JP2017143745A - Method and reagent for predicting coronary artery event - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a marker for prediction of an acute coronary syndrome based on a comprehensive genetic analysis.SOLUTION: According to the present invention, a method for providing data for predicting the presence or absence of a coronary artery event by using as a marker the expression of NGFR and/or DAPK1 in the peripheral blood leukocytes of a subject. Here, the occurrence of the coronary artery event is predicted when a decrease in the expression of NGFR and/or DAPK1 compared to a reference value is detected.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for predicting a coronary event and a detection reagent used in the method. In particular, the present invention relates to a method for predicting the presence or absence of secondary coronary events in patients after treatment of myocardial infarction.

  Acute coronary syndrome (ACS) is a disease caused by decreased or disrupted blood flow due to thrombus formation in the coronary arteries. Clinically, it includes angina pectoris, myocardial infarction, sudden cardiac death, and the like, and is one of the top causes of death due to disease worldwide. Known risk factors for ACS include smoking, diabetes, hypertension, and hypercholesterolemia.

  In addition to drug therapy with antiplatelet drugs, antithrombotic drugs, β-blockers, etc., ACS treatment includes percutaneous coronary intervention (PCI) and coronary artery bypass surgery. In particular, PCI has greatly improved the prognosis of patients with myocardial infarction over the past decades. In PCI, there has been a problem that restenosis occurs at the treatment site after stent placement. Such restenosis has been reduced by the use of a drug eluting stent (DES). Furthermore, oral administration of high dose statins has been shown to reduce secondary cardiovascular events.

  However, even when a drug-eluting stent is used, restenosis may occur at the treatment site, and a new lesion may occur at a location different from the treatment site. Therefore, continuous monitoring of patients after PCI is needed.

  Until now, as a biomarker for predicting the onset of coronary events such as myocardial infarction, serum of neutrophil and platelet-derived cytokines and soluble proteins from patients with atherosclerosis or patients in stable phase after ACS treatment The possibility of using the level has been reported (Non-Patent Documents 1-5). It has also been reported that microRNA in peripheral blood mononuclear cells can serve as a marker for prediction (Non-patent Documents 6-7).

Trial. J. Am. Coll. Cardiol. 2009; 54: 2358-2362 Circulation 2003; 108: 1440-1445 Eur. Heart J. 2008; 29: 1096-1102 Eur. Heart J. 2010; 31: 3024-3031 Int. J. Cardiol. 2015; 201: 499-507 Int. J. Cardiol. 2014; 172: 356-363 Int. J. Cardiol. 2014; 176: 375-385

However, no marker for ACS based on comprehensive genetic analysis has been reported, and a marker for predicting long-term prognosis (presence or absence of secondary coronary event) has not yet been established.
That is, there is a need for comprehensive profiling of gene expression in ACS patients that can be used as a predictor of ACS.

  In order to comprehensively analyze gene expression in peripheral blood lymphocytes (PBL) of patients with acute coronary syndrome, the present inventors conducted a five-year cohort study and analyzed the genes in PBL of acute patients. The correlation between expression and the presence or absence of secondary coronary events was examined. This trial is registered as UMIN clinical trial UMIN000001932.

  As a result of DNA microarray analysis, 83 genes were extracted as genes whose expression was significantly different between the group that caused the coronary secondary event and the group that did not, and 41 of these genes were compared with the control group. On the other hand, expression was increased, while 42 types had decreased expression. A group of genes found to be significantly different in expression has not been reported so far.

  Furthermore, when the identified gene group was examined separately between the case of restenosis at the treatment site and the case of new stenosis at a site different from the initial lesion, a difference was found in the significance of the expression change. That is, it has been clarified that the optimum predictive factor differs between the occurrence of restenosis and the occurrence of new stenosis.

  Based on these findings, the present inventors have identified markers that can predict restenosis after treatment of myocardial infarction and secondary coronary events such as new lesions.

