WO2020027569A1 - Composition for diagnosing alzheimer's disease by using alteration of cpg methylation in promoter of ide gene in skin cells, and use thereof - Google Patents

Composition for diagnosing alzheimer's disease by using alteration of cpg methylation in promoter of ide gene in skin cells, and use thereof Download PDF

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WO2020027569A1
WO2020027569A1 PCT/KR2019/009529 KR2019009529W WO2020027569A1 WO 2020027569 A1 WO2020027569 A1 WO 2020027569A1 KR 2019009529 W KR2019009529 W KR 2019009529W WO 2020027569 A1 WO2020027569 A1 WO 2020027569A1
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disease
alzheimer
methylation
cpg
methylation level
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안정혁
성혜윤
정지향
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이화여자대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to compositions, kits and methods for diagnosing Alzheimer's disease by detecting the methylation level of the CpG region of the Insulin Degrading Enzyme (IDE) gene promoter.
  • IDE Insulin Degrading Enzyme
  • Alzheimer's disease (or Alzheimer's dementia), a representative senile disease, is the most common form of dementia, with symptoms such as loss of cognition in time and space, delusions and personality changes, speech paralysis, and motor paralysis. It is a representative degenerative brain disease that progresses gradually over a long period and eventually leads to death.
  • Alzheimer's disease Although research to treat Alzheimer's disease has been ongoing for the last several decades, there is no fundamental treatment to date, but only the level of treatment to alleviate the symptoms or slow the progression of the lesion. Therefore, Alzheimer's disease has been suggested as the most effective management method to delay the onset of dementia through early diagnosis and proactive treatment.
  • MMSE mini-mental state examination
  • the age, education level, or intentional rejection of an individual significantly affect the accuracy of the test results.
  • brain imaging test using MRI or PET it is difficult to perform the test in the early stage without any special symptoms because expensive equipment is used.
  • the present inventors confirmed that the promoter of the Insulin Degrading Enzyme (IDE) gene is hypermethylated specifically for Alzheimer's disease in skin cells, and using this as a biomarker, the degree of methylation of the promoter of the IDE gene is measured. It was confirmed that the diagnosis can be completed the present invention.
  • IDE Insulin Degrading Enzyme
  • one embodiment of the present invention provides a composition for diagnosing Alzheimer's disease, including an agent for measuring the CpG methylation level of the IDE gene promoter.
  • Another example provides the use of an agent to measure the CpG methylation level of an IDE gene promoter from a skin sample for diagnosing Alzheimer's disease.
  • Another example provides an agent for measuring the CpG methylation level of an IDE gene promoter from a skin sample for use in diagnosing Alzheimer's disease.
  • Another example provides an Alzheimer's disease diagnostic kit comprising an agent for measuring the CpG methylation level of an IDE gene promoter.
  • Another example provides a method of diagnosing Alzheimer's disease by measuring the methylation level at the CpG site of the IDE gene promoter.
  • Another example provides a method of providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level at the CpG site of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
  • Another example provides a method of measuring the methylation level at the CpG site of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease to provide information necessary for diagnosing Alzheimer's disease.
  • Other examples include measuring the methylation level of the CpG region of the IDE gene promoter from skin samples of individuals suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
  • Another example includes measuring a methylation level of a CpG region of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And administering an Alzheimer's disease therapeutic agent to said individual determined to have Alzheimer's disease.
  • the present invention provides a molecular biological diagnostic method for diagnosing Alzheimer's disease by measuring the degree of methylation of a specific gene CpG region of genomic DNA collected from an individual's skin sample, which is simple, non-invasive and economical.
  • DNA methylation changes are easy to detect, and are stable and easy to analyze compared to conventional protein or RNA markers.
  • Figure 1 shows the DNA methylation changes of the IDE gene promoter CpG site in human skin fibroblasts treated with toxic beta amyloid (A ⁇ ) or DMSO.
  • Figure 2 shows the changes in IDE gene expression in human skin fibroblasts treated with toxic beta amyloid (A ⁇ ) or DMSO.
  • Figure 3 shows the DNA methylation changes of the IDE gene in human skin fibroblasts treated with 5-aza-2'-deoxycytidin or DMSO.
  • Figure 4 shows the expression changes of the IDE gene in human skin fibroblasts treated with 5-aza-2'-deoxycytidin or DMSO.
  • Figure 5 shows the IDE genes in (a) brain tissue (hippocampus), (b) brain tissue (cortex) and (c) skin tissue (skin fibroblasts) of normal rat (Wt) and Alzheimer's dementia mouse model (Tg). Expression change is shown.
  • the present invention is based on the discovery that the promoter of the Insulin Degrading Enzyme (IDE) gene is specifically hypermethylated in Alzheimer's disease in skin cells, by measuring the degree of specific DNA hypermethylation occurring at a specific CpG position of the promoter of the IDE gene. Molecular biological diagnostic techniques for diagnosing diseases are provided.
  • IDE Insulin Degrading Enzyme
  • DNA methylation changes are easy to detect because they are present in DNA, are stable compared to protein or RNA markers, and are easy to analyze because they occur at specific positions in a gene.
  • one embodiment provides a composition for diagnosing Alzheimer's disease, comprising an agent for measuring the CpG methylation level of an IDE gene promoter from a skin sample.
  • Another embodiment provides the use of an agent to measure the CpG methylation level of an IDE gene promoter from a skin sample for diagnosing Alzheimer's disease.
  • Another embodiment provides an agent for measuring the CpG methylation level of an IDE gene promoter from a skin sample for use in diagnosing Alzheimer's disease.
  • Another embodiment provides a kit for diagnosing Alzheimer's disease comprising the composition.
  • kits for diagnosing Alzheimer's disease comprising an agent for measuring the methylation level of the CpG region of an Insulin Degrading Enzyme (IDE) gene promoter from a skin sample.
  • IDE Insulin Degrading Enzyme
  • Another embodiment provides a method of diagnosing Alzheimer's disease by measuring the methylation level at the CpG site of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
  • Another embodiment provides a method of providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level at the CpG site of an IDE gene promoter from a biological sample of an individual suspected of Alzheimer's disease.
  • Another embodiment provides a method of measuring the methylation level at the CpG site of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease to provide information necessary for diagnosing Alzheimer's disease.
  • Another embodiment includes measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
  • Another embodiment includes measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And administering an Alzheimer's disease therapeutic agent to said individual determined to have Alzheimer's disease.
  • methylation refers to the attachment of a methyl group to the base constituting the DNA.
  • whether or not methylation in the present invention means whether or not methylation occurs in the cytosine of a specific CpG region of a specific gene.
  • methylation occurs, the binding of transcription factors is disturbed, thereby inhibiting the expression of specific genes.
  • demethylation or hypomethylation occurs, expression of specific genes increases.
  • 5-methylcytosine In 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. Methylation of 5-methylcytosine occurs only in C of CG dinucleotides (5'-mCG-3 '), called CpG, and methylation of CpG inhibits the expression of alu or transposons and genome repeats. In addition, since 5-mC of CpG is naturally deaminoated to easily become thymine (T), CpG is a site where most epigenetic changes occur frequently in mammalian cells.
  • 5-mC of CpG is naturally deaminoated to easily become thymine (T)
  • T thymine
  • the term "measurement of methylation level” refers to measuring methylation level of the CpG region of a gene, such as methylation specific PCR, for example, methylation-specific polymerase chain reaction (MSP), real-time methylation specificity. It can be measured by real time methylation-specific polymerase chain reaction (PCR), PCR using methylated DNA-specific binding proteins, or quantitative PCR. Alternatively, it may be measured by a method such as autobase analysis such as pyro sequencing or bisulfite sequencing, or may be used as DNA methylation microarray, immunoprecipitation using methylated CpG binding domain or anti-methylcytosine antibody. However, it is not limited thereto.
  • hypermethylation in the promoter of the IDE gene is specifically shown in a sample of an Alzheimer's disease individual, and it is possible to diagnose Alzheimer's disease according to whether the promoter is methylated.
  • the CpG site of the gene refers to the CpG site existing on the DNA of the gene.
  • the DNA of a gene is a concept that includes a series of structural units necessary for the gene to express and are operably linked to each other.
  • a promoter region, an open reading frame (ORF), and a terminator region Include. Therefore, the CpG region of the gene may be present in a promoter region, an open reading frame (ORF), or a terminator region of the gene.
  • measuring the methylation level of the CpG region of the IDE gene promoter in the present invention the promoter CpG region of the IDE gene, more specifically, the CpG region appearing in the 94334776 to 94334897 base sequence (SEQ ID NO: 1) of chromosome 10 May mean measuring the methylation level of cytosine. More specifically, it may mean measuring the methylation of cytosine located at 94334836 base (SEQ ID NO: 61 base) of chromosome 10.
  • the nucleotide sequence of the human genomic chromosome region was expressed according to the latest version of the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38 / hg38), but the specific sequence of the human genomic chromosome region is expressed as the genome sequence research results are updated. This may be somewhat altered, and such alterations may result in different expressions of the human genomic chromosome region of the present invention. Therefore, the human genomic chromosome region expressed according to the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38 / hg38) of the present invention is updated after the filing date of the present invention, so that the human reference sequence is updated to express the human genomic chromosome region. Even if this change is different from the present, it will be obvious that the scope of the present invention extends to the modified human genomic chromosome region. Such changes are those that can be easily understood by those of ordinary skill in the art.
  • IDE gene is a gene encoding an insulin degrading enzyme, the enzyme decomposes insulin in the blood to lose insulin activity, glucagon, amylin, bradykinin and calidine It is a zinc-containing proteolytic enzyme that is involved in intracellular peptide signaling by breaking down the same diverse peptides. Mice that have lost the IDE gene are known to raise insulin levels and improve glucose tolerance.
  • the human IDE gene is located on chromosome 10 and is registered as Gene ID: 3416 in the NCBI Entrez database.
  • diagnosis means identifying the presence or characteristic of a pathological condition.
  • diagnosis is to confirm the development of Alzheimer's disease, to measure the preclinical condition before the onset of symptoms, or to determine whether the disease is progressing or deepening.
  • the agent for measuring the methylation level of the CpG region is a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base, primers specific for the methylated allele sequence of the gene, specific for the unmethylated allele sequence.
  • Primers, methylated CpG binding domains, or methylated DNA antibodies that specifically bind to methylated DNA eg, antibodies that specifically bind to methylcytosine and the like.
  • the compound that modifies the unmethylated cytosine base may be, but is not limited to, bisulfite or a salt thereof, preferably sodium bisulfite.
  • Bisulfite-modified DNA can detect methylation through various methods such as sequencing or methylation-specific PCR, and the method of detecting methylation of genes by modifying unmethylated cytosine residues using such bisulfite is It is well known in the art (for example, WO01 / 26536; US2003 / 0148326A1).
  • the methylation sensitive restriction enzyme may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme capable of specifically detecting the methylation of the CpG site.
  • a restriction enzyme capable of specifically detecting the methylation of the CpG site.
  • Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I and the like are not limited thereto.
  • Methylation sensitive restriction enzymes other than the restriction enzymes are well known in the art.
  • a compound or methylation is obtained which obtains genomic DNA from a subject's skin sample and modifies unmethylated cytosine bases in the obtained DNA.
  • the treated DNA can be measured by amplifying by PCR using a primer and confirming the presence of the amplified result.
  • the formulations of the present invention may comprise primers specific for the methylated allele sequence of the gene and primers specific for the unmethylated allele sequence.
  • the term “primer” refers to a short nucleic acid sequence that can form base pairs with complementary templates with nucleic acid sequences having short free 3-terminal hydroxyl groups and that serves as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • primers, as sense and antisense nucleic acids having 7 to 50 nucleotide sequences may incorporate additional features that do not change the basic properties of the primers that serve as the starting point for DNA synthesis.
  • Primers of the present invention may be preferably designed according to the sequence of the specific CpG site to be analyzed for methylation, primer pairs that can specifically amplify cytosine that is methylated and not modified by bisulfite, And primer pairs that are not methylated to specifically amplify cytosine modified by bisulfite.
  • a methylated DNA antibody means the antibody which specifically binds to the methylated base in DNA.
  • an antibody having a property of recognizing and binding to methylated cytosine in a DNA chain such as an antibody to methylcytosine, may be mentioned.
  • methylated DNA antibodies it may be an antibody that can specifically recognize and specifically bind the DNA in the methylated state described herein.
  • a methylated DNA antibody can be produced by a conventional method using a methylated base, methylated DNA, or the like as an antigen.
  • a methylcytosine antibody an antibody is prepared by using DNA containing 5-methylcytidine, 5-methylcytosine, or 5-methylcytosine as an antigen, and then methyl methyltocin in the DNA. Specific binding of can be selected as an indicator.
  • methylated DNA is immunoprecipitated using them, followed by Southern blot, PCR, microarray, or sequence. Specific CpG sites can be identified through sequencing.
  • a substrate, a suitable buffer, a chromophore or fluorophore label, a secondary antibody labeled with a chromophore or fluorophore, and a chromophoric substrate can be used.
  • the substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like.
  • the chromophore may be a peroxidase or an alkaline force. Alkaline phosphatase may be used, and fluorescent materials may be FITC, RITC, or the like, and the colorant substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfur).
  • Phonic acid Phonic acid
  • OPD o-phenylenediamine
  • TMB tetramethyl benzidine
  • radioisotope labels latex bead labels
  • colloid labels colloid labels
  • biotin labels etc.
  • composition and kit may further include a polymerase agarose, a buffer solution for electrophoresis, and the like.
  • kit may be implemented in the form of DNA methylated microarray.
