KR101069593B1 - Markers for the diagnosis of acute myocardial infarction - Google Patents

Abstract

The present invention provides acute myocardial infarction diagnostic markers. It relates to a kit comprising an agent for measuring the expression pattern of the markers at the mRNA or protein level and a method for diagnosing acute myocardial infarction using the same.

Myocardial infarction, diagnosis, biomarkers

Description

Markers for the diagnosis of acute myocardial infarction

The present invention provides a kit for diagnosing acute myocardial infarction comprising a marker for diagnosing acute myocardial infarction and an agent for measuring the presence of the marker.

Acute myocardial infarction (AMI) is a symptom of heart failure due to necrosis of myocardial oxygen deficiency due to narrowing or occlusion of the coronary artery, with a severe mortality rate of 35-50% (death within 1 hour after infarction). Disease.

In the case of acute myocardial infarction, the mortality rate of the adult mortality rate in North America and Western Europe is one, and in Korea, the mortality rate has increased more than twice in recent decades. The incidence and mortality rates of cardiovascular vaginal changes are on the rise.

Medically, AMI diagnosis is based on the WHO standard, chest pain lasting more than 20 minutes, rapid change in electrocardiogram, increase in myocardial enzymes, and can be diagnosed as AMI if two or more of them are matched. However, a significant number of patients with chest pain are found to be non-AMI, and the sensitivity of AMI diagnosis to ECG findings is only 55-75%. In addition, more than two-thirds of the patients suspected of myocardial infarction are admitted to the hospital as non-myocardial infarction, which requires markers and diagnostic methods for accurate and rapid diagnosis of myocardial infarction.

Therefore, the accurate diagnosis of acute myocardial infarction of the patient, and the rapid development of diagnostic biomarkers, that is, the discovery of acute myocardial infarction-specific markers are required. To date, well-known AMI diagnostic markers used for diagnosing acute myocardial infarction (AMI) from the blood of a patient include myoglobin, CK-MB, Troponin I, and the like.

The characteristics of known markers are as follows:

Myoglobin: Myoglobin is secreted into the blood within 2 hours of myocardial injury, so early diagnosis is possible. However, myoglobin is present in skeletal muscle as well as myocardial tissue.

Creatine Kinase (CK) or Creatine Kinase Isoform MB (CK-MB): Mostly present in myocardial tissue, but some (about 5%) are present in skeletal muscle, making them less specific than cTn I. In addition, the calibration standard has the advantage that the deviation of the analysis value according to the manufacturer is small.

Cardiac Troponin T: Specificity for myocardial infarction is similar to cTn I, but has a problem of cross-reaction with some diseases such as kidney disease.

Cardiac Troponin I: The most specific protein for myocardial tissue damage. Due to the long residence time in the blood, late diagnosis is possible (120-216 hours). There is no standard calibration, so the manufacturer's measurements are up to 20 times higher.

Myocardial infarction, especially acute myocardial infarction, can lead to death if not treated within 6 hours after the onset of chest pain, so prompt diagnosis is very important. Nevertheless, since the markers are extremely low in blood and individual differences in patients, there is a need for the development of new markers that enable accurate and rapid diagnosis. In addition, the existing central laboratory equipment and inspection system has a limitation in rapid diagnosis, and it is required to develop new markers for accurate diagnosis and differential diagnosis of patients, and MEMS-based lab-on for easy diagnosis with high speed, high sensitivity, and specificity Requires development of a lab on a chip.

Microarrays integrate a set of oligonucleotides of complementary DNA (cDNA) or 20-25 base pairs in length on glass. cDNA microarrays are produced by labs in schools or by companies such as Agilent and Genomic Solutions, either mechanically immobilizing cDNA collections on a chip or by using ink jetting (Sellheyer, K and Belbin, TJ, J. Am). Acad.Dermatol. 51: 681-692, 2004). Oligonucleotide microarrays are made by Affymetrix by direct synthesis on a chip using photolithography, and by Agillent et al., Synthesized oligonucleotides are immobilized (Sellheyer, K.). and Belbin, TJ, J. Am. Acad.Drmatol. 51: 681-692, 2004).

Recently, genes expressed in specific tissues or cell lines by all chemicals, as well as drug and new drug candidates, are combined with high-throughput toxicological genomics (Toxicogenomics) research, which is an advanced technique using DNA microarray technology. Pattern analysis and quantitative analysis are now possible.

Thus, in order to analyze gene expression, RNA is obtained from a biological sample and hybridized with oligonucleotides in a DNA microarray, and the obtained RNA is labeled with fluorescence or isotope and converted to cDNA. Oligo microarrays are mainly labeled with two different fluorescences (eg, Cy3 and Cy5) to perform hybridization reactions simultaneously on the same chip by labeling the control and experimental groups, respectively, and then optically scanning the images to obtain the intensity of fluorescence and analyzing the results. Gene expression is determined according to the ratio of the two fluorescence intensities (Somasundaram, K., et al., Genomics Proteomics I: 1-10, 2002).

Therefore, by using the DNA microarray chip technology, by analyzing the frequency of expression of genes in specific cells and specific tissues, it is possible to search for a substance capable of diagnosing the onset of a disease and diagnose the same.

The present invention is to provide a biomarker that can effectively diagnose acute myocardial infarction.

The inventors of the present invention complained of acute heart myocardial infarction (AMI) in coronary angiogram and collected blood within 2 hours after chest pain and vasodilation. The aim of this study was to find markers showing rapid expression changes in the early stages of acute myocardial infarction.

As a biomarker capable of screening gene expression of acute myocardial infarction patients and efficiently diagnosing acute myocardial infarction, 149 marker genes shown in Table 3 were obtained.

