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WO2021172864A1 - Diagnostic et prédiction de la maladie d'alzheimer à l'aide d'une modification de méthylation épigénétique de gène - Google Patents

Diagnostic et prédiction de la maladie d'alzheimer à l'aide d'une modification de méthylation épigénétique de gène Download PDF

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WO2021172864A1
WO2021172864A1 PCT/KR2021/002309 KR2021002309W WO2021172864A1 WO 2021172864 A1 WO2021172864 A1 WO 2021172864A1 KR 2021002309 W KR2021002309 W KR 2021002309W WO 2021172864 A1 WO2021172864 A1 WO 2021172864A1
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Prior art keywords
alzheimer
disease
methylation
gene
dementia
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Korean (ko)
Inventor
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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Priority claimed from KR1020200024176A external-priority patent/KR102313454B1/ko
Priority claimed from KR1020200024180A external-priority patent/KR102313455B1/ko
Priority claimed from KR1020200024177A external-priority patent/KR102139314B1/ko
Priority claimed from KR1020200024179A external-priority patent/KR102313459B1/ko
Priority claimed from KR1020200024175A external-priority patent/KR102313453B1/ko
Priority claimed from KR1020200024178A external-priority patent/KR102313457B1/ko
Priority claimed from KR1020200024181A external-priority patent/KR102313456B1/ko
Application filed by Gwangju Institute of Science and Technology, Ewha Womans University filed Critical Gwangju Institute of Science and Technology
Publication of WO2021172864A1 publication Critical patent/WO2021172864A1/fr
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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

Definitions

  • the present invention detects or measures the methylation level of the gene CpG region, to diagnose Alzheimer's disease mild cognitive impairment, to diagnose Alzheimer's disease dementia, to diagnose Alzheimer's disease early, or to predict the risk of progression to Alzheimer's disease dementia Compositions, kits and methods for
  • Alzheimer's disease dementia is a representative neurodegenerative brain disease that starts with memory loss and progresses gradually over a long period of time with symptoms such as loss of time and space cognitive ability, delusions and personality changes, paralysis of speech and motor functions, and eventually death. am.
  • Alzheimer's disease dementia Although research for the treatment of Alzheimer's disease dementia has been continuously conducted for the past several decades, there is no fundamental treatment until now, and only a level of treatment that relieves symptoms or slows the progression of the lesion is being performed. Therefore, delaying the onset of dementia through early diagnosis and preemptive treatment for Alzheimer's disease is suggested as the most effective management method.
  • a new diagnostic marker that can represent clinical symptoms or accurately measure the Alzheimer's disease state is required.
  • a new diagnostic marker that can represent clinical symptoms or measure the preclinical state before symptoms appear for early diagnosis of Alzheimer's disease dementia.
  • the present inventors have identified FOXP1 (forkhead box P1), NAA60 (N-alpha-acetyltransferase 60, NatF catalytic subunit), SLC38A2 (solute carrier family 38 member 2), LOC391322 (D-dopachrome tautomerase-like), CD96 (CD96 molecule),
  • the present invention was completed by confirming that PLEC1 (plectin), and/or NHLRC4 (NHL repeat containing 4) genes exhibit specific methylation changes in Alzheimer's disease, and using this as a biomarker to diagnose Alzheimer's disease did it Specifically, the diagnosis of Alzheimer's disease includes diagnosing Alzheimer's disease mild cognitive impairment, diagnosing Alzheimer's disease dementia, early diagnosing Alzheimer's disease dementia, or predicting the risk of progression to Alzheimer's disease dementia.
  • an embodiment of the present invention provides a method of diagnosing Alzheimer's disease in an individual, a method of assisting in the diagnosis of Alzheimer's disease, a method of providing information for diagnosing Alzheimer's disease, or a method of detecting a biomarker for diagnosing Alzheimer's disease.
  • the method includes (i) FOXP1 (forkhead box P1), NAA60 (N-alpha-acetyltransferase 60, NatF catalytic subunit), SLC38A2 (solute carrier family 38 member 2), LOC391322 (D-dopachrome tautomerase-like ), CD96 (CD96 molecule), PLEC1 (plectin), and NHLRC4 (NHL repeat containing 4) measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of, and (ii) the above (i) comparing the level measured in the step to a reference value; Alzheimer's disease dementia is diagnosed when the CpG region of one or more gene promoters selected from the group consisting of FOXP1, NAA60, and SLC38A2 in the subject's sample is hypomethylated than the reference value; If the CpG region of the LOC391322 gene promoter in the sample of the subject is hypomethylated than the reference value, it is
  • kits for diagnosing Alzheimer's disease comprising an agent for measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of FOXP1, NAA60, SLC38A2, LOC391322, CD96, PLEC1, and NHLRC4.
  • Diagnosis provides a kit for diagnosing Alzheimer's disease mild cognitive impairment, diagnosing Alzheimer's disease dementia, diagnosing Alzheimer's disease dementia early, or predicting the risk of progression to Alzheimer's disease dementia.
  • compositions for diagnosing Alzheimer's disease comprising an agent for measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of FOXP1, NAA60, SLC38A2, LOC391322, CD96, PLEC1, and NHLRC4.
