KR102281644B1 - INSR Gene hypermethylation marker for diagnosis of delayed cerebral ischemia - Google Patents

Abstract

The present invention provides a composition for diagnosis of delayed ischemia comprising an agent capable of measuring the methylation level of an insulin receptor (INSR) gene, a kit for diagnosis of delayed ischemia comprising the composition, and diagnosis of delayed ischemia using the composition and kit It relates to a method of providing information for
According to the composition for diagnosing delayed ischemia, the kit for diagnosing delayed ischemia, and the method for providing information for diagnosing delayed ischemia of the present invention, the methylation level of the distal intergenic region upstream of the INSR gene is measured to delay occurrence after subarachnoid hemorrhage. It can be used for predicting or diagnosing the risk of developing sexual ischemia.

Description

INSR Gene hypermethylation marker for diagnosis of delayed cerebral ischemia

The present application provides a composition for diagnosing delayed ischemia comprising an agent capable of measuring the methylation level of an insulin receptor (INSR) gene, a kit for diagnosing delayed ischemia comprising the composition, and diagnosis of delayed ischemia using the composition and kit How to provide information.

Subarachnoid hemorrhage (SAH) due to rupture of a cerebral aneurysm is a life-threatening disease with a mortality rate of 45% within 30 days of onset. In addition, delayed cerebral ischemia (DCI) may occur within 30 to 40% of subarachnoid hemorrhage 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 arrhythmias, intestinal bleeding and urinary tract infection compared to patients without delayed ischemia, which leads to prolonged hospitalization and medical costs. increase, etc. The onset of this delayed ischemia is influenced by various clinical and radiological parameters. Well-known clinical risk factors for delayed ischemia are female sex, smoking, and low clinical grade at hospitalization. A high 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 (SNP) or genome-wide association studies (GWAS) are being conducted. However, only 5 to 10% of the susceptibility genetic variants were observed, suggesting that there are still unknown various genetic variants beyond the DNA sequence scale.

As one of the unknown novel pathways, there are epigenetic mechanisms including DNA methylation, histone modifications and non-coding RNAs. Among them, 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 studies have been conducted to confirm the epigenetic profile of delayed ischemia in patients with subarachnoid hemorrhage.

Accordingly, the present inventors have tried to develop a method for efficiently and easily diagnosing the onset of delayed ischemia, predicting the onset risk, and predicting the prognosis after subarachnoid hemorrhage. As a result, the methylation level of a specific gene or the gene expression level The present invention was completed by developing a diagnostic composition capable of diagnosing or predicting delayed ischemia by measuring.

Korean Patent Publication No. 10-2019-0008216

The present application provides a composition for diagnosing delayed ischemia comprising an agent capable of measuring the methylation level of an insulin receptor (INSR) gene, a kit for diagnosing delayed ischemia comprising the composition, and diagnosis of delayed ischemia using the composition and kit We want to provide a way to provide information.

The objects of the present application are not limited to the above-mentioned objects, and other objects and advantages of the present application that are not mentioned may be understood by the following description, and will be more clearly understood by the examples of the present application. It will also be readily apparent that the objects and advantages of the present application may be realized by means of the instrumentalities and combinations thereof indicated in the appended claims.

A first aspect of the present application is to provide a composition for diagnosing delayed ischemia, comprising an agent capable of measuring the methylation level of an insulin receptor (INSR).

A second aspect of the present application is to provide a composition for diagnosing delayed ischemia, comprising an agent capable of measuring the expression level of an insulin receptor (INSR).

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

A fourth aspect of the present application is to provide an information providing method for diagnosing delayed ischemia, comprising measuring the methylation level of an insulin receptor (INSR) gene from a biological sample isolated from an individual.

According to the composition for diagnosing delayed ischemia, the kit for diagnosing delayed ischemia, and the method for providing information for diagnosing delayed ischemia of the present invention, the methylation level of the INSR gene is measured to predict the risk of developing delayed ischemia after subarachnoid hemorrhage or can be used for diagnosis.