Acute myocardial infarction can be efficiently diagnosed by measuring mRNA or protein levels of one or more genes selected from the group consisting of marker genes listed in Table 3.

Hereinafter, the present invention will be described in detail.

The present invention provides 149 biomarkers that can efficiently diagnose the acute myocardial infarction described in Table 3. The biomarker is used to diagnose the onset of a disease from a biological sample of a subject suspected of acute myocardial infarction.

As used herein, the term “diagnostic marker”, “biomarker” or “marker” refers to a substance capable of diagnosing acute myocardial infarction, and a polypeptide or nucleic acid showing an increase or decrease by acute myocardial infarction compared to normal cells. Organic biomolecules such as (eg, mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, and the like). The diagnostic markers provided in the present invention are clearly shown in Table 3 with 149 genes identified as exhibiting rapid expression changes in the early stages of acute myocardial infarction. The present invention has identified a novel function as a diagnostic marker for acute myocardial infarction of the genes listed in Table 3. The markers may themselves indicate SNPs, latent splicing and other genetic defects.

The 149 markers provided by the present invention are genes with increased expression due to direct or indirect factors with the development of acute myocardial infarction, and the expression patterns of the genes in the biological samples of the subjects to be diagnosed are expressed at the mRNA or protein level. By measuring, the onset of acute myocardial infarction can be significantly confirmed.

In the present invention, the "biological sample" includes, but is not limited to, samples such as tissues, cells, whole blood, blood, plasma or urine, which can detect differences in the expression of marker genes by the onset of acute myocardial infarction. Preferably blood.

The present invention does not specifically limit the number and combination of markers selected from Table 3 for the diagnosis of acute myocardial infarction. That is, according to the purpose, a single gene may be used as a marker, or 2 to 149 plural genes may be selected in various combinations. That is, the present invention provides a wide range of markers that enable a variety of selection, and those skilled in the art select the type and number of genes in various combinations suitable for making medical judgment for various purposes, such as diagnosis of acute myocardial infarction, risk assessment Can be used.

Identification or detection of biomarkers at the level of mRNA or protein of the invention can include any method in the art. The biological sample to be tested can be compared with the corresponding biological sample from a healthy person. That is, "normal" expression level refers to the level of marker expression in individuals without acute myocardial infarction. Such samples may be present in standardized form.

The present invention relates to an acute myocardial infarction diagnostic kit comprising an agent for measuring the mRNA level of one or more acute myocardial infarction diagnostic biomarker selected from the group consisting of the genes listed in Table 3.

Agents for measuring mRNA levels of genes are preferably primer pairs or probes, and those skilled in the art can design primers or probes that specifically amplify specific regions of the gene based on the known sequence of the treaded marker.

An "primer", an example of an agent for measuring mRNA levels, is a nucleic acid sequence with a short free 3 'hydroxyl group that can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. It consists of primer pairs in the forward and reverse directions, and usually has a length of 7 to 50, more preferably 10 to 30 nucleic acids. Primers can initiate DNA synthesis in the presence of enzymes, reagents and four different nucleoside triphosphates for polymerization in the appropriate buffer and temperature. The primer may have additional features that do not change the basic properties of the primer that serve as the starting point for DNA synthesis.

In addition, the primer nucleic acid sequences of the present invention may, if necessary, include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg 33P), fluorescent molecules, chemical groups (eg biotin), and the like. have.

A person skilled in the art can determine a combination of primer pairs having high specificity and sensitivity without causing nonspecific amplification by considering variables such as the degree of hybridization between sequences in a target gene based on the gene sequence of known markers. As used herein, the term "nonspecific amplification" refers to nucleic acid amplification other than the target sequence that occurs by hybridizing a primer to a sequence other than the target sequence and acting as a substrate for primer extension.

Another example of an agent for measuring mRNA levels, “probe” refers to a nucleic acid fragment, such as RNA or DNA, that is short to several bases to hundreds of bases capable of specific binding to mRNA and is labeled and thus The presence of mRNA can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.

Primers or probes of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Moreover, it can transform into methylation, capping, etc. by a well-known method.

Analytical methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting, DNA microarray chips, and the like. The kit for measuring mRNA level of acute myocardial infarction biomarker is specifically a diagnostic kit for the RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting or DNA microarray chip, suitable for the analysis method One or more other component compositions, solutions or devices.

For example, kits for RT-PCR can contain test tubes or other appropriate containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymers, in addition to individual primer pairs specific for the marker gene. Enzymes such as enzymes and reverse transcriptases, DNAse, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as the quantitative control.

Also, for example, a kit for a DNA microarray chip may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The DNA microarray chip of the present invention can be produced by methods known to those skilled in the art. The method of manufacturing the microarray chip is as follows. In order to immobilize the searched biomarker as a probe DNA molecule on a substrate of a DNA microarray chip, a micropipetting method or a pin-shaped spotter using a piezoelectric method ), Etc. are used. The DNA microarray chip substrate may be coated with active groups such as amino-silane, poly-L-lysine, and aldehyde. Further, the substrate may be a slide glass, plastic, metal, silicon, nylon film, or nitrocellulose film. The kit may also consist of buffers used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTPs (premixed or separate feed), marker reagents such as chemical inducers of fluorescent dyes, wash buffers, and the like. Can be.

The present invention also relates to an acute myocardial infarction diagnostic kit comprising an agent for measuring the protein level of one or more acute myocardial infarction diagnostic biomarkers selected from the group consisting of the genes listed in Table 3.