  • the present invention relates to a method of diagnosing Alzheimer's disease by measuring the methylation level at the CpG site of an IDE gene promoter.
  • the present invention also relates to a method for detecting the methylation level of the CpG region of a gene from a skin sample of an individual suspected of Alzheimer's disease in order to provide information necessary for diagnosing Alzheimer's disease.
  • the present invention also relates to a method for providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level at the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
  • the present invention comprises the steps of measuring the methylation level of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; And comparing the methylation level with the methylation level of the corresponding gene promoter of the control sample, not Alzheimer's disease, to provide information for diagnosing Alzheimer's disease.
  • the present invention comprises the steps of measuring the methylation level of the CpG region of the IDE gene promoter from skin samples of individuals suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
  • the present invention comprises the steps of measuring the methylation level of the CpG region of the IDE gene promoter from skin samples of individuals suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And administering a treatment agent for Alzheimer's disease to the individual determined to have Alzheimer's disease.
  • the diagnosis method a method for providing information for diagnosis, and a treatment method will be described. .
  • skin sample includes skin tissues or skin cells having different methylation levels of genes due to Alzheimer's disease, and specifically, but not limited to skin fibroblasts and the like.
  • genomic DNA is obtained from skin samples of patients suspected of Alzheimer's disease to detect methylation levels.
  • SDS extraction Tinucleic acid
  • CTAB isolation Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980
  • a commercially available DNA extraction kit Can be done.
  • Detecting the methylation level of a specific CpG region of the gene may be achieved by using a restriction enzyme or bisulfite, using a difference between a methylated base and an unmethylated base, a methylated CpG binding domain or an anti-methylcytosine antibody.
  • Immunoprecipitation using eg, MIRA, MeDIP
  • DNA methylation may be carried out through a method using a microarray.
  • methylation-specific polymerase chain reaction for example, methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding proteins, quantitative PCR, Pyro sequencing, bisulfite sequencing, DNA methylation microarrays, or immunoprecipitation with methylated CpG binding domains or anti-methylcytosine antibodies can be used to measure or detect methylation levels.
  • measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease includes: (a) modifying the non-methylated cytosine base of genomic DNA in the sample obtained; Treating with a compound or methylation sensitive restriction enzyme; And (b) amplifying the treated DNA by PCR using a primer capable of amplifying a specific CpG site of the gene.
  • the compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite.
  • bisulfite preferably sodium bisulfite.
  • the methylation-sensitive restriction enzyme in step (a) may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme that can specifically detect methylation of a specific CpG site, as described above.
  • a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme that can specifically detect methylation of a specific CpG site, as described above.
  • Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I and the like but is not limited thereto.
  • Amplification in step (b) may be performed by a conventional PCR method.
  • the primers used may be preferably designed according to the sequence of the specific CpG region to be analyzed for methylation, as described above, and specifically amplify cytosine that is methylated and not modified by bisulfite. Possible primer pairs and primer pairs that are not methylated to specifically amplify cytosine modified by bisulfite.
  • step (c) confirming the presence of the result amplified in step (b) It may further comprise.
  • the presence of the result amplified in step (c) may be performed according to a method known in the art, for example, electrophoresis, according to whether a band of a desired position is detected.
  • primers capable of specifically amplifying two kinds of primer pairs used in step (a) namely, cytosine which was methylated and not modified by bisulfite, were used.
  • the degree of methylation can be determined according to the presence or absence of PCR results amplified by pairs and primer pairs that can specifically amplify cytosine modified by bisulfite, thereby not being methylated.
  • the methylation can be determined using a bisulfite genome sequencing method in which the sample genomic DNA is treated with bisulfite, the CpG region of the gene is amplified by PCR, and the nucleotide sequence of the amplified site is analyzed. .
  • CpG is determined to be methylated when there are PCR products in the DNA treated with restriction enzymes in a state known in the art, for example, when PCR products are displayed in mock DNA. If there is no PCR result in the enzyme-treated DNA, it can be determined whether or not the methylation according to the determination that CpG is unmethylated, which is obvious to those skilled in the art.
  • the mock DNA in the above means the sample DNA which is separated from the sample and is not treated at all.
  • an immunoprecipitation method using a methylated DNA antibody such as a methylated CpG binding domain (MBD) or an antibody against methylcytosine
  • a methylated DNA antibody such as a methylated CpG binding domain (MBD) or an antibody against methylcytosine
  • MBD methylated CpG binding domain
  • an antibody against methylcytosine can be exemplified.
  • specific CpG sites are identified by Southern blot, PCR, microarray, or sequencing. Can be.
  • the CpG region of the IDE gene in the target sample when the CpG region of the IDE gene in the target sample is detected with a high methylation state, it can be diagnosed or predicted as Alzheimer's disease.
  • the methylation change of a specific CpG region of the IDE gene is specifically shown in the skin samples of Alzheimer's disease individuals and affects the expression of the genes of the Alzheimer's disease individuals, so that the methylation changes of the genes are biomarkers. It can be used to diagnose Alzheimer's disease. For example, hypermethylation of specific CpG sites of the IDE gene promoter can be usefully used for diagnosing Alzheimer's disease using biomarkers.
  • Alzheimer's disease can be treated by administering an Alzheimer's disease therapeutic agent to the individual determined to have Alzheimer's disease.
  • the disease therapeutic agent may be administered in a therapeutically effective amount.
  • Alzheimer's disease therapeutic agents can use known therapeutic agents and include substances approved by the US Food and Drug Administration for the treatment of Alzheimer's disease.
  • Known Alzheimer's disease therapies include cholinergics / cholinesterase inhibitors, for example donepezil, rivastigmine, galantamine, or tacrine; As NMDA receptor antagonist, for example memantine; Examples of the antioxidants include, but are not limited to, selegiline, vitamin E, and the like.
  • Such therapeutic agents may be in pharmaceutically acceptable salt or ester form (eg, but not limited to, galantamine hydrobromide, rivastigmine tartrate, donepezil hydrochloric acid, and the like). In certain embodiments, these therapeutic agents may be used in combination of two or more.
  • pharmaceutically acceptable means a substance that can be effectively used for a desired use without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of the medical judgment.
  • pharmaceutically acceptable salts includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
  • pharmaceutically acceptable ester refers to esters that hydrolyze in vitro and include those that degrade directly in the human body to leave the parent compound or salt thereof.
  • Suitable ester groups include, for example, pharmaceutically acceptable aliphatic carboxylic acids, in particular those derived from alkano, alkeno, cyclicalkano and alkanedio acids, each alkyl or alkenyl powder advantageously It does not have more than six carbon atoms.
  • the therapeutic agents may additionally include a pharmaceutically acceptable carrier.
  • the dosage of the therapeutic agent depends on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous injection, and the like.
  • Normal human dermal fibroblasts (NHDF), primary skin dermal fibroblasts, were purchased from American type culture collection (ATCC no. PCS-201-012) and were treated with 2% (v / v) fetal bovine serum. , 1 ⁇ g / ml hydrocortisone, 10 ng / ml human epidermal growth factor, 3 ng / ml basic fibroblast growth factor, 10 ⁇ g / ml heparin Cultured in Medium 106 containing 100 U / ml penicillin, 100 ⁇ g / ml streptomycin, 30 ⁇ g / ml gentamicine and 15 ng / ml amphotericin B It was.
  • NHDF was treated with 1.5 ⁇ M toxic beta amyloid (A ⁇ 1-42 ) for 5 days to establish an Alzheimer's dementia cell model.
  • NHDF was treated with 1.5 ⁇ M dimethyl sulfoxide (DMSO) for 5 days instead of toxic beta amyloid.
  • DMSO dimethyl sulfoxide
  • NHDF was treated with 20 ⁇ M DNA methylation inhibitor 5-aza-2'-deoxycytidine (Sigma-Aldrich) for 3 days, and then the change of IDE gene expression was reverse transcription-quantitative polymerase chain reaction (RT). -qPCR).
  • RT reverse transcription-quantitative polymerase chain reaction
  • NHDF was treated with 20 ⁇ M DMSO for 3 days instead of 5-aza-2'-deoxycytidine.
  • DPBS Dulbecco's Phosphate-Buffered Saline
  • Fibroblasts were isolated by treatment at 300 rpm for 90 minutes at. The collagenase was exposed to 15% fetal bovine serum (FBS) and then centrifuged at 524 xg for 5 minutes. Centrifuged fibroblast precipitate was placed in DMEM / F12 medium with 15% FBS added and incubated in a 37% 5% CO 2 and 3% O 2 incubator. When the color of the medium turned yellow, half of the medium was removed and a new medium was added. After 14 days, the cells were incubated with EMEM medium containing 15% FBS, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin.
  • FBS fetal bovine serum
  • RNA and genomic DNA were extracted from mouse brain tissue, mouse skin fibroblast, and human skin fibroblast NHDF using RNeasy mini kit (Qiagen) and QIAmp mini kit (Qiagen), respectively. Extraction method was performed according to the manufacturer's manual. The extracted total RNA and genomic DNA were quantified using a spectrophotometer, and RNA and DNA states were electrophoresed on 1% agarose gel to confirm degradation.
  • a ⁇ 1-42 or NHDF treated with 5-aza-2'-deoxycytidine and NHDF treated with DMSO as a control group were subjected to genome-wide DNA methylation microarray using Infinium (®) Human Methylation EPIC BeadChip (Illumina).
  • the degree of DNA methylation is represented by a ⁇ value having a value of 0 to 1, and a ⁇ value of 0 means that the corresponding CpG site is completely unmethylated, and 1 means fully methylated.
  • Superscript II reverse transcriptase (Invitrogen) was used for cDNA synthesis. 1 ⁇ g of total RNA and 50 ng oligo dT were denatured at 70 ° C for 10 minutes, followed by 4 ⁇ l of 5X RT buffer, 2 ⁇ l of 0.1 mM DTT, 4 ⁇ l of 2.5 mM dNTP mixture, 200 units of Superscript II reverse transcriptase and RNase inhibitor 10 20 ⁇ l of the reaction solution mixed with the reaction solution containing the unit was reacted for 10 minutes at 25 °C, 50 minutes at 42 °C, 5 minutes at 95 °C to synthesize cDNA and dilute it 1: 4 to 2 ⁇ l Was used as a template.
  • qPCR contained 20 ⁇ l reaction solution containing 2 ⁇ l of cDNA, 10 ⁇ l of SYBR Premix EX Taq (Takara Bio), 0.4 ⁇ l of Rox reference dye (50 ⁇ , Takara Bio), and 200 nM of primers for each gene, and ABI 7500 fast sequence detection system (Applied Biosystems). After 30 seconds at 95 ° C, 40 cycles (3 seconds at 95 ° C, 30 seconds at 60 ° C) were repeatedly amplified. Specificity of the PCR product was confirmed by reacting for 15 seconds at 95 °C, 1 minute at 60 °C, 15 seconds at 95 °C. GAPDH mRNA expression was used as internal control, and IDE gene expression was corrected by ⁇ C T method to GAPDH expression level. Oligonucleotide primer sequences used are as follows.
  • Human skin fibroblast NHDF was cultured and treated with toxic beta amyloid (A ⁇ 1-42 ) for a long time (5 days at 1.5 ⁇ M), which is one of the pathogens of Alzheimer's dementia, to establish a cell model showing an Alzheimer's dementia specific phenotype.
  • a ⁇ 1-42 toxic beta amyloid
  • NHDF treated with A ⁇ 1-42 and NHDF treated with DMSO as a control were genome-wide using Infinium (®) Human Methylation EPIC BeadChip (Illumina). DNA methylation microarrays were performed. Bayesian t-test was used to select differentially methylated genes (DMGs) from both groups.
  • the CpG site with p value ⁇ 0.05 and the absolute value ⁇ difference ⁇ 0.3 was selected as the differentially methylated CpG site, and among these, the IDE gene whose methylation degree of the CpG site in the promoter region was changed. was finally selected as DMG (Table 2).
  • DNA methylation microarray analysis showed that in human skin fibroblasts treated with toxic beta amyloid, DNA methylation of specific CpG sites present in the IDE gene promoter was about 30% higher than that in control DMSO treated human skin fibroblasts. It was confirmed (Fig. 1).
  • the specific CpG site in the promoter of the IDE gene is the cytosine at 94334836 of chromosome 10 and the surrounding sequences are as follows:

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Abstract

The present invention relates to a composition, a kit and a method, which are for diagnosing Alzheimer's disease by detecting the methylation level of CpG sites of an insulin degrading enzyme (IDE) gene promoter.

Description

[규칙 제26조에 의한 보정 12.08.2019]　피부 세포에서 IDE 유전자 프로모터의 CpG 메틸화 변화를 이용한 알츠하이머 질환 진단용 조성물 및 이의 이용[Correction 12.08.2019] according to Rule 26. Composition for the diagnosis of Alzheimer's disease using CpG methylation of IDE gene promoter in skin cells and its use
본 발명은 IDE (Insulin Degrading Enzyme) 유전자 프로모터의 CpG 부위의 메틸화 수준을 검출함으로써, 알츠하이머 질환 (Alzheimer's disease)을 진단하기 위한 조성물, 키트 및 방법에 관한 것이다.The present invention relates to compositions, kits and methods for diagnosing Alzheimer's disease by detecting the methylation level of the CpG region of the Insulin Degrading Enzyme (IDE) gene promoter.
최근 급격한 산업화와 선진화 및 의학의 발전으로 국민 평균수명은 연장되었으나 젊은 층의 출산기피 현상으로 세계적으로 유례를 찾기 어려울 정도로 급속히 인구 노령화가 진행되고 있으며 이에 따른 노인성 질환이 크게 증가하고 있다. Recently, the average life expectancy has been extended due to the rapid industrialization, advancement and medical development, but population aging is rapidly progressing to the extent that it is hard to find the world's precedent due to the avoidance of childbirth of young people.