1 is a diagram illustrating a delayed ischemia diagnosis process using hypermethylation of an INSR gene.
FIG. 2 is a diagram showing the results of an anterior and posterior biogenomic association study for delayed ischemia.
3 is a diagram showing the nucleotide sequence of the hypermethylated region of the INSR gene and the positions of primers used for methylation-specific PCR.
4 is a diagram showing the nucleotide sequence of the hypermethylated region of the CDHR5 gene and the positions of primers used for methylation-specific PCR.
5 shows the results of methylation-specific PCR analysis of the INSR gene and the CDHR5 gene, specifically, hierarchical clustering according to the difference in methylation between patients with delayed ischemia and patients with non-delayed ischemia.
6 is a diagram showing the mRNA expression level of INSR according to delayed ischemia after subarachnoid hemorrhage.
7 is a diagram showing the mRNA expression level of CDHR5 according to delayed ischemia after subarachnoid hemorrhage.
8 is a diagram showing the hypermethylation levels of INSR and CDHR5 according to delayed ischemia after subarachnoid hemorrhage.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

A first aspect of the present application provides a composition for diagnosing delayed ischemia, comprising an agent capable of measuring the methylation level of an insulin receptor (INSR) gene.

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

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

As used throughout this specification, the term "INSR" is a gene encoding an insulin receptor, and isoforms of insulin receptor A (IR-A) and B (IR-B) by splicing of the gene. This can be translated.

In one embodiment of the present application, the region for measuring the methylation level of the INSR gene of the composition may specifically include the 7194868th to 7195079th sequence of human chromosome 19, and specifically may be the 7194996th, but this It is not limited.

In one embodiment of the present application, the composition may measure the methylation level of the CpG site ID: cg00441765 moiety.

In one embodiment of the present application, the composition may further include an agent capable of measuring the methylation level of a cadherin related family member 5 (CDHR5) gene.

In one embodiment of the present application, the composition may further include an agent capable of measuring the methylation level of the nucleotide comprising SEQ ID NO: 2, but is not limited thereto.

In one embodiment of the present application, the region for measuring the methylation level of the CDHR5 gene of the composition may specifically include the 618990th to 619260th sequence of human chromosome 11, and specifically may be the 619080th, but this It is not limited.

As used throughout this specification, the term "cadherin related family member 5 (CDHR5)" is one of the cadherin superfamily, and a sequence encoding calcium-binding motifs found in all cadherins. It is known to encode various isoform proteins by splicing.

In one embodiment of the present application, the composition may measure the methylation level of the CpG site ID: cg11464053 moiety.

In one embodiment of the present application, the composition includes an agent capable of measuring the methylation level of one or more moieties selected from the group consisting of CpG site ID: cg08969578, cg15090337, cg02180699, cg26306080, cg26826512, cg09217327, cg23222472, and cg19751670. may include.

In one embodiment of the present application, the composition may further include an agent capable of measuring the methylation level of one or more genes selected from the group consisting of ILDR1, NOL4L, ZNF706, IR640, MAP3K13, ASCC2, LSMEM1 and LOC389641 there is.

As used throughout this specification, the term "methylation" or "DNA methylation" refers to the attachment of a methyl group to a base constituting DNA. For example, whether the methylation is a specific CpG (Cytosine) of a specific gene It may mean whether methylation occurs in the cytosine of the phosphate guanine) site. In general, when methylation or hypermethylation occurs, the binding of transcription factors is disturbed and the expression of a specific gene may be suppressed. Conversely, if unmethylation or hypomethylation occurs, the expression of a specific gene may increase. there is.

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

As used throughout this specification, the term "measurement of methylation level" refers to measuring the methylation level of the CpG region of a specific gene, specifically, the INSR gene and/or the CDHR5 gene, and includes methylation-specific PCR, for example, methylation-specific PCR. (methylation-specific polymerase chain reaction, MSP), real time methylation-specific polymerase chain reaction (PCR), PCR using a methylated DNA-specific binding protein, or quantitative PCR. Alternatively, pyrosequencing, MS-HRM (Methylation-Sensitive High-Resolution Melting Analysis) and methylation determination using methylation-sensitive restriction enzymes, automatic base analysis such as DNA chip and bisulfite sequencing It can be measured by a method such as, but is not limited thereto.

According to one embodiment of the present application, "an agent capable of measuring the methylation level" means a molecule that can be used to measure the methylation level of a specific DNA region, specifically methylation for INSR gene and/or CDHR5 gene- It may include a primer or a probe for performing specific PCR, but is not limited thereto.

The primer used for measuring the methylation level may include a primer specific for the methylated allele sequence of the gene and a primer specific for the unmethylated allele sequence of the gene, wherein the primer is a target for analyzing whether methylation is A pair of primers that can be desirably designed according to the sequence of a specific CpG site, each capable of amplifying methylated cytosines that have not been modified by bisulfite, and cytosines that are not methylated and modified by bisulfite It may be a pair of primers capable of specifically amplifying.