That is, the present invention provides the following.
1. The presence or absence of coronary event using NGFR (tumor necrosis factor receptor superfamily member 16 precursor (low affinity nerve growth factor receptor)) and / or DAPK1 (cell death-related kinase 1) in the peripheral blood leukocytes of subjects A method for providing data for prediction, wherein the occurrence of a coronary event is predicted when a decrease in expression of NGFR and / or DAPK1 compared to a reference value is detected.
2. 2. The method according to 1 above, wherein the coronary event is a new lesion, and the detection of decreased expression of one or more genes shown in Table 1 is used as an index.
3. 2. The method according to 1 above, wherein the coronary event is restenosis of a myocardial infarction treatment site, and the detection of decreased expression of one or more genes shown in Table 2 is used as an index.
4). Data for detecting the expression of marker genes in peripheral blood leukocytes collected from patients with acute myocardial infarction, and predicting the presence or absence of secondary coronary events based on the increase or decrease in expression of the marker genes compared to the reference value The method described above, wherein the marker gene is one or more genes shown in Table 3 or 4.
5). 5. The method according to 4 above, wherein the peripheral blood is collected before treatment of an acute myocardial infarction patient.
6). 6. The method according to 4 or 5 above, wherein the data is based on a decrease in the expression of the gene shown in Table 3.
7). 6. The method according to 4 or 5 above, wherein the data is based on increased expression of the genes shown in Table 4.
8). 6. The method according to 4 or 5 above, wherein the marker gene comprises NGFR and / or DAPK1.
9. A detection reagent for use in the method according to any one of 1 to 8 above, which enables detection of the expression of any of the genes shown in Tables 1 to 4.
10. 10. The detection reagent according to 9 above, which is DNA or RNA for hybridization with mRNA in a sample.
11. 10. The detection reagent according to 9 above, which is an antibody for specific binding to a protein in a sample.
12 A kit used for predicting a coronary event, comprising the detection reagent shown in any one of 9 to 11 above.

  Independent of conventional risk factors, markers have been identified that can predict the risk of developing or recurrent acute coronary syndromes. The present invention provides a very useful method, especially for prognostic assessment of patients after PCI.

Expression of 83 genes (P <0.005) extracted from 7,785 genes by hierarchical clustering analysis and class comparison analysis was applied to the group with non-lethal coronary event (NFE) (right side) and the group without event (left side) Classify and show in heat map. The results for each sample from 30 patients are shown in columns and the results for each gene are shown in rows. Expression data is illustrated as a color image, with red and green showing up- and down-regulated genes, respectively, and gray showing unavailable data. Figure 2 shows a Kaplan-Meier curve comparing the cumulative incidence of new lesions over 30 years of follow-up over 30 years according to the expression level of NGFR. “NGFR Low”, “NGFR Medium”, and “NGFR High” indicate patients in a low expression group, a moderate expression group, and a high expression group of NGFR, respectively. FIG. 5 shows a Kaplan-Meier curve comparing the cumulative incidence of restenosis over a follow-up period of more than 30 years according to the expression level of DAPK1 in 30 patients. “DAPK1 Low”, “DAPK1 Medium”, and “DAPK1 High” indicate patients in the low expression group, moderate expression group, and high expression group of DAPK1, respectively.

  As a first embodiment, the present invention provides NGFR (tumor necrosis factor receptor superfamily member 16 precursor (low affinity nerve growth factor receptor)) (Tumor necrosis factor receptor superfamily member 16) in peripheral blood leukocytes of a subject. A method for providing data for predicting the presence or absence of coronary events using a precursor (Low-affinity nerve growth factor receptor) and / or DAPK1 (Death-associated protein kinase 1) as a marker. Providing a method as described above, wherein the occurrence of a coronary event is predicted when a decrease in expression of NGFR and / or DAPK1 compared to a reference value is detected.

  Herein, the tumor necrosis factor receptor superfamily member 16 precursor (low affinity nerve growth factor receptor) is referred to as “NGFR” for simplicity. NGFR is a receptor for neutrophin and is known to belong to the tumor necrosis factor receptor superfamily. Nerve growth factor has effects such as regulation of cell proliferation and differentiation in the nervous system, but its relationship with coronary events is not known. NGFR is located on chromosome 17 (17q21-q22) in humans. The nucleotide sequence and amino acid sequence of NGFR mRNA are registered in the NCBI database as NCBI reference sequences: NM_002507.3 and NP_002498.1, respectively.

