KR102496589B1 - VHL Gene hypermethylation marker for diagnosis of delayed cerebral ischemia - Google Patents

Abstract

The present invention relates to a composition for diagnosing delayed cerebral ischemia comprising an agent capable of measuring the methylation level of a von Hippel-Lindau (VHL) gene, a kit for diagnosing delayed cerebral ischemia comprising the composition, and delayed cerebral ischemia using the composition and kit It relates to a method of providing information for diagnosis.
According to the composition for diagnosing delayed cerebral ischemia, the kit for diagnosing delayed cerebral ischemia, and the method for providing information for diagnosing delayed cerebral ischemia of the present invention, the risk of delayed cerebral ischemia occurring after subarachnoid hemorrhage is predicted by measuring the methylation level of the VHL gene or can be used for diagnosis.

Description

VHL Gene hypermethylation marker for diagnosis of delayed cerebral ischemia}

The present application provides a composition for diagnosing delayed cerebral ischemia comprising an agent capable of measuring the methylation level of a von Hippel-Lindau (VHL) gene, a kit for diagnosing delayed cerebral ischemia comprising the composition, and diagnosing delayed cerebral ischemia using the composition and kit It is about how to provide information for

Subarachnoid hemorrhage (SAH) due to ruptured cerebral aneurysm is a life-threatening disease, with a mortality rate of 45% within 30 days of onset. In addition, delayed cerebral ischemia (DCI) can develop within a range of 30-40% after subarachnoid hemorrhage, which is associated with a major cause of mortality. Patients with subarachnoid hemorrhage with delayed ischemia are more likely to have various complications, such as pneumonia, cardiac arrhythmia, intestinal bleeding and urinary tract infection, compared to patients without delayed ischemia, resulting in prolonged hospitalization and medical costs. may increase, etc. The onset of delayed ischemia is influenced by various clinical and radiological variables. Well-known clinical risk factors for delayed ischemia are female sex, smoking, and low clinical grade at hospitalization. A higher Hunt-Hess-grade (Grade IV and V) or World Federation of Neurosurgical Societies (WFNS) grade increases the likelihood of developing delayed ischemia.

In relation to the onset of delayed ischemia, various genetic studies such as single nucleotide polymorphisms (SNPs) or genome-wide association studies (GWAS) have been conducted. However, only 5 to 10% of sensitive genetic variants were observed, implying that there are still unknown genetic variants beyond the DNA sequence scale.

As one of the unknown novel pathways, there are epigenetic mechanisms including DNA methylation, histone modification, and non-coding RNA. Among these, DNA methylation is a process in which a methyl group binds to a cytosine nucleotide in front of guanine, and is generally known to inhibit gene transcription. To date, no study has been conducted to confirm the epigenetic profile of delayed ischemia in patients with subarachnoid hemorrhage.

Therefore, the present inventors have tried to develop a method that can efficiently and easily diagnose the onset of delayed cerebral ischemia, predict the risk of onset, and predict the prognosis of delayed cerebral ischemia that occurs after subarachnoid hemorrhage. Alternatively, the present invention was completed by developing a predictable diagnostic composition.

Korean Patent Publication No. 10-2019-0008216

The present application provides a composition for diagnosing delayed cerebral ischemia comprising an agent capable of measuring the methylation level of a von Hippel-Lindau (VHL) gene, a kit for diagnosing delayed cerebral ischemia comprising the composition, and diagnosing delayed cerebral ischemia using the composition and kit We want to provide a method of providing information for

Objects of the present application are not limited to the above-mentioned purposes, and other objects and advantages of the present application not mentioned above can be understood by the following description and will be more clearly understood by the examples of the present application. Further, it will be readily apparent that the objects and advantages of the present application may be realized by means of the instrumentalities and combinations indicated in the claims.

A first aspect of the present application is to provide a composition for diagnosing delayed cerebral ischemia, including an agent capable of measuring the methylation level of a von Hippel-Lindau ( VHL ) gene.

A second aspect of the present application is to provide a kit for diagnosing delayed cerebral ischemia, comprising the composition for diagnosing delayed cerebral ischemia.

A third aspect of the present application is to provide an information providing method for diagnosing delayed cerebral ischemia, comprising measuring the methylation level of von Hippel-Lindau ( VHL ) gene from a biological sample isolated from an individual.

According to the composition for diagnosing delayed cerebral ischemia, the kit for diagnosing delayed cerebral ischemia, and the method for providing information for diagnosing delayed cerebral ischemia of the present invention, the risk of delayed cerebral ischemia occurring after subarachnoid hemorrhage is predicted by measuring the methylation level of the VHL gene or can be used for diagnosis.