As used throughout this specification, the term "primer" refers to a nucleotide sequence having a short free 3' hydroxyl group, which can form a complementary template and a base pair and copy the template strand. It refers to a short sequence that serves as a starting point for Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. In this case, PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

The probe used to measure the methylation level may refer to a hybridization probe, and refers to an oligonucleotide capable of sequence-specific binding to a complementary strand of a nucleic acid. The probe may be used in a kit or a prediction method for diagnosing delayed ischemia by measuring the methylation level. Probes of interest may be labeled so that they can be detected, for example with radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. Appropriate labeling of the probe as described above is a technique well known in the art, and can be performed by 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 may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoros. amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In one embodiment of the present application, the agent capable of measuring the methylation level may be a primer set that can be used for methylation-specific PCR, specifically, a methylation-specific primer set and a non-methylation-specific primer set. may include.

In one embodiment of the present application, the methylation-specific primer set for measuring the methylation level of the INSR gene may include a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, and the non-methylation-specific primer set The red primer set may include a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6.

In one embodiment of the present application, the methylation-specific primer set for measuring the methylation level of the CDHR5 gene may include a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8, and the non-methylation-specific primer set The red primer set may include a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10.

According to an embodiment of the present application, as a result of confirming the methylation level of the INSR gene in patients with delayed ischemia and non-patients using methylation-specific PCR, it was confirmed that the methylation level was higher in patients with delayed ischemia. Therefore, based on the above information, the methylation level of the INSR gene, specifically the methylation level of the region containing the nucleotide of SEQ ID NO: 1, is checked to diagnose or develop delayed ischemia, specifically delayed ischemia after subarachnoid hemorrhage. risk can be predicted.

According to an embodiment of the present application, as a result of confirming the methylation level of the CDHR5 gene in patients with delayed ischemia and non-patients using methylation-specific PCR, it was confirmed that the methylation level was higher in patients with delayed ischemia. Therefore, based on the above, the methylation level of the CDHR5 gene, specifically the methylation level of the region containing the nucleotide of SEQ ID NO: 2, is checked to diagnose or develop delayed ischemia, specifically, delayed ischemia that occurs after subarachnoid hemorrhage. risk can be predicted.

According to one embodiment of the present application, the composition for diagnosis of delayed ischemia may include an agent capable of measuring the methylation level of the nucleotide comprising SEQ ID NO: 1.

According to one embodiment of the present application, the composition for diagnosis of delayed ischemia may further include an agent capable of measuring the methylation level of the nucleotide comprising SEQ ID NO: 2.

According to one embodiment of the present application, the INSR gene may be included in a marker composition for diagnosis of delayed ischemia.

According to one embodiment of the present application, the CDHR5 gene may be included in a marker composition for diagnosis of delayed ischemia.

As used throughout this specification, the term “marker” refers to a biomarker, and it is possible to detect a change in a living organism and objectively measure the normal or pathological state of the organism, the degree of response to a drug, and the like.

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

As used throughout this specification, the term “diagnosis” refers to determining whether delayed ischemia has already occurred in a specific individual.

The term "prediction" used throughout the present specification refers to whether a specific individual is likely to develop delayed ischemia, and if there is a possibility of developing delayed ischemia, it is determined whether the likelihood of developing delayed ischemia is relatively high compared to a large number of unspecified individuals. means to do

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

According to an embodiment of the present application, the INSR gene in a patient with delayed ischemia shows a higher methylation level than that of a patient without delayed ischemia. It can be seen that the onset of the disease, the possibility of onset of delayed ischemia, and the like can be diagnosed.

According to an embodiment of the present application, the composition may be a composition for diagnosing delayed ischemia in Koreans and Asians.

A second aspect of the present application provides a composition for diagnosing delayed ischemia, comprising an agent capable of measuring the expression level of an insulin receptor (INSR). The content overlapping with the first aspect also applies to the composition for diagnosis of delayed ischemia of the second aspect.

According to an embodiment of the present application, it was confirmed that the INSR gene region is hypermethylated, resulting in a decrease in the expression level of INSR, and delayed ischemia may occur therefrom. The methylation level of the INSR gene and/or INSR By checking the expression level of delayed ischemia, it is possible to diagnose whether or not the onset of delayed ischemia, specifically, delayed ischemia caused by subarachnoid hemorrhage, or predict the risk of the onset.

