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WO2024019556A1 - Marqueur de diagnostic prédictif pour la maladie d'alzheimer à l'aide d'un changement de méthylation d'adn dans le gène krt32 - Google Patents

Marqueur de diagnostic prédictif pour la maladie d'alzheimer à l'aide d'un changement de méthylation d'adn dans le gène krt32 Download PDF

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WO2024019556A1
WO2024019556A1 PCT/KR2023/010461 KR2023010461W WO2024019556A1 WO 2024019556 A1 WO2024019556 A1 WO 2024019556A1 KR 2023010461 W KR2023010461 W KR 2023010461W WO 2024019556 A1 WO2024019556 A1 WO 2024019556A1
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alzheimer
disease
methylation
dementia
cognitive impairment
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Korean (ko)
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안정혁
이현주
정지향
박희경
성혜윤
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Gwangju Institute of Science and Technology
Ewha Womans University
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Gwangju Institute of Science and Technology
Ewha Womans University
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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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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

Definitions

  • the present invention relates to a composition, kit, and method for predicting the risk of progression to Alzheimer's disease dementia by detecting the methylation level of the CpG region of the gene.
  • Dementia is a representative disease that causes a large socioeconomic burden, and among them, Alzheimer's disease dementia is the most common type of dementia.
  • Alzheimer's disease Dementia is a representative neurodegenerative brain disease that begins with memory loss and gradually progresses over a long period of time to symptoms such as loss of cognitive ability in time and space, delusions and personality changes, paralysis of language function, and paralysis of motor function, ultimately leading to death. am.
  • Alzheimer's disease dementia Although research into the treatment of Alzheimer's disease dementia has been continuously conducted for the past several decades, there is currently no fundamental cure, and only treatments that alleviate symptoms or slow down the progression of the lesion are being carried out. Therefore, delaying the onset of Alzheimer's disease dementia through early diagnosis and preemptive treatment has been suggested as the most effective management method.
  • the neurocognitive tests currently used to diagnose Alzheimer's disease dementia have the disadvantage that the patient's age, level of education, or intentional refusal to answer can significantly affect the accuracy of the test results, and brain imaging tests using MRI or PET have the disadvantage of significantly affecting the accuracy of the test results.
  • the use of expensive equipment has the disadvantage of making it difficult to perform tests in the early stages when no special symptoms are present.
  • Alzheimer's disease dementia includes a method of detecting beta amyloid protein in cerebrospinal fluid or serum, a method of measuring the concentration of tau protein, and a glial fibrillary acidic protein (GFAP)-antibody.
  • Biochemical methods such as detection methods have been proposed, but problems such as efficiency and accuracy of clinical application of diagnostic methods have been raised (International Patent Publication No. 92/17152; US Patent No. 4,666,829; International Patent Publication No. 89/17152). No. 06242; U.S. Patent No. 5,231,000, etc.)
  • the present inventors confirmed that the KRT32 (Keratin 32) gene is specifically hypermethylated in mild cognitive impairment in Alzheimer's disease, and used this as a biomarker to measure the degree of methylation of the KRT32 gene to detect mild cognitive impairment in Alzheimer's disease.
  • the present invention was completed by confirming that it is possible to diagnose or predict the risk of progression to Alzheimer's disease dementia.
  • an example of the present invention provides a composition for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, including an agent for measuring the methylation level of the CpG region of the KRT32 gene promoter.
  • Another example provides the use of an agent that measures the level of CpG methylation of the KRT32 gene promoter to diagnose mild cognitive impairment in Alzheimer's disease or predict the risk of progression to dementia in Alzheimer's disease.
  • Another example provides an agent that measures CpG methylation levels in the KRT32 gene promoter for use in diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another example provides a kit for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, including an agent for measuring the methylation level of the CpG region of the KRT32 gene promoter.
  • Another example provides a method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, comprising measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual.
  • Another example provides a method of providing information for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, which includes measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual.
  • Another example provides a method of measuring the methylation level at the CpG region of the KRT32 gene promoter from a sample of an individual to provide information necessary for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another example includes measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample other than mild cognitive impairment from Alzheimer's disease or comparing it with a predetermined threshold (cut-off); And if the methylation level measured from the sample of the subject is higher than the value measured from the control sample or a threshold value, determining that the subject has mild cognitive impairment in Alzheimer's disease or is at risk of progressing to dementia in Alzheimer's disease. , provides a method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another example includes measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample other than mild cognitive impairment from Alzheimer's disease or comparing it with a predetermined threshold (cut-off); If the methylation level measured from the sample of the subject is higher than the value measured from the control sample or a threshold value, determining that the subject has mild cognitive impairment in Alzheimer's disease or is at risk of progressing to Alzheimer's disease dementia; and administering a therapeutic agent for Alzheimer's disease to the individual determined to have mild cognitive impairment in Alzheimer's disease or to be at risk for progression to dementia in Alzheimer's disease.