대표적 노인성 질환인 알츠하이머 질환 (또는 알츠하이머성 치매)은 치매의 가장 흔한 형태로, 기억력 감퇴로 시작하여 시간과 공간의 인지 능력 상실, 망상 및 성격변화, 언어 기능 마비, 및 운동 기능 마비 등의 증상으로 장기간에 걸쳐 점진적으로 진행하여 결국 사망에 이르는 대표적인 퇴행성 뇌질환이다. Alzheimer's disease (or Alzheimer's dementia), a representative senile disease, is the most common form of dementia, with symptoms such as loss of cognition in time and space, delusions and personality changes, speech paralysis, and motor paralysis. It is a representative degenerative brain disease that progresses gradually over a long period and eventually leads to death.
알츠하이머 질환을 치료하기 위한 연구가 지난 수십년간 지속적으로 진행되어 왔음에도 불구하고 현재까지 근본적인 치료제는 없으며 단지 증상을 완화하거나 병변의 진행을 늦추는 수준의 치료만이 행해지고 있는 실정이다. 따라서, 알츠하이머 질환은 조기 진단 및 선제적 치료를 통해 치매의 발병시기를 지연시키는 것이 가장 효과적인 관리 방법으로 제시되고 있다.Although research to treat Alzheimer's disease has been ongoing for the last several decades, there is no fundamental treatment to date, but only the level of treatment to alleviate the symptoms or slow the progression of the lesion. Therefore, Alzheimer's disease has been suggested as the most effective management method to delay the onset of dementia through early diagnosis and proactive treatment.
그러나, 현재 알츠하이머 질환 진단에 사용되고 있는 간이 정신 상태 검사 (mini-mental state examination: MMSE)의 경우 개체의 연령, 교육수준, 또는 의도적 대답 거부 등이 검사 결과의 정확도에 크게 영향을 미치는 단점이 있고, MRI나 PET을 이용한 뇌영상검사의 경우, 고가의 장비를 시용하기 때문에 특별한 증상을 나타내지 않는 초기단계에 검사를 시행하기가 어려운 단점이 있다.However, in the case of the mini-mental state examination (MMSE), which is currently used for diagnosing Alzheimer's disease, the age, education level, or intentional rejection of an individual significantly affect the accuracy of the test results. In case of brain imaging test using MRI or PET, it is difficult to perform the test in the early stage without any special symptoms because expensive equipment is used.
이에, 알츠하이머 질환의 진단을 위한 다른 방법으로, 뇌척수액 또는 혈청에서 베타 아밀로이드 단백질을 검출하는 방법, tau 단백질의 농도를 측정하는 방법, 신경아교원섬유 산성 단백질(glial fibrillary acidic protein, GFAP)-항체를 검출하는 방법 등의 생화학적 방법들이 제안되었으나, 진단의 간편성과 정확도 등의 문제가 제기되고 있다 (국제공개특허 제92/17152호; 미국특허 제4,666,829호; 국제공개특허 제89/06242호; 미국특허 제5,231,000호 등).Thus, as another method for diagnosing Alzheimer's disease, a method for detecting beta amyloid protein in cerebrospinal fluid or serum, a method for measuring the concentration of tau protein, and detecting a glial fibrillary acidic protein (GFAP) -antibody Biochemical methods have been proposed, but problems such as simplicity and accuracy of diagnosis have been raised (International Patent No. 92/17152; US Patent No. 4,666,829; International Publication No. 89/06242; US Patent). 5,231,000, etc.).
따라서, 알츠하이머 질환의 조기진단을 위해 임상증상을 대변하거나, 증상이 나타나기 이전의 전임상 상태를 측정할 수 있는 새로운 진단 마커가 절실히 요구된다.Therefore, there is an urgent need for new diagnostic markers to represent clinical symptoms or to measure preclinical conditions before symptoms appear for early diagnosis of Alzheimer's disease.
본 발명자들은 피부 세포에서 IDE (Insulin Degrading Enzyme) 유전자의 프로모터가 알츠하이머 질환에 특이적으로 고메틸화(hypermethylation)되는 것을 확인하고, 이를 바이오마커로 이용하여 IDE 유전자의 프로모터의 메틸화 정도를 측정함으로써 알츠하이머 질환을 진단할 수 있다는 것을 확인하여 본 발명을 완성하게 되었다.The present inventors confirmed that the promoter of the Insulin Degrading Enzyme (IDE) gene is hypermethylated specifically for Alzheimer's disease in skin cells, and using this as a biomarker, the degree of methylation of the promoter of the IDE gene is measured. It was confirmed that the diagnosis can be completed the present invention.
이에, 본 발명의 일예는 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제를 포함하는 알츠하이머 질환 진단용 조성물을 제공한다.Thus, one embodiment of the present invention provides a composition for diagnosing Alzheimer's disease, including an agent for measuring the CpG methylation level of the IDE gene promoter.
다른 예는 알츠하이머 질환을 진단하기 위한, 피부 시료로부터 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제의 용도를 제공한다.Another example provides the use of an agent to measure the CpG methylation level of an IDE gene promoter from a skin sample for diagnosing Alzheimer's disease.
다른 예는 알츠하이머 질환을 진단하는데 사용하기 위한, 피부 시료로부터 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제를 제공한다.Another example provides an agent for measuring the CpG methylation level of an IDE gene promoter from a skin sample for use in diagnosing Alzheimer's disease.
다른 예는 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제를 포함하는 포함하는 알츠하이머 질환 진단용 키트를 제공한다.Another example provides an Alzheimer's disease diagnostic kit comprising an agent for measuring the CpG methylation level of an IDE gene promoter.
다른 예는 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하여 알츠하이머 질환을 진단하는 방법을 제공한다.Another example provides a method of diagnosing Alzheimer's disease by measuring the methylation level at the CpG site of the IDE gene promoter.
다른 예는 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하는 단계를 포함하는, 알츠하이머 질환 진단을 위한 정보 제공 방법을 제공한다.Another example provides a method of providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level at the CpG site of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
다른 예는 알츠하이머 질환 진단에 필요한 정보를 제공하기 위하여, 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하는 방법을 제공한다.Another example provides a method of measuring the methylation level at the CpG site of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease to provide information necessary for diagnosing Alzheimer's disease.
다른 예는 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 및 상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계를 포함하는, 알츠하이머 질환의 진단 방법을 제공한다.Other examples include measuring the methylation level of the CpG region of the IDE gene promoter from skin samples of individuals suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
다른 예는, 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계; 및 알츠하이머 질환를 가진 것으로 결정된 상기 개체에 알츠하이머 질환 치료제를 투여하는 단계를 포함하는, 알츠하이머 질환의 치료 방법을 제공한다.Another example includes measuring a methylation level of a CpG region of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And administering an Alzheimer's disease therapeutic agent to said individual determined to have Alzheimer's disease.
본 발명은 개체의 피부 시료에서 채취한 게놈 DNA의 특정 유전자 CpG 부위의 메틸화 정도를 측정하여 알츠하이머 질환을 진단하는 분자생물학적 진단법을 제공하며, 이는 간편하고 비침습적이며 경제적인 장점이 있다. 또한, DNA 메틸화 변화는 검출이 용이하고, 종래 단백질이나 RNA 마커들에 비해 안정적이며 분석이 쉬운 장점이 있다. 또한, 피부 세포나 피부 조직 시료를 통한 손쉬운 진단이 가능한 장점이 있다.The present invention provides a molecular biological diagnostic method for diagnosing Alzheimer's disease by measuring the degree of methylation of a specific gene CpG region of genomic DNA collected from an individual's skin sample, which is simple, non-invasive and economical. In addition, DNA methylation changes are easy to detect, and are stable and easy to analyze compared to conventional protein or RNA markers. In addition, there is an advantage that can be easily diagnosed through skin cells or skin tissue samples.
도 1은 독성 베타 아밀로이드(Aβ) 또는 DMSO를 처리한 인간 피부 섬유아세포에서 IDE 유전자 프로모터 CpG 부위의 DNA 메틸화 변화를 나타낸 것이다.Figure 1 shows the DNA methylation changes of the IDE gene promoter CpG site in human skin fibroblasts treated with toxic beta amyloid (Aβ) or DMSO.
도 2는 독성 베타아밀로이드(Aβ) 또는 DMSO를 처리한 인간 피부 섬유아세포에서 IDE 유전자 발현 변화를 나타낸 것이다.Figure 2 shows the changes in IDE gene expression in human skin fibroblasts treated with toxic beta amyloid (Aβ) or DMSO.
도 3은 5-aza-2'-deoxycytidin 또는 DMSO를 처리한 인간 피부 섬유아세포에서 IDE 유전자의 DNA 메틸화 변화를 나타낸 것이다.Figure 3 shows the DNA methylation changes of the IDE gene in human skin fibroblasts treated with 5-aza-2'-deoxycytidin or DMSO.
도 4는 5-aza-2'-deoxycytidin을 또는 DMSO를 처리한 인간 피부 섬유아세포에서 IDE 유전자의 발현 변화를 나타낸 것이다.Figure 4 shows the expression changes of the IDE gene in human skin fibroblasts treated with 5-aza-2'-deoxycytidin or DMSO.
도 5는 정상 쥐(Wt) 및 알츠하이머성 치매 생쥐 모델(Tg)의 (a) 뇌 조직(해마), (b) 뇌 조직(피질) 및 (c) 피부 조직(피부 섬유아세포)에서 IDE 유전자의 발현 변화를 나타낸 것이다.Figure 5 shows the IDE genes in (a) brain tissue (hippocampus), (b) brain tissue (cortex) and (c) skin tissue (skin fibroblasts) of normal rat (Wt) and Alzheimer's dementia mouse model (Tg). Expression change is shown.
본 발명은 피부 세포에서 IDE (Insulin Degrading Enzyme) 유전자의 프로모터가 알츠하이머 질환에 특이적으로 고메틸화된다는 발견에 기초한 것으로, IDE 유전자의 프로모터의 특정 CpG 위치에서 일어나는 특이적인 DNA 고메틸화 정도를 측정하여 알츠하이머 질환을 진단하는 분자생물학적 진단 기술을 제공한다. The present invention is based on the discovery that the promoter of the Insulin Degrading Enzyme (IDE) gene is specifically hypermethylated in Alzheimer's disease in skin cells, by measuring the degree of specific DNA hypermethylation occurring at a specific CpG position of the promoter of the IDE gene. Molecular biological diagnostic techniques for diagnosing diseases are provided.
DNA 메틸화 변화는 DNA에 존재하기 때문에 검출이 용이하고, 단백질이나 RNA 마커들에 비해 안정적이며, 한 유전자의 특정 위치에서 일어나므로 분석이 손쉬운 장점을 가진다. DNA methylation changes are easy to detect because they are present in DNA, are stable compared to protein or RNA markers, and are easy to analyze because they occur at specific positions in a gene.
또한, 본 발명에서는, 놀랍게도, 피부 섬유아세포에서의 IDE 유전자의 비정상적인 메틸화의 변화가 알츠하이머 질환 모델의 뇌병변 시료에서 얻은 게놈 DNA의 메틸화 변화와 상당한 유사성을 보이는 것으로 확인되었으므로, 피부 세포나 피부 조직 시료를 통한 손쉬운 진단이 가능하다는 장점이 있다.In addition, in the present invention, it was surprisingly found that abnormal methylation of IDE genes in skin fibroblasts showed considerable similarity to methylation changes of genomic DNA obtained from brain lesion samples of Alzheimer's disease model. There is an advantage that easy diagnosis is possible through.
따라서, 일 구체예는 피부 시료로부터 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제를 포함하는 알츠하이머 질환 진단용 조성물을 제공한다.Accordingly, one embodiment provides a composition for diagnosing Alzheimer's disease, comprising an agent for measuring the CpG methylation level of an IDE gene promoter from a skin sample.
다른 구체예는 알츠하이머 질환을 진단하기 위한, 피부 시료로부터 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제의 용도를 제공한다.Another embodiment provides the use of an agent to measure the CpG methylation level of an IDE gene promoter from a skin sample for diagnosing Alzheimer's disease.
다른 구체예는 알츠하이머 질환을 진단하는데 사용하기 위한, 피부 시료로부터 IDE 유전자 프로모터의 CpG 메틸화 수준을 측정하는 제제를 제공한다.Another embodiment provides an agent for measuring the CpG methylation level of an IDE gene promoter from a skin sample for use in diagnosing Alzheimer's disease.
다른 구체예는 상기 조성물을 포함하는 알츠하이머 질환 진단용 키트를 제공한다.Another embodiment provides a kit for diagnosing Alzheimer's disease comprising the composition.
다른 구체예는 피부 시료로부터 IDE (Insulin Degrading Enzyme) 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 제제를 포함하는 알츠하이머 질환 진단용 키트를 제공한다.Another embodiment provides a kit for diagnosing Alzheimer's disease comprising an agent for measuring the methylation level of the CpG region of an Insulin Degrading Enzyme (IDE) gene promoter from a skin sample.
다른 구체예는 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하여 알츠하이머 질환을 진단하는 방법을 제공한다.Another embodiment provides a method of diagnosing Alzheimer's disease by measuring the methylation level at the CpG site of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
다른 구체예는 알츠하이머 질환이 의심되는 개체의 생물학적 시료로부터 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하는 단계를 포함하는, 알츠하이머 질환 진단을 위한 정보 제공 방법을 제공한다.Another embodiment provides a method of providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level at the CpG site of an IDE gene promoter from a biological sample of an individual suspected of Alzheimer's disease.