Agents for measuring protein levels of genes are preferably antibodies, which can be readily prepared using techniques well known in the art.

In the present invention, an antibody includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, the antibody of the present invention is not only a complete form having two full-length light chains and two full-length heavy chains, but also antigen binding of antibody molecules such as Fab, F (ab '), F (ab') 2 and Fv. It may be a functional fragment that holds a function.

Methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein Chips and the like, but is not limited thereto. Kits for measuring the protein level of acute myocardial infarction biomarker specifically the ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, a kit for protein chips.

For example, a kit for ELISA may comprise reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with antibodies) and substrates thereof. have. It may also include antibodies specific for quantitative control proteins. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid support, and attaches to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibodies and antigens, a label that recognizes the antibody after reacting with another antibody that recognizes the antigen in a complex of the antibody and the antigen attached to a solid support Kits for a variety of ELISAs, including indirect sandwich ELISA using secondary antibodies.

Also, for example, the present invention relates to diagnostic markers for protein chips in which corresponding antibodies to one or more markers are arranged at fixed locations on a substrate and immobilized at high density. The protein chip kit of the present invention may be composed of a buffer solution, a marker reagent, a wash buffer solution, and the like used for hybridization.

Kits for diagnosing acute myocardial infarction provided herein may require certain additional equipment, such as a PCR cycler, microarray reader, or FACS device.

In addition, the present invention comprises the steps of measuring the mRNA or protein of one or more acute myocardial infarction diagnostic biomarkers selected from the group consisting of the genes listed in Table 3; And comparing the increase in mRNA or protein levels with the mRNA or protein levels in a normal control sample.

Isolation of mRNA or protein from a biological sample can be carried out using known processes and mRNA or protein levels can be measured by the various methods discussed above. mRNA or protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.

The gene expression level in the normal control group and the gene expression level in patients with suspected acute myocardial infarction can be compared at the mRNA or protein level, and the actual expression level of the suspected acute myocardial infarction was judged by the significant increase in the expression level in the acute myocardial infarction marker gene. Acute myocardial infarction can be diagnosed.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited by the following examples.

Example 1 Total RNA Extraction from Whole Blood Obtained from MI Patients

1-1: Sample Preparation and RNA Extraction

Among the patients who visited the Catholic University of Gangnam St. Mary's Hospital during the period from April 2006 to December 2006, 10 patients (experimental group) and 10 normal patients confirmed with acute myocardial infarction, recovered after 1 week of cardiovascular surgery. 5 ml of blood samples were obtained from 10 patients each.

1-2: Preparation of Oligonucleotide DNA Microarrays

At the Catholic University Microdissection Genomics Research Center (MGRC), Compugen / Sigma Human Oligolibrary (60-mers) representing 18,664 LEADS clusters (Compugen / Sigma-Genosys, Woodland, TX) was spotted on poly-L-lysine coated glass microscope slides It was.

1-3: RNA isolation

The blood sample obtained in Example 1-1 was dissolved using a lysis buffer, whole RNA was isolated using the Trizol reagent (Invitrogen) according to the manufacturer's method, or purified using Paxgene kit (BD science). It was.

The quantity and quality of each total RNA sample was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) (FIG. 2).

Example 2: Microarray Experiment

Microarray experiments were performed using the isolated and purified RNA. In this case, the microarray analysis used is a conventional microarray chip analysis method. When the mRNA extracted from whole blood samples of two different groups (control group and experimental group) is used as a template, reverse transcriptase by reverse transcriptase results in different fluorescent substances. CDNA was synthesized by adding Cy3 (red) and Cy5 (green), followed by mixing the two groups of cDNA in the same amount to bind to the microarray chip. In contrast, the human reference RNA (Stratagene) sold as a control, containing about 1900 human genetic information It is also possible to use a method of binding to an oligo microarray chip.

2-1: Sample Preparation and RNA Extraction

CDNA was prepared from the total RNA (20 μg) obtained in Example 1 for oligo microarray analysis. Labeling used a generally used direct labeling method. Specifically, 20 μg of the total RNA obtained and 2 μg (1 μg / μl) of oligo (dT) primer were mixed and primed by reacting at 65 ° C. for 10 minutes, and then a reagent was prepared as follows for Reverse Transcript reaction. Mixed. (Please tell us the ingredients of the reagent.)

Configuration Volume (μl) 0.1 M DTT 4 μl 20X dNTP (10 mM dATP, dCTP, dGTP and 4 mM dTTP) 2 μl SuperScript II (200U.ul) 3 μl Cy-3 or Cy-5 dUTP (1mM) 4 μl

The obtained RNA was converted to cDNA by reverse transcription and mixed with cDNA labeled by reverse transcription from human reference control RNA and applied to the microarray. All samples were subjected to hybridization on the microarray chip with genes labeled from human reference RNA to quantify the level of expression, and to compare the normal, myocardial infarction, and recovered patient groups. An analysis method for deriving a large group of genes.

2-2: hybridization reaction

Hybridization and washing were performed using the Catholic Medical College's MAUI system. Specifically, hybridization including hybridization buffer DIG (2x hybridization buffer), Herring Sperm DNA (2 μg / μl), and sample and distilled water was performed in a 42 ° C. water bath for at least 18 hours. Then wash twice for 5 minutes in 2 x SSC / 0.1% SDS, then again for 5 minutes in 1x SSC / 0.1% SDS, 5 minutes in 0.2 x SSC, 5 minutes in 0.05 x SSC, and 5 minutes in distilled water. The slide was then dried by centrifugation at 1000 rpm for 5 minutes.