1 is a diagram showing a diagnosis process of delayed cerebral ischemia using hypermethylation of VHL gene.
2 is a view showing the results of functional enrichment analysis of hypermethylated genes between patients with and without delayed cerebral ischemia after subarachnoid hemorrhage. Yellow boxes indicate statistical significance.
3 is a view showing the results of functional enrichment analysis of hypomethylated genes between patients with and without delayed cerebral ischemia after subarachnoid hemorrhage. Yellow boxes indicate statistical significance.
Figure 4 is a diagram showing protein-protein interaction networks of hypermethylated genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.
5 is a diagram showing protein-protein interaction networks of hypomethylated genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.
6 is a view showing results of modular cluster analysis of hypermethylated genes and enrichment analysis of related genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.
7 is a view showing results of modular cluster analysis of hypermethylated genes and enrichment analysis of related genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.
8 is a view showing results of modular cluster analysis of hypermethylated genes and enrichment analysis of related genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.
9 is a view showing results of modular cluster analysis of hypomethylated genes and enrichment analysis of related genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.
10 is a view showing results of modular cluster analysis of hypomethylated genes and enrichment analysis of related genes between delayed cerebral ischemia patients (DCI) and non-delayed cerebral ischemia patients (non-DCI) after subarachnoid hemorrhage.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

A first aspect of the present application provides a composition for diagnosing delayed ischemia, including an agent capable of measuring the methylation level of a von Hippel-Lindau ( VHL ) gene.

As used throughout this specification, the term “delayed cerebral ischemia (DIC)” refers to the occurrence of focal neurological disorders (e.g., hemiparesis, aphasia, apraxia, hemianopia or neglect), or the Glasgow Coma Scale (total score or one of its individual components), the onset of delayed cerebral ischemia may be due to various clinical and radiological variables. As used herein, the "delayed cerebral ischemia" refers to "DIC", " It can be named interchangeably with "delayed ischemia".

In one embodiment of the present application, the composition may include an agent capable of measuring the methylation level of the promoter region of the VHL gene.

In one embodiment of the present application, the composition may include an agent capable of measuring the methylation level of nucleotides including SEQ ID NO: 1, but is not limited thereto.

In one embodiment of the present application, SEQ ID NO: 1 may include a nucleotide sequence around the methylation region of the VHL gene.

In one embodiment of the present application, the composition is KIF3A (kinesin family member 3A), KIFAP3 ( kinesin associated protein 3), RACGAP1 ( Rac GTPase Activating Protein 1), OPRM1 ( Opioid Receptor Mu 1), ALB (albumin) and IL5 (Interleukin 5) may further include an agent capable of measuring the methylation level of one or more genes selected from the group consisting of.

In one embodiment of the present application, the composition may further include an agent capable of measuring the methylation level of nucleotides containing at least one of the nucleotide sequences of SEQ ID NOs: 2 to 9, but is not limited thereto.

In one embodiment of the present application, SEQ ID NOs: 1 to 9 may each contain the nucleotide sequence of the methylation region of the following genes, and the methylation region in the nucleotide sequence of each SEQ ID NO. It is listed in Table 3.

- SEQ ID NO: 1: VHL (von Hippel-Lindau), chromosome 3 10182523-10182524

- SEQ ID NO: 2: KIF3A (kinesin family member 3A). Chromosome 5 132040050- 132040051

- SEQ ID NO: 3: KIFAP3 ( kinesin associated protein 3), chromosome 1 170044709- 170044710

- SEQ ID NO: 4: RACGAP1 ( Rac GTPase Activating Protein 1), chromosome 12 50397469- 50397470

- SEQ ID NO: 5: RACGAP1 ( Rac GTPase Activating Protein 1), chromosome 12 50397685- 50397685

- SEQ ID NO: 6: OPRM1 ( Opioid Receptor Mu 1), chromosome 6 154410759- 154410760

- SEQ ID NO: 7: ALB (albumin), chromosome 4 74269995-74269996

- SEQ ID NO: 8: IL5 (Interleukin 5), chromosome 5 131879309- 131879310

- SEQ ID NO: 9: IL5 (Interleukin 5), chromosome 5 131891673- 131891674

As used throughout the present specification, the term "methylation" or "DNA methylation" means that a methyl group is attached to a base constituting DNA. It may mean whether or not methylation occurs at cytosine at the phosphate Guanine) site. In general, when methylation or hypermethylation occurs, transcription factor binding is hindered and expression of a specific gene may be suppressed. Conversely, when unmethylation or hypomethylation occurs, expression of a specific gene may increase. there is.