다른 구체예는 알츠하이머 질환 진단에 필요한 정보를 제공하기 위하여, 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하는 방법을 제공한다.Another embodiment provides a method of measuring the methylation level at the CpG site of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease to provide information necessary for diagnosing Alzheimer's disease.
다른 구체예는 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 및 상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계를 포함하는, 알츠하이머 질환의 진단 방법을 제공한다.Another embodiment includes measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
다른 구체예는, 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계; 및 알츠하이머 질환를 가진 것으로 결정된 상기 개체에 알츠하이머 질환 치료제를 투여하는 단계를 포함하는, 알츠하이머 질환의 치료 방법을 제공한다.Another embodiment includes measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And administering an Alzheimer's disease therapeutic agent to said individual determined to have Alzheimer's disease.
본 발명에서 용어, "메틸화" 또는 "메틸레이션"은 DNA를 구성하는 염기에 메틸기가 부착되는 것을 말한다. 바람직하게, 본 발명에서 메틸화 여부는 특정 유전자의 특정 CpG 부위의 사이토신에서 일어나는 메틸화 여부를 의미한다. 메틸화가 일어난 경우 그로 인하여 전사인자의 결합이 방해를 받게 되어 특정 유전자의 발현이 억제되며, 반대로, 비메틸화 또는 저메틸화가 일어나는 경우 특정 유전자의 발현이 증가하게 된다.In the present invention, the term "methylation" or "methylation" refers to the attachment of a methyl group to the base constituting the DNA. Preferably, whether or not methylation in the present invention means whether or not methylation occurs in the cytosine of a specific CpG region of a specific gene. When methylation occurs, the binding of transcription factors is disturbed, thereby inhibiting the expression of specific genes. In contrast, when demethylation or hypomethylation occurs, expression of specific genes increases.
포유동물 세포의 게놈 DNA 에는 A, C, G 및 T 에 더하여, 사이토신 링의 다섯번째 탄소에 메틸 그룹이 부착된 5-메틸사이토신(5-methylcytosine, 5-mC)이라는 5번째 염기가 존재한다. 5-메틸사이토신의 메틸화는 CpG라고 불리는 CG 디뉴클레오티드(5'-mCG-3')의 C에서만 일어나고, CpG의 메틸화는 alu 또는 트랜스포존과 게놈의 반복서열이 발현되는 것을 억제한다. 또한, 상기 CpG의 5-mC가 자연적으로 탈아미노화하여 티민(T)이 되기 쉽기 때문에, CpG는 포유동물 세포에서 대부분의 후생유전학적 변화가 자주 일어나는 부위이다.In 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. Methylation of 5-methylcytosine occurs only in C of CG dinucleotides (5'-mCG-3 '), called CpG, and methylation of CpG inhibits the expression of alu or transposons and genome repeats. In addition, since 5-mC of CpG is naturally deaminoated to easily become thymine (T), CpG is a site where most epigenetic changes occur frequently in mammalian cells.
본 발명에서 용어, "메틸화 수준의 측정"은 유전자의 CpG 부위의 메틸화 수준을 측정하는 것으로서, 메틸화 특이적인 PCR, 예를 들어 메틸화 특이적 PCR(methylation-specific polymerase chain reaction, MSP), 실시간 메틸화 특이적 PCR(real time methylation-specific polymerase chain reaction), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 또는 정량 PCR 등을 통해 측정할 수 있다. 또는, 파이로시퀀싱 또는 바이설파이트 시퀀싱과 같은 자동염기분석 등의 방법으로 측정하거나, DNA 메틸레이션 마이크로어레이, 또는 메틸화된 CpG 결합 도메인 또는 항-메틸사이토신 항체를 이용한 면역침강법 등을 사용할 수 있으나, 이에 제한되는 것은 아니다. 알츠하이머 질환이 아닌 사람의 시료에 비하여, 알츠하이머 질환 개체의 시료에서는 IDE 유전자의 프로모터에서의 고메틸화가 특이적으로 나타나는 것을 통해서, 프로모터의 메틸화 여부에 따라 알츠하이머 질환 여부를 진단할 수 있다.As used herein, the term "measurement of methylation level" refers to measuring methylation level of the CpG region of a gene, such as methylation specific PCR, for example, methylation-specific polymerase chain reaction (MSP), real-time methylation specificity. It can be measured by real time methylation-specific polymerase chain reaction (PCR), PCR using methylated DNA-specific binding proteins, or quantitative PCR. Alternatively, it may be measured by a method such as autobase analysis such as pyro sequencing or bisulfite sequencing, or may be used as DNA methylation microarray, immunoprecipitation using methylated CpG binding domain or anti-methylcytosine antibody. However, it is not limited thereto. Compared to a sample of a person who is not Alzheimer's disease, hypermethylation in the promoter of the IDE gene is specifically shown in a sample of an Alzheimer's disease individual, and it is possible to diagnose Alzheimer's disease according to whether the promoter is methylated.
본 발명에서, 유전자의 CpG 부위란, 유전자의 DNA 상에 존재하는 CpG 부위를 말한다. 유전자의 DNA 는, 유전자가 발현하는데 필요하며 서로 작동가능하게 연결되어 있는 일련의 구성 단위를 모두 포함하는 개념으로, 예를 들어, 프로모터 영역, 단백질 코딩 영역(open reading frame, ORF) 및 터미네이터 영역을 포함한다. 따라서, 유전자의 CpG 부위는 해당 유전자의 프로모터 영역, 단백질 코딩 영역(open reading frame, ORF) 또는 터미네이터 영역 등에 존재할 수 있다.In the present invention, the CpG site of the gene refers to the CpG site existing on the DNA of the gene. The DNA of a gene is a concept that includes a series of structural units necessary for the gene to express and are operably linked to each other. For example, a promoter region, an open reading frame (ORF), and a terminator region Include. Therefore, the CpG region of the gene may be present in a promoter region, an open reading frame (ORF), or a terminator region of the gene.
일예로, 본 발명에서 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 것은, IDE 유전자의 프로모터 CpG 부위, 보다 상세하게는 10 번 염색체의 94334776 내지 94334897 번째 염기서열(서열번호 1) 중에 나타나는 CpG 부위의 사이토신의 메틸화 수준을 측정하는 것을 의미할 수 있다. 보다 구체적으로, 10 번 염색체의 94334836 번째 염기(서열번호 1의 61번째 염기)에 위치한 사이토신의 메틸화를 측정하는 것을 의미할 수 있다. In one embodiment, measuring the methylation level of the CpG region of the IDE gene promoter in the present invention, the promoter CpG region of the IDE gene, more specifically, the CpG region appearing in the 94334776 to 94334897 base sequence (SEQ ID NO: 1) of chromosome 10 May mean measuring the methylation level of cytosine. More specifically, it may mean measuring the methylation of cytosine located at 94334836 base (SEQ ID NO: 61 base) of chromosome 10.
본 발명에서는 상기 인간 게놈 염색체 부위의 염기서열은 최신 버전인 GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38) 에 따라 표현하였지만 상기 인간 게놈 염색체 부위의 구체적 서열은 게놈 서열 연구 결과가 업데이트됨에 따라서 그 표현이 다소 변경될 수 있으며, 이러한 변경에 따라 본 발명의 상기 인간 게놈 염색체 부위의 표현이 상이해질 수 있다. 따라서, 본 발명의 GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38) 에 따라 표현된 인간 게놈 염색체 부위는 본 발명의 출원일 이후 인간 표준 염기서열(human reference sequence)이 업데이트되어 상기 인간 게놈 염색체 부위의 표현이 지금과 다르게 변경된다고 하여도, 본 발명의 범위가 상기 변경된 인간 게놈 염색체 부위에 미치게 됨은 자명하다고 할 것이다. 이러한 변경 내용은 본 발명이 속하는 기술분야의 통상의 지식을 가진 자라면 누구라도 용이하게 알 수 있는 사항이다.In the present invention, the nucleotide sequence of the human genomic chromosome region was expressed according to the latest version of the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38 / hg38), but the specific sequence of the human genomic chromosome region is expressed as the genome sequence research results are updated. This may be somewhat altered, and such alterations may result in different expressions of the human genomic chromosome region of the present invention. Therefore, the human genomic chromosome region expressed according to the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38 / hg38) of the present invention is updated after the filing date of the present invention, so that the human reference sequence is updated to express the human genomic chromosome region. Even if this change is different from the present, it will be obvious that the scope of the present invention extends to the modified human genomic chromosome region. Such changes are those that can be easily understood by those of ordinary skill in the art.
본 발명에서 용어, "IDE 유전자" 는 인슐린 분해 효소(Insulin Degrading Enzyme)를 코딩하는 유전자로, 상기 효소는 혈액에서 인슐린을 분해하여 인슐린 활성을 소실시키며, 글루카곤, 아밀린, 브래디키닌 및 칼리딘과 같은 다앙한 펩타이드들을 분해함으로써 세포간 펩타이드 시그널링에 관여하는, 아연-함유 단백질 분해 효소이다. IDE 유전자를 결실시킨 마우스들은 인슐린 수치가 상승하고 당 내성이 향상되는 것으로 알려져 있다. 인간 IDE 유전자는 10번 염색체에 위치하며, NCBI Entrez database 에 Gene ID: 3416 로 등록되어 있다.In the present invention, the term "IDE gene" is a gene encoding an insulin degrading enzyme, the enzyme decomposes insulin in the blood to lose insulin activity, glucagon, amylin, bradykinin and calidine It is a zinc-containing proteolytic enzyme that is involved in intracellular peptide signaling by breaking down the same diverse peptides. Mice that have lost the IDE gene are known to raise insulin levels and improve glucose tolerance. The human IDE gene is located on chromosome 10 and is registered as Gene ID: 3416 in the NCBI Entrez database.
본 발명에서 용어, "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 알츠하이머 질환 발병 여부를 확인하거나, 증상이 나타나기 이전의 전임상 상태를 측정하거나, 나아가 질환의 진행 여부 또는 심화 여부를 확인하는 것이다.As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the development of Alzheimer's disease, to measure the preclinical condition before the onset of symptoms, or to determine whether the disease is progressing or deepening.
본 발명에서, CpG 부위의 메틸화 수준을 측정하는 제제는 비메틸화 사이토신 염기를 변형시키는 화합물 또는 메틸화 민감성 제한효소, 유전자의 메틸화된 대립형질 서열에 특이적인 프라이머, 비메틸화된 대립형질 서열에 특이적인 프라이머, 메틸화된 CpG 결합 도메인, 또는 메틸화 DNA에 특이적으로 결합하는 메틸화 DNA 항체 (예를 들어, 메틸사이토신에 특이적으로 결합하는 항체) 등을 포함할 수 있다.In the present invention, the agent for measuring the methylation level of the CpG region is a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base, primers specific for the methylated allele sequence of the gene, specific for the unmethylated allele sequence. Primers, methylated CpG binding domains, or methylated DNA antibodies that specifically bind to methylated DNA (eg, antibodies that specifically bind to methylcytosine) and the like.
상기 비메틸화 사이토신 염기를 변형시키는 화합물은 바이설파이트(bisulfite) 또는 이의 염일 수 있으나 이에 제한되지 않으며, 바람직하게는 소듐 바이설파이트일 수 있다. 바이설파이트 변형된 DNA 는 서열분석 또는 메틸화 특이적 PCR 등 다양한 방법을 통하여 메틸화를 검출할 수 있으며, 이러한 바이설파이트를 이용하여 비메틸화 사이토신 잔기를 변형시켜 유전자의 메틸화 여부를 검출하는 방법은 당 업계에 널리 공지되어 있다(예를 들어, WO01/26536; US2003/0148326A1).The compound that modifies the unmethylated cytosine base may be, but is not limited to, bisulfite or a salt thereof, preferably sodium bisulfite. Bisulfite-modified DNA can detect methylation through various methods such as sequencing or methylation-specific PCR, and the method of detecting methylation of genes by modifying unmethylated cytosine residues using such bisulfite is It is well known in the art (for example, WO01 / 26536; US2003 / 0148326A1).
또한, 상기 메틸화 민감성 제한효소는 CpG 부위의 메틸화를 특이적으로 검출할 수 있는 제한효소로서 제한효소의 인식부위로 CG를 함유하는 제한효소일 수 있다. 예를 들면, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI 등이 있으며 이에 제한되지 않는다. 상기 제한효소 인식부위의 C에서의 메틸화 또는 비메틸화에 따라 제한효소에 의한 절단 여부가 달라지고 이를 PCR 또는 서던블롯(Southern Blot) 분석을 통해 검출할 수 있게 된다. 상기 제한효소 이외의 다른 메틸화 민감성 제한효소는 당 업계에 잘 알려져 있다.In addition, the methylation sensitive restriction enzyme may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme capable of specifically detecting the methylation of the CpG site. For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I and the like are not limited thereto. Depending on the methylation or demethylation of the restriction enzyme recognition site in C, whether the cleavage by the restriction enzyme is changed and it can be detected by PCR or Southern blot analysis. Methylation sensitive restriction enzymes other than the restriction enzymes are well known in the art.
