KR20150014039A - Composition for determining of sudden cardiac death and method for determining of sudden cardiac death - Google Patents

Abstract

The present invention relates to a method for determining sudden cardiac death by using HBA1/2, HBB, or PDK4 gene as a marker. According to the present invention, a sudden cardiac death can be easily determined in case that the anatomical determination is different in an autopsy. Moreover, provided in the present invention is a method for determining a sudden cardiac death for an autopsy, which comprises the steps of: contacting a composition for determining a sudden cardiac death to a biological sample; and comparing the level of HBA1/2, HBB and/or PDK4 gene in the biological sample with the corresponding level of HBA1/2, HBB and/or PDK4 gene of a control sample which is not in the case of the death by a sudden cardiac death.

Description

Technical Field [0001] The present invention relates to a composition for the determination of acute cardiac death and a method for determining the sudden cardiac death.

The present invention relates to a method for discriminating acute cardiac death.

Recently, the field of genetics in the forensic field, especially the law, has been developed very rapidly by the progress of accumulation and analysis techniques of molecular biological knowledge. Since it is stable for a considerable period of time after death, it has a short half-life and is rapidly degraded by RNA degradation enzymes as compared with DNA that is easy to manipulate. However, there are reports that RNA is much more stable than was feared. With the development of analytical techniques, quantitative analysis of trace RNA samples has become possible, and the development and use of post-RNA analysis technology has recently been promoted (Castensson et al. Genome Res 10: 1219, 2000; Trotter et al., Brain Res 942: 120, 2002). In particular, the development of reverse transcription-polymerase chain reaction (RT-PCR) technology has greatly improved the sensitivity and accuracy of quantitative RNA analysis from autopsy tissues, and analysis of the post-transcriptional RNA expression pattern is drawing attention.

Immediate early genes, hypoxia-inducible factor-1, vascular endothelial growth factor, glucose transporter-1, DUSP 1 Recent results have shown that post-expression patterns of many genes such as dual specificity phosphatase 1 and heat shock proteins (HSP) can reflect specific death conditions (Zhao et al., Forensic Sci Int 177: 176, 2008 Mol Cells < RTI ID = 0.0 > et al., ≪ / RTI > Int J Legal Med 126: 581, 2012; Chung et al. 34: 473, 2012).

However, sensitivity and standardization problems remain in the practical use of molecular pathologic examination techniques based on the expression pattern of post-transcriptional gene expression, and there is a lack of established forensic RNA molecule markers.

On the other hand, acute cardiac death is defined as "an anatomic cardiac condition that can not be predicted at all, regardless of the presence or absence of a cardiac event, resulting in an acute episode of unconsciousness within 1 hour, " Endogenous sudden death from heart disease is a common cause of 20% of the total number of autopsy cases of the National Institute of Scientific Investigation. Among these, acute myocardial infarction (AMI), which can confirm the infarction of the myocardium, and ischemic changes of the coronary arteries and moderate myocardial infarction (AMI), which can be confirmed by the closure of the lumen narrowed by coronary atherosclerosis, In the case of chronic coronary disease (CCD), which can be easily distinguished anatomically, there are many cases that are not. In the case of coronary atherosclerosis, both coronary arteries are severely narrowed, but only one coronary artery is severely narrowed, and there is a considerably large individual difference in relation to the duration of ischemic heart disease. In addition, it is sometimes difficult to specify the exact sign even after performing autopsy.

Although cardiac troponin T is known as a marker for discriminating acute cardiac death, troponin T is difficult to be practically used because it is possible only under limited conditions of 12 hours after death.

In addition, the association of hemoglobin A 1 or 2, hemoglobin B, and pyruvate dehydrogenase kinase 4 gene with acute cardiac discrimination is not known.

1. U.S. Patent Application Publication No. 2009-0317905

Accordingly, an object of the present invention is to provide a method for discriminating acute cardiac death using HBA1 / 2, HBB and / or PDK4 as a marker.