According to one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of cadherin related family member 5 (CDHR5).

According to an embodiment of the present application, it was confirmed that the CDHR5 gene region is hypermethylated, thereby reducing the expression level of CDHR5, and delaying ischemia may occur therefrom. The methylation level of the CDHR5 gene and/or CDHR5 By checking the expression level of delayed ischemia, it is possible to diagnose whether or not the onset of delayed ischemia, specifically, delayed ischemia caused by subarachnoid hemorrhage, or predict the risk of the onset.

As used throughout the present specification, the term "measurement of expression level" refers to measuring the expression or level of expression of a specific gene or protein encoded by the gene, specifically mRNA of the INSR gene or protein encoded by the gene. may be to measure the expression level of the CDHR5 gene or may be to measure the expression level of the protein encoded by the additional CDHR5 gene, but is not limited thereto.

According to one embodiment of the present application, the method for measuring the mRNA level is reverse transcriptase polymerization (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT -PCR), quantitative RT-PCR, RNase protection method, Northern blotting, or DNA chip technology.

According to one embodiment of the present application, the method for measuring the level of the protein is Western blotting (Western blotting), ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: radioimmunoassay), radioimmuno-diffusion (radical immunodiffusion), Ouchterlony immunodiffusion, rocket immunoeletrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence ), immunochromatography, FACS analysis (fluorescenceactivated cell sorter analysis), or protein chip technology.

According to one embodiment of the present application, "an agent capable of measuring the expression level" refers to a molecule that can be used to determine the expression level of a specific gene or protein encoded by the gene, specifically the gene or the It may include an agent capable of detecting and/or amplifying a protein, but is not limited thereto.

As used throughout this specification, the term "agent capable of detecting a specific gene or protein" refers to an agent capable of specifically binding to and recognizing the specific gene or protein, or detecting and amplifying, " An agent capable of amplifying a specific gene or protein" refers to an agent capable of increasing the number by repeating the replication of the specific gene or protein, for example, a polynucleotide containing the gene can be specifically amplified. It may mean a primer or a probe capable of specifically binding, but is not limited thereto.

According to one embodiment of the present application, the agent capable of measuring the expression level is a primer pair, probe or antisense nucleotide that specifically binds to the INSR gene or additionally CDHR5 gene, or to a protein encoded by the genes. It may be a specific antibody, but is not limited thereto.

The primers used for measuring the expression level of the gene are prepared under suitable conditions (for example, 4 different nucleoside triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature. -Can be a single-stranded oligonucleotide that can serve as a starting point for directing DNA synthesis, and the appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the polynucleotide of the region for measuring the expression level of the gene, but should be sufficiently complementary to hybridize.

According to one embodiment of the present application, the agent capable of measuring the expression level may be a primer set that can be used for quantitative real-time PCR (qPCR) for measuring the expression level of mRNA.

In one embodiment of the present application, the primer set for measuring the expression level of INSR may include a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12.

In one embodiment of the present application, the primer set for measuring the expression level of CDHR5 may include a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14.

According to an embodiment of the present application, as a result of confirming the mRNA expression levels of INSR and CDHR5 in patients with delayed ischemia and non-patients using quantitative real-time PCR, it was found that the mRNA expression levels of INSR and CDHR5 were lower in patients with delayed ischemia. Confirmed. Therefore, by checking the mRNA expression level of INSR and/or CDHR5 based on the above, it is possible to diagnose whether delayed ischemia, specifically, delayed ischemia that occurs after subarachnoid hemorrhage, or to predict the onset risk.

A third aspect of the present application provides a kit for diagnosing delayed ischemia, comprising the composition for diagnosing delayed ischemia. The contents overlapping with the first aspect and the second aspect are equally applied to the third aspect of the delayed ischemia diagnostic kit.

According to one embodiment of the present application, the kit may be a PCR kit, RT-PCR kit, qRT-PCR kit, methylation-specific PCR kit, DNA chip kit, Western blot kit, or ELISA kit, but is limited thereto no.

According to one embodiment of the present application, the kit may include polynucleotides, cDNAs, mRNAs, polypeptides, etc. of the present application, as well as other component compositions, solutions or devices suitable for the analysis method.

According to one embodiment of the present application, the PCT kit may be a kit including essential elements necessary for performing PCR. The PCR kit contains, in addition to polynucleotides, primers or probes specific for the genes or methylation regions, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), and 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 necessary for performing the DNA chip, and the DNA chip kit is a polynucleotide, primer or probe specific for the genes or methylation region. includes 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 including essential elements necessary for performing RT-PCR, and the RT-PCR kit is specific for the genes or methylation region. In addition to primers, test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors , DEPC-water, sterile water, and the like.

