in Examples.
FIG. 2 is a table summarizing 112 gene polymorphisms examined in screening-related analysis in the same example.
FIG. 3 shows primers used to determine the genotype in Examples (SEQ ID NOs: 19, 20, 21, 16, 17, 18, 25, 26, 27, 31, 32, 33 in order from the top). , 34, 35, 36, 28, 29, 30, 13, 14, 15, 22, 23, 24, 10, 11, 12), probe (SEQ ID NOs: 37, 38, 39, 40 in order from the top) and other It is the table | surface which put together the conditions. In the figure, FITC represents fluorescein isothiocyanate, TxR represents Texas Red, and Biotin represents biotin.
FIG. 4 is a table summarizing single nucleotide polymorphisms examined in the association analysis of Examples.
FIG. 5 is a table summarizing background data of 1215 males and 973 females subjected to association analysis in Examples. Each data of age and body mass index is expressed as mean ± standard deviation. * 1 in the table is P <0.0001, * 2 is P <0.05, * 3 is P <0.001, * 4 is P Each represents <0.01.
FIG. 6 is a table summarizing the results of genetic polymorphisms and multinomial logistic regression analysis that were the targets of association analysis. The lower P value in each SNP is shown in bold.
FIG. 7 is a diagram showing the genotype distribution of gene polymorphisms associated with coronary artery spasm.
FIG. 8 is a table showing the results of the step forward selection method of multinomial logistic regression analysis for gene polymorphisms associated with coronary artery spasm.
FIG. 9 is a table showing the results of a genetic risk diagnosis of coronary artery spasm using 4 combination gene polymorphisms in men.
FIG. 10 is a table showing the results of a genetic risk diagnosis of coronary artery spasm using four combination gene polymorphisms in women.
FIG. 11 is a graph showing the relationship between the cumulative odds ratio and the number of single nucleotide polymorphisms in the risk diagnosis of coronary artery spasm. (A) shows the relationship in men and (B) shows the relationship in women. Each SNP in (A) is SNP1: TNFα (-863C → A) polymorphism, SNP2: p22 Fox (242C → T) polymorphism, SNP3: ApoC-III (-482C → T) polymorphism, SNP4: ApoE ( -219G → T) Polymorphism. Each SNP in (B) is SNP1: stromelysin 1 (-1171 / 5A → 6A) polymorphism, SNP2: IL-6 (-634C → G) polymorphism, SNP3: FABP2 (2445G → A) polymorphism, SNP4: ApoE (4070C → T) polymorphism, SNP5: ENOS (-786T → C) polymorphism.

Claims (4)

Coronary artery spasm risk testing method comprising the following step (a):
(A) a step of analyzing polymorphisms (1) to (3) below in a nucleic acid sample:
(1) tumor necrosis factor α gene at the base number -863 of polymorphism (197 th nucleotide in the nucleotide sequence of SEQ ID NO: 1),
(2) NADH / NADPH oxidase p22 Fox gene nucleotide numbers 242 of polymorphism (684 th nucleotide in the nucleotide sequence of SEQ ID NO: 2),
(3) apolipoprotein E nucleotide numbers -219 of genetic polymorphism (801 th nucleotide in the nucleotide sequence of SEQ ID NO: 3). Coronary artery spasm risk test method comprising the following steps (i) to (iii):
(i) a step of analyzing polymorphisms (1) to (3) below in a nucleic acid sample:
(1) tumor necrosis factor α gene at the base number -863 of polymorphism (197 th nucleotide in the nucleotide sequence of SEQ ID NO: 1),
(2) NADH / NADPH oxidase p22 Fox gene nucleotide numbers 242 of polymorphism (684 th nucleotide in the nucleotide sequence of SEQ ID NO: 2),
(3) apolipoprotein E nucleotide numbers -219 of genetic polymorphism (801 th nucleotide in the nucleotide sequence of SEQ ID NO: 3),
(ii) determining the genotype of the nucleic acid sample from the polymorphism information obtained by the step, and
(iii) A step of determining a genetic risk of coronary artery spasm from the determined genotype. The following (1) to (3) coronary artery spasm risk test kit comprising the nucleic acid of,
(1) a nucleic acid for analyzing polymorphism of tumor necrosis factor α gene nucleotide numbers -863 position (197 th nucleotide in the nucleotide sequence of SEQ ID NO: 1), complementary to the constant region containing the polymorphic site A nucleic acid that specifically hybridizes to said region,
(2) NADH / NADPH oxidase p22 Fox gene nucleotide numbers 242 of a nucleic acid for analyzing polymorphism (684 th nucleotide in the nucleotide sequence of SEQ ID NO: 2), complementary to the constant region containing the polymorphic site A nucleic acid comprising a specific sequence and specifically hybridizing to said region ,
(3) a nucleic acid for apolipoprotein E nucleotide number -219 of genetic polymorphisms (801 th nucleotide in the nucleotide sequence of SEQ ID NO: 3) analysis, complementary constant region containing the polymorphic site A nucleic acid comprising a sequence and specifically hybridizing to said region . The following (1) to (3) immobilizing a nucleic acid for testing nucleic acids of the coronary artery spasm comprising fixed to an insoluble support risks,
(1) a nucleic acid for analyzing polymorphism of tumor necrosis factor α gene nucleotide numbers -863 position (197 th nucleotide in the nucleotide sequence of SEQ ID NO: 1), complementary to the constant region containing the polymorphic site A nucleic acid that specifically hybridizes to said region,
(2) NADH / NADPH oxidase p22 Fox gene nucleotide numbers 242 of a nucleic acid for analyzing polymorphism (684 th nucleotide in the nucleotide sequence of SEQ ID NO: 2), complementary to the constant region containing the polymorphic site A nucleic acid comprising a specific sequence and specifically hybridizing to said region ,
(3) a nucleic acid for apolipoprotein E nucleotide number -219 of genetic polymorphisms (801 th nucleotide in the nucleotide sequence of SEQ ID NO: 3) analysis, complementary constant region containing the polymorphic site A nucleic acid comprising a sequence and specifically hybridizing to said region .

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