  On the other hand, in this specification, cell death-related protein kinase 1 is described as “DAPK1” for the sake of brevity. DAPK1 is a calcium / calmodulin-dependent serine / threonine kinase that induces apoptosis in various cells by stimulating Fas, IFN-γ, TNF-α, etc., but it may exert anti-apoptotic action depending on the situation. It has been reported. There is no known association between DAPK1 and coronary events. DAPK1 is located in chromosome 9 (9q21.33) in humans, and the nucleotide sequence and amino acid sequence of the mRNA are registered in the NCBI database as NCBI reference sequences: NM_001288729.1 and NP_001275658.1, respectively.

  As used herein, a “subject” is a human subject that can develop a coronary event. Preferably, the subject is a patient who has developed myocardial infarction, in particular an acute myocardial infarction patient.

  “Subject's peripheral blood leukocytes” are peripheral blood leukocytes collected from the subject. If the subject is a patient with myocardial infarction, collection is for drug administration, percutaneous coronary intervention (PCI), etc. It is preferable to be before treatment.

  The term “coronary event” is a term commonly used in the art and can be easily understood by those skilled in the art. In this specification, the term “coronary event” refers to stenosis and occlusion of coronary arteries such as coronary artery stenosis, sudden cardiac death, fatal and non-fatal myocardial infarction, unstable angina, and symptoms caused thereby. It is intended to include.

  In addition, “(coronary artery) secondary event” is an event that occurs several months to several years after the treatment of myocardial infarction, etc., and in this field, the event such as restenosis at the treatment site, It is understood to include both events such as stenosis that occur at different sites. However, as described above, the present inventors have found that a gene that can be a predictive marker for a coronary artery event can vary depending on the expression change in these restenosis and stenosis at different sites. Therefore, in the present specification, an event such as stenosis that occurs at a site different from the treatment site is referred to as a “new lesion”.

  The “reference value” is a numerical value that can be used as a reference for determining an increase or decrease in expression in the method of the present invention. For example, an average value of expression in a non-ACS subject, for example, a healthy person, or a patient with ACS It can be the mean value of expression in subjects who have not developed secondary events.

  In addition, in DNA microarray analysis, the expression value of each gene may be obtained as a ratio to the expression of a reference RNA (expression value in a universal control sample). Thus, the reference value can also be the ratio of gene expression to reference RNA expression in a non-ACS subject, eg, a healthy person, or a subject who has not developed a secondary event in an ACS-onset patient. Further, the reference value may be a value obtained by converting the obtained ratio value into, for example, a log2 scale.

  The reference value as described above can be obtained by detecting the expression of the control sample simultaneously with the detection of the expression in the subject subject, and the expression can be compared. Alternatively, the expression of the control sample can be measured in advance and prepared as a standard expression level, and in some cases as an expression level within a certain range. A person skilled in the art can understand a method for obtaining a reference value for detecting a disease using a genetic marker as an index.

  As used herein, “gene” refers to a polynucleotide encoding a protein, ie, DNA or RNA, as generally understood in the art, but in addition to this, it is not necessarily a protein. Polynucleotides that can be transcribed into RNA in vivo without coding are included.

  In the present specification, “expression” of a gene includes both expression of RNA (transcription product) complementary to the gene and expression of protein (translation product) encoded by the gene. Therefore, in this specification, the expression level of a gene refers to the expression level or expression intensity of a transcription product or translation product. The expression level can usually be analyzed based on the production amount of a transcription product corresponding to a gene, or the production amount, activity, etc. of its translation product.

  The gene expression level may be expressed as an absolute value or a relative value. Thus, “increased expression” and “decreased expression” can be expressed as expression levels that are increased or decreased relative to a reference value, or alternatively, as “fold-change” in expression. In the method of the present invention, the fluctuation of expression in “increased expression” and “decreased expression” suitable for predicting coronary events is, for example, 2 times or more, 3 times or more, 4 times or more. Therefore, the “fold change” is preferably 1.25 times or more for an increase in expression and 0.80 times or less (less than) for a decrease in expression.

  The expression level may be measured by measuring a gene transcription product, ie, mRNA, or by measuring a gene translation product, ie, protein. Preferably, it is carried out by measuring a gene transcription product. A gene transcription product also includes cDNA obtained by reverse transcription from mRNA.