In addition to A, C, G, and T, in the genomic DNA of mammalian cells, there is a fifth base called 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. do. Methylation of 5-methylcytosine occurs only at the C of CG dinucleotide (5'-mCG-3'), called CpG, and methylation of CpG inhibits the expression of alu or transposon and genomic repeats. In addition, since 5-mC of the CpG is easily deaminated naturally to become thymine (T), CpG is a site where most epigenetic changes frequently occur in mammalian cells.

As used throughout the present specification, the term "measurement of methylation level" refers to specific genes, specifically VHL (von Hippel-Lindau), KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1), OPRM1 (Opioid Receptor Mu 1), ALB (albumin), and / or IL5 (Interleukin 5) as measuring the methylation level of the CpG region of the gene, it may be to use the difference between methylated and unmethylated bases, , methylation-specific polymerase chain reaction (MSP), real-time methylation-specific polymerase chain reaction (PCR), PCR using methylated DNA-specific binding proteins, or quantitative PCR. can Alternatively, pyrosequencing, MS-HRM (Methylation-Sensitive High-Resolution Melting Analysis) and methylation using methylation-sensitive restriction enzymes, automated sequencing such as DNA chip and bisulfite sequencing or the like, or DNA methylation microarray, or immunoprecipitation using methylated CpG binding domains or anti-methylcytosine antibodies, etc. may be used, but is not limited thereto.

According to one embodiment of the present application, "agent capable of measuring methylation level" refers to a molecule that can be used to measure the methylation level of a specific DNA region, specifically VHL (von Hippel-Lindau), KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1), OPRM1 (Opioid Receptor Mu 1), ALB (albumin) and / or IL5 (Interleukin 5) Methylation-specific PCR for genes It may include primers or probes for performing, but is not limited thereto.

The primers used to measure the methylation level may include a primer specific for a methylated allelic sequence of a gene and a primer specific for an unmethylated allelic sequence, and the primer is a subject to be analyzed for methylation. A pair of primers that can be preferably designed according to the sequence of a specific CpG site and can specifically amplify cytosine that is methylated and not modified by bisulfite, respectively, and cytosine that is not methylated and modified by bisulfite It may be a primer pair that can specifically amplify.

The term "primer" used throughout the present specification is a base sequence having a short free 3' terminal hydroxyl group, capable of forming a base pair with a complementary template, and copying the template strand. refers to a short sequence that serves as a starting point for A primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. At this time, PCR conditions and lengths of sense and antisense primers may be modified based on those known in the art.

The probe used to measure the methylation level may mean a hybridization probe, and mean an oligonucleotide capable of sequence-specifically binding to a complementary strand of a nucleic acid. The probe may be used in a kit or prediction method for diagnosing delayed cerebral ischemia by measuring the methylation level. The probe of interest may be labeled so as to be detectable, for example labeled with a radioactive isotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme. Properly labeling the probe as described above is a technique widely known in the art and can be performed through a conventional method.

According to one embodiment of the present application, the primer or probe may be chemically synthesized using a phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologues of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In one embodiment of the present application, as an agent capable of measuring the methylation level, the agent for measuring the methylation level of the CpG site is a compound that modifies unmethylated cytosine base, a methylated sensitized restriction enzyme, or a methylated allele of a gene Sequence-specific primers, primers specific to unmethylated allelic sequences, methylated CpG binding domains, or methylated DNA antibodies that specifically bind to methylated DNA (e.g., antibodies that specifically bind to methylcytosine) ) and the like.

The compound that transforms the unmethylated cytosine base may be bisulfite or a salt thereof, but is not limited thereto, and may preferably be sodium bisulfite. Bisulfite-modified DNA can detect methylation through various methods such as sequencing or methylation-specific PCR. It is well known in the art.

In addition, the methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of a CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, and NotI. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, cleavage by the restriction enzyme is different, and this can be detected through PCR or Southern blot analysis. Other methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

On the other hand, a methylated DNA antibody refers to an antibody that specifically binds to a methylated base in DNA. Specifically, antibodies having the property of recognizing and binding to methylated cytosine in the DNA chain, such as antibodies against methylcytosine, are exemplified. In addition, among commercially available "methylated" DNA antibodies, the antibody capable of specifically recognizing and specifically binding to DNA in the "methylated" state described herein may be used.

A methylated DNA antibody can be prepared by a conventional method using a methylated base, a methylated DNA, or the like as an antigen. For example, in order to prepare a methylcytosine antibody, an antibody is prepared using 5-methylcytidine, 5-methylcytosine, or DNA containing 5-methylcytosine as an antigen, and then methylcytosine in DNA The specific binding of can be selected as an indicator.