In order to identify markers for acute cardiac discrimination, the present inventors selected control and experimental groups, extracted RNAs from control and experimental groups, and analyzed genes by microarray analysis. In particular, as a control group, death due to trauma (traumatic death; TD), death due to chronic coronary disease (CCD), and acute myocardial infarction (AMI) The cases of death were selected. As a result, a total of 10 genes were differentially expressed in the experimental group as compared with the control group (FIG. 1). These results suggest that Sudden cardiac death (SCD), which is suspected of being a direct sign of cardiac dysfunction but difficult to determine its cause, is applied to extend the use of acute cardiac death in cases of chronic ischemic heart disease or acute myocardial infarction, The mRNA levels of the total 10 genes in the SCD group were analyzed. As a result, the level of mRNA of the hemoglobin subtypes HGB1 (hemoglobin alpha 1), HBA2 (hemoglobin alpha 2), or HBB (hemoglobin beta) gene was higher in the acute cardiac group than in the control group TD and the pyruvate dehydrogenase And that the mRNA level of the kinase subtype PDK4 (pyruvate dehydrogenase kinase 4) gene was low, thereby completing the present invention (FIG. 2).

Accordingly, the present invention provides a composition for the determination of acute cardiac injury, which comprises a probe for one or more genes selected from the group consisting of HBA 1/2 (hemoglobin alpha 1/2), HBB and PDK4.

In the present invention, acute cardiac death is an acute attack in which a patient can not expect any time or pattern of death regardless of the presence of an anatomic cardiac lesion. If the cardiac function is lost due to loss of consciousness and external causes within 1 hour Including coronary artery disease, cardiovascular system, electrocardiographic disorders, chronic ischemic heart disease, or acute myocardial infarction. That is, in the present invention, an acute cardiac death eventually includes, without limitation, the case of death from a fatal heart arrhythmia such as cardiac contraction or ventricular fibrillation. HBA1 and HBA2 are different hemoglobin subtypes, but because the base sequences are the same, HBA1 and HBA2 genes are represented by HBA1 / 2 in the present invention. As mentioned above, in the acute cardiac group, the mRNA levels of the HBA1 / 2 and HBB genes are high and the mRNA level of the PDK4 gene is low, so that acute cardiac death can be identified using these as markers.

In the present invention, a probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or several hundreds of nucleotides which can be specifically bound to mRNA. And the presence of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, and an RNA probe, which can be used to confirm the presence or absence of a specific mRNA, and a reverse transcription polymerase chain reaction reverse transcription-polymerase chain reaction, RT-PCR). The primers or probes of the present invention can be chemically synthesized using known methods.

In one embodiment of the invention, the probe may be a sense and / or antisense primer complementary to the mRNA of the HBA1 / 2, HBB or PDK4 gene. Sense or antisense primer may be included alone or in combination with a sense and antisense primer.

In the present invention, a primer is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form a base pair with a complementary template and function as a starting point for template strand copying ≪ / RTI > The primer can initiate DNA synthesis in the presence of a reagent (polymerase or reverse transcriptase) for polymerization and a different four different dNTPs (deoxynuleoside triphosphate) at the appropriate buffer solution and temperature. The primer of the present invention may be composed of a sense (forward) and antisense (backward) nucleic acid, preferably a primer having 7 to 50 nucleotide sequences, specific for each marker, HBA1 / 2, HBB or PDK4 gene. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. In addition, the primer nucleic acid sequence of the present invention may comprise a label which can be detected directly or indirectly, if necessary, by spectroscopic, photochemical, biochemical, immunochemical or chemical means.

In one embodiment of the invention, the composition for the determination of acute cardiac arrest may comprise probes for genes of HBA1 / 2, HBB and PDK4. As described later, when all three markers are included, the sensitivity and specificity of the discrimination of acute cardiac death can be improved.

The present invention also provides an acute cardiac catheterization kit or discrimination system comprising probes for HBA1 / 2, HBB and / or PDK4 genes.

In one embodiment of the invention, the acute cardiac catheterization kit includes, without limitation, a diagnostic kit based on conventional mRNA quantitation analysis. For example, an RT-PCR kit or a microarray kit. In the case of RT-PCR kits, in addition to each primer pair specific for the marker gene, an enzyme such as a test tube or other appropriate container, reaction buffer, deoxynucleotide (dNTP), Taq polymerase and reverse transcriptase, DNase, RNase Inhibitors, sterile water, and the like.