  The expression level of the transcript may be measured by measuring the level of gene expression in the sample using, for example, nucleotides containing all or part of the base sequence of mRNA as a probe or primer. For example, a microarray (microchip) is used. It is possible to measure by the conventional method, Northern blot method, quantitative PCR method and the like. Quantitative PCR methods include agarose gel electrophoresis, fluorescent probe method, RT-PCR method, real-time PCR method, ATAC-PCR method (Kato, K. et al., Nucl. Acids Res., 25, 4694-4696. 1997), Taqman PCR method (SYBR (registered trademark) green method) (Schmittgen TD, Methods 25, 383-385, 2001), Body Map method (Gene, 174, 151-158 (1996)), Serial analysis of gene expression (SAGE ) Method (US Pat. Nos. 527,154, 544,861, European Patent Publication No. 0761822), MAGE method (Micro-analysis of Gene Expression) (Japanese Patent Laid-Open No. 2000-232888), etc. are known and used appropriately can do. Using these methods, the amount of mRNA can be measured by the use of nucleotide probes or primers that hybridize to the mRNA. The base length of the probe or primer used for the measurement is 10 to 50 bp, preferably 15 to 25 bp.

  When measuring the expression of a plurality of genes, particularly the expression of many types of genes at the same time, it is preferable to use a DNA microarray. A DNA microarray can be prepared by immobilizing a nucleotide comprising a nucleotide sequence of a gene or a nucleotide comprising a partial sequence thereof on an appropriate substrate. Examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer. Examples of the size of the substrate include 3.5 mm × 5.5 mm, 18 mm × 18 mm, 22 mm × 75 mm, etc., which are variously set according to the number of probe spots on the substrate and the size of the spots. be able to. As a method for immobilizing a polynucleotide or a fragment thereof, it is possible to electrostatically bind to a solid phase carrier surface-treated with a polycation such as polylysine, polyethyleneimine, polyalkylamine, etc. A nucleotide having a functional group such as an amino group, an aldehyde group, an SH group, or biotin can be covalently bonded to a solid phase surface into which a functional group such as an aldehyde group or an epoxy group has been introduced. Immobilization may be performed using an array machine. At least one gene or a fragment thereof is immobilized on a substrate to prepare a DNA microarray. The DNA microarray is contacted with mRNA or cDNA derived from a subject labeled with a fluorescent substance, hybridized, and fluorescent on the DNA microarray. By measuring the intensity, the type and amount of mRNA can be determined.

  When using a DNA microarray, a sample derived from a subject containing the target mRNA and the control sample can be simultaneously hybridized to the microarray. Thereby, the expression level of the gene whose expression is fluctuating in the sample derived from the subject compared to the control sample can be obtained as a relative expression change. The mRNA derived from the subject and optionally the mRNA derived from the control sample can be labeled, and the labeling is not particularly limited, but commercially available fluorescent substances such as Cy3 and Cy5 can be used.

Moreover, a commercially available product can be used as appropriate for the DNA microarray. Examples of usable DNA microarrays include 3D-Gene ^™ Human Oligo chip 25k (manufactured by Toray Industries, Inc.).

  The expression level of the translation product can be measured by quantifying the translated protein or measuring the activity of the protein. Alternatively, protein quantification can be performed using antibodies specific for the protein.

  The antibody can be prepared by a known method. Alternatively, antibodies having reactivity with human NGFR, human DAPK1, etc. are provided by, for example, Invitrogen, Santa Cruz Biotechnology, Sigma-Aldrich, etc., and these can be appropriately obtained and used. . The antibody for detection may be a polyclonal antibody or a monoclonal antibody.

  Although the detection of the protein using an antibody is not limited, it can be performed by, for example, Western blotting, ELISA, flow cytometry and the like. The antibody can be labeled with a fluorescent label, a radioactive label, an enzyme, biotin or the like, and a secondary antibody for detection labeled as such can also be used.

  The method of the present invention, in the first embodiment, the above-mentioned coronary event is a new lesion, and particularly comprises the detection of the expression of NGFR. In this embodiment, in addition to NGFR, detection of a decrease in the expression of one or more genes shown in Table 1 can be used as an indicator. Table 1 shows 10 genes whose expression was significantly decreased in the group with new lesions when compared with the presence or absence of new lesions during the 5-year follow-up period found by the present inventors. , P-values are listed in ascending order. Therefore, in addition to NGFR, this aspect is selected from RBP7, PDXK, C6orf166, CDA, C9orf164, RRAGD, PTHR2, KIAA0319L, MRGPRF, 1, 2, 3, 4, 5, If a decrease in expression of 7, 8, or 9 genes is detected, it indicates that there is a high probability that a new lesion will occur. Detection of expression of genes other than NGFR can also be performed as described above.