알츠하이머 질환이 의심되는 개체의 유전자의 특정 CpG 부위에서의 메틸화 수준을 측정하는 대표적인 일예로서, 개체의 피부 시료에서 게놈 DNA를 수득하고, 수득한 DNA에 메틸화되지 않은 사이토신 염기를 변형시키는 화합물 또는 메틸화 민감성 제한효소를 처리한 후, 상기 처리된 DNA를 프라이머를 이용하여 PCR에 의해 증폭시키고 그 증폭된 결과물의 존부를 확인하는 것을 통해 측정할 수 있다.As a representative example of measuring the methylation level at a particular CpG site of a gene of a subject suspected of Alzheimer's disease, a compound or methylation is obtained which obtains genomic DNA from a subject's skin sample and modifies unmethylated cytosine bases in the obtained DNA. After treating the restriction enzyme, the treated DNA can be measured by amplifying by PCR using a primer and confirming the presence of the amplified result.
따라서, 본 발명의 제제는 유전자의 메틸화된 대립형질 서열에 특이적인 프라이머 및 비메틸화된 대립형질 서열에 특이적인 프라이머를 포함할 수 있다. 본 발명에서, 용어 "프라이머"는 짧은 자유 3 말단 수산화기를 가지는 핵산 서열로 상보적인 주형(template)과 염기쌍을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 중합효소 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다. 또한, 프라이머는, 7개 내지 50개의 뉴클레오타이드 서열을 가진 센스 및 안티센스 핵산으로서, DNA 합성의 개시점으로 작용하는 프라이머의 기본 성질을 변화시키지 않는 추가의 특징을 혼입할 수 있다.Thus, the formulations of the present invention may comprise primers specific for the methylated allele sequence of the gene and primers specific for the unmethylated allele sequence. In the present invention, the term “primer” refers to a short nucleic acid sequence that can form base pairs with complementary templates with nucleic acid sequences having short free 3-terminal hydroxyl groups and that serves as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In addition, primers, as sense and antisense nucleic acids having 7 to 50 nucleotide sequences, may incorporate additional features that do not change the basic properties of the primers that serve as the starting point for DNA synthesis.
본 발명의 프라이머는 메틸화 여부를 분석하는 대상이 되는 특정 CpG 부위의 서열에 따라 바람직하게 디자인될 수 있으며, 각각 메틸화되어 바이설파이트에 의해 변형되지 않았던 사이토신을 특이적으로 증폭할 수 있는 프라이머쌍, 및 메틸화되지 않아 바이설파이트에 의해 변형된 사이토신을 특이적으로 증폭할 수 있는 프라이머쌍일 수 있다. Primers of the present invention may be preferably designed according to the sequence of the specific CpG site to be analyzed for methylation, primer pairs that can specifically amplify cytosine that is methylated and not modified by bisulfite, And primer pairs that are not methylated to specifically amplify cytosine modified by bisulfite.
한편, 메틸화 DNA 항체란, DNA 중의 메틸화된 염기에 특이적으로 결합하는 항체를 말한다. 구체적으로는, 메틸사이토신에 대한 항체와 같이, DNA 사슬 중 메틸화된 사이토신을 인식하여 결합하는 성질을 가진 항체를 들 수 있다. 또한, 시판되고 있는 메틸화 DNA 항체 중에서, 본원에 기재된 메틸화 상태의 DNA를 특이적으로 인식하여 특이적으로 결합할 수 있는 항체일 수 있다. In addition, a methylated DNA antibody means the antibody which specifically binds to the methylated base in DNA. Specifically, an antibody having a property of recognizing and binding to methylated cytosine in a DNA chain, such as an antibody to methylcytosine, may be mentioned. In addition, among commercially available methylated DNA antibodies, it may be an antibody that can specifically recognize and specifically bind the DNA in the methylated state described herein.
메틸화 DNA 항체는 메틸화된 염기, 메틸화 DNA 등을 항원으로 하여 통상의 방법에 의해 제작할 수 있다. 예를 들어, 메틸사이토신 항체를 제조하기 위해서는, 5-메틸사이티딘, 5-메틸사이토신, 또는 5-메틸사이토신을 포함하는 DNA 등을 항원으로 하여 항체를 제조한 후 DNA 중의 메틸사이토신으로의 특이적인 결합을 지표로서 선별할 수 있다. A methylated DNA antibody can be produced by a conventional method using a methylated base, methylated DNA, or the like as an antigen. For example, to prepare a methylcytosine antibody, an antibody is prepared by using DNA containing 5-methylcytidine, 5-methylcytosine, or 5-methylcytosine as an antigen, and then methyl methyltocin in the DNA. Specific binding of can be selected as an indicator.
또한, 메틸화된 CpG 결합 도메인(CpG binding domain: MBD) 또는 메틸화 DNA 항체를 이용하여 메틸화 수준을 측정하는 경우, 이들을 이용하여 메틸화된 DNA 를 면역침강시킨 후, 서던블롯, PCR, 마이크로어레이, 또는 서열분석(sequencing) 등을 통해 특정 CpG 부위를 확인할 수 있다. 또한, 항체의 면역학적 검출 또는 정량을 위하여 기질, 적당한 완충용액, 발색 효소 또는 형광물질 표지, 발색 효소 또는 형광물질로 표지된 2차 항체, 및 발색 기질 등을 사용할 수 있다. 상기에서 기질은 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스틸렌 수지로 합성된 96 웰 플레이트 및 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등이 사용될 수 있고, 형광 물질은 FITC, RITC 등이 사용될 수 있고, 발색기질액은 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)) 또는 OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘)가 사용될 수 있고, 그 외 방사성 동위원소 표지, 라텍스 비드 표지, 콜로이드 표지, 비오틴 표지 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.In addition, when the methylation level is measured using a methylated CpG binding domain (MBD) or a methylated DNA antibody, the methylated DNA is immunoprecipitated using them, followed by Southern blot, PCR, microarray, or sequence. Specific CpG sites can be identified through sequencing. In addition, for immunological detection or quantification of the antibody, a substrate, a suitable buffer, a chromophore or fluorophore label, a secondary antibody labeled with a chromophore or fluorophore, and a chromophoric substrate can be used. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase or an alkaline force. Alkaline phosphatase may be used, and fluorescent materials may be FITC, RITC, or the like, and the colorant substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfur). Phonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine), and other radioisotope labels, latex bead labels, colloid labels, biotin labels, etc. may be used, but are not limited thereto. no.
상기 조성물 및 키트에는 상기 제제 이외에도, 중합효소 아가로스, 전기영동에 필요한 완충용액 등이 추가로 포함될 수 있다. 또한, 상기 키트는 DNA 메틸화 마이크로어레이 형태로 구현될 수 있다.In addition to the preparation, the composition and kit may further include a polymerase agarose, a buffer solution for electrophoresis, and the like. In addition, the kit may be implemented in the form of DNA methylated microarray.
다른 구체예로, 본 발명은 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하여 알츠하이머 질환을 진단하는 방법에 관한 것이다.In another embodiment, the present invention relates to a method of diagnosing Alzheimer's disease by measuring the methylation level at the CpG site of an IDE gene promoter.
또한, 본 발명은 알츠하이머 질환 진단에 필요한 정보를 제공하기 위하여, 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 유전자의 CpG 부위의 메틸화 수준을 검출하는 방법에 관한 것이다.The present invention also relates to a method for detecting the methylation level of the CpG region of a gene from a skin sample of an individual suspected of Alzheimer's disease in order to provide information necessary for diagnosing Alzheimer's disease.
또한, 본 발명은 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위에서의 메틸화 수준을 측정하는 단계를 포함하는, 알츠하이머 질환 진단을 위한 정보 제공 방법에 관한 것이다.The present invention also relates to a method for providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level at the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
예를 들어, 본 발명은 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 메틸화 수준을 측정하는 단계; 및 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계를 포함하는, 알츠하이머 질환 진단을 위한 정보를 제공하는 방법을 제공한다.For example, the present invention comprises the steps of measuring the methylation level of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease; And comparing the methylation level with the methylation level of the corresponding gene promoter of the control sample, not Alzheimer's disease, to provide information for diagnosing Alzheimer's disease.
또한, 본 발명은 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 및 상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계를 포함하는, 알츠하이머 질환의 진단 방법을 제공한다.In addition, the present invention comprises the steps of measuring the methylation level of the CpG region of the IDE gene promoter from skin samples of individuals suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
또한, 본 발명은 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계; 및 알츠하이머 질환를 가진 것으로 결정된 상기 개체에 알츠하이머 질환 치료제를 투여하는 단계를 포함하는, 알츠하이머 질환의 치료 방법을 제공한다.이하, 상기 진단 방법, 진단을 위한 정보를 제공하는 방법 및 치료방법에 관하여 설명한다.In addition, the present invention comprises the steps of measuring the methylation level of the CpG region of the IDE gene promoter from skin samples of individuals suspected of Alzheimer's disease; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And administering a treatment agent for Alzheimer's disease to the individual determined to have Alzheimer's disease. Hereinafter, the diagnosis method, a method for providing information for diagnosis, and a treatment method will be described. .
본 발명에서 용어 "피부 시료"란 알츠하이머 질환에 의해 유전자의 메틸화 수준이 차이 나는 피부 조직 또는 피부 세포를 포함하고, 구체적으로는 피부 섬유아세포 등을 포함하나, 이에 제한되지 않는다.In the present invention, the term "skin sample" includes skin tissues or skin cells having different methylation levels of genes due to Alzheimer's disease, and specifically, but not limited to skin fibroblasts and the like.
먼저, 알츠하이머 질환이 의심되는 환자의 피부 시료로부터 게놈 DNA를 수득하여 메틸화 수준을 검출하기 위하여, 게놈 DNA의 수득은 당 업계에서 통상적으로 사용되는 페놀/클로로포름 추출법, SDS 추출법(Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB 분리법(Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) 또는 상업적으로 판매되는 DNA 추출 키트를 이용하여 수행할 수 있다.First, in order to obtain genomic DNA from skin samples of patients suspected of Alzheimer's disease to detect methylation levels, the obtaining of genomic DNA is performed by phenol / chloroform extraction, SDS extraction (Tai et al., Plant commonly used in the art). Mol. Biol. Reporter, 8: 297-303, 1990), CTAB isolation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or using a commercially available DNA extraction kit Can be done.
상기 유전자의 특정 CpG 부위의 메틸화 수준을 검출하는 단계는, 메틸화된 염기와 메틸화되지 않은 염기의 차이를 이용한, 제한효소 또는 바이설파이트를 이용하는 방법, 메틸화된 CpG 결합 도메인 또는 항-메틸사이토신 항체를 이용한 면역침강방법(예, MIRA, MeDIP), DNA 메틸레이션 마이크로어레이를 이용한 방법 등을 통해 수행될 수 있다. 예를 들어, 메틸화 특이적 중합효소반응(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 중합효소반응(real time methylation-specific polymerase chain reaction), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, 파이로시퀀싱, 바이설파이트 시퀀싱, DNA 메틸레이션 마이크로어레이, 또는 메틸화된 CpG 결합 도메인 또는 항-메틸사이토신 항체를 이용한 면역침강법 등을 사용하여 메틸화 수준을 측정 내지 검출할 수 있다.Detecting the methylation level of a specific CpG region of the gene may be achieved by using a restriction enzyme or bisulfite, using a difference between a methylated base and an unmethylated base, a methylated CpG binding domain or an anti-methylcytosine antibody. Immunoprecipitation using (eg, MIRA, MeDIP), DNA methylation may be carried out through a method using a microarray. For example, methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding proteins, quantitative PCR, Pyro sequencing, bisulfite sequencing, DNA methylation microarrays, or immunoprecipitation with methylated CpG binding domains or anti-methylcytosine antibodies can be used to measure or detect methylation levels.
일예로, 본원의 방법에서 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계는, (a) 수득된 시료 내 게놈 DNA를 비메틸화 사이토신 염기를 변형시키는 화합물 또는 메틸화 민감성 제한효소로 처리하는 단계; 및 (b) 상기 처리된 DNA를 해당 유전자의 특정 CpG 부위를 증폭할 수 있는 프라이머를 이용하여 PCR에 의해 증폭하는 단계를 포함할 수 있다.For example, in the method of the present invention, measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease, includes: (a) modifying the non-methylated cytosine base of genomic DNA in the sample obtained; Treating with a compound or methylation sensitive restriction enzyme; And (b) amplifying the treated DNA by PCR using a primer capable of amplifying a specific CpG site of the gene.
상기 (a) 단계에서 비메틸화 사이토신 염기를 변형시키는 화합물은 바이설파이트일 수 있으며, 바람직하게는 소듐 바이설파이트일 수 있다. 이러한 바이설파이트를 이용하여 비메틸화 사이토신 잔기를 변형시켜 유전자의 메틸화 여부를 검출하는 방법은 당 업계에 널리 공지되어 있다. The compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite. The use of such bisulfites to modify unmethylated cytosine residues to detect whether the gene is methylated is well known in the art.
또한, 상기 (a) 단계에서 메틸화 민감성 제한효소는 상기에서 설명한 바와 같이, 특정 CpG 부위의 메틸화를 특이적으로 검출할 수 있는 제한효소로서 제한효소의 인식부위로 CG를 함유하는 제한효소일 수 있으며, 예를 들면, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI 등이 있으며, 이에 제한되지 않는다. In addition, the methylation-sensitive restriction enzyme in step (a) may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme that can specifically detect methylation of a specific CpG site, as described above. For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I and the like, but is not limited thereto.
상기 (b) 단계에서 증폭은 통상적인 PCR 방법에 의해 수행될 수 있다. 이때 사용되는 프라이머는 상기에서 설명한 바와 같이, 메틸화 여부를 분석하는 대상이 되는 특정 CpG 부위의 서열에 따라 바람직하게 디자인될 수 있으며, 각각 메틸화되어 바이설파이트에 의해 변형되지 않았던 사이토신을 특이적으로 증폭할 수 있는 프라이머쌍 및 메틸화되지 않아 바이설파이트에 의해 변형된 사이토신을 특이적으로 증폭할 수 있는 프라이머 쌍일 수 있다.Amplification in step (b) may be performed by a conventional PCR method. At this time, the primers used may be preferably designed according to the sequence of the specific CpG region to be analyzed for methylation, as described above, and specifically amplify cytosine that is methylated and not modified by bisulfite. Possible primer pairs and primer pairs that are not methylated to specifically amplify cytosine modified by bisulfite.