The present invention also relates to a method for determining the level of HBA1 / 2, HBB and / or PDK4 gene in a biological sample by contacting a biological sample with a composition for the determination of acute cardiac death, With a corresponding HBA1 / 2, HBB and / or PDK4 gene levels. Acute cardiac death can be determined by comparing one or more gene levels of HBA1 / 2, HBB, and PDK4 in the biological sample and the control sample. For example, comparing the HBA1 / 2 gene level of the biological sample with the HBA1 / 2 gene level of the control sample, the HBB gene level of the biological sample with the HBB gene level of the control sample, The level of PDK4 gene in the sample can be compared. We also compared the HBA1 / 2 and HBB gene levels of biological samples with the HBA1 / 2 and HBB gene levels of the control samples or the HBA1 / 2 and PDK4 gene levels of the biological samples with the HBA1 / 2 and PDK4 gene levels of the control samples Alternatively, HBB and PDK4 gene levels of the biological sample can be compared with HBB and PDK4 gene levels of the control sample. In addition, the HBA1 / 2, HBB and PDK4 gene levels of the biological sample can be compared with the HBA1 / 2, HBB and PDK4 gene levels of the control sample. In the present specification, a control sample, which is not a case of death from acute cardiac death, means a case where an unexpected death cause of acute cardiac death is evident. This includes, for example, death due to traumatic injury without intrinsic or extrinsic cardiac damage.

MRNA levels can be measured by one method for measuring HBA1 / 2, HBB and / or PDK4 gene levels. In this case, RT-PCR, competitive RT-PCR, RNase protection assay, northern blotting, DNA chip, nano-string, or RNA analysis using RNA sequence can be used as an assay method. Through the above detection methods, it is possible to confirm the amount of mRNA expression in the biological sample of the acute cardiac death candidate group and the amount of mRNA expression in the control sample, and compare the degree of expression thereof to determine whether or not the patient died of acute cardiac death.

In one embodiment of the invention, the biological sample may be myocardial tissue. To investigate the tissue specificity of HBA1 / 2, HBB or PDK4 gene expression as an acute cardiac markers, we compared the mRNA levels of HBA1 / 2, HBB or PDK4 gene from myocardial and cerebral tissues of TD group in acute cardiac arrest group and control group Quantitative RT-PCR was performed and analyzed. As a result, in the myocardial tissue, the level of mRNA of HBA1 / 2 and HBB gene was high and the level of mRNA of PDK4 gene was low in the acute cardiac group compared with the control group as in the microarray analysis, Of the total population. From these results, it can be seen that the mRNA levels of the above-mentioned markers are specific in myocardial tissues (FIG. 3).

In one embodiment of the invention, the case where the HBA1 / 2 and / or HBB gene level of the biological sample is higher than the HBA1 / 2 and / or HBB gene level of the control sample can be determined as acute cardiac death. For example, if the HBA1 / 2 gene level of the biological sample is higher than the HBA1 / 2 gene level of the control sample, the HBB gene level of the biological sample is higher than the HBB gene level of the control sample, The level of HBB gene in the biological sample is higher than that of the control sample and the level of the HBB gene in the biological sample is higher than the level of the HBB gene in the control sample.

In one embodiment of the invention, acute cardiac death can be determined if the level of HBA1 / 2 and / or HBB gene in the biological sample is at least two times higher than the level of HBA1 / 2 and / or HBB gene in the control sample. For example, if the HBA1 / 2 gene level of the biological sample is two times higher than the HBA1 / 2 gene level of the control sample, the HBB gene level of the biological sample is at least two times higher than the HBB gene level of the control sample, The HBA1 / 2 gene level of the sample is two times higher than the HBA1 / 2 gene level of the control sample, and the HBV gene level of the biological sample is two times higher than the HBB gene level of the control sample, .

In one embodiment of the present invention, a case in which the PDK4 gene level of the biological sample is lower than that of the control sample is determined as acute cardiac death. For example, a case in which the level of PDK4 gene in a biological sample is less than 1/2 of the PDK4 gene level in a control sample can be determined as acute cardiac death.