  Here, the “new lesion” includes both a lesion in the first coronary event and a lesion in a secondary event that occurs at a different site from the lesion in the first coronary event.

  The gene names and symbols shown in the following Tables 1 to 4 are notation clearly understood in the art, and NCBI No is the reference sequence number of mRNA in the NCBI database described above. is there. A person skilled in the art can know various characteristics of each gene based on this information, and easily obtain information on DNA sequences and mRNA sequences, and amino acid sequences of proteins encoded by these genes. Can do.

  The method of the present invention, in the second embodiment, the above-mentioned coronary artery event is restenosis after myocardial infarction treatment, and in particular comprises detection of DAPK1 expression. In this embodiment, in addition to DAPK1, detection of a decrease in the expression of one or more genes shown in Table 2 can be used as an index. Table 2 shows 10 genes whose expression was significantly decreased in the group with restenosis when compared with the presence or absence of restenosis during the follow-up period of 5 years found by the present inventors. The p-values are listed in ascending order. Therefore, in addition to DAPK1, this aspect is one, two, three, four, five, six selected from DDI2, SETD2, BHLHB8, NR2E3, SLC9A1, DIDO1, IGHV3-13, USF1, ABHD4. When a decrease in expression of species, 7, 8, or 9 genes is detected, it is indicated that restenosis is likely to occur at the treatment site. Detection of the expression of genes other than DAPK1 can also be performed as described above.

  As can be seen from Tables 1 and 2, it was surprisingly found that the gene group whose expression changes is different when a new lesion occurs and when restenosis occurs at the treatment site. This has never been known before, and the change in expression between predicting the possibility of a new lesion and predicting the possibility of a lesion such as restenosis at the treatment site. Means that the genes to be considered are different.

  As a second embodiment, the present invention also detects expression of a marker gene in peripheral blood leukocytes collected from a patient with acute myocardial infarction, and based on an increase or decrease in expression of the marker gene compared to a reference value. Provided is a method for providing data for predicting the presence or absence of a secondary coronary event, wherein the marker gene is one or more genes shown in Table 3 or 4. The peripheral blood is not particularly limited, but is preferably collected before treatment of an acute myocardial infarction patient.

  Tables 3 and 4 show that each of the 20 genes whose change in expression should be detected when predicting the prognosis in patients who developed ACS without distinguishing between new lesions and restenosis, whether there is a secondary event Are described in ascending order of the p-value when compared.

  In the first embodiment, the data is based on a decrease in the expression of the genes shown in Table 3. For example, at least one selected from NFAM1, RFX2, DAPK1, NGFR, LILRA2, NIN, C9orf164, NP_569736.1, ELMO1, GRB2, PHLPP, WAS, RRAGD, HEBP2, TREML2, BHLHB8, SLC35E3, TUFT1, EXTL3, NCF4 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 decreased gene expression may be detected when coronary secondary events occur Shown high.

  In a second embodiment, the data is based on increased expression of the genes shown in Table 4. One or more types selected from DUT, DUSP14, LPIN1, BIVM, NSG1_HUMAN, ITGB7, COQ3, SH2B1, SEGEF, WDR70, Q96NS8_HUMAN, RAI16, PFKP, LAT, CD320, CCDC92, SLC25A19, CD8B, CBFA2T2, LRRC8C 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 increased expression of genes is detected, indicating that there is a high possibility of secondary coronary events It is.

  In a third embodiment, the data is based on a decrease in the expression of the genes shown in Table 3 and an increase in the expression of the genes shown in Table 4.

  As shown in Table 3, both NGFR and DAPK1, which should be particularly considered in the first embodiment, are ranked higher as genes showing a significant decrease in expression. Therefore, in this embodiment, it is preferred that the marker gene comprises NGFR and / or DAPK1.

  In the third embodiment, the present invention is a detection reagent for use in the above-described method of the present invention, which enables detection of the expression of any of the genes shown in Tables 1 to 4. I will provide a.