In addition, when measuring the level of methylation using a methylated CpG binding domain (MBD) or methylated DNA antibody, after immunoprecipitation of methylated DNA using these, Southern blot, PCR, microarray, or sequence A specific CpG site can be identified through sequencing or the like. In addition, for immunological detection or quantification of the antibody, a substrate, an appropriate buffer solution, a chromogenic enzyme or fluorescent label, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, and a chromogenic substrate may be used. In the above, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used as the substrate, and the coloring enzyme may be peroxidase, alkaline phosphatase Alkaline phosphatase, etc. may be used, FITC, RITC, etc. may be used as the fluorescent material, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sul Ponic acid)) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) may be used, and other radioactive isotope labels, latex bead labels, colloid labels, biotin labels, etc. may be used, but are not limited thereto. no.

According to one embodiment of the present application, the VHL gene may be included in the marker composition for diagnosing delayed cerebral ischemia, and specifically, the hypermethylated state of the VHL gene may be included in the marker composition for diagnosing delayed cerebral ischemia.

According to one embodiment of the present application, at least one gene selected from the group consisting of kinesin family member 3A (KIF3A), kinesin associated protein 3 (KIFAP3), Rac GTPase Activating Protein 1 (RACGAP1) and Opioid Receptor Mu 1 (OPRM1) is delayed It may be included in the marker composition for diagnosing sexual cerebral ischemia, and specifically, the hypermethylation state of the gene may be included in the marker composition for diagnosing delayed cerebral ischemia.

According to one embodiment of the present application, one or more genes selected from the group consisting of albumin (ALB) and interleukin 5 (IL5) may be included in the marker composition for diagnosing delayed cerebral ischemia, and specifically, the hypomethylation state of the gene is delayed. It may be included in the marker composition for diagnosing sexual cerebral ischemia.

As used throughout the present specification, the term "marker" refers to a biomarker and can detect changes in living organisms, thereby objectively measuring the normal or pathological state of living organisms, the degree of response to drugs, and the like.

According to one embodiment of the present application, the composition for diagnosing delayed cerebral ischemia of the present application may diagnose the onset of delayed cerebral ischemia, predict the risk of delayed cerebral ischemia, or predict the prognosis of delayed cerebral ischemia, but is limited thereto no.

The term "diagnosis" used throughout the present specification means determining whether or not delayed cerebral ischemia has already occurred for a specific individual.

The term "prediction" used throughout the present specification determines whether there is a possibility of developing delayed cerebral ischemia for a specific individual, and if there is a possibility of developing delayed cerebral ischemia, the likelihood of developing delayed cerebral ischemia is relatively higher than that of an unspecified number of people means to do

According to one embodiment of the present application, the delayed cerebral ischemia may occur after subarachnoid hemorrhage, but is not limited thereto.

According to one embodiment of the present application, the VHL gene in a patient with delayed cerebral ischemia shows a higher methylation level than a patient without delayed cerebral ischemia. It can be seen that it is possible to diagnose whether or not the disease has occurred and the possibility of developing delayed cerebral ischemia.

According to one embodiment of the present application, the KIF3A, KIFAP3, RACGAP1 and / or OPRM1 genes in patients with delayed cerebral ischemia show higher methylation levels than those in patients without delayed cerebral ischemia, thus methylation of the genes It can be seen that the occurrence of delayed cerebral ischemia and the possibility of delayed cerebral ischemia can be diagnosed by checking the level.

According to one embodiment of the present application, the ALB (albumin) and / or IL5 (Interleukin 5) genes in patients with delayed cerebral ischemia show lower methylation levels than those in patients without delayed cerebral ischemia. It can be seen that the occurrence of delayed cerebral ischemia and the possibility of delayed cerebral ischemia can be diagnosed through the confirmation of the methylation level of the gene.

In one embodiment of the present application, the composition may be a composition for diagnosing delayed ischemia in Koreans and Asians.

According to an embodiment of the present application, the genetic methylation level of patients with delayed cerebral ischemia after subarachnoid hemorrhage was analyzed using bioinformatics based on epigenome-wide association studies (EWAS) data Results, VHL (von Hippel-Lindau), KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1), and OPRM1 (Opioid Receptor Mu 1) genes in patients with delayed cerebral ischemia was confirmed to be hypermethylated than non-delayed cerebral ischemia patients, and in the case of ALB (albumin) and IL5 (Interleukin 5) genes, it was confirmed that patients with delayed cerebral ischemia were hypomethylated than patients with delayed cerebral ischemia, the results Based on this, delayed cerebral ischemia can be diagnosed using each of the above genes or a combination thereof as a methylation marker.