  • Diagnosis provides a composition for diagnosing Alzheimer's disease mild cognitive impairment, diagnosing Alzheimer's disease dementia, diagnosing Alzheimer's disease dementia early, or predicting the risk of progression to Alzheimer's disease dementia.
  • the present invention measures the degree of methylation of a specific gene CpG region of genomic DNA collected from a patient's sample to diagnose Alzheimer's disease mild cognitive impairment, Alzheimer's disease dementia, early diagnosis of Alzheimer's disease dementia, or Alzheimer's disease
  • a molecular biological diagnostic method for predicting the risk of progression to dementia is provided, which is simple, non-invasive, and economically advantageous.
  • DNA methylation changes are easy to detect, stable and easy to analyze compared to conventional protein or RNA markers.
  • FIG. 2 shows the results of a receiver-operating characteristic curve (ROC curve) analysis result for evaluating the effectiveness of discriminating an Alzheimer's disease dementia patient group from a normal control group using a change in DNA methylation of the FOXP1 gene.
  • ROC curve receiver-operating characteristic curve
  • Figure 3 shows the difference in the DNA methylation degree of the NAA60 gene in the normal control group and Alzheimer's disease dementia patient group.
  • FIG. 4 shows the results of a receiver-operating characteristic curve (ROC curve) analysis for evaluating the effectiveness of discriminating an Alzheimer's disease dementia patient group from a normal control group using a change in DNA methylation of the NAA60 gene.
  • ROC curve receiver-operating characteristic curve
  • 5 shows the difference in DNA methylation degree of the SLC38A2 gene in the normal control group and the Alzheimer's disease dementia patient group.
  • FIG. 6 shows the results of a receiver-operating characteristic curve (ROC curve) analysis result for evaluating the effectiveness of discriminating an Alzheimer's disease dementia patient group from a normal control group using a change in DNA methylation of the SLC38A2 gene.
  • ROC curve receiver-operating characteristic curve
  • the first figure of FIG. 9 shows the results of the receiver-operating characteristic curve (ROC curve) analysis for evaluating the effectiveness of distinguishing the Alzheimer's disease mild cognitive impairment patient group from the normal control group using the DNA methylation change of the LOC391322 gene
  • the second The figure shows the results of ROC curve analysis to evaluate the effectiveness of distinguishing the Alzheimer's disease dementia patient group from the normal control group using the DNA methylation change of the LOC391322 gene.
  • ROC curve receiver-operating characteristic curve
  • ROC curve receiver-operating characteristic curve
  • ROC curve receiver-operating characteristic curve
  • ROC curve receiver-operating characteristic curve
  • the present invention provides FOXP1 (forkhead box P1), NAA60 (N-alpha-acetyltransferase 60, NatF catalytic subunit), SLC38A2 (solute carrier family 38 member 2), LOC391322 (D-dopachrome tautomerase-like), CD96 (CD96 molecule), It is based on the fact that the PLEC1 (plectin) and NHLRC4 (NHL repeat containing 4) genes show specific methylation changes in Alzheimer's disease. Molecular biological diagnostic technology is provided.
  • DNA methylation changes are present in DNA, they are easy to detect, more stable than protein or RNA markers, and have the advantages of easy analysis because they occur at a specific location in a gene.
  • genomic DNA is extracted from the samples of the normal control group (normal cognitive function group without amyloid pathology), Alzheimer's disease mild cognitive impairment patient group, and Alzheimer's disease dementia patient group, and DNA methylation mutation analysis is performed.
  • the DNA methylation profile was analyzed, and compared with the normal control group, genes whose DNA methylation was changed by 30% or more in the CpG region of the gene promoter region of patients with Alzheimer's disease mild cognitive impairment and Alzheimer's disease dementia were selected.
  • FOXP1, NAA60 and SLC38A2 genes were reduced by about 48%, about 34%, and about 45%, respectively, of DNA methylation of the CpG region of the promoter in the Alzheimer's disease patient group compared to the normal control group.
  • ROC curve receiveriver operating characteristics curve
  • the DNA methylation of the CpG region of the promoter was decreased by about 31% and about 33% in the Alzheimer's disease mild cognitive impairment patient group and the Alzheimer's disease dementia patient group compared to the normal control group in the CD96 and NHLRC4 genes, and the PLEC1 gene was increased by about 35%.
  • one embodiment provides a method of diagnosing Alzheimer's disease in a subject, a method of assisting in the diagnosis of Alzheimer's disease, a method of providing information for diagnosing Alzheimer's disease, or a method of detecting a biomarker for diagnosing Alzheimer's disease
  • the method includes (i) FOXP1 (forkhead box P1), NAA60 (N-alpha-acetyltransferase 60, NatF catalytic subunit), SLC38A2 (solute carrier family 38 member 2), LOC391322 (D-dopachrome tautomerase-like) , CD96 (CD96 molecule), PLEC1 (plectin), and NHLRC4 (NHL repeat containing 4) measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of, and (ii) the step (i) comparing the measured level with a reference value; Alzheimer's disease dementia is diagnosed when the CpG region of one or more gene promoters selected from the
  • kits for diagnosing Alzheimer's disease comprising an agent for measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of FOXP1, NAA60, SLC38A2, LOC391322, CD96, PLEC1, and NHLRC4.