A fourth aspect of the present application provides an information providing method for diagnosing delayed ischemia, comprising measuring the methylation level of an insulin receptor (INSR) gene from a biological sample isolated from an individual. The contents overlapping with the first to third aspects are equally applied to the information providing method for the diagnosis of delayed ischemia of the fourth aspect.

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

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

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

As used throughout this specification, the term "sample" refers to a material derived from an individual who has or is likely to develop delayed ischemia, and specifically, tissues, cells, 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 a nucleic acid, for example, DNA, mRNA, or cDNA synthesized from mRNA, and the DNA methylation level or expression of a specific gene/protein therefrom. 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, a Korean or an Asian, but is not limited thereto.

According to one embodiment of the present application, in order to perform risk prediction or diagnosis of the individual, the method further comprises analyzing non-genetic information, wherein the non-genetic information includes gender, age, hypertension, diabetes, It may be one or more selected from the group consisting of hyperlipidemia and smoking.

According to one embodiment of the present application, the method comprises: measuring the methylation level of the INSR gene from a biological sample isolated from a control; and comparing the methylation level of the subject and the control group.

As used throughout this specification, the term “control group” may refer to a general individual or a non-patient group that has not developed delayed ischemia.

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

In one embodiment of the present application, the method may further comprise measuring the methylation level of the CDHR5 gene.

According to one embodiment of the present application, the method comprises the steps of diagnosing the subject as having developed delayed ischemia or predicting the risk of developing it at a high level when the methylation level of the CDHR5 gene of the subject is higher than the methylation level of the control group It may be to further include.

A fifth aspect of the present application provides an information providing method for diagnosing delayed ischemia, comprising measuring an expression level of an insulin receptor (INSR) from a biological sample isolated from an individual. The contents overlapping with the first to fourth aspects are equally applied to the information providing method for the delayed ischemia diagnosis of the fifth aspect.

According to one embodiment of the present application, the method comprises the steps of: measuring the expression level of INSR from a biological sample isolated from a control; And it may further comprise the step of comparing the expression level of the INSR of the subject and the control.

According to one embodiment of the present application, when the expression level of the INSR of the subject is lower than the expression level of the INSR of the control, the subject is diagnosed as having delayed ischemia or the risk of onset is predicted at a high level It may be to further include a step.

According to one embodiment of the present application, the method may further comprise measuring the expression level of CDHR5.

According to one embodiment of the present application, the method includes measuring the expression level of CDHR5 from a biological sample isolated from a control; and comparing the expression levels of the genes and/or proteins of the individual and the control group.

According to one embodiment of the present application, when the expression level of CDHR5 of the subject is lower than the expression level of CDHR5 of the control, the subject is diagnosed as having delayed ischemia or the risk of onset is predicted at a high level It may be to further include a step.

[Example]

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

Example 1: Cohort Collection and Study

The cohort for performing the present application was derived from the Chuncheon Sacred Heart Hospital stroke database. In this database, the subarachnoid hemorrhage patients for the Examples and Experimental Examples herein met the following conditions: 1) an adult over 18 years old, and 2) a subarachnoid due to aneurysmal rupture associated with a saccular appearance. bleeding. In addition, patients with the following conditions were excluded: 1) spinal, anatomical, traumatic, and infectious aneurysms, 2) concomitant cerebrovascular disease such as arteriovenous malformation or dural arteriovenous fistula, and 3) Angiogram-negative subarachnoid hemorrhage. This application was carried out in two stages: a discovery stage and a replication stage. In the discovery stage, epigenetic association studies were used to identify sensitive epigenetic markers in 40 patients with subarachnoid hemorrhage. In addition, in the replication phase, methylation-specific PCR was performed in 36 independent cohorts.

The present application developed an epigenetic pattern of blood expression potential markers for predicting delayed ischemia after subarachnoid hemorrhage. Delayed ischemia has been defined as a novel neurological deficit including motor disturbances, sensory changes, aphasia and reduced levels of consciousness with concomitant cerebral vasospasm. Clinical demographics of radiographically detected sex, age, hypertension (HTN), diabetes (diabetes mellitus, DM), hyperlipidemia, smoking and aneurysms were reviewed. Our study was approved by the Institutional Audit Committee (No. 2016-3 and 2017-9 and 2018-6).