또한, 본원의 방법에서 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계는, (c) 상기 (b) 단계에서 증폭된 결과물의 존부를 확인하는 단계를 추가로 포함할 수 있다. 상기 (c) 단계에서 증폭된 결과물의 존부는 당 업계에 공지된 방법, 예를 들면 전기영동을 수행하여 원하는 위치의 밴드의 검출 여부에 따라서 수행될 수 있다. 예를 들면, 비메틸화 사이토신 잔기를 변형시키는 화합물을 사용한 경우 상기 (a) 단계에서 사용한 두 종류의 프라이머쌍, 즉 메틸화되어 바이설파이트에 의해 변형되지 않았던 사이토신을 특이적으로 증폭할 수 있는 프라이머쌍 및 메틸화되지 않아 바이설파이트에 의해 변형된 사이토신을 특이적으로 증폭할 수 있는 프라이머쌍에 의해 각각 증폭된 PCR 결과물의 존부에 따라 메틸화 정도를 판단할 수 있다. 바람직하게, 샘플 게놈 DNA를 바이설파이트로 처리하고, 해당 유전자의 CpG 부위를 PCR로 증폭하고, 증폭된 부위의 염기서열을 분석하는 바이설파이트 게놈 시퀀싱 방법을 사용하여 메틸화 여부를 판단할 수 있다. In addition, measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease in the method of the present invention, (c) confirming the presence of the result amplified in step (b) It may further comprise. The presence of the result amplified in step (c) may be performed according to a method known in the art, for example, electrophoresis, according to whether a band of a desired position is detected. For example, in the case of using a compound that modifies an unmethylated cytosine residue, primers capable of specifically amplifying two kinds of primer pairs used in step (a), namely, cytosine which was methylated and not modified by bisulfite, were used. The degree of methylation can be determined according to the presence or absence of PCR results amplified by pairs and primer pairs that can specifically amplify cytosine modified by bisulfite, thereby not being methylated. Preferably, the methylation can be determined using a bisulfite genome sequencing method in which the sample genomic DNA is treated with bisulfite, the CpG region of the gene is amplified by PCR, and the nucleotide sequence of the amplified site is analyzed. .
또한, 제한효소를 이용한 경우에도 당 업계에 공지된 방법, 예를 들어 mock DNA에서 PCR 결과물이 나타난 상태에서, 제한효소로 처리된 DNA에서 PCR 결과물이 있는 경우는 CpG가 메틸화된 것으로 판단하고, 제한효소로 처리된 DNA에서 PCR 결과물이 없는 경우는 CpG가 비메틸화 한 것으로 판단하는 것에 따라 그 메틸화 여부를 판단할 수 있으며, 이는 당업자에게 자명하다. 상기에서 mock DNA란 시료에서 분리되고 아무런 처리를 하지 않은 상태의 시료 DNA를 의미한다.Also, even when restriction enzymes are used, CpG is determined to be methylated when there are PCR products in the DNA treated with restriction enzymes in a state known in the art, for example, when PCR products are displayed in mock DNA. If there is no PCR result in the enzyme-treated DNA, it can be determined whether or not the methylation according to the determination that CpG is unmethylated, which is obvious to those skilled in the art. The mock DNA in the above means the sample DNA which is separated from the sample and is not treated at all.
유전자의 특정 CpG 부위의 메틸화 수준을 검출하는 다른 예로, 메틸화 DNA 항체, 예를 들어 메틸화된 CpG 결합 도메인(CpG binding domain: MBD) 또는 메틸사이토신에 대한 항체를 이용한 면역침강방법을 예시할 수 있다. 예를 들어, 메틸화된 CpG 결합 도메인 또는 5-메틸사이토신을 특이적으로 인지하는 항체를 이용하여 면역침강한 후 서던블롯, PCR, 마이크로어레이, 또는 서열분석(sequencing)등을 통해 특정 CpG 부위를 확인할 수 있다.As another example of detecting the methylation level of a particular CpG region of a gene, an immunoprecipitation method using a methylated DNA antibody, such as a methylated CpG binding domain (MBD) or an antibody against methylcytosine, can be exemplified. . For example, after immunoprecipitation using an antibody that specifically recognizes a methylated CpG binding domain or 5-methylcytosine, specific CpG sites are identified by Southern blot, PCR, microarray, or sequencing. Can be.
이와 같은 방법에 따라, 대상 시료 내 IDE 유전자의 CpG 부위가 고메틸화 상태로 검출되는 경우, 알츠하이머 질환이라고 진단 또는 예측할 수 있는 것이다.According to such a method, when the CpG region of the IDE gene in the target sample is detected with a high methylation state, it can be diagnosed or predicted as Alzheimer's disease.
따라서, 상기 본 발명에 따를 경우, IDE 유전자의 특정 CpG 부위의 메틸화 변화는 알츠하이머 질환 개체의 피부 시료에서 특이적으로 나타나며 알츠하이머 질환 개체의 유전자의 발현에 영향을 미치므로, 유전자의 메틸화 변화를 바이오마커로 사용하여 알츠하이머 질환 진단에 유용하게 사용될 수 있다. 예를 들어, IDE 유전자 프로모터의 특정 CpG 부위의 고메틸화를 바이오마커로 사용하여 알츠하이머 질환 진단에 유용하게 사용될 수 있다.Therefore, according to the present invention, the methylation change of a specific CpG region of the IDE gene is specifically shown in the skin samples of Alzheimer's disease individuals and affects the expression of the genes of the Alzheimer's disease individuals, so that the methylation changes of the genes are biomarkers. It can be used to diagnose Alzheimer's disease. For example, hypermethylation of specific CpG sites of the IDE gene promoter can be usefully used for diagnosing Alzheimer's disease using biomarkers.
나아가, 본 발명에서, 알츠하이머 질환를 가진 것으로 결정된 상기 개체에 알츠하이머 질환 치료제를 투여함으로써 알츠하이머 질환을 치료할 수 있다. 상기 질환 치료제는 치료적 유효량으로 투여될 수 있다.Furthermore, in the present invention, Alzheimer's disease can be treated by administering an Alzheimer's disease therapeutic agent to the individual determined to have Alzheimer's disease. The disease therapeutic agent may be administered in a therapeutically effective amount.
알츠하이머 질환 치료제는 공지의 치료제들을 사용할 수 있으며, 알츠하이머병의 치료를 위해 미국 식품 의약품 안전청에 의해 승인된 물질들을 포함한다. 공지된 알츠하이머 질환 치료제로는 콜린 용제(cholinergics)/콜린에스테라아제 저해제로서, 예를 들어, 도네페질, 리바스티그민, 갈란타민, 또는타크린; NMDA 수용체 길항제로서, 예를 들어, 메만틴; 항산화제로서, 예를 들어 셀레길린, 또는 비타민 E 등을 예시할 수 있으나 이에 제한되지 않는다. 이러한 치료제들은 약학적으로 허용가능한 염 또는 에스테르 형태일 수 있으나, 이에 제한되지 않는다 (예로, 갈란타민 하이드로브로마이드, 리바스티그민 타르트레이트, 도네페질 염산 등). 소정의 구현예들에서, 이들 치료제는 2 이상이 병용될 수 있다. Alzheimer's disease therapeutic agents can use known therapeutic agents and include substances approved by the US Food and Drug Administration for the treatment of Alzheimer's disease. Known Alzheimer's disease therapies include cholinergics / cholinesterase inhibitors, for example donepezil, rivastigmine, galantamine, or tacrine; As NMDA receptor antagonist, for example memantine; Examples of the antioxidants include, but are not limited to, selegiline, vitamin E, and the like. Such therapeutic agents may be in pharmaceutically acceptable salt or ester form (eg, but not limited to, galantamine hydrobromide, rivastigmine tartrate, donepezil hydrochloric acid, and the like). In certain embodiments, these therapeutic agents may be used in combination of two or more.
용어, "약학적으로 허용되는"은 의약학적 판단의 범위 내에서, 과도한 독성, 자극, 알레르기 반응 등을 유발하지 않고 원하는 용도에 효과적으로 사용 가능한 물질을 의미한다. 본 발명에서 용어, "약학적으로 허용되는 염" 이란 약학적으로 허용되는 무기산, 유기산, 또는 염기로부터 유도된 염을 포함한다. 용어 "약학적으로 허용가능한 에스테르"는 본 명세서에서 사용되는 바 시험관내에서 가수분해하는 에스테르들을 말하고, 부모 화합물 또는 그의 염을 이탈하도록 인간 신체에서 바로 분해되는 것들을 포함한다. 적합한 에스테르기들은, 예를 들면 약학적으로 허용가능한 지방족 카르복실산들, 상세하게 알카노, 알케노, 고리알카노 및 알칸디오 산들로부터 유래한 것들을 포함하고, 각각의 알킬 또는 알케닐 분체는 유리하게 6개 이상의 탄소 원자들을 가지지 않는다. 상기 치료제들은 추가적으로 약학적으로 허용가능한 담체를 포함할 수 있다. 치료제의 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 정맥, 근육, 피하주사 등에 의해 투여될 수 있다.The term "pharmaceutically acceptable" means a substance that can be effectively used for a desired use without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of the medical judgment. As used herein, the term "pharmaceutically acceptable salts" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. The term "pharmaceutically acceptable ester" as used herein refers to esters that hydrolyze in vitro and include those that degrade directly in the human body to leave the parent compound or salt thereof. Suitable ester groups include, for example, pharmaceutically acceptable aliphatic carboxylic acids, in particular those derived from alkano, alkeno, cyclicalkano and alkanedio acids, each alkyl or alkenyl powder advantageously It does not have more than six carbon atoms. The therapeutic agents may additionally include a pharmaceutically acceptable carrier. The dosage of the therapeutic agent depends on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous injection, and the like.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples are merely to illustrate the invention, the present invention is not limited by the following Examples.
실시예 1. 인간 피부 섬유아세포 배양 및 처리Example 1 Human Skin Fibroblast Culture and Treatment
사람의 일차(primary) 피부 섬유아세포인 NHDF (normal human dermal fibroblast) 는 American type culture collection (ATCC no. PCS-201-012)에서 구입하여 2% (v/v) 우태아혈청(fetal bovine serum), 1 μg/ml 하이드로코르티손(hydrocortisone), 10 ng/ml 인간 표피성장인자(human epidermal growth factor), 3 ng/ml 염기성 섬유아세포 성장인자(basic fibroblast growth factor), 10 μg/ml 헤파린(heparin), 100 U/ml 페니실린(penicillin), 100 μg/ml 스트렙토마이신(streptomycin), 30 μg/ml 겐타마이신(gentamicine) 및 15 ng/ml 암포테리신 B(amphotericin B)를 포함하고 있는 Medium 106 에서 배양하였다. Normal human dermal fibroblasts (NHDF), primary skin dermal fibroblasts, were purchased from American type culture collection (ATCC no. PCS-201-012) and were treated with 2% (v / v) fetal bovine serum. , 1 μg / ml hydrocortisone, 10 ng / ml human epidermal growth factor, 3 ng / ml basic fibroblast growth factor, 10 μg / ml heparin Cultured in Medium 106 containing 100 U / ml penicillin, 100 μg / ml streptomycin, 30 μg / ml gentamicine and 15 ng / ml amphotericin B It was.
배양된 NHDF를 1.5 μM 독성 베타 아밀로이드(Aβ1-42)로 5일간 처리하여 알츠하이머성 치매 세포 모델을 확립하였다. 대조군으로서, 독성 베타 아밀로이드 대신 1.5 μM 디메틸 설폭시드(Dimethyl sulfoxide: DMSO)로 NHDF를 5일간 처리하였다.Cultured NHDF was treated with 1.5 μM toxic beta amyloid (Aβ 1-42 ) for 5 days to establish an Alzheimer's dementia cell model. As a control, NHDF was treated with 1.5 μM dimethyl sulfoxide (DMSO) for 5 days instead of toxic beta amyloid.
또한, 배양된 NHDF를 20 μM DNA 메틸화 억제제인 5-aza-2'-deoxycytidine (Sigma-Aldrich)로 3일간 처리한 후 IDE 유전자 발현 변화를 역전사-정량적 PCR (Reverse transcription-quantitative polymerase chain reaction: RT-qPCR)을 이용하여 측정하였다. 대조군으로서, 5-aza-2'-deoxycytidine 대신 20 μM DMSO로 NHDF를 3일간 처리하였다.In addition, the culture of NHDF was treated with 20 μM DNA methylation inhibitor 5-aza-2'-deoxycytidine (Sigma-Aldrich) for 3 days, and then the change of IDE gene expression was reverse transcription-quantitative polymerase chain reaction (RT). -qPCR). As a control, NHDF was treated with 20 μM DMSO for 3 days instead of 5-aza-2'-deoxycytidine.