  • the present invention provides a molecular biological diagnostic method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease by measuring the degree of methylation of the CpG region of a specific gene in genomic DNA collected from a patient's sample, which is simple and It has the advantage of being non-invasive and economical. Additionally, DNA methylation changes are easy to detect and have the advantage of being more stable and easier to analyze than conventional protein or RNA markers.
  • Figure 1 shows the difference in the degree of DNA methylation of the KRT32 gene in a group of patients with mild cognitive impairment without amyloid pathology (“MCI (non-AD)”) and a group of patients with mild cognitive impairment from Alzheimer’s disease (“Prodromal AD”) without amyloid pathology in DNA methylation mutation analysis. It is shown.
  • MCI mild cognitive impairment without amyloid pathology
  • Prodromal AD Alzheimer’s disease
  • Figure 2 shows the difference in the degree of DNA methylation of the KRT32 gene in a group of patients with mild cognitive impairment without amyloid pathology (“MCI (non-AD)”) and a group of patients with mild cognitive impairment from Alzheimer’s disease (“Prodromal AD”) without amyloid pathology in pyrosequencing analysis. It is shown.
  • MCI mild cognitive impairment without amyloid pathology
  • Prodromal AD Alzheimer’s disease
  • Figure 3 shows the effectiveness of distinguishing between a group of patients with mild cognitive impairment without amyloid pathology (“MCI (non-AD)”) and a group of patients with mild cognitive impairment from Alzheimer’s disease (“Prodromal AD”) using DNA methylation changes in the KRT32 gene.
  • MCI mild cognitive impairment without amyloid pathology
  • Prodromal AD a group of patients with mild cognitive impairment from Alzheimer’s disease
  • ROC curve receiver operating characteristics curve
  • the present invention is based on the discovery that the KRT32 gene is specifically hypermethylated in mild cognitive impairment in Alzheimer's disease.
  • the present invention measures the degree of specific DNA hypermethylation that occurs at a specific CpG position in the KRT32 gene to diagnose mild cognitive impairment in Alzheimer's disease or Alzheimer's disease. It provides molecular biological diagnostic technology to predict the risk of progression to dementia.
  • DNA methylation changes are easy to detect because they exist in DNA, are more stable than protein or RNA markers, and have the advantage of being easy to analyze because they occur at a specific location in a gene.
  • genomic DNA was extracted from samples of a patient group with mild cognitive impairment without amyloid pathology and a patient group with mild cognitive impairment with Alzheimer's disease, DNA methylation profile was analyzed through DNA methylation mutation analysis, and amyloid pathology was analyzed. Compared to the mild cognitive impairment patient group without any findings, genes with more than 20% DNA methylation change in the CpG region of the gene promoter region of the Alzheimer's disease mild cognitive impairment patient group were selected.
  • one embodiment provides a composition for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, including an agent for measuring the methylation level of the CpG region of the KRT32 gene promoter.
  • Another embodiment provides the use of an agent that measures the level of CpG methylation of the KRT32 gene promoter for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another embodiment provides an agent for measuring CpG methylation levels in the KRT32 gene promoter for use in diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another embodiment provides a kit for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, including an agent for measuring the methylation level of the CpG region of the KRT32 gene promoter.
  • Another embodiment provides a method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, comprising measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual.
  • Another embodiment provides a method of providing information for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, which includes measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual.
  • Another embodiment provides a method of measuring the methylation level at the CpG region of the KRT32 gene promoter from a sample of an individual to provide information necessary for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another specific example includes measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample other than mild cognitive impairment from Alzheimer's disease or comparing it with a predetermined threshold (cut-off); And if the methylation level measured from the sample of the subject is higher than the value measured from the control sample or a threshold value, determining that the subject has mild cognitive impairment in Alzheimer's disease or is at risk of progressing to dementia in Alzheimer's disease. , Provides a method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • Another specific example includes measuring the methylation level of the CpG region of the KRT32 electronic promoter from a sample of an individual; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample other than mild cognitive impairment from Alzheimer's disease or comparing it with a predetermined threshold (cut-off); If the methylation level measured from the sample of the subject is higher than the value measured from the control sample or a threshold value, determining that the subject has mild cognitive impairment in Alzheimer's disease or is at risk of progressing to dementia in Alzheimer's disease; and administering an Alzheimer's disease therapeutic agent to the individual determined to have mild cognitive impairment of Alzheimer's disease or to be at risk of progression to Alzheimer's disease dementia.