Example 2: Extraction and Quantitation of Genomic DNA

To extract genomic DNA (gDNA), a whole blood sample was centrifuged at 3000 g for 4 minutes at room temperature to obtain a smoke film, and the smoke film fraction sample was stored at -80°C. gDNA was extracted from the smoke screen using the FlexiGene DNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration and purity of the extracted gDNA was confirmed by measuring the absorbance ratio (A260/A280) using a UV spectrophotometer Biospectrophotometer (Eppendorf, Hamburg, Germany).

Example 3: Epigenome-wide association study (EWAS)

To evaluate the DNA methylation profile at the genome scale, the following experiments were performed.

First, to perform Infinium MethylationEPIC assay on genomic DNA, genomic DNA was treated with sodium bisulfite using EZ-96 DNA methylation kit (Zymo Research, CA, USA) according to the manufacturer's instructions. did. DNA methylation was quantified using Infinium MethylationEPIC (EPIC) BeadChip (Illumina, CA, USA).

Potentially present raw quality probes in the raw data were filtered using the minfi package (version 1.24.0.). First, samples with insignificant mean detection P-values (>0.05) were excluded. A functional normalization algorithm (preprocessFunnorm) for Illumina methylated microarrays implemented in the minfi package was used to remove unwanted mutations by returning the variability to the control probes present in the methylated microarray. Finally, differentially methylated CpG sites (DMCpGs) were identified with a q-value cutoff of 0.00005 using minfi (dmfFinder). To report methylation levels, methylation and non-methylation signals were used to calculate beta values and M-values.

Example 4: Methylation-specific PCR

Hydrogen sulfite modification was performed on approximately 2 μg of DNA using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly describing the above process, gDNA was mixed with hydrogen sulfite mix and DNA protection buffer, and then converted to a SimpliAmp Thermal cycler (Thermo Fisher Scientific, MA, USA). The conversion cycling conditions were as follows: 95° C., 5 minutes, 60° C., 25 minutes 95° C., 5 minutes, 60° C., 85 minutes, 95° C., 5 minutes, and 60° C., 175 minutes. Finally, hydrogen sulfite-modified DNA was collected using 20 μl of EB buffer.

In addition, the sequence of the primer for performing methylation-specific PCR, the size of the PCR product, and the target region to be amplified by PCR are described in Table 1, and the PCR reaction for both non-methylated and methylated primers was performed under the following conditions. 45 cycles: 95° C., 15 sec, 55° C., 15 sec and 72° C., 30 sec, using 1.25 U Taq (iTaq, Intron Biotechnology, Seoul, Korea), final volume 20 μl. The PCR product obtained in the above reaction was analyzed by electrophoresis on a 3% agarose gel and visualized in UV.

primer sequence (5'-3') product
Size (bp) location SEQ ID NO: INSR-MF GGATAATGTTAAATTTGGATGTTTC 233 chr19:7194868-7195079 3 INSR-MR ACTAATACAAATATAAACCACCGCG 4 INSR-UF GGATAATGTTAAATTTGGATGTTTT 231 5 INSR-UR TAATACAAATATAAACCACCACACC 6 CDHR5-MF TGGGTTTTGGATTAGTATTATTTTCGA 248 chr11:618990-619260 7 CDHR5-MR CCACACCGAAAAACATCGAC 8 CDHR5-UF TGGGTTTTGGATTAGTATTATTTTTGA 248 9 CDHR5-UR TCCCACACCAAAAAACATCAAC 10

*a: U indicates primers for unmethylated alleles.

*b: M indicates primers for methylated alleles

*c: The primer position of qPCR indicates the base from the start codon. The first nucleotide of the start codon is defined as position 1.

Example 5: Quantitative real-time PCR

To measure gene expression levels, quantitative real-time PCR (qPCR) was performed using Power SYBR Green PCR master Mix (Thermo Fisher Scientific, MA, USA). The qPCR primer sequences are shown in Table 2 below, and the PCR reaction was performed for 45 cycles under the following conditions: 94°C, 15 sec, 55°C, 30 sec and 70°C, 30 sec, Rotor-Gene Q (Qiagen, Hilden, Germany) used.

primer sequence (5'-3') product
Size (bp) SEQ ID NO: INSR-F AGACGTCCCGTCAAATATTGC 341 11 INSR-R ACTCGTTGACCGTCTTCACC 12 CDHR5-F GAGGGAGAGGTTGTGCTGAC 281 13 CDHR5-R CTCAGAGTTGTGCCAGAGGG 14