The term 'epigenome' used throughout the present specification refers to a pattern of chemical groups that are attached to a DNA sequence and affect its activity. As a result of the largest research ever to explore the human epigenome, an epigenetic tag (marker) related to a difference in body mass index (BMI) was discovered, drawing attention from the academic world. The epigenome-wide association studies (EWAS) using the anteroposterior genome were inspired by genome-wide association studies (GWAS). GWAS aims to find disease-related mutations by comparing and examining genetic variations in healthy people and patients. The goal of EWAS is basically the same as GWAS, only the target has changed from genome to epigenome. EWAS aims to find markers associated with diseases or mutations by comparatively examining the distribution of methyl groups attached to thousands of specific DNA nucleotides in the whole genome of healthy people and patients.

A major research subject of epigenetics is methylation, which refers to the phenomenon in which a methyl group is attached to a gene. Methylation, which tends to suppress gene activity, plays an important role during embryogenesis. In other words, methylation plays a role in helping embryonic stem cells differentiate into specialized tissues by directing the expression of genes. However, methylation also occurs in response to environmental changes, and genetic modifications resulting from this may be passed on to offspring. Methylation also contributes to the development of cancer and type 2 diabetes. Because these epigenetic alterations may occur throughout a person's lifetime, they can provide clues about diseases that genetics cannot detect.

The term "bioinformatics" used throughout the present specification is a discipline that mainly deals with biological problems at the molecular level using applied mathematics, information science, statistics, computer science, artificial intelligence, chemistry, and biochemistry. Research fields in computational biology often overlap with systems biology. Major research areas are diverse, including sequence alignment, gene search, gene combination, protein structure alignment, protein structure prediction, gene expression prediction, protein interactions, and evolution models. As a sub-field of bioinformatics, genomics (personal genomics, functional genomics, comparative genomics), variant genomics, proteomics, interactomics and multiomics, etc. are included.

A second aspect of the present application provides a kit for diagnosing delayed cerebral ischemia, comprising the composition for diagnosing delayed cerebral ischemia. Content overlapping with the first aspect is also applied to the kit for diagnosis of delayed cerebral ischemia of the second aspect.

According to one embodiment of the present application, the kit measures the methylation level of the CpG region of the gene, and a methylation-specific PCR kit, for example, a methylation-specific polymerase chain reaction (MSP) kit, real-time Methylation-specific PCR (real time methylation-specific polymerase chain reaction) kit, PCR kit using methylated DNA-specific binding protein, or quantitative pyrosequencing kit, Methylation-Sensitive High-Resolution Melting Analysis (MS-HRM) It may be a high-resolution melting curve analysis) kit, methylation determination using methylation sensitive restriction enzyme, DNA chip kit, DNA methylation microarray kit, or immunoprecipitation kit using methylated CpG binding domain or anti-methylcytosine antibody. , but is not limited thereto.

According to one embodiment of the present application, the PCR kit may be a kit including essential elements required to perform PCR. The PCR kit contains, in addition to polynucleotides, primers or probes specific for the above genes or methylated regions, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymer enzymes such as enzymes and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.

According to one embodiment of the present application, the DNA chip kit may be a kit including essential elements required to perform DNA chip, and the DNA chip kit may include polynucleotides, primers or probes specific for the genes or methylation regions. and a substrate to which is attached, and the substrate may include a nucleic acid corresponding to a quantitative control gene or a fragment thereof.

According to one embodiment of the present application, the RT-PCR kit may be a kit that includes essential elements required to perform RT-PCR, and the RT-PCR kit is a kit for each specific gene or methylation region. In addition to primers, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors , DEPC-water, sterile water, and the like.

A third aspect of the present application provides an information-providing method for diagnosing delayed cerebral ischemia, comprising measuring the methylation level of von Hippel-Lindau ( VHL ) gene from a biological sample isolated from an individual. The contents overlapping with the first and second aspects are also applied to the information provision method for diagnosing delayed cerebral ischemia of the third aspect.

According to one embodiment of the present application, the method may include measuring the methylation level of a nucleotide including SEQ ID NO: 1 in a biological sample isolated from the subject.

According to one embodiment of the present application, the diagnosis of delayed cerebral ischemia herein may be diagnosing the onset of delayed ischemia, predicting the risk of developing delayed cerebral ischemia, or diagnosing the prognosis of delayed cerebral ischemia, and the delayed cerebral ischemia It may occur after subarachnoid hemorrhage, but is not limited thereto.

As used throughout the present specification, the term "subject" refers to any organism that has or is likely to develop delayed cerebral ischemia, and specific examples include mice, monkeys, cows, pigs, mini-pigs, livestock, humans, and the like. It may include mammals, farmed fish, etc., but is not limited thereto.

As used throughout the present specification, the term "sample" refers to a material derived from an individual who has or is likely to develop delayed cerebral ischemia, and specifically, tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, It may include urine and the like, but is not limited thereto. In addition, a genetic sample may be obtained from these samples, and the genetic sample may include nucleic acid, for example, DNA, mRNA, or cDNA synthesized from mRNA, from which DNA methylation level or expression of a specific gene / protein As long as the level can be confirmed, the type is not limited thereto.