실시예 2. 생쥐 피부 섬유아세포 (skin fibroblast) 분리 및 배양Example 2. Isolation and Culture of Mouse Skin Fibroblasts
알츠하이머성 치매 모델로서, APP Swedish 돌연변이와 9번째 엑손 결손 돌연변이를 동시에 발현하는 12개월령의 APP swe/PS1 형질전환 쥐 (B6C3-Tg(APP695)85Dbo Tg(PSEN1)85Dbo)를 사용하였으며, 형질전환 쥐와 대조군으로서 littermate 정상 쥐의 뇌조직 (전두엽 피질(frontal cortex)과 해마(hippocampus)) 과 피부 조직으로부터 피부 섬유아세포를 각각 분리하여 유전자 발현의 변화를 조사하였다.As an Alzheimer's dementia model, 12-month-old APP swe / PS1 transgenic mice (B6C3-Tg (APP695) 85Dbo Tg (PSEN1) 85Dbo) expressing an APP Swedish mutation and a 9th exon deficient mutation were used. As a control, fibroblasts were isolated from the brain tissues (frontal cortex, hippocampus) and skin tissues of littermate normal rats, and the changes of gene expression were investigated.
구체적으로, 형질전환 쥐와 littermate 정상 쥐의 피부 조직에서 섬유아세포를 무균 환경 하에 분리하기 위하여, 쥐에서 적출된 피부를 Dulbecco's Phosphate-Buffered Saline (DPBS) 에 넣어 4 ℃에서 이동 및 보관하였고, 포셉으로 피부에 있는 DPBS 를 최대한 제거한 후 페트리 디쉬에 옮겨 잘게 잘라주었다. 잘게 자른 조각을 0.14 units/ml의 Liberase DH collagenase (Sigma-Aldrich)를 포함한 배지 (100 U/mL 페니실린, 100 μg/mL 스트렙토마이신 및 30 μg/ml 겐타마이신을 포함한 DMEM/F12) 로 옮겨 37℃에서 90분 간 300 rpm으로 처리하여 섬유아세포를 분리하였다. Collagenase를 불활성시키기 위해 15% 우태아혈청 (FBS)에 노출시킨 뒤 524 xg에서 5분간 원심분리하였다. 원심분리된 섬유아세포 침전물을 15% FBS가 첨가된 DMEM/F12 배지에 넣고 37℃의 5% CO2 및 3% O2 배양기에서 배양하였다. 배지의 색이 노란색으로 변하면 절반의 배지를 빼내고 새 배지를 추가하였다. 14일 후 15% FBS, 100 U/ml 페니실린 및 100 μg/ml 스트렙토마이신을 포함한 EMEM 배지로 바꾸어서 배양하였다. Specifically, in order to separate fibroblasts from skin tissues of transgenic mice and littermate normal mice under sterile environment, the skins extracted from rats were placed in Dulbecco's Phosphate-Buffered Saline (DPBS) and transferred and stored at 4 ° C. After removing as much DPBS from the skin, it was chopped into a Petri dish. Transfer the chopped pieces to medium containing 0.14 units / ml Liberase DH collagenase (Sigma-Aldrich) (DMEM / F12 containing 100 U / mL penicillin, 100 μg / mL streptomycin and 30 μg / ml gentamicin) at 37 ° C. Fibroblasts were isolated by treatment at 300 rpm for 90 minutes at. The collagenase was exposed to 15% fetal bovine serum (FBS) and then centrifuged at 524 xg for 5 minutes. Centrifuged fibroblast precipitate was placed in DMEM / F12 medium with 15% FBS added and incubated in a 37% 5% CO 2 and 3% O 2 incubator. When the color of the medium turned yellow, half of the medium was removed and a new medium was added. After 14 days, the cells were incubated with EMEM medium containing 15% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin.
실시예 3. Total RNA 와 genomic DNA추출Example 3 Total RNA and Genomic DNA Extraction
생쥐 뇌조직, 생쥐 피부 섬유아세포, 및 인간 피부 섬유아세포 NHDF에서 각각 RNeasy mini kit (Qiagen)와 QIAmp mini kit (Qiagen)을 이용하여 total RNA와 genomic DNA를 추출하였다. 추출방법은 제조사의 매뉴얼을 따라 실시하였다. 추출된 total RNA와 genomic DNA는 분광광도계(spectrophotometer)를 이용하여 정량하였으며, RNA 와 DNA 상태는 1% 아가로스 젤에서 전기영동하여 degradation 여부를 확인하였다.Total RNA and genomic DNA were extracted from mouse brain tissue, mouse skin fibroblast, and human skin fibroblast NHDF using RNeasy mini kit (Qiagen) and QIAmp mini kit (Qiagen), respectively. Extraction method was performed according to the manufacturer's manual. The extracted total RNA and genomic DNA were quantified using a spectrophotometer, and RNA and DNA states were electrophoresed on 1% agarose gel to confirm degradation.
실시예 4. DNA 메틸레이션 마이크로어레이Example 4 DNA Methylation Microarrays
Aβ1-42 또는 5-aza-2'-deoxycytidine으로 처리한 NHDF와, 대조군으로 DMSO로 처리한 NHDF 세포를, Infinium(®) Human Methylation EPIC BeadChip (Illumina)을 사용하여 genome-wide DNA 메틸레이션 마이크로어레이를 수행하였다. DNA 메틸화 정도는 0~1 값을 갖는 β값으로 표시되며 β값 0은 해당 CpG 부위가 완전히 비메틸화 된 것을 의미하며, 1은 완전히 메틸화된 것을 의미한다. Aβ 1-42 or NHDF treated with 5-aza-2'-deoxycytidine and NHDF treated with DMSO as a control group were subjected to genome-wide DNA methylation microarray using Infinium (®) Human Methylation EPIC BeadChip (Illumina). The degree of DNA methylation is represented by a β value having a value of 0 to 1, and a β value of 0 means that the corresponding CpG site is completely unmethylated, and 1 means fully methylated.
두 군에서 차별적으로 메틸화되어 있는 유전자 (Differentially methylated genes, DMGs)를 확인하기 위하여 Bayesian t-test 방법을 사용하였다. 최종적으로 p value<0.05이면서 절대값 β 차이≥0.3 인 CpG 부위를 차별적으로 메틸화되어 있는 CpG 부위(differentially methylated CpG site)로 선정하고, 이 중에서 프로모터 부분에 존재하는 CpG 부위의 메틸화 정도가 변한 유전자를 DMG로 선정하였다.Bayesian t-test was used to identify differentially methylated genes (DMGs) in both groups. Finally, the CpG site with p value <0.05 and the absolute value β difference ≥0.3 was selected as the differentially methylated CpG site, and among these, the gene whose methylation degree of the CpG site in the promoter region was changed was selected. DMG was selected.
실시예 5. 역전사 정량 PCT (RT-qPCR)Example 5 Reverse Transcription Quantitative PCT (RT-qPCR)
cDNA 합성을 위해 Superscript II reverse transcriptase (Invitrogen)을 사용하였다. 1 μg의 total RNA 와 50 ng oligo dT를 70℃에서 10분간 denature한 후 여기에 5X RT buffer 4 μl, 0.1 mM DTT 2 μl, 2.5 mM dNTP mixture 4 μl, Superscript II 역전사효소 200 units 및 RNase inhibitor 10 units을 포함한 반응액에 혼합한 20 μl 반응액을 25℃에서 10분, 42℃에서 50분, 95℃에서 5분 동안 반응하여 cDNA를 합성한 후 이것을 1:4로 희석하여 이 중 2 μl를 취하여 주형으로 사용하였다. qPCR은 cDNA 2 μl, SYBR Premix EX Taq (Takara Bio) 10 μl, Rox reference dye (50Υ, Takara Bio) 0.4 μl 및 각 유전자의 프라이머 200 nM를 포함한 20 μl 반응액을 ABI 7500fast sequence detection system (Applied Biosystems)을 이용하여 95℃에서 30초 반응한 후, 40 cycles (95℃에서 3초, 60℃에서 30초) 반복하여 증폭하였다. PCR 산물의 specificity는 95℃에서 15초, 60℃에서 1분, 95℃에서 15초 동안 반응하여 확인하였다. GAPDH mRNA 발현을 internal control로 사용하였으며, IDE 유전자의 발현은 GAPDH 발현 수준으로 ΔΔCT 방법으로 보정되었다. 사용된 올리고뉴클레오티드 프라이머 서열은 다음과 같다.Superscript II reverse transcriptase (Invitrogen) was used for cDNA synthesis. 1 μg of total RNA and 50 ng oligo dT were denatured at 70 ° C for 10 minutes, followed by 4 μl of 5X RT buffer, 2 μl of 0.1 mM DTT, 4 μl of 2.5 mM dNTP mixture, 200 units of Superscript II reverse transcriptase and RNase inhibitor 10 20 μl of the reaction solution mixed with the reaction solution containing the unit was reacted for 10 minutes at 25 ℃, 50 minutes at 42 ℃, 5 minutes at 95 ℃ to synthesize cDNA and dilute it 1: 4 to 2 μl Was used as a template. qPCR contained 20 μl reaction solution containing 2 μl of cDNA, 10 μl of SYBR Premix EX Taq (Takara Bio), 0.4 μl of Rox reference dye (50Υ, Takara Bio), and 200 nM of primers for each gene, and ABI 7500 fast sequence detection system (Applied Biosystems). After 30 seconds at 95 ° C, 40 cycles (3 seconds at 95 ° C, 30 seconds at 60 ° C) were repeatedly amplified. Specificity of the PCR product was confirmed by reacting for 15 seconds at 95 ℃, 1 minute at 60 ℃, 15 seconds at 95 ℃. GAPDH mRNA expression was used as internal control, and IDE gene expression was corrected by ΔΔC T method to GAPDH expression level. Oligonucleotide primer sequences used are as follows.
프라이머primer 서열order 서열번호SEQ ID NO:
human IDE (forward)human IDE (forward) 5'- AGCAGGCTTGAGCTATGATCT -3'5'- AGCAGGCTTGAGCTATGATCT -3 ' 22
human IDE (reverse)human IDE (reverse) 5'- GTTCAGCCCGGAAATTGTTAAGA -3'5'- GTTCAGCCCGGAAATTGTTAAGA -3 ' 33
human GAPDH (forward)human GAPDH (forward) 5'- AATCCCATCACCATCTTCCA -3'5'- AATCCCATCACCATCTTCCA -3 ' 44
human GAPDH (reverse)human GAPDH (reverse) 5'- TGGACTCCACGACGTACTCA -3'5'- TGGACTCCACGACGTACTCA -3 ' 55
mouse IDE (forward)mouse IDE (forward) 5'- CAGAAGGACCTCAAGAATGGGT -3'5'- CAGAAGGACCTCAAGAATGGGT -3 ' 66
mouse IDE (reverse)mouse IDE (reverse) 5'- GCCTCGTGGTCTCTCTTTATCT -3'5'- GCCTCGTGGTCTCTCTTTATCT -3 ' 77
mouse GAPDH (forward)mouse GAPDH (forward) 5'- AGGTCGGTGTGAACGGATTTG -3'5'- AGGTCGGTGTGAACGGATTTG -3 ' 88
mouse GAPDH (reverse)mouse GAPDH (reverse) 5'- TGTAGACCATGTAGTTGAGGTCA -3'5'- TGTAGACCATGTAGTTGAGGTCA -3 ' 99
실험결과1. 독성 베타 아밀로이드를 처리한 인간 피부 섬유아세포의 DNA 메틸화 변화 분석Experimental Results 1. Analysis of DNA Methylation Changes in Human Skin Fibroblasts Treated with Toxic Beta Amyloid
인간 피부 섬유아세포 NHDF를 배양하여 알츠하이머성 치매의 병인 물질의 하나로 알려진 독성 베타 아밀로이드 (Aβ1-42)로 장기간 (1.5 μM로 5일) 처리하여 알츠하이머성 치매 특이적 표현형을 나타내는 세포 모델을 확립하였다. 확립된 알츠하이머성 치매 세포 모델의 DNA 메틸화 변화를 분석하기 위해 Aβ1-42 로 처리한 NHDF와 대조군으로 DMSO로 처리한 NHDF 세포를 Infinium(®) Human Methylation EPIC BeadChip (Illumina)을 사용하여 genome-wide DNA 메틸레이션 마이크로어레이를 시행하였다. 두 군에서 차별적으로 메틸화 되어 있는 유전자 (Differentially methylated genes, DMGs)를 선별하기 위하여 Bayesian t-test 방법을 사용하였다. 최종적으로 p value<0.05이면서 절대값 β 차이≥0.3 인 CpG 부위를 차별적으로 메틸화되어 있는 CpG 부위(differentially methylated CpG site)로 선정하고, 이 중에서 프로모터 부분에 존재하는 CpG 부위의 메틸화 정도가 변한 IDE 유전자를 DMG로 최종 선정하였다 (표 2).Human skin fibroblast NHDF was cultured and treated with toxic beta amyloid (Aβ 1-42 ) for a long time (5 days at 1.5 μM), which is one of the pathogens of Alzheimer's dementia, to establish a cell model showing an Alzheimer's dementia specific phenotype. . To analyze DNA methylation changes in established Alzheimer's dementia cell models, NHDF treated with Aβ 1-42 and NHDF treated with DMSO as a control were genome-wide using Infinium (®) Human Methylation EPIC BeadChip (Illumina). DNA methylation microarrays were performed. Bayesian t-test was used to select differentially methylated genes (DMGs) from both groups. Finally, the CpG site with p value <0.05 and the absolute value β difference ≥0.3 was selected as the differentially methylated CpG site, and among these, the IDE gene whose methylation degree of the CpG site in the promoter region was changed. Was finally selected as DMG (Table 2).