Example 6: Statistical Analysis

Differentially methylated CpG sites (DMCpGs) were adjusted by cutting off a P-value of 0.00005. To compare methylation profiles between delayed ischemia and non-delayed ischemia groups, scatter plots for probe methylation values and box plots for the genomic features of each group were generated by R script. The relative intensity of methylation was expressed as the median value with the interquartile range. Relative methylation strength was calculated as the degree of methylation divided by the combined strength of methylation and unmethylation. Data regarding relative degree of methylation and mRNA expression were compared using the Mann-Whitney U test. A P-value < 0.05 was considered statistically significant, and the analysis was performed using MedCalc software (Medcalc, Mariakerke, Belgium).

Experimental Example 1: Confirmation of clinical characteristics of the cohort group

The clinical characteristics of the cohort participating in the present experiment described in Example 1 were not significantly different according to female sex, hypertension, diabetes, hyperlipidemia, and smoking in delayed ischemia. However, patients with subarachnoid hemorrhage at the discovery stage tended to be younger than those with subarachnoid hemorrhage at the replication stage.

At the discovery stage, higher HH grades were observed more frequently in delayed ischemia than in non-delayed ischemia, but it was not statistically significant (p=0.311), and coil embolization was performed in most patients (80%), Of the 13 patients with delayed ischemia, 2 (15.4%) underwent chemical angiogenesis to reverse cerebral vasospasm.

For the replication phase, higher H-H grades and Fisher grades were observed in patients with delayed ischemia, but were not statistically significant (p=0.095, p=0.421, respectively).

In addition, most aneurysms (81%) were located in the anterior cerebral circulation.

Experimental Example 2: Confirmation of DNA methylation and mRNA expression level of differentially methylated genes in the discovery stage

An anterior and posterior biogenomic association study for delayed ischemia in 40 patients with subarachnoid hemorrhage was performed. A total of 35 CpG sites passed the cut-off (Fig. 2). Among the identified DMCpGs, cg00441765 and cg11464053 were non -The top two CpG sites showing hypermethylation were identified in patients with delayed ischemia compared with patients with delayed ischemia (Table 3).

CpG site ID P-value Q-value Chr Position Distance to TSS (bp) Gene Name Gene Description cg00441765 8.55E-11 1.90E-05 chr19 7194996 99317 INSR insulin receptor cg11464053 1.01E-10 1.90E-05 chr11 619080 5986 CDHR5 cadherin related family member 5 cg08969578 3.16E-10 2.54E-05 chr3 121717880 23247 ILDR1 immunoglobulin-like domain containing receptor 1 cg15090337 3.32E-10 2.54E-05 chr20 31165773 7102 NOL4L nucleolar protein 4 like cg02180699 4.02E-10 2.54E-05 chr8 102219149 -857 ZNF706 zinc finger protein 706 cg26306080 4.01E-10 2.54E-05 chr19 19550743 4871 MIR640 microRNA 640 cg26826512 5.05E-10 2.73E-05 chr3 185046528 -34308 MAP3K13 mitogen-activated protein kinase kinase kinase 13 cg09217327 9.73E-10 3.68E-05 chr22 30234641 -348 ASCC2 activating signal cointegrator 1 complex subunit 2 cg23222472 1.06E-09 3.68E-05 chr7 112135961 14895 LSMEM1 leucine rich single-pass membrane protein 1 cg19751670 8.56E-10 3.68E-05 chr8 23088262 5528 LOC389641 uncharacterized LOC389641

Next, as a result of hierarchical clustering of CpG sites based on differences in DNA methylation in patients with delayed ischemia, the top two CpG sites, cg00441765 and cg11464053, were the second intron of the insulin receptor (INSR) and the cadherin-related family member (CDHR5), respectively. 5) was confirmed to be located in exon 13 ( FIGS. 3 to 5 ).

Next, RT-PCR was performed to confirm the mRNA expression level of the top 10 genes in Table 3. As a result, it was confirmed that the mRNA level of INSR was decreased in patients with delayed ischemia compared to patients with non-delayed ischemia (0.00020 [0.00012-0.00030] vs. 0.00050 [0.00030-0.00068]; p=0.006) (Fig. 6). Also, for CDHR5, decreased mRNA levels were observed in patients with delayed ischemia compared with patients with non-delayed ischemia (0.114 [0.053-0.143] vs. 0.170 [0.110-0.212]; p=0.010) (Fig. 7). In addition, the association between the degree of methylation and the corresponding mRNA expression was not inversely proportional in other genes following delayed lingual development.