2-3: Fluorescence Image Acquisition

Scanning with a Genepix 4000B (Axon Instruments, CA) to obtain hybridized images of slides obtained from Example 2-2. Specifically, the chip washed with the non-binding gene through the hybridization reaction is read by a laser fluorescence scanner (laser fluorescence scanner), where green is expressed in the control group, red is specifically expressed in the experimental group (acute myocardial infarction) It will show the activity of the gene, and yellow is the complementary color of green and red means that there is no significant difference between the two groups. As such, in order to obtain the expression ratio of the gene from the scanned images, it was analyzed using GenePix 4.1 software (Axon Instruments, CA).

First, unsupervised mean clustering analysis was performed to perform their entire gene expression profiling to show a total of 9031 gene expression profiles in FIG. 3.

Next, large-scale characteristic MI-related molecular markers were isolated by T-test and folds-change analysis, out of the 9031 genes present on the oligo chip, among the 4903 genes with a p value of 0.05 or less. Only those genes whose expression of Cy5 / Cy3 were overexpressed 1.5 fold were classified. As a result, it was confirmed that 1056 genes increased expression.

Representative signal pathways reproduced from the 1056 MI-specific genes are shown in Table 1 below, except that they are classified only when the number of genes is 10 or more.

In addition, by identifying the characteristic MI-related genetic elements by T-test and fold-change analysis, among 4903 genes having a p value of 0.05 or less, the genes overexpressing Cy5 / Cy3 more than 2 times were classified. , 149 genes were confirmed to increase the expression (Fig. 5).

The biological processes reproduced from the MI-related genes with increased expression are summarized in Table 2 below, except p ≧ 0.1.

Information on 149 acute myocardial infarction-specific genes obtained by RNA extraction from the blood of acute myocardial infarction patients, normal subjects and recovered acute myocardial infarction patients using the whole genome DNA microarray technique is as follows. same.