According to one embodiment of the present application, the subject may be a human, specifically Korean or Asian, but is not limited thereto.

According to one embodiment of the present application, the method may further include comparing the methylation level of the gene of the individual with a predetermined cut-off.

According to one embodiment of the present application, the method comprises measuring a methylation level of a VHL gene from a biological sample separated from a control group; and comparing methylation levels of the individual and the control group.

As used throughout the present specification, the term "control group" may mean a general subject or non-patient group in which delayed cerebral ischemia does not occur.

According to one embodiment of the present application, the method, when the methylation level of the VHL gene of the subject is higher than the methylation level of the control group, determining the subject as having delayed cerebral ischemia or predicting the risk of developing at a high level It may be to further include.

In one embodiment of the present application, the method is performed on biological samples isolated from subjects and controls, A) KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1) and OPRM1 ( Measuring the methylation level of one or more genes selected from the group consisting of Opioid Receptor Mu 1); B) comparing the methylation levels of the individual and the control group; And C) if the methylation level of the gene measured in step A of the individual is higher than the methylation level of the control group, determining that the individual has delayed cerebral ischemia or predicting a high risk of developing it. may include

In one embodiment of the present application, the method comprises the steps of measuring the methylation level of one or more genes selected from the group consisting of ALB (albumin) and IL5 (Interleukin 5) from biological samples isolated from individuals and controls; E) comparing the methylation levels of the subject and the control group; And F) if the methylation level of the gene measured in step D of the subject is lower than the methylation level of the control group, determining that the subject has delayed cerebral ischemia or predicting a high risk of developing it. may include

In one embodiment of the present application, the method may include measuring the methylation level of nucleotides including at least one of the nucleotide sequences of SEQ ID NOs: 2 to 9 in a biological sample isolated from the subject.

[Example]

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: DNA methylation data and processing

We analyzed data from epigenome-wide association studies (EWAS) of patients with subarachnoid hemorrhage (SAH) from a regional stroke center in Chuncheon. The data correlated with anteroposterior genomic methylation profiles of CpG sites in 40 patients with aneurysmal SAH using the Infinium MethylationEPIC (EPIC) BeadChip (Illumina, CA, USA). Raw quality probes of the raw data were filtered using the minfi package (version 1.24.0). Specifically, samples with a detection P-value (>0.05) were first removed. Next, the remaining probes were normalized using the preprocessFunnorm algorithm implemented in the minfi package. The method removes undesirable mutations by regressing variability using control probes present in the methylation microarray. Finally, a differentially methylated CpG site (DMCpG) was identified with a q-value cutoff of 0.01. Among the identified DMCpGs, CpG sites with beta (β) values greater than 0.2 were used for further analysis. The degree of DNA methylation is represented by a β value having a value of 0 to 1. A β value of 0 means that the corresponding CpG site is completely unmethylated, and 1 means that it is completely methylated. Finally, genes with DMCpG located in the promoter region (±2 kb from the transcription start site) were selected and used to infer the potential function of DMCpG in gene regulatory terms.

Example 2: Bioinformatics analysis

Functional enrichment analysis was performed using a web-based gene enrichment analysis and visualization tool ( http://cbl-gorilla.cs.technion.ac.il ). Next, a protein-protein interaction (PPI) was constructed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). The cut-off criterion was set at an interaction score of 0.4. The Cluster-ONE application was used for module analysis. A module was defined as the number of connected interactions greater than 10. Hub genes were defined as genes with high connectivity in the top 10% of candidate modules.

Experimental Example 1: Functional enrichment analysis result

Bioinformatics analysis was performed on methylated genes of patients with delayed cerebral ischemia after the subarachnoid hemorrhage described in Example 1 above.

Specifically, functional enrichment analysis of hypermethylated or hypomethylated genes between patients with and without delayed cerebral ischemia after subarachnoid hemorrhage was performed.

As a result, among the gene ontology (GO) biological process (BP) terms retrieved from hypermethylation, organic cyclic compound biosynthesis process, nucleobase-containing compound (nucleobase-containing Compound) biosynthetic process, heterocyclic compound biosynthetic process, aromatic compound biosynthetic process, and cellular nitrogen compound biosynthetic process-related terms were judged to be the most significant (Table 1, Figure 1). 2). Gene Ontology life metabolism terms retrieved from hypomethylation are carbohydrate metabolic process, regulation of cell size, and detection of stimulus.

GO molecular function (MT) terms are alpha-amylase activity, amylase activity, amylase activity (maltohexaose release), hydrolase activity, and O-glyco It is determined by hydrolyzing O-glycosyl compounds (Table 1 and FIG. 3).