  In the first embodiment, the detection reagent is DNA or RNA for hybridization with mRNA in the sample. DNA or RNA is a probe capable of detecting hybridization with a fluorescent label or the like. Alternatively, DNA or RNA is a primer that can be used to amplify mRNA.

  In a second embodiment, the detection reagent is an antibody for specific binding to a protein in the sample.

Here, the “sample” refers to a sample derived from a subject in which the mRNA or protein to be detected may be present and a sample for comparison, for detection from the peripheral blood leukocytes and peripheral blood leukocytes of the subject. The above-mentioned operations such as cell lysis, centrifugation, filtration, extraction, etc. may be included, and various reagents, buffers and the like may be included.
The detection reagent can be appropriately labeled with a label necessary for detection.

  In the fourth embodiment, the present invention provides a kit used for predicting a coronary event comprising the detection reagent of the present invention described above. In addition to the detection reagent, the kit of the present invention may contain other reagents necessary for detection, such as buffers, various nucleotides, other reagents necessary for hybridization or antibody binding.

  Furthermore, as described above, according to the present invention, it is found that the expression of NGFR and / or DAPK1 as well as the genes shown in Tables 1 to 3 are decreased in subjects who are likely to develop a coronary event. Has been. Therefore, in such a subject, NGFR and / or DAPK1, or in addition to this, a protein encoded by the gene shown in Tables 1 to 3, or a polynucleotide having the same base sequence as the gene, in an appropriate form, for example, By incorporating the gene-encoded protein as a cell surface antigen or by incorporating it into a vector normally used in the art, such as a viral vector or a plasmid vector, and administering it to a subject as a therapeutic agent, It may be possible to prevent the onset.

On the other hand, in order to decrease the expression of genes whose expression is increased in subjects who are likely to develop coronary events, for example, the genes shown in Table 4, base sequences complementary to the base sequences of these genes are used. It is envisaged that antibodies specific to interfering RNA such as siRNA and proteins encoded by these genes are administered as therapeutic agents.
The method for administering the therapeutic agent is not particularly limited, but for example, intravenous administration is preferred.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. The statistical method used in this example is easily understood by those skilled in the art, and SPSS statistical software (version 19, manufactured by IBM) was used for analysis.

[Clinical characteristics of the subject]
Kanazawa University Hospital, Matsuto Ishikawa Central Hospital, and Toyama Prefectural Central Hospital have been approved by each ethics committee for clinical research since 2007.
The subjects to be analyzed included 30 patients with acute myocardial infarction (ACS group) who underwent percutaneous coronary intervention (PCI), and 15 non-ACS subjects (control group).

  Table 5 lists some of the clinical baseline characteristics of 30 subjects in the ACS group and 15 subjects in the control group whose results are described in this example. The age group of the ACS group and the control group was almost the same, but the incidence of diabetes was 50% in the ACS group. Also, compared to the control group, the ACS group had higher values of glycosylated hemoglobin and fasting plasma glucose, while the high specific gravity lipoprotein cholesterol level was lower.

  The 30 patients in the ACS group were treated with PCI and then followed for a 5-year cohort follow-up. As a result, 36% (11 cases) had a secondary event, that is, a non-fatal coronary event (NFE) judged to require revascularization. NFE included restenosis at the treatment site and new lesions at sites different from the treatment site, and 7 cases had both restenosis and 7 new lesions. Table 6 lists some of the clinical baseline characteristics of patients with and without NFE.

[Obtaining a sample from a subject]
Peripheral blood was collected from 30 ACS group patients prior to PCI and 5 ml of blood was collected into two 2.5 ml PAXgene ^™ blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) and -80 ° C until RNA isolation Saved with.

RNA was isolated from the resulting blood using the PAXgene ^™ system. Specifically, the PAXgene ^™ blood RNA tube was centrifuged to collect the pellet, washed and resuspended in buffer. To the resuspended pellet, lysis buffer of PAXgene ^™ blood RNA kit (Qiagen, Valencia, CA, USA) was added, and RNA was purified and extracted. The extracted RNA was stored at −80 ° C. until DNA microarray analysis.