  • methylation refers to the attachment of a methyl group to a base constituting DNA.
  • methylation refers to whether methylation occurs at cytosine in the CpG region of a specific gene promoter. When methylation occurs, the binding of transcription factors is disrupted and the expression of a specific gene is suppressed. Conversely, when unmethylation or hypomethylation occurs, the expression of a specific gene increases.
  • the genomic DNA of mammalian cells contains a fifth base called 5-methylcytosine (5-mC), which has a methyl group attached to the fifth carbon of the cytosine ring. do.
  • Methylation of 5-methylcytosine occurs only at the C of CG dinucleotide (5'-mCG-3'), called CpG, and methylation of CpG suppresses the expression of alu or transposon and repetitive sequences of the genome.
  • CpG is the site where most epigenetic changes frequently occur in mammalian cells.
  • methylation level measurement refers to measuring the methylation level of a gene CpG region, using methylation-specific PCR, for example, methylation-specific polymerase chain reaction (MSP), real-time methylation-specific PCR. It can be measured through PCR (real time methylation-specific polymerase chain reaction), PCR using a methylated DNA-specific binding protein, or quantitative PCR. Alternatively, it can be measured by methods such as automatic base analysis such as pyrosequencing or bisulfite sequencing, DNA methylation microarray, or immunoprecipitation using a methylated CpG binding domain or anti-methylcytosine antibody. However, it is not limited to this.
  • the risk of progression to Alzheimer's disease dementia can be predicted by specifically showing hypermethylation in the KRT32 gene in patient samples compared to samples from people without Alzheimer's disease or compared to a predetermined threshold (cut-off). there is.
  • the human KRT32 gene is located on chromosome 17 and is registered in the NCBI Entrez database as Gene ID: 3882.
  • the CpG site of a gene refers to a CpG site present on the DNA of the gene.
  • the DNA of a gene is a concept that includes a series of structural units that are required for gene expression and are operably linked to each other, for example, a promoter region, a protein coding region (open reading frame, ORF), and a terminator region. Includes. Therefore, the CpG site of a gene may be present in the promoter region, protein coding region (open reading frame, ORF), or terminator region of the gene.
  • the CpG site where disease-specific hypermethylation occurs in the KRT32 gene may be present in the promoter of the gene.
  • the methylation level of the CpG region of the KRT32 gene promoter is measured by measuring the methylation level of the promoter CpG region of the KRT32 gene, more specifically, the CpG region appearing in the 41467764 to 41467885 base sequence of chromosome 17 (SEQ ID NO: 1). It may include measuring the methylation level of cytosine:
  • it may include measuring the methylation of cytosine located at base 41467824 of chromosome 17 (base 61 of SEQ ID NO: 1).
  • the base sequence of the human genome chromosome region is expressed according to the latest version of GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38), but the specific sequence of the human genome chromosome region is expressed as the results of genome sequence research are updated. This may be slightly changed, and depending on this change, the expression of the human genomic chromosomal region of the present invention may be different. Therefore, the human genome chromosomal region expressed according to the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38) of the present invention is a human reference sequence that has been updated after the filing date of the present invention to represent the human genomic chromosomal region. Even if it is changed differently from what it is now, it is clear that the scope of the present invention extends to the changed human genome chromosomal region. These changes can be easily known by anyone with ordinary knowledge in the technical field to which the present invention pertains.
  • Alzheimer's disease is a representative neurodegenerative brain disease with memory decline as an initial symptom and subsequent decline in overall cognitive function.
  • AD Alzheimer's disease
  • Alzheimer's disease is based on postmortem brain autopsy findings, but as pathological accumulation of amyloid protein and tau protein, which are known to be representative features of brain autopsy findings, can be indirectly confirmed through PET imaging or cerebrospinal fluid examination, clinical Compared to the era when diagnosis was based solely on symptoms, the accuracy of diagnosis has improved dramatically.
  • Biomarkers for defining Alzheimer's disease include beta-amyloid-related indicators (e.g., binding of amyloid-PET ligand in the cerebral cortex or reduction of A ⁇ 42 in cerebrospinal fluid) and tau-related indicators in the form of neurofibrillary tangles (e.g. (e.g., increased phosphorylated tau in cerebrospinal fluid or binding of tau-PET ligands in the brain cortex), or indicators of neurodegeneration (e.g., increased cerebrospinal fluid total tau or brain atrophy on MRI and brain hypometabolism on FDG-PET). ), etc.