유전자gene Methylation Chip IDMethylation Chip ID Location of CpG siteLocation of CpG site Differentially methylated valueDifferentially methylated value 1)One) 메틸레이션 상태Methylation status
IDEIDE cg18197594cg18197594 TSS1500TSS1500 0.330130.33013 고메틸화Hypermethylation
1) 베타 아밀로이드(Aβ1-42)로 처리한 세포에서의 β 값과 DMSO 로 처리한 세포에서의 β값의 차이를 말함1) The difference between β values in cells treated with beta amyloid (Aβ 1-42 ) and β values in cells treated with DMSO.
2. 독성 베타 아밀로이드를 처리한 인간 피부 섬유아세포에서 IDE 유전자의 프로모터 CpG 메틸화 변화2. Changes in Promoter CpG Methylation of the IDE Gene in Human Skin Fibroblasts Treated with Toxic Beta Amyloid
DNA 메틸레이션 마이크로어레이 분석 결과, 독성 베타 아밀로이드를 처리한 인간 피부 섬유아세포에서 IDE 유전자의 프로모터에 존재하는 특정 CpG 부위의 DNA 메틸화가, 대조군 DMSO를 처리한 인간 피부 섬유아세포에 비해 30% 정도 높아져 있는 것을 확인하였다 (도 1). IDE 유전자의 프로모터에 존재하는 특정 CpG 부위는 10번 염색체의 94334836 번째에 위치하는 사이토신이며 주변 염기서열은 다음과 같다:DNA methylation microarray analysis showed that in human skin fibroblasts treated with toxic beta amyloid, DNA methylation of specific CpG sites present in the IDE gene promoter was about 30% higher than that in control DMSO treated human skin fibroblasts. It was confirmed (Fig. 1). The specific CpG site in the promoter of the IDE gene is the cytosine at 94334836 of chromosome 10 and the surrounding sequences are as follows:
CAAGTATTGCTTTAAATGGGAATTCAGTAGGAGGGTTCCTTAATCAAGTTGATGCTTTAT C GAAGACGTACCCACGATCTTTTTAACAGTGCGGTTTCACCTAAAGGCAAACCTTTGGATGA (서열번호 1: 10 번 염색체의 94334776 내지 94334897 번째 염기서열)CAAGTATTGCTTTAAATGGGAATTCAGTAGGAGGGTTCCTTAATCAAGTTGATGCTTTAT C G AAGACGTACCCACGATCTTTTTAACAGTGCGGTTTCACCTAAAGGCAAACCTTTGGATGA (SEQ ID NOs 1: 94334776 to 94334897 nucleotide sequences of Chromosome 10)
또한, 독성 베타아밀로이드를 처리한 인간 피부 섬유아세포에서 IDE 유전자의 발현이 대조군에 비해 현저히 감소한 것을 RT-qPCR로 확인하였다 (도 2).In addition, it was confirmed by RT-qPCR that the expression of the IDE gene in human skin fibroblasts treated with toxic beta amyloid was significantly reduced compared to the control (Fig. 2).
3. DNA 메틸화에 의한 IDE 유전자의 발현 조절3. Regulation of IDE Gene Expression by DNA Methylation
인간 피부 섬유아세포에 탈 DNA 메틸레이션 제제인 5-aza-2'-deoxycytidin을 3일간 처리한 후 IDE 유전자의 발현 변화를 조사한 결과, DNA 메틸화가 줄어들면서 IDE 유전자의 발현이 증가하는 것을 확인할 수 있었다. 이것은 DNA 메틸화에 의해 IDE 유전자의 발현이 억제되는 것을 보여주며 IDE 유전자의 발현이 DNA 메틸화에 의해 조절되는 것을 의미한다 (도 3 및 도 4).After 3-day treatment of 5-aza-2'-deoxycytidin, a de-methylation methylation agent in human skin fibroblasts, the expression of IDE genes was examined and the expression of IDE genes was increased as DNA methylation decreased. . This shows that the expression of the IDE gene is inhibited by DNA methylation and means that the expression of the IDE gene is regulated by DNA methylation (FIGS. 3 and 4).
4. 알츠하이머성 치매 모델의 피부 섬유아세포에서 IDE 유전자의 발현 감소4. Reduction of IDE Gene Expression in Skin Fibroblasts of Alzheimer's Dementia Model
12개월령 알츠하이머성 치매 모델 APP swe/PS1 형질전환쥐 (Borchelt; B6C3-Tg(APP695)85Dbo/Tg(PSEN1)85Dbo)의 해마(hippocampus) 및 피질(cortex) 부위에서 IDE 유전자의 발현이 정상 쥐와 비교하여 감소되어 있었다 (도 5). 또한, 동일 쥐의 피부에서 추출 배양한 피부 섬유아세포에서도 뇌조직과 유사하게 IDE 유전자의 발현이 감소되어 있는 것을 확인할 수 있었다 (도 5). 따라서, 알츠하이머성 치매 동물 모델을 통하여 IDE 유전자의 발현 감소는 알츠하이머성 치매에서 특징적으로 나타나는 현상임을 확인하였다. 본 실험 결과를 통하여 IDE 유전자의 발현 감소는 뇌조직 뿐만 아니라 피부에서도 발견되는 알츠하이머성 치매의 특성임을 확인할 수 있었다.12-month-old Alzheimer's dementia model APP swe / PS1 transgenic rats (Borchelt; B6C3-Tg (APP695) 85Dbo / Tg (PSEN1) 85Dbo) in the hippocampus and cortex region of the IDE gene expression in normal rats Compared to that (FIG. 5). In addition, it was confirmed that the expression of the IDE gene is reduced in the skin fibroblasts extracted from the same mouse skin and cultured similarly to the brain tissue (FIG. 5). Therefore, it was confirmed through the animal model of Alzheimer's dementia that the expression of the IDE gene is a characteristic phenomenon in Alzheimer's dementia. Through the experimental results, it was confirmed that the decrease in the expression of the IDE gene is a characteristic of Alzheimer's dementia found in not only brain tissue but also skin.
결론conclusion
결론적으로, 독성 베타 아밀로이드를 처리한 피부 섬유아세포에서 알츠하이머성 치매 개체의 뇌세포와 유사하게 IDE의 발현이 저하되어 있는 것을 확인하였고, IDE 유전자의 발현이 독성 베타 아밀로이드에 의해서 유도된 DNA 메틸화 변화에 의해 조절됨을 확인하였다. 본 실시예의 결과는 알츠하이머성 치매 개체의 뇌세포에서 일어나는 생화학적 변화를 피부 섬유아세포에서 확인할 수 있음을 시사하며, 피부 섬유아세포를 이용하여 IDE 유전자의 특정 CpG 부위에서 나타나는 특이적인 DNA 메틸레이션의 고메틸화를 측정함으로써 비침습적인 방법으로 알츠하이머성 치매를 진단하는 바이오마커로 활용될 수 있음을 나타내는 것이다.In conclusion, we found that the expression of IDE was decreased in brain fibroblasts treated with toxic beta amyloid, similar to the brain cells of Alzheimer's dementia, and that the expression of IDE gene was related to the DNA methylation changes induced by toxic beta amyloid. It was confirmed that it is controlled by. The results of this example suggest that the biochemical changes in brain cells of Alzheimer's dementia individuals can be identified in skin fibroblasts, and that specific DNA methylation at specific CpG sites of IDE genes can be identified using skin fibroblasts. By measuring methylation, it can be used as a biomarker to diagnose Alzheimer's dementia in a non-invasive way.

Claims (14)

  1. 피부 시료로부터 IDE (Insulin Degrading Enzyme) 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 제제를 포함하는 알츠하이머 질환 진단용 조성물.A composition for diagnosing Alzheimer's disease, comprising an agent for measuring the methylation level of the CpG region of an IDE (Insulin Degrading Enzyme) gene promoter from a skin sample.
  2. 제1항에 있어서, 상기 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 제제는, 비메틸화 사이토신 염기를 변형시키는 화합물 또는 메틸화 민감성 제한효소, 상기 유전자 프로모터의 CpG 부위의 메틸화된 서열에 특이적인 프라이머, 비메틸화된 서열에 특이적인 프라이머, 메틸화된 CpG 결합 도메인, 또는 메틸사이토신에 특이적으로 결합하는 항체를 포함하는 것인 조성물.According to claim 1, wherein the agent for measuring the methylation level of the CpG region of the gene promoter is a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base, primers specific for the methylated sequence of the CpG region of the gene promoter , An antibody that specifically binds to a primer specific for an unmethylated sequence, a methylated CpG binding domain, or methylcytosine.
  3. 제2항에 있어서, 상기 비메틸화 사이토신 염기를 변형시키는 화합물은 바이설파이트(bisulfite) 또는 이의 염인 조성물.The composition of claim 2, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.
  4. 제2항에 있어서, 상기 메틸화 민감성 제한효소는 SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI 또는 NotI인 조성물.The composition of claim 2, wherein the methylation sensitive restriction enzyme is Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI or Not I.
  5. 제1항 내지 제4항 중 어느 한 항에 있어서, 상기 IDE 유전자 프로모터의 CpG 는 서열번호 1(10 번 염색체의 94334776 내지 94334897 번째)의 염기서열 중에 나타나는 CpG 를 포함하는 것인 조성물.The composition according to any one of claims 1 to 4, wherein the CpG of the IDE gene promoter comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 1 (94334776 to 94334897 of chromosome 10).
  6. 제1항 내지 제5항 중 어느 한 항의 조성물을 포함하는, 알츠하이머 질환 진단용 키트.A kit for diagnosing Alzheimer's disease, comprising the composition of any one of claims 1 to 5.
  7. 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계를 포함하는, 알츠하이머 질환 진단을 위한 정보를 제공하는 방법.A method for providing information for diagnosing Alzheimer's disease, comprising measuring the methylation level of a CpG region of an IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease.
  8. 제7항에 있어서, 상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터에서의 메틸화 수준과 비교하는 단계를 더욱 포함하는 방법.8. The method of claim 7, further comprising comparing the methylation level with the methylation level in the corresponding gene promoter of a control sample that is not Alzheimer's disease.
  9. 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; Measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease;
    상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계; 및 Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease; And
    상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계를 포함하는, 알츠하이머 질환의 진단 방법.And determining that the subject has Alzheimer's disease when the methylation level measured from the sample of the subject is higher than that measured from the control sample.
  10. 알츠하이머 질환이 의심되는 개체의 피부 시료로부터 IDE 유전자 프로모터의 CpG 부위의 메틸화 수준을 측정하는 단계; Measuring the methylation level of the CpG region of the IDE gene promoter from a skin sample of an individual suspected of Alzheimer's disease;
    상기 메틸화 수준을 알츠하이머 질환이 아닌 대조군 시료의 해당 유전자 프로모터의 메틸화 수준과 비교하는 단계;Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample that is not Alzheimer's disease;
    상기 개체의 시료로부터 측정된 메틸화 수준이 대조군 시료로부터 측정된 것보다 높을 경우 개체가 알츠하이머 질환을 가진 것으로 결정하는 단계; 및Determining that the subject has Alzheimer's disease if the methylation level measured from the sample of the subject is higher than that measured from the control sample; And
    알츠하이머 질환를 가진 것으로 결정된 상기 개체에 알츠하이머 질환 치료제를 투여하는 단계를 포함하는, 알츠하이머 질환의 치료 방법.A method of treating Alzheimer's disease, comprising administering an Alzheimer's disease therapeutic agent to said individual determined to have Alzheimer's disease.
  11. 제7항 내지 제10항 중 어느 한 항에 있어서, 상기 유전자 프로모터의 CpG 부위의 메틸화 수준의 측정 단계는 The method of any one of claims 7 to 10, wherein the step of measuring the methylation level of the CpG region of the gene promoter is
    (a) 수득된 시료 내 게놈 DNA를 비메틸화 사이토신 염기를 변형시키는 화합물 또는 메틸화 민감성 제한효소로 처리하는 단계; 및 (a) treating genomic DNA in the obtained sample with a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base; And
    (b) 상기 처리된 DNA를 IDE 유전자 프로모터의 CpG 부위를 증폭할 수 있는 프라이머를 이용하여 PCR에 의해 증폭하는 단계를 포함하는 방법.(b) amplifying the treated DNA by PCR using a primer capable of amplifying the CpG region of the IDE gene promoter.
  12. 제11항에 있어서, 상기 비메틸화 사이토신 염기를 변형시키는 화합물은 바이설파이트(bisulfite) 또는 이의 염인 방법.The method of claim 11, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.
  13. 제7항 내지 제12항 중 어느 한 항에 있어서, 상기 IDE 유전자 프로모터의 CpG 는 서열번호 1(10 번 염색체의 94334776 내지 94334897 번째)의 염기서열 중에 나타나는 CpG 를 포함하는 것인 방법.The method according to any one of claims 7 to 12, wherein the CpG of the IDE gene promoter comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 1 (94334776 to 94334897th of chromosome 10).
  14. 제7항 내지 제13항 중 어느 한 항에 있어서, 상기 메틸화 수준을 검출하는 방법은 메틸화 특이적 중합효소반응(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 중합효소반응(real time methylation-specific polymerase chain reaction), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, 파이로시퀀싱, 바이설파이트 시퀀싱, DNA 메틸레이션 마이크로어레이, 및 메틸화된 CpG 결합 도메인 또는 항-메틸사이토신 항체를 이용한 면역침강법으로 구성된 군에서 선택되는 것인 방법.The method according to any one of claims 7 to 13, wherein the method for detecting the methylation level is methylation-specific polymerase chain reaction, real time methylation-specific reaction. polymerase chain reaction, PCR with methylated DNA specific binding proteins, quantitative PCR, pyro sequencing, bisulfite sequencing, DNA methylation microarrays, and immunoprecipitation with methylated CpG binding domains or anti-methylcytosine antibodies Method selected from the group consisting of laws.
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