[DNA microarray analysis]
0.5 μg of total RNA was used for each sample, amplified using Amino Allyl MessageAmp ^™ II aRNA Amplificaion kit (Life Technologies, Carlsbad, Calif., USA) and labeled with Cy3. Labeled RNA sample and Cy5 labeled reference aRNA (Human Universal Reference Total RNA: hURR # 636538, Clontech, TAKARA BIO INC.) On 3D-Gene ^TM Human Oligo chip 25k (Toray Industries, Inc.) at 37 ° C Hybridized for 16 hours.

After hybridization, each DNA chip was washed and dried, and then hybridization signals derived from Cy3 and Cy5 were detected using Scan Array Express (PerkinElmer, Waltham, MA, USA). The detection result of the scan image was analyzed using GenePix Pro (Molecular Devices, Sunnyvale, CA, USA). The ratio of the expression intensity of each gene relative to the control (hURR) was calculated for each sample and converted to a log ₂ scale.

[A differentially expressed gene in ACS predicts non-fatal coronary events]
The 3D-Gene ^™ Human Oligo chip 25k (manufactured by Toray Industries, Inc.) can analyze the expression of 24,267 genes. In order to efficiently compare gene expression, the inventors first selected 7,785 genes excluding genes that did not change between samples.

  As a result of the cohort study, in order to examine genes that are expressed differently in the group with and without the secondary event, BRB-Array tools (version 4.4.0) (NCBI, NIH, Bethesda, MD, USA) Class comparison analysis was performed using class comparison tool based on univariate t-test. From the selected 7,785 genes, 83 genes were further extracted (41 up-regulated species and 42 down-regulated 42 species), assuming that a gene showing a p value of less than 0.005 was significant. As shown in FIG. 1, the increase or decrease in the expression of these genes is clearly determined depending on the presence or absence of secondary events.

[Marker genes for predicting the onset of secondary coronary events]
The expression ratio of the 83 genes selected as differentially expressed genes in the group with and without the secondary event to the reference aRNA was calculated on a log2 scale, and the average value of each group for each gene was calculated for each gene. Calculated as (with event) / (without event) as the fold-change.

  Table 7 shows the 20 genes whose expression increased in the group that had the secondary event in ascending order of p-value. Table 8 shows the 20 genes whose expression decreased in the group with the secondary event in ascending order of p-value.

  Increases and / or decreases in the expression of these genes are associated with the presence or absence of secondary events, and thus the genes shown in Tables 7 and 8 can be used alone or in combination, respectively, for secondary events in ACS patients. It became clear that it can be used to predict the onset.

[Marker genes for predicting the development of new lesions after ACS treatment]
In the gene group whose expression was decreased in the group that had the secondary event, several genes that were functionally interesting were found, and further analysis was performed on the expression of these genes.

  The state generally referred to as a secondary event is understood to include both events such as restenosis at a treatment site and events such as stenosis occurring at a site different from the treatment site. Surprisingly, however, the genes that can predict these events differ between stenosis, etc. (new lesion) at a site different from the previous treatment site, and restenosis, etc. at the previous treatment site. It was found.

  Table 9 lists the 10 genes whose expression was significantly decreased in the group with new lesions in ascending order of p-value when compared with the presence or absence of new lesions during the 5-year follow-up period. It is a thing. Therefore, it was revealed that the genes shown in Table 9 can be used alone or in combination to predict the onset of new lesions. These genes may be able to predict both lesions in the first coronary event and lesions in secondary events that occurred at a different site than the lesion in the first coronary event.

[Marker gene for predicting the onset of restenosis after ACS treatment]
Table 10 lists the 10 genes whose expression was significantly reduced in the group with restenosis in ascending order of p-value when compared with the presence or absence of restenosis during the 5-year follow-up period. It is a thing. Therefore, it was revealed that the genes shown in Table 10 can be used alone or in combination to predict the onset of restenosis at the treatment site.

[NGFR expression level in ACS]
As a single marker candidate for predicting coronary artery events, NGFR (Table 9) that was most significantly down-regulated (with the lowest P-value) in the new lesion generation group from the extracted 83 genes was examined. The 30 ACS groups were divided into 3 groups (10 each), based on the NGFR expression level in the sample, low expression group, moderate expression group and high expression group, and the cumulative incidence of new lesions in each group Were evaluated using the Kaplan-Meier method and compared using the log-rank test.