As mentioned above, acute cardiac death can be discriminated by using each of the HBA1 / 2, HBB or PDK4 genes as a marker. Alternatively, two or more of the HBA1 / 2, HBB and PDK4 genes may be used as markers or HBA1 / And PDK4 as markers can be used to identify acute cardiac death. Acute cardiac death, including all three, can improve the sensitivity and specificity of acute cardiac discrimination.

Therefore, acute cardiac death can be identified when the HBA1 / 2 and HBB gene levels of the biological sample are higher than the HBA1 / 2 and HBB gene levels of the control sample and the PDK4 gene level of the biological sample is lower than the PDK4 gene level of the control sample.

In one embodiment of the present invention, the case where the gene level of HBA1 / 2, HBB and PDK4 in the sample satisfies i), ii) and iii) is determined as acute cardiac death.

[Formula 1]

B is the gene level of the HBB of the biological sample, b is the gene level of HBB of the control sample, C is the gene level of HBA1 / 2 of the biological sample, Is the gene level of PDK4 in the biological sample and c is the gene level of PDK4 in the control sample.

In one embodiment of the invention, the case where the HBA1 / 2, HBB and PDK4 gene levels in the sample satisfy one or more of i) and ii) of the following formula 2 can be determined as acute cardiac death.

[Formula 2]

D is the relative gene expression level of HBA1 / 2 of the biological sample relative to the control sample, E is the relative gene expression level of the HBB of the biological sample relative to the control sample, F is the relative level of expression of PDK4 of the biological sample relative to the control sample Relative gene expression level.

The rate of RNA degradation after death can vary from person to person and can vary depending on the surrounding environment, such as time lapse, temperature, or humidity. Therefore, the relative mRNA expression ratio of HBA1 / 2 or HBB and PDK4, that is, the relative mRNA level of HBA1 / 2 or HBB relative to the control sample, compared with the mRNA level of HBA1 / 2, HBB or PDK4, And the relative mRNA level of PDK4 is used as an index, the specificity and sensitivity of discriminating acute cardiac infarction can be increased by reducing the error according to the condition (FIG. 4).

According to the present invention, acute cardiac death can be easily discriminated even in the case of autopsy, in which it is difficult to discriminate an acute cardiac anatomy anatomically.

FIG. 1 shows the results of microarray analysis of mRNA expression patterns of myocardial tissues in the damaged heart (TD), chronic ischemic heart disease (CCD), and acute myocardial infarction (AMI). FIGS. 1A and 1B show genes that are differentially expressed in the CCD and AMI groups belonging to acute cardiac pathology compared to TD, and FIG. 1C shows the expression patterns thereof in a bar graph.
FIG. 2 shows the results of analyzing gene mRNA levels in the acute cardiac death (SCD) group as compared to the TD group. Figure 2a is a bar graph with mean and standard deviation, and Figure 2c is a box-whisker plot with median and quadratic distributions.
FIG. 3 shows the results of quantitative RT-PCR analysis of expression patterns of HBA1 / 2, HBB and PDK4 in myocardial and cerebral cortical tissues in the TD and SCD groups.
Figure 4 shows the ratio between the relative mRNA levels of HBA1 / 2 or HBB and the relative mRNA levels of PDK4 mRNA in the TD and SCD groups.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention. These embodiments are provided so that the disclosure of the present invention is not limited thereto and that those skilled in the art will fully understand the scope of the present invention and that the present invention is not limited by the scope of the claims Only.

[ Example ]

[ Example  1] Acute cardiac arrest Marker  Selection of candidates

In order to select candidates for acute cardiac discrimination markers, RNA was extracted from traumatic injury, death due to chronic ischemic heart disease, and death from acute myocardial infarction. And the number of differentially expressed genes in the case of death from heart disease were analyzed.