  • beta-amyloid-related indicators e.g., binding of amyloid-PET ligand in the cerebral cortex or reduction of A ⁇ 42 in cerebrospinal fluid
  • tau-related indicators in the form of neurofibrillary tangles e.g. (e.g., increased phosphorylated tau in cerebrospinal fluid or binding of tau-PET
  • Alzheimer's Disease Research Diagnostic Criteria Jack CR According to , et al., NIA-AA Research Framework: toward a biological definition of Alzheimer's disease.
  • Alzheimers Dement 2018;14:535-562 Alzheimer's disease is a pathological characteristic of Alzheimer's disease even before clinical symptoms of dementia appear.
  • Alzheimer's disease biomarkers as described above
  • they are included in the diagnostic criteria for Alzheimer's disease.
  • Alzheimer's disease is largely subdivided into preclinical Alzheimer's disease, prodromal Alzheimer's disease, and symptomatic Alzheimer's disease. It is explained with the concept of continuum.
  • Alzheimer's disease dementia shows pathological findings of Alzheimer's disease in biological marker tests, symptoms of dementia develop, and objective cognitive function declines. As a result, the ability to live independently is impaired.
  • AD with dementia or “Symptomatic AD” are also used.
  • Alzheimer's disease mild cognitive impairment in the present invention is premised on representing the pathological characteristics (biomarkers) of Alzheimer's disease, but is a condition that is not dementia because there is objective cognitive decline but the ability to perform daily life is preserved. This refers to the stage before the onset of Alzheimer's disease or dementia.
  • MCI cognitive impairment
  • the general "mild cognitive impairment (MCI)” includes cases where cognitive ability is clinically determined to be reduced due to not only Alzheimer's disease but also other neurodegenerative diseases or various factors. While 1-2% of the general elderly population progresses to dementia each year, it is known that 5-20% of elderly people with mild cognitive impairment progress to dementia per year, and mild cognitive impairment is considered a high-risk group for dementia. However, mild cognitive impairment may progress not to Alzheimer's disease dementia but to other dementia diseases such as fronto-temporal dementia or vascular dementia, and 20 to 30% of cases recover to normal or do not progress to dementia but similar to mild cognitive impairment. It is known to be maintained.
  • Alzheimer's disease mild cognitive impairment refers to mild cognitive impairment caused by Alzheimer's disease, that is, mild cognitive impairment that shows the pathological characteristics (biomarkers) of Alzheimer's disease. Compared to general mild cognitive impairment, Alzheimer's disease dementia There is a very high possibility that it will proceed. According to previous reports, in the case of mild cognitive impairment not caused by Alzheimer's disease, the rate of progression to Alzheimer's disease dementia within 3 years was only about 5%, but in the case of mild cognitive impairment of Alzheimer's disease according to the NIA-AA diagnostic criteria, the rate of progression to Alzheimer's disease within 3 years was high. The rate of progression to dementia was significantly higher at approximately 59% (Brain 2015: 138; 1327-1338).
  • the methylation marker of the present invention is specifically highly methylated in the mild cognitive impairment patient group with Alzheimer's disease compared to the mild cognitive impairment patient group without amyloid pathology, and through ROC curve analysis, the methylation marker of the present invention is more methylated in the mild cognitive impairment patient group without amyloid pathology and the Alzheimer's disease patient group. Since its effectiveness in distinguishing mild cognitive impairment patient groups has been confirmed, it can be used as a diagnostic marker for diagnosing mild cognitive impairment in Alzheimer's disease or as a predictive diagnostic marker for Alzheimer's disease dementia that predicts the risk of progression from mild cognitive impairment in Alzheimer's disease to dementia in Alzheimer's disease. It can be.
  • composition, kit, and method of the present invention can predict the risk of Alzheimer's dementia in advance, thereby selecting patients with a high likelihood of progressing to Alzheimer's disease dementia and delaying the worsening of the disease through active preventive management and early treatment.
  • early detection can delay the onset and reduce the prevalence of dementia, ultimately improving the quality of life for patients and their families and greatly reducing socioeconomic costs borne by the nation. You can.
  • diagnosis means confirming the existence of a disease or the presence or characteristics of a disease pathological state. Comprehensively, diagnosis includes determining whether a specific disease currently exists, measuring the condition before symptoms appear, determining the prognosis of the disease, and further determining whether the disease has progressed or worsened. can do.