Experimental Example 2: PPI Network and Module Analysis

In hypermethylation, a total of 367 nodes and 756 edges were set, and in hypomethylation, 360 nodes and 644 edges were set. The PPI networks for hypermethylation and hypomethylation are shown in Figures 4 and 5, respectively. Three modules of hypermethylated genes (Figs. 6 to 8) and two modules of hypomethylated genes (Figs. 9 and 10) were constructed with statistical significance. Enrichment analysis showed that the hypermethylation module was mainly responsible for antigen processing (Ubiquitination & Proteasome degradation), Golgi-to-ER retrograde transport and G alpha (i). signaling event (G alpha (i) signaling event)]. Regarding the two hypermethylation modules, post-translational protein phosphorylation and regulation of natural killer cell chemotaxis were closely related.

In addition, when comparing patients with delayed cerebral ischemia after subarachnoid hemorrhage and patients with non-delayed cerebral ischemia, the 5 hub genes related to hypermethylation in patients with delayed cerebral ischemia were VHL (von Hippel-Lindau), KIF3A (kinesin family) member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1) and OPRM1 (Opioid Receptor Mu 1), two hub genes associated with hypomethylation in patients with delayed cerebral ischemia are albumin (ALB) and Interleukin 5 (IL5) (Table 2), it can be seen that the hub genes are hypermethylated or hypomethylated in patients with delayed cerebral ischemia after subarachnoid hemorrhage.

In addition, methylation regions of 7 types of genes hypermethylated or hypomethylated in patients with delayed cerebral ischemia after subarachnoid hemorrhage were specifically identified, and the results are shown in Table 3 below.

Taken together, in the case of patients with delayed cerebral ischemia after subarachnoid hemorrhage, five hub genes related to hypermethylation, VHL (von Hippel-Lindau), KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3) , Hypermethylation of RACGAP1 (Rac GTPase Activating Protein 1) or OPRM1 (Opioid Receptor Mu 1) genes was observed, or hypomethylation of ALB (albumin) or IL5 (Interleukin 5) genes, which are two hub genes related to hypomethylation, was observed Accordingly, it can be seen that delayed cerebral ischemia occurring after subarachnoid hemorrhage can be effectively diagnosed or the prognosis can be predicted by analyzing the degree of hypermethylation or hypomethylation of each of the seven genes or a combination thereof.

So far, the present invention has been mainly looked at with respect to preferred embodiments. Those skilled in the art to which the present invention belongs will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from a descriptive point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

As described above, although the present invention has been described by the limited embodiments and drawings, the present invention is not limited thereto, and the technical spirit of the present invention and the following by those skilled in the art to which the present invention belongs Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

<110> Industry Academic Cooperation Foundation, Hallym University <120> VHL Gene hypermethylation marker for diagnosis of delayed cerebral ischemia <130> 1 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 129 <212> DNA <213> artificial sequence <220> <223> VHL_hypermethylation <400> 1 ccttgtgatc agcccacttc agcctcccaa agtactggga ttacaggcat gagccactgc 60 acccggccat agttgctatt ttatttgtgt gattgattac cgtatgacgc tttattgaa 120 gtgcagtga 129 <210> 2 <211> 119 <212> DNA <213> artificial sequence <220> <223> KIF3A_hypermethylation <400> 2 atatcatcat acactaaagg aacaactaaa atatcttgtt attatgtcaa actcaggacc 60 cgaaggctcc ctaagtcaga gtaaacatgc caagtgtcaa atataagtag aaatactac 119 <210> 3 <211> 119 <212> DNA <213> artificial sequence <220> <223> KIFAP3_hypermethylation <400> 3 gcacgtagtt gctctgctgc ttttttcaaa tgtagtacat tgatgcaatt attgattatc 60 cgttatgtgt aaaacaccat gttaaggcac tgcatggtat attaaatata gaaatacag 119 <210> 4 <211> 119 <212> DNA <213> artificial sequence <220> <223> RACGAP1_hypermethylation_1 <400> 4 gactgtatca ggagatgacc ttttatgcag aagcatcact gtaagttaaa agaagtgtgc 60 cgccccataa aagcagggca acaaatagtt taacatgtta accattgttg atgaaagca 119 <210> 5 <211> 119 <212> DNA <213> artificial sequence <220> <223> RACGAP1_hypermethylation_2 <400> 5 gaaccaaact aattcagaaa taaggcacaa gtctatttaa gagcagcaaa taaaccctga 60 cgacatacag tcttgctaat atttttatta ttagctgggg ctttaataag tcaaaatat 119 <210> 6 <211> 119 <212> DNA <213> artificial sequence <220> <223> OPRM1_hypermethylation <400> 6 taacagggca agatcctatc caaaaaaaaa aaaaaaggaa gaaactcaac aaagcagcat 60 cgttgctatt attgcagcta tttagccaat aggtacatca ttgacatcat tgtaaatag 119 <210> 7 <211> 119 <212> DNA <213> artificial sequence <220> <223> ALB_hypomethylation <400> 7 gagtctagtt aataatctac aattattggt taaagaagta tattagtgct aatttccctc 60 cgtttgtcct agcttttctc ttctgtcaac cccacacgcc tttggcacaa tgaagtggg 119 <210> 8 <211> 119 <212> DNA <213> artificial sequence <220> <223> IL5_hypomethylation_1 <400> 8 gtctttgagg aaatgaataa tttctaacaa tcagatagag aatgcctaat aatggcatat 60 cgtgaaacta aatgtttcca atgccttata tcttaaaaaa taaatttact tttatcttt 119 <210> 9 <211> 119 <212> DNA <213> artificial sequence <220> <223> IL5_hypomethylation_2 <400> 9 ataattttaa tgttttgttc cattagttat tttaactatt ttgtaataac tgacactttc 60 cggttttttt taacctggta cagcaagaga ctatttctca ctggtcttcc ccacttgca 119