Gene registration number gene Abbreviation SYNONYMS NM_001032220.1 Homo sapiens solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1 (SLC11A1), transcript variant 2, mRNA. SLC11A1 LSH; NRAMP; NRAMP1 NM_003855.2 Homo sapiens interleukin 18 receptor 1 (IL18R1), mRNA. IL18R1 IL1RRP; CDw218a; IL-1Rrp NM_033423.2 Homo sapiens granzyme H (cathepsin G-like 2, protein h-CCPX) (GZMH), mRNA. GZMH CCP-X; CGL-2; CSP-C; CTLA1; CTSGL2 NM_003245.2 Homo sapiens transglutaminase 3 (E polypeptide, protein-glutamine-gamma-glutamyltransferase) (TGM3), mRNA. TGM3 TGE; MGC126249; MGC126250 NM_000342.1 Homo sapiens solute carrier family 4, anion exchanger, member 1 (erythrocyte membrane protein band 3, Diego blood group) (SLC4A1), mRNA. SLC4A1 DI; FR; SW; WD; WR; AE1; WD1; BND3; EPB3; CD233; EMPB3; RTA1A; MGC116750; MGC116753; MGC126619; MGC126623 NM_003255.4 Homo sapiens TIMP metallopeptidase inhibitor 2 (TIMP2), mRNA. TIMP2 CSC-21K NM_022718.2 Homo sapiens matrix metallopeptidase 25 (MMP25), transcript variant 2, mRNA. MMP25 MMP20; MT-MMP6; MT6-MMP NM_018643.2 Homo sapiens triggering receptor expressed on myeloid cells 1 (TREM1), mRNA. TREM1 TREM-1 NM_016006.3 Homo sapiens abhydrolase domain containing 5 (ABHD5), mRNA. ABHD5 CDS; CGI58; IECN2; NCIE2; MGC8731 NM_002104.2 Homo sapiens granzyme K (granzyme 3; tryptase II) (GZMK), mRNA. GZMK TRYP2 BC070337 Homo sapiens T cell receptor alpha locus, mRNA (cDNA clone MGC: 88342 IMAGE: 30352166), complete cds CR596519 full-length cDNA clone CS0DI056YK21 of Placenta Cot 25-normalized of Homo sapiens (human) NM_032412.2 Homo sapiens putative nuclear protein ORF1-FL49 (ORF1-FL49), mRNA. ORF1-FL49 NM_007115.2 Homo sapiens tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), mRNA. TNFAIP6 TSG6 NM_006006.4 Homo sapiens zinc finger and BTB domain containing 16 (ZBTB16), transcript variant 1, mRNA. ZBTB16 PLZF; ZNF145 BX098605 BX098605 Soares fetal liver spleen 1NFLS Homo sapiens cDNA clone IMAGp998E21524, mRNA sequence NM_006212.2 Homo sapiens 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2 (PFKFB2), transcript variant 1, mRNA. PFKFB2 DKFZp781D2217; PFK-2 / FBPase-2 NM_001995.2 Homo sapiens acyl-CoA synthetase long-chain family member 1 (ACSL1), mRNA. ACSL1 ACS1; LACS; FACL1; FACL2; LACS1; LACS2 NM_005091.1 Homo sapiens peptidoglycan recognition protein 1 (PGLYRP1), mRNA. PGLYRP1 PGRP; TAG7; PGRPS; PGLYRP; PGRP-S; TNFSF3L; MGC126894; MGC126896 NM_022746.2 Homo sapiens MOCO sulphurase C-terminal domain containing 1 (MOSC1), mRNA. MOSC1 FLJ22390; RP11-295M18.1 NM_005810.3 Homo sapiens killer cell lectin-like receptor subfamily G, member 1 (KLRG1), mRNA. KLRG1 2F1; MAFA; MAFA-L; MAFA-2F1; MGC13600 NM_004458.1 Homo sapiens acyl-CoA synthetase long-chain family member 4 (ACSL4), transcript variant 1, mRNA. ACSL4 ACS4; FACL4; LACS4; MRX63 NM_012483.1 Homo sapiens granulysin (GNLY), transcript variant 519, mRNA. GNLY 519; LAG2; NKG5; LAG-2; D2S69E; TLA519; lymphokine NM_015444.1 Homo sapiens Ras-induced senescence 1 (RIS1), mRNA. RIS1 p40BBP; DKFZp586E1621 NM_014358.1 Homo sapiens C-type lectin domain family 4, member E (CLEC4E), mRNA. CLEC4E MINCLE; CLECSF9 NM_001785.1 Homo sapiens cytidine deaminase (CDA), mRNA. CDA CDD; hscd NM_000419.2 Homo sapiens integrin, alpha 2b (platelet glycoprotein IIb of IIb / IIIa complex, antigen CD41B) (ITGA2B), mRNA. ITGA2B GTA; CD41; GP2B; HPA3; CD41B; GPIIb XM_930820.1 PREDICTED: Homo sapiens leucine-rich repeat kinase 2, transcript variant 2 (LRRK2), mRNA. LRRK2 XM_942570.1 PREDICTED: Homo sapiens RALBP1 associated Eps domain containing 2 (REPS2), mRNA. REPS2 NM_006609.2 Homo sapiens mitogen-activated protein kinase kinase kinase 2 (MAP3K2), mRNA. MAP3K2 MEKK2; MEKK2B NM_138557.1 Homo sapiens toll-like receptor 4 (TLR4), transcript variant 4, mRNA. TLR4 TOLL; hToll NM_004666.1 Homo sapiens vanin 1 (VNN1), mRNA. VNN1 Tiff66; MGC116930; MGC116931; MGC116932; MGC116933 NM_018050.2 Homo sapiens MANSC domain containing 1 (MANSC1), mRNA. MANSC1 FLJ10298; LOH12CR3; 9130403P13Rik NM_003355.2 Homo sapiens uncoupling protein 2 (mitochondrial, proton carrier) (UCP2), nuclear gene encoding mitochondrial protein, mRNA. UCP2 UCPH; SLC25A8 NM_003749.2 Homo sapiens insulin receptor substrate 2 (IRS2), mRNA. IRS2 NM_005384.2 Homo sapiens nuclear factor, interleukin 3 regulated (NFIL3), mRNA. NFIL3 E4BP4; IL3BP1; NFIL3A; NF-IL3A NM_001024460.1 Homo sapiens vanin 3 (VNN3), transcript variant 3, mRNA. VNN3 HSA238982 NM_006864.1 Homo sapiens leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 3 (LILRB3), mRNA. LILRB3 HL9; ILT5; LIR3; CD85A; LIR-3 NM_020980.2 Homo sapiens aquaporin 9 (AQP9), mRNA. AQP9 SSC1; HsT17287 NM_006456.1 Homo sapiens ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3) -N-acetylgalactosaminide alpha-2,6-sialyltransferase 2 (ST6GALNAC2), mRNA. ST6GALNAC2 STHM; SIAT7; SIAT7B; SIATL1; ST6GalNAII NM_005620.1 Homo sapiens S100 calcium binding protein A11 (calgizzarin) (S100A11), mRNA. S100A11 MLN70; S100C NM_006931.1 Homo sapiens solute carrier family 2 (facilitated glucose transporter), member 3 (SLC2A3), mRNA. SLC2A3 GLUT3; FLJ90380 NM_005601.3 Homo sapiens natural killer cell group 7 sequence (NKG7), mRNA. NKG7 GIG1 NM_003853.2 Homo sapiens interleukin 18 receptor accessory protein (IL18RAP), mRNA. IL18RAP ACPL; CDw218b; MGC120589; MGC120590 NM_007219.2 Homo sapiens ring finger protein 24 (RNF24), mRNA. RNF24 G1L NM_004350.1 Homo sapiens runt-related transcription