Preparation of sample

In this example, among the autopsy cases provided by the National Institute of Investigation, from non-infarcted left ventricular myocardium and cerebral cortical tissue Were collected. The signs and symptoms of this case were 1) traumatic death (TD, n = 16), 2) chronic coronary disease (CCD, n = 19) and 3) acute myocardial infarction myocardial infarction, AMI, n = 15) were confirmed. Information on the age, cardiac weight, coronary atherosclerosis and left ventricular wall thickness of the specific experimental group are shown in Table 1 below. The TD group is a case of traumatic injuries that do not have serious cardiac disease, which is enough to be a sign. CCD is a case of ischemic heart disease and the cause of death is presumed to be caused by aggravation of ischemic heart disease, but there is no necrosis of the myocardium and the occlusion of the coronary artery is moderate. AMI is a case of marked coronary necrosis and coronary arterial thrombosis with elevation of the coronary arteries. The extracted tissues were immediately treated with RNAlater ^™ solution (QIAGEN) at 4 ° C for 16 hours and then stored frozen at -70 ° C.

RNA  extraction

50-100 mg of myocardial tissue stored at -70 ° C was placed in a cryo tube, left in liquid nitrogen for 10 minutes, cooled rapidly, and then tissue was pulverized using SKMILL-200 (Tokken Inc.). Subsequently, total RNA was isolated from the milled tissue according to the manufacturer's standard protocol using micro RNeasy mini kit (QIAGEN). The isolated total RNA was determined by absorbance measurement.

Microarray  analysis

Analysis of large-scale mRNA expression patterns using microarrays was basically carried out according to the method previously reported by the present inventor (Chung et al., Mol Cells 34: 473, 2012). In TD, CCD, and AMI experimental groups, one sample was prepared by mixing the same amount of myocardial RNA extracted from 4 ~ 5 specimens in order to offset individual deviations. Respectively. Microarray experiments for the same mixed sample were repeated twice independently. Microarray analysis was performed according to the Affymetrix GeneChip Expression Analysis Manual, where 200 ng of total RNA per reaction was amplified and labeled. The microarray used in this example is Affymetrix GeneChip Human Gene 1.0 ST Arrays.

As a result, ten genes that are differentially expressed in the CCD and AMI groups belonging to cardiac pathogens were selected as candidates (FIGS. 1A and 1B), and their expression patterns were shown in a bar graph (FIG. 1C). The expression levels of the six genes, OGN, ASPN, MFAP5, HBB and HBA1 / 2, were 1.5 times higher in the CCD and AMI groups compared to the 5 genes of PPP1R1A, AREG, PDK4, B3GNT5 and NR4A2 As shown in Fig. Since HBA1 and HBA2 have the same nucleotide sequence, they were treated as a single index in the present invention.

All gene names are represented by official genetic symbols used in the GenBank database.

Differences in mRNA levels between each experimental group were statistically verified by Student's t-test and statistically significant at p <0.05.

[ Example  2] Acute cardiac arrest Marker  Confirm

The relative mRNA levels in the sudden cardiac death (SCD) group were measured using the TD group as a control group for the 10 candidate genes selected in Example 1.

As in the case of Example 1, the specimen was collected from non-infarcted left ventricular myocardial tissue by infarction from the dead body within 4 days after the autopsy provided by National Institute of Investigation. The control group, TD, and the experimental group, were classified as sudden cardiac death (SCD, n = 23) by morphological, biochemical, and toxicological findings. SCD is a case of acute cardiac death because of the absence of anatomical, toxicologic, and pharmacologically distinct signs, although some liver cirrhosis is found. The extracted tissues were immediately treated with RNAlater ^™ solution (QIAGEN) at 4 ° C for 16 hours and then stored frozen at -70 ° C.

RNA was extracted and microarray analysis was performed as in Example 1 (Fig. 2). Relative mRNA levels in the SCD group relative to the mean measurements of the TD group were plotted as a bar graph (mean and standard error notation, Figure 2a) or box-whisker plot (median and quartile notation, Figure 2b) As a result, mRNA expression of hemoglobin subtype HBA1 / 2 (2.273 ± 0.197) and HBB (2.371 ± 0.200) mRNA was significantly higher in the SCD group compared to that of the PDB4 subtype of pyruvate dehydrogenase enzyme (0.500 ± 0.169 MRNA expression was significantly lower than that of control.

These results indicate that HBA1 / 2, HBB and PDK4 can be used as markers for acute cardiac discrimination.