Claims (11)

Including an agent capable of measuring the methylation level of the VHL (von Hippel-Lindau) gene,
Measuring the methylation level of the von Hippel-Lindau ( VHL ) gene is to measure the methylation level of any one or more of the 62nd to 65th nucleotides of SEQ ID NO: 1,
A composition for diagnosing delayed cerebral ischemia.
delete According to claim 1,
The composition is from the group consisting of KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1), OPRM1 (Opioid Receptor Mu 1), ALB (albumin) and IL5 (Interleukin 5) Further comprising an agent capable of measuring the methylation level of one or more selected genes,
Measuring the methylation level of the KIF3A (kinesin family member 3A) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 2,
Measuring the methylation level of the kinesin associated protein 3 (KIFAP3) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 3,
Measuring the methylation level of the RACGAP1 (Rac GTPase Activating Protein 1) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides in the sequence of SEQ ID NO: 4 or 5,
Measuring the methylation level of the OPRM1 (Opioid Receptor Mu 1) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 6,
Measuring the methylation level of the ALB (albumin) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 7,
Measuring the methylation level of the IL5 (Interleukin 5) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides in the sequence of SEQ ID NO: 8 or 9,
A composition for diagnosing delayed cerebral ischemia.
According to claim 1,
The delayed cerebral ischemia is a composition for diagnosing delayed cerebral ischemia, which occurs after subarachnoid hemorrhage.
A kit for diagnosing delayed cerebral ischemia, comprising the composition of claim 1.
Measuring the methylation level of the von Hippel-Lindau ( VHL ) gene from a biological sample isolated from the subject,
Measuring the methylation level of the VHL (von Hippel-Lindau) gene is to measure the methylation level of any one or more of the 62nd to 65th nucleotides of SEQ ID NO: 1,
Information provision method for diagnosis of delayed cerebral ischemia.
7. The method of claim 6, wherein the method
Measuring the methylation level of the VHL gene from the biological sample isolated from the control group; and
Comparing the methylation level of the subject and the control group
Which further comprises, information providing method for the diagnosis of delayed cerebral ischemia.
delete The method of claim 6, wherein the method comprises from biological samples isolated from individuals and controls,
At least one selected from the group consisting of KIF3A (kinesin family member 3A), KIFAP3 (kinesin associated protein 3), RACGAP1 (Rac GTPase Activating Protein 1), OPRM1 (Opioid Receptor Mu 1), ALB (albumin), and IL5 (Interleukin 5) Further comprising measuring the methylation level of the gene,
Measuring the methylation level of the KIF3A (kinesin family member 3A) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 2,
Measuring the methylation level of the KIFAP3 (kinesin associated protein 3) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 3,
Measuring the methylation level of the RACGAP1 (Rac GTPase Activating Protein 1) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides in the sequence of SEQ ID NO: 4 or 5,
Measuring the methylation level of the OPRM1 (Opioid Receptor Mu 1) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 6,
Measuring the methylation level of the ALB (albumin) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides of SEQ ID NO: 7,
Measuring the methylation level of the IL5 (Interleukin 5) gene is to measure the methylation level of any one or more of the 59th to 62nd nucleotides in the sequence of SEQ ID NO: 8 or 9,
Information provision method for diagnosis of delayed cerebral ischemia.
delete 7. The method of claim 6, wherein the method
A method for providing information for diagnosing delayed cerebral ischemia, comprising measuring the methylation level of a nucleotide comprising SEQ ID NO: 1 from a biological sample isolated from an individual.

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