WO2020027569A1 - Composition pour le diagnostic de la maladie d'alzheimer par utilisation d'une altération de la méthylation des cpg dans le promoteur du gène ide dans des cellules de la peau, et son utilisation - Google Patents

Composition pour le diagnostic de la maladie d'alzheimer par utilisation d'une altération de la méthylation des cpg dans le promoteur du gène ide dans des cellules de la peau, et son utilisation 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
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    • 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

La présente invention concerne une composition, une trousse et un procédé, qui sont destinés au diagnostic de la maladie d'Alzheimer par détection du niveau de méthylation des sites CpG d'un promoteur du gène de l'enzyme de dégradation de l'insuline (IDE).
PCT/KR2019/009529 2018-07-31 2019-07-31 Composition pour le diagnostic de la maladie d'alzheimer par utilisation d'une altération de la méthylation des cpg dans le promoteur du gène ide dans des cellules de la peau, et son utilisation WO2020027569A1 (fr)

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KR101965380B1 (ko) * 2018-07-31 2019-04-03 이화여자대학교 산학협력단 피부 세포에서 IDE 유전자 프로모터의 CpG 메틸화 변화를 이용한 알츠하이머 질환 진단용 조성물 및 이의 이용
KR102313452B1 (ko) 2020-02-27 2021-10-14 이화여자대학교 산학협력단 알츠하이머병 치매 치료를 위한 분자 진단 테스트
KR102313458B1 (ko) 2020-02-27 2021-10-14 이화여자대학교 산학협력단 알츠하이머병 치매 관련 유전자 바이오마커 및 이의 이용
KR102313454B1 (ko) * 2020-02-27 2021-10-15 이화여자대학교 산학협력단 유전자 CpG 메틸화 변화를 이용한 알츠하이머병 치매 진단용 조성물 및 이의 이용
WO2021172864A1 (fr) * 2020-02-27 2021-09-02 이화여자대학교 산학협력단 Diagnostic et prédiction de la maladie d'alzheimer à l'aide d'une modification de méthylation épigénétique de gène
WO2021172863A1 (fr) * 2020-02-27 2021-09-02 이화여자대학교 산학협력단 Test de diagnostic moléculaire pour la maladie d'alzheimer
KR102313453B1 (ko) * 2020-02-27 2021-10-15 이화여자대학교 산학협력단 유전자의 dna 메틸레이션 변화를 이용한 알츠하이머병 경도인지장애의 진단 마커

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101302173B1 (ko) * 2012-12-07 2013-08-30 이화여자대학교 산학협력단 Ｈｍｏｘ１ 유전자의 메틸화 변화를 이용한 알츠하이머 질환의 진단용 조성물 및 이를 이용한 알츠하이머 질환의 진단방법
EP2746402A1 (fr) * 2013-03-12 2014-06-25 Academisch Medisch Centrum Procédé de pronostic de la maladie d'Alzheimer et substrats pour utilisation associée
KR20160080165A (ko) * 2014-12-29 2016-07-07 주식회사 휴젠바이오 퇴행성 뇌질환 진단용 조성물 및 이를 이용한 퇴행성 뇌질환의 진단 방법
KR101965380B1 (ko) * 2018-07-31 2019-04-03 이화여자대학교 산학협력단 피부 세포에서 IDE 유전자 프로모터의 CpG 메틸화 변화를 이용한 알츠하이머 질환 진단용 조성물 및 이의 이용

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4666829A (en) 1985-05-15 1987-05-19 University Of California Polypeptide marker for Alzheimer's disease and its use for diagnosis
CA1339014C (fr) 1987-10-08 1997-03-25 Ronald E. Majocha Anticorps pour peptide a4-amyloide
US5231000A (en) 1987-10-08 1993-07-27 The Mclean Hospital Antibodies to A4 amyloid peptide
US5297562A (en) 1991-04-01 1994-03-29 President And Fellows Of Harvard College Method for detecting and treating Alzheimer's disease
KR101596359B1 (ko) * 2014-12-01 2016-02-22 이화여자대학교 산학협력단 SORT1 유전자 프로모터의 CpG 메틸화 변화를 이용한 모야모야병 진단용 조성물 및 이의 이용
KR101597812B1 (ko) * 2015-10-13 2016-02-25 이화여자대학교 산학협력단 후성유전학적 변화 및 전사체 통합분석을 통한 후성 유전학적 진단법 개발

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101302173B1 (ko) * 2012-12-07 2013-08-30 이화여자대학교 산학협력단 Ｈｍｏｘ１ 유전자의 메틸화 변화를 이용한 알츠하이머 질환의 진단용 조성물 및 이를 이용한 알츠하이머 질환의 진단방법
EP2746402A1 (fr) * 2013-03-12 2014-06-25 Academisch Medisch Centrum Procédé de pronostic de la maladie d'Alzheimer et substrats pour utilisation associée
KR20160080165A (ko) * 2014-12-29 2016-07-07 주식회사 휴젠바이오 퇴행성 뇌질환 진단용 조성물 및 이를 이용한 퇴행성 뇌질환의 진단 방법
KR101965380B1 (ko) * 2018-07-31 2019-04-03 이화여자대학교 산학협력단 피부 세포에서 IDE 유전자 프로모터의 CpG 메틸화 변화를 이용한 알츠하이머 질환 진단용 조성물 및 이의 이용

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEAL, M. C.: "Transcriptional regulation of insulin-degrading enzyme modulates mitochondrial amyloid beta (Abeta) peptide catabolism and functionality", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 288, 3 May 2013 (2013-05-03), pages 18, XP055682365, DOI: 10.1074/jbc.M112.424820 *
ZUO, X.: "Promoter polymorphisms which modulate insulin degrading enzyme expression may increase susceptibility to Alzheimer's disease", BRAIN RESEARCH, vol. 1249, 2009, pages 1 - 8, XP025860175, DOI: 10.1016/j.brainres.2008.10.034 *

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