factor 3 (RUNX3), transcript variant 2, mRNA. RUNX3 AML2; CBFA3; PEBP2aC NR_002205.1 Homo sapiens ferritin, heavy polypeptide-like 12 (FTHL12) on chromosome 9. FTHL12 NM_004117.2 Homo sapiens FK506 binding protein 5 (FKBP5), mRNA. FKBP5 P54; FKBP51; FKBP54; PPIase; Ptg-10; MGC111006 NM_004665.2 Homo sapiens vanin 2 (VNN2), transcript variant 1, mRNA. VNN2 FOAP-4; GPI-80 NM_031950.2 Homo sapiens Ksp37 protein (KSP37), mRNA. KSP37 NM_002243.3 Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15), transcript variant 2, mRNA. KCNJ15 KIR1.3; KIR4.2; MGC13584 NM_021935.2 Homo sapiens prokineticin 2 (PROK2), mRNA. PROK2 BV8; PK2; MIT1 NM_001462.3 Homo sapiens formyl peptide receptor-like 1 (FPRL1), transcript variant 1, mRNA. FPRL1 ALXR; HM63; FMLPX; FPR2A; FPRH1; FPRH2; LXA4R; FMLP-R-II NM_022733.1 Homo sapiens stromal membrane-associated protein 1-like (SMAP1L), mRNA. SMAP1L RP1-228H13.3 NM_000607.1 Homo sapiens orosomucoid 1 (ORM1), mRNA. ORM1 ORM; AGP1; AGP-A NM_002304.1 Homo sapiens lunatic fringe homolog (Drosophila) (LFNG), mRNA. LFNG SCDO3 NM_014918.3 Homo sapiens carbohydrate (chondroitin) synthase 1 (CHSY1), mRNA. CHSY1 KIAA0990 NM_020698.1 Homo sapiens transmembrane and coiled-coil domain family 3 (TMCC3), mRNA. TMCC3 KIAA1145 NM_001013725.1 Homo sapiens hypothetical gene supported by BC044942 (LOC441268), mRNA. LOC441268 NM_000574.2 Homo sapiens CD55 antigen, decay accelerating factor for complement (Cromer blood group) (CD55), mRNA. CD55 CR; TC; DAF NM_170776.3 Homo sapiens G protein-coupled receptor 97 (GPR97), mRNA. GPR97 Pb99; PGR26; GPR-97 NM_170776.3 Homo sapiens G protein-coupled receptor 97 (GPR97), mRNA. GPR97 Pb99; PGR26; GPR-97 NM_002346.1 Homo sapiens lymphocyte antigen 6 complex, locus E (LY6E), mRNA. LY6E RIGE; SCA2; RIG-E; SCA-2; TSA-1 NM_004177.3 Homo sapiens syntaxin 3A (STX3A), mRNA. STX3A NM_003137.3 Homo sapiens SFRS protein kinase 1 (SRPK1), mRNA. SRPK1 SFRSK1 NM_182898.2 Homo sapiens cAMP responsive element binding protein 5 (CREB5), transcript variant 1, mRNA. CREB5 CRE-BPA NM_000045.2 Homo sapiens arginase, liver (ARG1), mRNA. ARG1 NM_174918.1 Homo sapiens mast cell-expressed membrane protein 1 (MCEMP1), mRNA. MCEMP1 NM_001011666.1 Homo sapiens cAMP responsive element binding protein 5 (CREB5), transcript variant 4, mRNA. CREB5 CRE-BPA NM_000878.2 Homo sapiens interleukin 2 receptor, beta (IL2RB), mRNA. IL2RB CD122; P70-75 NM_005442.2 Homo sapiens eomesodermin homolog (Xenopus laevis) (EOMES), mRNA. EOMES TBR2 NM_000896.1 Homo sapiens cytochrome P450, family 4, subfamily F, polypeptide 3 (CYP4F3), mRNA. CYP4F3 CPF3; CYP4F; LTB4H NM_020820.2 Homo sapiens phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1 (PREX1), mRNA. PREX1 KIAA1415 NM_014320.2 Homo sapiens heme binding protein 2 (HEBP2), mRNA. HEBP2 PP23; SOUL; C6orf34; C6ORF34B; KIAA1244; RP3-422G23.1 NM_005746.1 Homo sapiens pre-B-cell colony enhancing factor 1 (PBEF1), transcript variant 1, mRNA. PBEF1 PBEF; MGC117256 NM_006144.2 Homo sapiens granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) (GZMA), mRNA. GZMA HFSP; CTLA3 NM_145032.2 Homo sapiens F-box and leucine-rich repeat protein 13 (FBXL13), mRNA. FBXL13 Fbl13; FLJ38068; MGC21636 NM_000578.2 Homo sapiens solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1 (SLC11A1), transcript variant 1, mRNA. SLC11A1 LSH; NRAMP; NRAMP1 NM_020531.2 Homo sapiens chromosome 20 open reading frame 3 (C20orf3), mRNA. C20orf3 BSCv; APMAP; C20ORF2 NM_006725.2 Homo sapiens CD6 antigen (CD6), mRNA. CD6 TP120 NM_002863.3 Homo sapiens phosphorylase, glycogen; liver (Hers disease, glycogen storage disease type VI) (PYGL), mRNA. PYGL NM_001550.2 Homo sapiens interferon-related developmental regulator 1 (IFRD1), transcript variant 1, mRNA. IFRD1 PC4; TIS7 NM_052972.2 Homo sapiens leucine-rich alpha-2-glycoprotein 1 (LRG1), mRNA. LRG1 LRG; HMFT1766 NM_002029.3 Homo sapiens formyl peptide receptor 1 (FPR1), mRNA. FPR1 FPR; FMLP NM_005980.2 Homo sapiens S100 calcium binding protein P (S100P), mRNA. S100P MIG9 NM_001768.4 Homo sapiens CD8 antigen, alpha polypeptide (p32) (CD8A), transcript variant 1, mRNA. CD8A CD8; MAL; p32; Leu2 NM_000804.2 Homo sapiens folate receptor 3 (gamma) (FOLR3), mRNA. FOLR3 FR-G; FR-gamma; gamma-hFR NM_201525.1 Homo sapiens G protein-coupled receptor 56 (GPR56), transcript variant 3, mRNA. GPR56 BFPP; TM7LN4; TM7XN1 NM_001738.1 Homo sapiens carbonic anhydrase I (CA1), mRNA. CA1 NM_004226.2 Homo sapiens serine / threonine kinase 17b (apoptosis-inducing) (STK17B), mRNA. STK17B DRAK2 NM_007246.2 Homo sapiens kelch-like 2, Mayven (Drosophila) (KLHL2), mRNA. KLHL2 MAYVEN; ABP-KELCH NM_003494.2 Homo sapiens dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive) (DYSF), mRNA. DYSF FER1L1; LGMD2B; FLJ00175; dysferlin NM_004567.2 Homo sapiens 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mRNA. PFKFB4 NM_015292.1 Homo sapiens family with sequence similarity 62 (C2 domain containing), member A (FAM62A), mRNA. FAM62A MBC2; KIAA0747 NM_015171.1 Homo sapiens exportin 6 (XPO6), mRNA. XPO6 EXP6; RANBP20; FLJ22519; KIAA0370 NM_007199.1 Homo sapiens interleukin-1 receptor-associated kinase 3 (IRAK3), mRNA. IRAK3 IRAK-M NM_002923.1 Homo sapiens regulator of G-protein signaling 2, 24kDa (RGS2), mRNA. RGS2 G0S8 NM_012387.1 Homo sapiens peptidyl arginine deiminase, type IV (PADI4), mRNA. PADI4 PAD; PDI4; PDI5; PADI5 NM_173822.1 Homo sapiens hypothetical protein MGC39518 (MGC39518), mRNA. MGC39518 