  As a result, as shown in FIG. 2, no new lesion was observed in the NGFR high expression group (▲), whereas in the NGFR low expression group (●), it was 50% new after 5 years. Lesions had occurred.

  To identify factors that correlate with the presence or absence of secondary events during the 5 years after treatment, univariate analysis using the Cox model for patients with low NGFR expression and baseline characteristics of patients or clinical biomarkers (multiple branches) Multivariate analysis adjusted for lesions, fasting immunoreactive insulin, and the presence or absence of diabetes. Multivariate analysis used a stepwise selection method with a p-value of 0.25 as a criterion for incorporation into the calculation. As a result, as shown in Table 11, it was revealed that the lower NGFR expression in PBL showed a high hazard ratio and was an independent risk factor for secondary events. As shown in Table 6, smoking and LDL-C serum levels could not be correlated with the presence or absence of secondary events.

[DAPK1 expression level in ACS]
As another single marker candidate for predicting coronary events, DAPK1 (Table 10) that was most significantly down-regulated (with the lowest P-value) in the restenosis generation group was examined. The 30 ACS groups were divided into three groups (10 each), based on the level of DAPK1 expression in the sample, a low expression group, a moderate expression group, and a high expression group. Cumulative onset of restenosis in each group Rates were evaluated using the Kaplan-Meier method and compared using the log-rank test.

  As a result, as shown in FIG. 3, in the DAPK1 high expression group (▲), no new lesion was observed, whereas in the DAPK1 low expression group (●), it exceeded 50% after 5 years. Restenosis occurred.

  To identify factors that correlate with the presence or absence of secondary events during the 5-year post-treatment period, univariate analysis using the Cox model for patients with low DAPK1 expression and baseline characteristics of patients or clinical biomarkers (multiple branches) Multivariate analysis adjusted for lesions, fasting immunoreactive insulin, and the presence or absence of diabetes. Multivariate analysis used a stepwise selection method with a p-value of 0.25 as a criterion for incorporation into the calculation. As a result, as shown in Table 12, it was revealed that lower DAPK1 expression in PBL showed a high hazard ratio and was an independent risk factor for secondary events.

  The present invention provides new detection reagents and methods that can be used to predict coronary events. The marker gene detected in the method of the present invention is likely to be a risk factor independent of the previously reported risk factors for ACS. Since ACS is a disease in which early response is very important, the method of the present invention that allows prediction of the long-term prognosis of patients especially after PCI treatment can be very beneficial.

Claims (12)

  The presence or absence of coronary event using NGFR (tumor necrosis factor receptor superfamily member 16 precursor (low affinity nerve growth factor receptor)) and / or DAPK1 (cell death-related kinase 1) in the peripheral blood leukocytes of subjects A method for providing data for prediction, wherein the occurrence of a coronary event is predicted when a decrease in expression of NGFR and / or DAPK1 compared to a reference value is detected.   The method according to claim 1, wherein the coronary event is a new lesion, and the detection of decreased expression of one or more genes shown in Table 1 is used as an index.   The method according to claim 1, wherein the coronary event is restenosis of a myocardial infarction treatment site, and the detection of decreased expression of one or more genes shown in Table 2 is used as an index.   Data for detecting the expression of marker genes in peripheral blood leukocytes collected from patients with acute myocardial infarction, and predicting the presence or absence of secondary coronary events based on the increase or decrease in expression of the marker genes compared to the reference value The method described above, wherein the marker gene is one or more genes shown in Table 3 or 4.   The method according to claim 4, wherein the peripheral blood is collected before treatment of an acute myocardial infarction patient.   The method according to claim 4 or 5, wherein the data is based on a decrease in the expression of the genes shown in Table 3.   The method according to claim 4 or 5, wherein the data is based on an increase in the expression of the genes shown in Table 4.   The method according to claim 4 or 5, wherein the marker gene comprises NGFR and / or DAPK1.   A detection reagent for use in the method according to any one of claims 1 to 8, which enables detection of the expression of any of the genes shown in Tables 1 to 4.   The detection reagent according to claim 9, which is DNA or RNA for hybridization with mRNA in a sample.   The detection reagent according to claim 9, which is an antibody for specific binding to a protein in a sample.   A kit used for predicting a coronary event, comprising the detection reagent according to any one of claims 9 to 11.

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