[ Example  3] Quantitative in the myocardium and cerebral cortex RT - PCR

Expression patterns of the HBA1 / 2, HBB and PDK4 candidate genes from the myocardial tissue and the cerebral tissue RNA of the TD group and the SCD group in order to finally confirm that the HBA1 / 2, HBB and PDK4 identified in Example 2 were acute cardiac markers Quantitative RT-PCR was performed for each sample. Like the myocardial tissue in Example 1, 50-100 mg of the cerebral tissue was placed in a cryo tube, allowed to stand for 10 minutes in liquid nitrogen, and rapidly cooled. Then, using SKMILL-200 (Tokken Inc) The tissue was ground. Subsequently, total RNA was isolated from the milled tissue according to the manufacturer's standard protocol using micro RNeasy mini kit (QIAGEN). The isolated total RNA was determined by absorbance measurement.

Quantitative RT-PCR was performed according to the method previously reported by the present inventor (Chung et al., Mol Cells 34: 473, 2012). Using MMLV reverse transcriptase (Promega), 1 μg of total RNA was subjected to reverse transcription by random priming to synthesize cDNA. Quantitative real-time PCR was performed using the SYBR Green I PCR master mixture (Life Technologies) and gene-specific primers for the cDNA samples, and the level of the mRNA contained in the cDNA sample was measured. The PCR reaction was carried out at 50 ° C for 2 minutes and at 90 ° C for 10 minutes, followed by 50 cycles of 95 ° C for 15 seconds, 62 ° C for 30 seconds, and 72 ° C for 30 seconds. The relative levels of HBA1 / 2, HBB, and PDK4 mRNA were normalized by the level of GAPDH. The PCR products were confirmed by gene sequencing analysis. The nucleotide sequences of the primers used in this PCR reaction are as follows.

SEQ ID NO: 1: 5'-TTA AGG GCC ACG GCA AGA AG-3 '(HBA1 / 2-F)

SEQ ID NO: 2: 5'-GCA GTG GCT TAG GAG CTT GA-3 '(HBA1 / 2-R)

SEQ ID NO: 3: 5'-GTG AAC GTG GAT GAA GTT GG-3 '(HBB-F)

SEQ ID NO: 4: 5'-ACT TTC TTG CCA TGA GCC TT-3 '(HBB-R)

SEQ ID NO: 5: 5'-GCT GAT GAA CCA GCA CAT TC-3 '(PDK4-F)

SEQ ID NO: 6: 5'-CAC TCA AAG GCA TCT TGG AC-3 '(PDK4-R)

SEQ ID NO: 7: 5'-ATG TTC GTC ATG GGT GTG AA-3 '(GAPDH-F)

SEQ ID NO: 8: 5'-GGT GCT AAG CAG TTG GTG GT-3 '(GAPDH-R)

In this process, mRNA expression patterns of the three candidate genes in the cerebrum were also analyzed by quantitative RT-PCR for samples in which the cranial cortex tissue was available in the experimental group. In the cerebral tissues, 13 samples in the TD group and 15 samples in the SCD group were available. Each gene expression was normalized to a housekeeping gene, GAPDH, and the relative expression level was expressed as the mean of the control group, TD group, as 1 (FIG. 3)

In the case of myocardial tissue as shown in FIG. 3A, quantitative RT-PCR also showed a pattern similar to that of microarray analysis. HBA1 / 2 and HBB mRNA levels were significantly higher in the SCD group than in the TD group, and PDK4 mRNA expression level was lower in the SCD group. The increase / decrease width of each gene was measured by 1) HBA1 / 2: 4.183 ± 1.111 times, 2) HBB: 6.262 ± 1.897 times, and 3) PDK4: 0.269 ± 0.111 times. In contrast, all of the three genes in the cerebral cortex showed no differential expression according to the sign (FIG. 3b), indicating that the differential expression pattern of the three genes found in myocardial tissue was tissue specific.

The difference in mRNA levels between each experimental group was statistically verified by Student's t-test and statistically significant at p <0.05.

[ Example  4] Hemoglobin and Pyruvic acid  Dehydrogenase Kinase  Verification of integrated index of subtype expression pattern

As shown in Example 3, the HBA1 / 2 and HBB mRNA expression levels of the present invention were significantly higher in the SCD group, whereas the PDK4 mRNA levels were lower in the same samples. Based on this, quantitative RT-PCR was performed using the value obtained by dividing the relative mRNA level of HBA1 / 2 and HBB genes by the relative level of PDK4 as an index in order to utilize a more practical and efficient integrated index (FIG. 4).