  • the term "predicting the risk of progression to Alzheimer's disease dementia” refers to assessing and predicting in advance the risk of developing Alzheimer's disease dementia or progressing to Alzheimer's disease dementia at a stage before symptoms of Alzheimer's disease dementia appear.
  • the agent for measuring the methylation level of a CpG site is a compound that modifies an unmethylated cytosine base, a methylation-sensitive restriction enzyme, a primer specific for the methylated allele sequence of the gene, or a compound specific for the unmethylated allele sequence. It may include a primer, a methylated CpG binding domain, or a methylated DNA antibody that specifically binds to methylated DNA (for example, an antibody that specifically binds to methylcytosine).
  • the compound that modifies the unmethylated cytosine base may be bisulfite or a salt thereof, but is not limited thereto, and is preferably sodium bisulfite.
  • Methylation of bisulfite-modified DNA can be detected through various methods such as sequencing or methylation-specific PCR.
  • the method of detecting methylation of a gene by modifying unmethylated cytosine residues using bisulfite is It is well known in the art (e.g. WO01/26536; US2003/0148326A1).
  • the methylation-sensitive restriction enzyme is a restriction enzyme that can specifically detect methylation of a CpG site and may be a restriction enzyme that contains CG as a recognition site of the restriction enzyme. Examples include Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, etc., but are not limited thereto. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, cleavage by the restriction enzyme varies and can be detected through PCR or Southern Blot analysis. Other methylation sensitive restriction enzymes other than the above restriction enzymes are well known in the art.
  • genomic DNA is obtained from a patient's sample, and the obtained DNA is treated with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme. Afterwards, the treated DNA can be amplified by PCR using primers and measured by confirming the presence or absence of the amplified product.
  • the agent of the present invention may include 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 having a short free 3 terminal hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. Additionally, primers, both sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides, may incorporate additional features that do not change the basic nature of the primer, which serves as an initiation point for DNA synthesis.
  • the primers of the present invention can be preferably designed according to the sequence of the specific CpG site to be analyzed for methylation, and each primer pair is capable of specifically amplifying a cytosine that is methylated and has not been modified by bisulfite, And it may be a primer pair that can specifically amplify cytosine that is not methylated and thus modified by bisulfite.
  • a methylated DNA antibody refers to an antibody that specifically binds to a methylated base in DNA.
  • antibodies that have the property of recognizing and binding to methylated cytosine in the DNA chain such as antibodies to methylcytosine, can be cited.
  • methylated DNA antibodies there may be an antibody that can specifically recognize and specifically bind to DNA in the methylated state described herein.
  • Methylated DNA antibodies can be produced by conventional methods using methylated bases, methylated DNA, etc. as antigens.
  • methylated bases methylated DNA, etc.
  • an antibody is prepared using 5-methylcytidine, 5-methylcytosine, or DNA containing 5-methylcytosine as an antigen, and then methylcytosine in the DNA is used as an antigen.
  • the specific combination of can be selected as an indicator.
  • methylated DNA is immunoprecipitated using these, and then analyzed by Southern blot, PCR, microarray, or sequence analysis. Specific CpG sites can be confirmed through sequencing.
  • a substrate, an appropriate buffer solution, a chromogenic enzyme or a fluorescent substance label, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate can be used.
  • the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass
  • the coloring enzyme may be peroxidase or alkaline phosphatase. Fatase (alkaline phosphatase), etc. can be used, the fluorescent substance can be FITC, RITC, etc., and the coloring substrate solution is ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphate).
  • Ponic acid o-phenylenediamine
  • TMB tetramethyl benzidine
  • other radioactive isotope labels, latex bead labels, colloid labels, biotin labels, etc. can be used, but are limited to these. no.
  • compositions and kits may additionally include polymerase agarose, buffer solutions required for electrophoresis, etc. Additionally, the kit may be implemented in the form of a DNA methylation microarray.
  • the present invention relates to a method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease by measuring the methylation level at the CpG region of the KRT32 gene promoter.
  • the present invention relates to a method for detecting the methylation level of the CpG region of a gene promoter from a sample of an individual in order to provide information necessary for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • the present invention relates to a method of providing information for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease, comprising measuring the methylation level at the CpG region of the KRT32 gene promoter from a sample of an individual. will be.
  • the present invention includes the steps of measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual; And comparing the methylation level with the methylation level of the corresponding gene in a control sample other than Alzheimer's disease mild cognitive impairment or comparing it with a predetermined threshold (cut-off) for diagnosis of Alzheimer's disease mild cognitive impairment or Alzheimer's disease dementia. It concerns a method of providing information for predicting the risk of progression.