NM_015266.1 Homo sapiens solute carrier family 9 (sodium / hydrogen exchanger), member 8 (SLC9A8), mRNA. SLC9A8 NHE8; KIAA0939; DKFZp686C03237 NM_024829.4 Homo sapiens hypothetical protein FLJ22662 (FLJ22662), mRNA. FLJ22662 NM_004994.2 Homo sapiens matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) (MMP9), mRNA. MMP9 GELB; CLG4B NM_005041.3 Homo sapiens perforin 1 (pore forming protein) (PRF1), mRNA. PRF1 P1; PFP; HPLH2; MGC65093 NM_006904.6 Homo sapiens protein kinase, DNA-activated, catalytic polypeptide (PRKDC), mRNA. PRKDC HYRC; p350; DNAPK; DNPK1; HYRC1; XRCC7 NM_001620.1 Homo sapiens AHNAK nucleoprotein (desmoyokin) (AHNAK), transcript variant 1, mRNA. AHNAK AHNAKRS; MGC5395 NM_006317.3 Homo sapiens brain abundant, membrane attached signal protein 1 (BASP1), mRNA. BASP1 CAP23; NAP22; CAP-23; NAP-22; MGC8555 NM_016337.2 Homo sapiens Enah / Vasp-like (EVL), mRNA. EVL RNB6 NM_032536.1 Homo sapiens netrin G2 (NTNG2), mRNA. NTNG2 Lmnt2; NTNG1; KIAA0625; KIAA1857; MGC21884; bA479K20.1 NM_004356.3 Homo sapiens CD81 antigen (target of antiproliferative antibody 1) (CD81), mRNA. CD81 S5.7; TAPA1; TSPAN28 NM_000876.1 Homo sapiens insulin-like growth factor 2 receptor (IGF2R), mRNA. IGF2R MPRI; CD222; CIMPR; M6P-R NM_080387.4 Homo sapiens C-type lectin domain family 4, member D (CLEC4D), mRNA. CLEC4D MCL; Mpcl; CLEC-6; CLECSF8; MGC40078 NM_024693.2 Homo sapiens enoyl Coenzyme A hydratase domain containing 3 (ECHDC3), mRNA. ECHDC3 FLJ20909 XM_937928.1 PREDICTED: Homo sapiens similar to H3 histone, family 3B (LOC347376), mRNA. LOC347376 NM_001629.2 Homo sapiens arachidonate 5-lipoxygenase-activating protein (ALOX5AP), mRNA. ALOX5AP FLAP NM_002129.2 Homo sapiens high-mobility group box 2 (HMGB2), mRNA. HMGB2 HMG2 XM_930111.1 PREDICTED: Homo sapiens similar to H3 histone, family 3B (LOC644914), mRNA. LOC644914 NM_080491.1 Homo sapiens GRB2-associated binding protein 2 (GAB2), transcript variant 1, mRNA. GAB2 KIAA0571 NM_006800.2 Homo sapiens male-specific lethal 3-like 1 (Drosophila) (MSL3L1), transcript variant 3, mRNA. MSL3L1 DKFZP586J1822 NM_000478.2 Homo sapiens alkaline phosphatase, liver / bone / kidney (ALPL), mRNA. ALPL HOPS; TNAP; TNSALP; AP-TNAP XM_933030.1 PREDICTED: Homo sapiens similar to hypothetical protein LOC284701, transcript variant 1 (LOC643313), mRNA. LOC643313 NM_052839.2 Homo sapiens pannexin 2 (PANX2), mRNA. PANX2 hPANX2; MGC119432 NM_014982.1 Homo sapiens pecanex homolog (Drosophila) (PCNX), mRNA. PCNX PCNXL1; KIAA0805; KIAA0995 NM_006748.1 Homo sapiens Src-like-adaptor (SLA), mRNA. SLA SLA1; SLAP NM_001706.2 Homo sapiens B-cell CLL / lymphoma 6 (zinc finger protein 51) (BCL6), transcript variant 1, mRNA. BCL6 BCL5; LAZ3; BCL6A; ZNF51; ZBTB27 NM_001304.3 Homo sapiens carboxypeptidase D (CPD), mRNA. CPD NM_004776.2 Homo sapiens UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5 (B4GALT5), mRNA. B4GALT5 gt-V; B4Gal-T5; beta4Gal-T5; beta4GalT-V; BETA4-GALT-IV NM_173343.1 Homo sapiens interleukin 1 receptor, type II (IL1R2), transcript variant 2, mRNA. IL1R2 IL1RB; CD121b; MGC47725 NM_012413.3 Homo sapiens glutaminyl-peptide cyclotransferase (glutaminyl cyclase) (QPCT), mRNA. QPCT QC; GCT NM_005195.2 Homo sapiens CCAAT / enhancer binding protein (C / EBP), delta (CEBPD), mRNA. CEBPD CELF; CRP3; C / EBP-delta; NF-IL6-beta NM_000717.2 Homo sapiens carbonic anhydrase IV (CA4), mRNA. CA4 CAIV; RP17 NM_005345.4 Homo sapiens heat shock 70kDa protein 1A (HSPA1A), mRNA. HSPA1A HSP72; HSPA1; HSPA1B; HSP70-1 NM_005153.1 Homo sapiens ubiquitin specific peptidase 10 (USP10), mRNA. USP10 UBPO; KIAA0190 NM_001031711.1 Homo sapiens endoplasmic reticulum-golgi intermediate compartment (ERGIC) 1 (ERGIC1), transcript variant 1, mRNA. ERGIC1 ERGIC32; ERGIC-32; KIAA1181; MGC14345 NM_007288.1 Homo sapiens membrane metallo-endopeptidase (neutral endopeptidase, enkephalinase, CALLA, CD10) (MME), transcript variant 2a, mRNA. MME NEP; CD10; CALLA; MGC126681; MGC126707 NM_181717.1 Homo sapiens HLA complex group 27 (HCG27), mRNA. HCG27 FLJ40123; bCX101P6.9; bPG299F13.9; bQB115I13.2 NM_001828.4 Homo sapiens Charcot-Leyden crystal protein (CLC), mRNA. CLC LGALS10; LPPL_HUMAN NM_024298.2 Homo sapiens leukocyte receptor cluster (LRC) member 4 (LENG4), mRNA. LENG4 BB1 NM_198040.1 Homo sapiens polyhomeotic-like 2 (Drosophila) (PHC2), transcript variant 1, mRNA. PHC2 PH2; EDR2; HPH2 NM_001776.3 Homo sapiens ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), mRNA. ENTPD1 CD39; ATPDase; NTPDase-1 NM_013416.2 Homo sapiens neutrophil cytosolic factor 4, 40kDa (NCF4), transcript variant 2, mRNA. NCF4 NCF; MGC3810; P40PHOX; SH3PXD4 NM_031476.1 Homo sapiens cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2), mRNA. CRISPLD2 CRISP11; LCRISP2; MGC74865; DKFZP434B044 NM_000022.2 Homo sapiens adenosine deaminase (ADA), mRNA. ADA AK092074 Homo sapiens cDNA FLJ34755 fis, clone NHNPC1000034 NM_144673.2 Homo sapiens CKLF-like MARVEL transmembrane domain containing 2 (CMTM2), mRNA. CMTM2 CKLFSF2; MGC39436 NM_002357.2 Homo sapiens MAX dimerization protein 1 (MXD1), mRNA. MXD1 MAD; MAD1; MGC104659 AL049435 Homo sapiens mRNA; cDNA DKFZp586B0220 (from clone DKFZp586B0220) NM_182757.2 Homo sapiens IBR domain containing 2 (IBRDC2), mRNA. IBRDC2 p53RFP; KIAA0161; MGC71786; bA528A10.3 NM_176894.1 Homo sapiens purinergic receptor P2Y, G-protein coupled, 13 (P2RY13), transcript variant 2, mRNA. P2RY13 GPCR1; GPR86; GPR94; P2Y13; SP® FKSG77