The results of PT-PCR are shown in Fig. The gray point is the value measured in the individual sample, the black dot means the mean, and the deviation means the 95% confidence interval of each distribution. As a result, it can be deduced that if the value of HBA1 / 2 or HBB mRNA relative to the control sample divided by the relative amount of PDK4 mRNA was 10 or more, it died due to cardiac dysfunction (FIG. 4).

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Composition for determining of sudden cardiac death and method          for determining of sudden cardiac death <130> P130684 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBA 1/2 forward primer <400> 1 ttaagggcca cggcaagaag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBA 1/2 reverse primer <400> 2 gcagtggctt aggagcttga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBB forward primer <400> 3 gtgaacgtgg atgaagttgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBB reverse primer <400> 4 actttcttgc catgagcctt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDK4 forward primer <400> 5 gctgatgaac cagcacattc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDK4 reverse primer <400> 6 cactcaaagg catcttggac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 7 atgttcgtca tgggtgtgaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 8 ggtgctaagc agttggtggt 20

Claims (14)

A composition for the diagnosis of acute cardiac death, comprising a probe for at least one gene selected from the group consisting of HBA1 / 2, HBB and PDK4 in a cadaveric autopsy.
The method according to claim 1,
Wherein the probe is a sense and / or antisense primer complementary to the mRNA of the HBA1 / 2, HBB or PDK4 gene.
The method according to claim 1,
A probe for the genes of HBA 1/2, HBB and PDK4.
An acute cardiac catheterization kit comprising the composition of any one of claims 1 to 3.
5. The method of claim 4,
An RT-PCR kit or microarray kit, an acute cardiac catheterization kit.
Contacting the composition of any one of claims 1 to 3 with a biological sample; And
Comparing said HBA1 / 2, HBB and / or PDK4 gene levels in said biological sample with HBA1 / 2, HBB and / or PDK4 gene levels of a control sample rather than death from acute cardiac death.
The method according to claim 6,
The biological sample is myocardial tissue, which is an acute cardiac discrimination method.
The method according to claim 6,
A method wherein the level of HBA1 / 2 and / or HBB gene in the biological sample is higher than the level of HBA1 / 2 and / or HBB gene in the control sample.
9. The method of claim 8,
A method wherein the level of HBA1 / 2 and / or HBB gene in a biological sample is at least two times higher than the level of HBA1 / 2 and / or HBB gene in a control sample.
The method according to claim 6,
A method wherein the PDK4 gene level in the biological sample is lower than the level of the PDK4 gene in the control sample is determined as acute cardiac death.
11. The method of claim 10,
A method wherein the PDK4 gene level of the biological sample is less than 1/2 of the PDK4 gene level of the control sample is determined as acute cardiac death.
The method according to claim 6,
A method of determining acute cardiac death when the HBA1 / 2 and HBB gene levels of the biological sample are higher than the HBA1 / 2 and HBB gene levels of the control sample and the PDK4 gene level of the biological sample is lower than the PDK4 gene level of the control sample.
13. The method of claim 12,
Method for determining acute cardiac death when gene levels of HBA1 / 2, HBB and PDK4 in the sample satisfy i), ii) and iii) of the following formula 1:
[Formula 1]

B is the gene level of the HBB of the biological sample, b is the gene level of HBB of the control sample, C is the gene level of HBA1 / 2 of the biological sample, Is the gene level of PDK4 in the biological sample and c is the gene level of PDK4 in the control sample.
The method according to claim 6,
Method for determining acute cardiac death when the HBA1 / 2, HBB and PDK4 gene levels in the sample satisfy one or more of i) and ii) of the following formula 2:
[Formula 2]

D is the relative gene expression level of HBA1 / 2 of the biological sample relative to the control sample, E is the relative gene expression level of the HBB of the biological sample relative to the control sample, F is the relative level of expression of PDK4 of the biological sample relative to the control sample Relative gene expression level.

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