  • the present invention includes the steps of measuring the methylation level of the CpG region of the KRT32 gene promoter from a sample of an individual; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample other than mild cognitive impairment from Alzheimer's disease or comparing it with a predetermined threshold (cut-off); And if the methylation level measured from the sample of the subject is higher than the value measured from the control sample or a threshold value, determining that the subject has mild cognitive impairment in Alzheimer's disease or is at risk of progressing to dementia in Alzheimer's disease. , relates to a method for diagnosing mild cognitive impairment in Alzheimer's disease or predicting the risk of progression to dementia in Alzheimer's disease.
  • the present invention includes measuring the methylation level of the CpG region of the KRT32 electronic promoter from a sample of an individual; Comparing the methylation level with the methylation level of the corresponding gene promoter in a control sample other than mild cognitive impairment from Alzheimer's disease or comparing it with a predetermined threshold (cut-off); If the methylation level measured from the sample of the subject is higher than the value measured from the control sample or a threshold value, determining that the subject has mild cognitive impairment in Alzheimer's disease or is at risk of progressing to Alzheimer's disease dementia; and administering a therapeutic agent for Alzheimer's disease to an individual determined to have mild cognitive impairment in Alzheimer's disease or to be at risk for progression to dementia in Alzheimer's disease.
  • sample includes samples such as cells, tissues, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum, or urine in which the methylation level of genes is different due to Alzheimer's disease, but is not limited to the above examples and includes DNA. Any sample that can be extracted can be used. Preferred embodiments include skin tissue or skin cells, for example, but are not limited to skin fibroblasts.
  • genomic DNA was obtained using the phenol/chloroform extraction method and SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB separation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980), or a commercially available DNA extraction kit.
  • the step of detecting the methylation level of a specific CpG site of the gene includes a method using restriction enzymes or bisulfite, using the difference between methylated bases and unmethylated bases, a methylated CpG binding domain, or an anti-methylcytosine antibody. It can be performed through immunoprecipitation methods (e.g., MIRA, MeDIP), methods using DNA methylation microarray, etc.
  • 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 protein, quantitative PCR,
  • the methylation level can be measured or detected using pyrosequencing, bisulfite sequencing, DNA methylation microarray, or immunoprecipitation using a methylated CpG binding domain or anti-methylcytosine antibody.
  • the step of measuring the methylation level of the CpG region of the gene promoter includes (a) treating genomic DNA in the obtained sample with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme; and (b) amplifying the treated DNA by PCR using primers capable of amplifying a specific CpG region of the gene.
  • the compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite.
  • a method for detecting methylation of a gene by modifying unmethylated cytosine residues using bisulfite is widely known in the art.
  • the methylation sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of a specific CpG site, as described above, and may be a restriction enzyme containing CG as the recognition site of the restriction enzyme.
  • examples include Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, etc., but are not limited thereto.
  • Amplification in step (b) may be performed by a conventional PCR method.
  • the primers used at this time can be preferably designed according to the sequence of the specific CpG site to be analyzed for methylation, and each specifically amplifies cytosines that have been methylated and have not been modified by bisulfite. It may be a primer pair that can specifically amplify cytosine that is not methylated and thus modified by bisulfite.
  • the step of measuring the methylation level of a specific CpG region of the gene may further include the step of (c) confirming the presence or absence of the product amplified in step (b).
  • the presence or absence of the amplified product in step (c) can be determined depending on whether a band at a desired position is detected by a method known in the art, for example, electrophoresis.
  • a method known in the art for example, electrophoresis.
  • two types of primer pairs are used in step (a), that is, a primer that can specifically amplify cytosine that is methylated and has not been modified by bisulfite.
  • the degree of methylation can be determined based on the presence or absence of PCR results each amplified by a pair of primers that are not methylated and can specifically amplify cytosine modified by bisulfite.
  • methylation can be determined using a bisulfite genome sequencing method in which sample genomic DNA is treated with bisulfite, the CpG region of the gene is amplified by PCR, and the base sequence of the amplified region is analyzed. .
  • a method known in the art for example, when a PCR result is obtained from mock DNA and a PCR result is obtained from DNA treated with a restriction enzyme, it is determined that the CpG is methylated, and the restriction enzyme is determined to be methylated. If there is no PCR result from the enzyme-treated DNA, whether the CpG is methylated can be determined by determining that it is unmethylated, and this is obvious to those skilled in the art.
  • mock DNA refers to sample DNA that has been isolated from the sample and has not undergone any treatment.
  • Another example of detecting the methylation level of a specific CpG region of a gene may be an immunoprecipitation method using a methylated DNA antibody, for example, an antibody against a methylated CpG binding domain (MBD) or methylcytosine.