Since acute myocardial infarction can be easily and efficiently diagnosed using the acute myocardial infarction marker disclosed in the present invention, it is expected to be widely used in various medical fields.

FIG. 1 illustrates an experimental procedure in which total RNA is obtained using tririzol from blood obtained from peripheral blood of a patient and then applied to DNA microarrays.

Figure 2 shows the quality and quantity of the total RNA extracted into the blood of the patient, the purity of the RNA extracted from the patient is applied to the DNA microarray by identifying ribosmal RNA bands (18S & 28S) through the BioAnalyzer It shows that high quality RNA can be obtained.

3 is a comparison of 9031 gene expression profiles obtained by extracting RNA from patient blood, performing DNA microarray experiments, and performing data correction. In the figure, two groups were shown on the dendrogram in the hierarchical clustering result, which are the myocardial infarction group (MI), normal (N) and recovery (FU) group, respectively. It can be seen that the gene expression in blood of myocardial infarction patients is different from the normal one. Especially, since the recovery group (FU) recovered from the same patient exists as the normal group, many gene changes related to myocardial infarction disease can be seen. From this, a gene group capable of discriminating myocardial infarction was secured.

Figure 4 shows that the expression of the gene group due to myocardial infarction is a specific expression compared to the normal or recovery group is expressed differently by the PCA analysis method.

FIG. 5 can accurately classify and identify myocardial infarction patients on the basis of the preceding results of gene expression specific to myocardial infarction through comparative analysis of gene expression from blood of myocardial infarction patients and blood of normal or recovered patients. In order to secure genetic markers, Volcanoa plot and T-test were combined to derive at least 149 genes that showed at least two-fold changes in gene expression compared to normal subjects. Shows that this is done through hierarchical clustering.

Claims (6)

Biomarker for diagnosing acute myocardial infarction consisting of the genes listed in Table 3. Acute myocardial infarction diagnostic kit comprising a primer pair or a probe for measuring the mRNA level of the biomarker for diagnosis of acute myocardial infarction consisting of the genes described in Table 3. The diagnostic kit of claim 2, wherein the probe is an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. The diagnostic kit of claim 2, wherein the kit is for RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting or DNA microarray chip. Acute myocardial infarction diagnostic kit comprising an antibody, for measuring the protein level of the biomarker for diagnosis of acute myocardial infarction consisting of the genes described in Table 3. The diagnostic kit of claim 5, wherein the kit is for Western blot, ELISA, radioisotope immunoassay, immunoprecipitation assay, complement fixation assay, FACS or protein chip.

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