Combining the above results, it can be seen that hypermethylation occurs in some regions of the INSR gene and CDHR5 gene and the mRNA expression level is reduced in patients with delayed ischemia compared to patients with non-delayed ischemia. It can be seen that delayed ischemia can be effectively diagnosed based on the methylation level and mRNA expression level of the gene or CDHR5 gene.

Experimental Example 3: Confirmation of DNA methylation and mRNA expression level of differentially methylated genes in the discovery stage

In order to determine whether the methylation of the INSR gene and the CDHR5 gene identified in Experimental Example 2 differs between patients with delayed ischemia and patients with non-delayed ischemia, the methylation status of the same region as annotated in the Infinium MethylationEPIC assay was evaluated. Methylation-specific PCR was used in each of 36 subarachnoid hemorrhage patients.

As a result, it was confirmed that the methylation level was higher in patients with delayed ischemia than in patients with non-delayed ischemia ( FIG. 8 ).

- INSR: 0.855 (0,779-0.913) patients with delayed ischemia; 0.582 (0.565-0.689) patients with non-delayed ischemia.

- CDHR5: 0.786 (0.708-0.904) patients with delayed ischemia; 0.632 (0.610-0.679) patients with non-delayed ischemia.

Based on the above results, it can be seen that hypermethylation occurs in some regions of the INSR gene and CDHR5 gene in patients with delayed ischemia compared with patients with non-delayed ischemia.

So far, the present invention has been focused on preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive point of view. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within an equivalent scope should be construed as being included in the present invention.

As described above, although the present invention has been described with reference to limited embodiments and drawings, the present invention is not limited thereto, and the technical idea of the present invention and the following by those of ordinary skill in the art to which the present invention pertains. 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> INSR Gene hypermethylation marker for diagnosis of delayed cerebral ischemia <130> 1 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> INSR <400> 1 aggacaatgc taaacctgga tgtctcgagg acaggggatg gggtttttgt gtcatctatg 60 ttctgatgct ttttcattta atacgagaac aggtttccta tgatttggca cactgggaca 120 ttcgacatgt gtttgttgaa tgaaaaaaag aaaaaagaga aatgctaaca atttgttgaa 180 tagtccataa aagagcaaag ctggcctggc gcggtggctc acacctgtac cagc 234 <210> 2 <211> 256 <212> DNA <213> Artificial Sequence <220> <223> CDHR5 <400> 2 tgggccctgg accagcacca cttccgaggt ccccagaccc cctgagccct cccagggacc 60 ctccacgacc agctctgggg gaggcacagg ccctcatcca ccctctggca caactctgag 120 gccaccaacc tcgtccacac ccggggggcc cccgggtgca gaaaacagca cctccccacca 180 accagccact cccggtgggg acacagcaca gaccccaaag ccaggaacct ctcagccgat 240 gccccccggt gtggga 256 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> INSR-MF <400> 3 ggataatgtt aaatttggat gtttc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> INSR-MR <400> 4 actaatacaa atataaacca ccgcg 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> INSR-UF <400> 5 ggataatgtt aaatttggat gtttt 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> INSR-UR <400> 6 taatacaaat ataaaccacc acacc 25 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CDHR5-MF <400> 7 tgggttttgg attagtatta ttttcga 27 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDHR5-MR <400> 8 ccacaccgaa aaacatcgac 20 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CDHR5-UF <400> 9 tgggttttgg attagtatta tttttga 27 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CDHR5-UR <400> 10 tcccacacca aaaaacatca ac 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> INSR-F <400> 11 agacgtcccg tcaaatattg c 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> INSR-R <400> 12 actcgttgac cgtcttcacc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDHR5-F <400> 13 gagggagagg ttgtgctgac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDHR5-R <400> 14 ctcagagttg tgccagaggg 20

Claims (11)

delete delete delete delete A composition for diagnosis of delayed ischemia, comprising an agent capable of measuring the expression level of an insulin receptor (INSR).
6. The method of claim 5,
The composition further comprises an agent capable of measuring the expression level of CDHR5 (cadherin related family member 5), a composition for diagnosis of delayed ischemia.
A kit for diagnosing delayed ischemia, comprising the composition of claim 5 or 6. delete delete delete delete

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