  • a methylated DNA antibody for example, an antibody against a methylated CpG binding domain (MBD) or methylcytosine.
  • MBD methylated CpG binding domain
  • specific CpG sites can be identified through Southern blot, PCR, microarray, or sequencing. You can.
  • the risk of progression to Alzheimer's disease dementia can be predicted in advance.
  • methylation changes in specific CpG regions of genes appear specifically in individual samples, and therefore, methylation changes in genes can be used as a biomarker to predict Alzheimer's disease dementia.
  • hypermethylation of a specific CpG region of the KRT32 gene can be used as a biomarker to predict the risk of progression to Alzheimer's disease dementia.
  • the Alzheimer's disease patient group includes three groups: preclinical Alzheimer's disease (AD), prodromal Alzheimer's disease, and Symptomatic Alzheimer's disease.
  • the control group includes normal control group (normal cognitive function and no amyloid pathology), mild cognitive impairment (MCI) due to non-AD, and dementia not due to Alzheimer's disease. It consists of three groups: dementia due to non-AD.
  • the medical history was taken through a structured interview by a neurologist, along with the research subjects and their guardians.
  • the degree of decline in current cognitive function and daily living skills were determined through neuropsychological testing.
  • three stages of cognitive decline were determined: normal cognitive function, mild cognitive impairment, and dementia.
  • normal cognitive function group apart from complaints of subjective cognitive decline, all items of the objective neuropsychological test showed test results of standard scores (z score ⁇ -1.5) or higher, so there was no objective finding of cognitive decline and daily living ability. There is no damage.
  • mild cognitive impairment the patient or informant complains of memory decline, and neuropsychological tests show a standard score of 1.5 or less (z score ⁇ -1.5), indicating objective memory decline, but overall cognitive function and This is a group where daily living skills are maintained.
  • the dementia group is a group that shows objective decline (standard score of 1.5 or less) in two or more cognitive domains, has a slowly progressive clinical pattern, and requires care from others due to impairment of daily living abilities.
  • blood tests brain structural imaging (computed tomography, CT or magnetic resonance imaging, MRI), and brain functional imaging (functional MRI, amyloid positron emission tomography, amyloid PET) are performed to detect decline in cognitive function.
  • Other possible causes were ruled out, and the accumulation of amyloid pathology in the brain was confirmed. Ruling out other causes that may cause decline in cognitive function is an important step in diagnosing Alzheimer's disease. It is important to rule out these possibilities by checking blood tests for thyroid hormone dysfunction, vitamin B12 or folic acid deficiency, and neurosyphilis.
  • alcoholism, drug addiction, major depressive disorder, bipolar disorder, schizophrenia, and convulsive disorders were also excluded because they may be associated with cognitive decline.
  • amyloid PET is considered as a control group and according to clinical symptoms, normal control group (group with normal cognitive function and no amyloid pathology), mild cognitive impairment (MCI) not caused by Alzheimer's disease. ) due to non-AD), or dementia not caused by Alzheimer's disease (dementia due to non-AD).
  • a skin biopsy was taken from the patient's inner thigh using a cylindrical blade with a diameter of 2 mm.
  • the collected skin biopsy was divided into 6 parts, placed in a 24-well cell culture plate coated with 0.1% gelatin with medium (DMEM/20% FBS) sufficient to submerge the skin biopsy pieces, and cells were observed extending from the edges of the skin biopsy pieces for 7 days. observed.
  • the medium was replenished every 2-3 days to prevent drying of the skin biopsy pieces. After 7 days, the amount of medium was increased to 500 ⁇ L and the medium was changed every 2-3 days. After 14 days, when the cells grew around the skin biopsy piece and the outer edge of the culture well was filled with cells, it was transferred to a 35 mm culture dish and cultured on the right side of the medium.
  • Genomic DNA was extracted from skin fibroblasts cultured in Example 2 using the QIAmp DNA mini kit (Qiagen). The extraction method was performed according to the manufacturer's manual. The extracted genomic DNA was quantified using a spectrophotometer, and the state of the DNA was checked for degradation by electrophoresis on a 1% agarose gel.
  • DNA methylation mutation analysis involves extracting DNA from skin fibroblasts of patient and control groups, performing bisulfite conversion to change unmethylated cytosine to uracil, and then converting unmethylated cytosine to uracil using an Infinium MethylationEPIC bead chip (Illumina).
  • the degree of methylation was measured for 850,000 CpG sites.
  • the degree of DNA methylation is expressed as a ⁇ value ranging from 0 to 1.
  • a ⁇ value of 0 means that the corresponding CpG site is completely unmethylated, and 1 means that it is completely methylated.
  • DMGs differentially methylated genes
  • PCR Pyrosequencing analysis was performed to measure the degree of methylation of a specific CpG region (cg25598400, Chr17: 41467824) of the KRT32 (Keratin 32) gene.
  • PCR was performed using bisulfite-treated gDNA as a template.
  • PCR primers were designed using the PSQ assay design program (Qiagen, USA). The base sequences of the designed PCR primers are as follows:
  • Biotinylated reverse primer 5'- CCTCTAAAATTCCTATTCCTTTTCTACT -3' (SEQ ID NO: 3)
  • the PCR reaction solution contained 20 ng or less of bisulfite-converted gDNA, 10 ⁇ l 2' Hot/Start PCR premix (Enzynomics), 1 ⁇ l forward primer (10 pmole/ ⁇ l), and 1 ⁇ l biotinylated reverse primer (10 ⁇ l). It was prepared so that the total volume, including pmole/ ⁇ l), was 20 ⁇ l.
  • the PCR reaction goes through an initial denaturation process at 95°C for 10 minutes, followed by a denaturation process at 95°C for 30 seconds, annealing process at 56°C for 30 seconds, and extension process at 72°C. This was performed for 30 seconds and this process was repeated 50 times.
  • the final PCR product was prepared as an ssDNA template using Streptavidin Sepharose HP beads (Amersham Biosciences).
  • the reaction solution was prepared for each sample to have a total volume of 80 ⁇ l, including 1-2 ⁇ l beads, 40 ⁇ l binding buffer, 16-18 ⁇ l PCR product, and high purity water, and then dispensed into each well of the PCR plate. After enclosing the plate, it was mixed well at 1400 rpm for 15-20 minutes using an orbital shaker.
  • the DNA methylation percentage of a specific CpG site (cg25598400, Chr17:41467824) of the KRT32 gene was calculated as follows.
  • the base sequence of the generated PCR product is 5'- AGTAGTGGTTGTTTTTTTTAATGAAAGTATTTTTTATGGGAAGAGGTTTGGTTATTTAATAAGGAAATGTGTGGGTGGTAGAAATTGTTT Y GATGTTGGTAGCGGTATAAATTTAGTAGAAAGGAATAGGAATTTTAGAGG -3' (SEQ ID NO: 5), and the ratio of cytosine bases at the position indicated by Y (C or T) was calculated as a percentage: C/C+T * 100 (%)
  • Genomic DNA was obtained by culturing skin fibroblasts from skin tissue of 20 patients with mild cognitive impairment without amyloid pathology and 28 patients with mild cognitive impairment with Alzheimer's disease in patients who visited Ewha Mans University Mokdong Hospital from 2018 to 2019. was extracted.
  • the DNA methylation profile was analyzed using the Illumina Human MethylationEPIC bead chip, and compared to the mild cognitive impairment patient group without amyloid pathology, more than 20% of DNA was found in the specific CpG region of the gene promoter in skin fibroblasts of the Alzheimer's disease mild cognitive impairment patient group. Genes with changed methylation were selected.
  • the KRT32 (Keratin 32) gene was confirmed to have a 21% increase in DNA methylation at the specific promoter CpG region in skin fibroblasts from the Alzheimer's disease mild cognitive impairment patient group compared to the mild cognitive impairment patient group without amyloid pathology. .
  • KRT32 was found in the Alzheimer's disease mild cognitive impairment patient group compared to the mild cognitive impairment patient group without amyloid pathology. It was confirmed that DNA methylation of the gene-specific promoter CpG site (cg25598400) was increased by 21% ( Figure 2).
  • the receiver operating characteristic curve was used to determine whether it was possible to distinguish between a group of patients with mild cognitive impairment without amyloid pathology and a group of patients with mild cognitive impairment from Alzheimer's disease.
  • AUC area under the curve

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Abstract

La présente invention concerne une composition, un kit et un procédé permettant de prédire le risque de progression de la maladie d'Alzheimer (MA) en détectant le niveau de méthylation de la région CpG du promoteur d'un gène de la kératine 32 (KRT32), et présente l'effet de représenter les symptômes cliniques pour un diagnostic précoce de la MA ou de faire le point sur l'état préclinique avant l'apparition des symptômes.
PCT/KR2023/010461 2022-07-20 2023-07-20 Marqueur de diagnostic prédictif pour la maladie d'alzheimer à l'aide d'un changement de méthylation d'adn dans le gène krt32 Ceased WO2024019556A1 (fr)

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