  • Diagnosis of disease diagnosing Alzheimer's disease mild cognitive impairment, diagnosing Alzheimer's disease dementia, early diagnosis of Alzheimer's disease dementia, or predicting the risk of progression to Alzheimer's disease dementia provides a kit.
  • Another embodiment is a composition for diagnosing Alzheimer's disease, comprising an agent for measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of FOXP1, NAA60, SLC38A2, LOC391322, CD96, PLEC1, and NHLRC4.
  • Disease diagnosis provides a composition for diagnosing Alzheimer's disease mild cognitive impairment, diagnosing Alzheimer's disease dementia, early diagnosing Alzheimer's disease dementia, or predicting the risk of progression to Alzheimer's disease dementia.
  • methylation refers to the attachment of a methyl group to a base constituting DNA.
  • methylation means whether methylation occurs in the cytosine of the CpG region of a specific gene promoter. In the case of methylation, the binding of transcription factors is disturbed and the expression of a specific gene is suppressed. Conversely, when unmethylation or hypomethylation occurs, the expression of a specific gene is increased.
  • 5-methylcytosine In the genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base called 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. do. 5-methylcytosine methylation occurs only at C of a CG dinucleotide called CpG (5'-mCG-3'), and CpG methylation inhibits expression of alu or transposon and genomic repeats. In addition, since 5-mC of CpG is easily deamidated to thymine (T), CpG is a site where most epigenetic changes frequently occur in mammalian cells.
  • 5-mC of CpG is easily deamidated to thymine (T)
  • T thymine
  • the term "measurement of methylation level” refers to measuring the methylation level of the CpG region of a gene, and methylation-specific PCR, for example, methylation-specific polymerase chain reaction (MSP), real-time methylation-specific It can be measured through real time methylation-specific polymerase chain reaction (PCR), PCR using a methylated DNA-specific binding protein, or quantitative PCR.
  • MSP methylation-specific polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR PCR using a methylated DNA-specific binding protein
  • quantitative PCR quantitative PCR.
  • measurement by methods such as automatic sequencing such as pyrosequencing or bisulfite sequencing, or immunoprecipitation using DNA methylation microarray, methylated CpG binding domain or anti-methylcytosine antibody, etc. can be used.
  • the present invention is not limited thereto.
  • FOXP1, NAA60 and/or FOXP1, NAA60 and/or in a sample from a patient compared to a baseline e.g., a sample from a healthy subject or a sample from a subject not diagnosed with Alzheimer's disease, or compared to a predetermined cut-off
  • a baseline e.g., a sample from a healthy subject or a sample from a subject not diagnosed with Alzheimer's disease, or compared to a predetermined cut-off
  • a baseline e.g., a sample from a healthy subject or a sample from a subject not diagnosed with Alzheimer's disease, or compared to a predetermined cut-off
  • hypomethylation in the LOC391322 gene in the patient's sample compared to a baseline e.g., a sample from a healthy individual or a sample from a subject not diagnosed with Alzheimer's disease, or compared to a predetermined cut-off, is Through the specific appearance, Alzheimer's disease mild cognitive impairment or Alzheimer's disease dementia can be diagnosed early.
  • a decrease in the CD96 and NHLRC4 genes in the patient's sample Through specific methylation or high methylation in the PLEC1 gene, it is possible to diagnose Alzheimer's disease mild cognitive impairment or predict the risk of progression to Alzheimer's disease dementia.
  • the human FOXP1 gene is located on chromosome 3 and is registered as Gene ID: 27086 in the NCBI Entrez database.
  • the human NAA60 gene is located on chromosome 16 and is registered as Gene ID: 79903 in the NCBI Entrez database.
  • the human SLC38A2 gene is located on chromosome 12 and is registered as Gene ID: 54407 in the NCBI Entrez database.
  • the human LOC391322 gene is located on chromosome 22 and is registered as Gene ID: 391322 in the NCBI Entrez database.
  • the human CD96 gene is located on chromosome 3 and is registered as Gene ID: 10225 in the NCBI Entrez database.
  • the human PLEC1 gene is located on chromosome 8 and is registered as Gene ID: 5339 in the NCBI Entrez database.
  • the human NHLRC4 gene is located on chromosome 16 and is registered as Gene ID: 283948 in the NCBI Entrez database.
  • the CpG region of a gene refers to a CpG region present on the DNA of a gene.
  • DNA of a gene is a concept that includes all a series of structural units that are necessary 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.
  • the CpG region of a gene may exist in a promoter region, a protein coding region (open reading frame, ORF) or a terminator region of the gene.
  • the CpG region in which disease-specific hypomethylation occurs may be present in the promoter of the gene, and the CpG region in which disease-specific hypermethylation occurs in the PLEC1 gene is the promoter of the gene. may exist in
  • measuring the methylation level of the CpG region of the FOXP1 gene promoter is the CpG region of the promoter of the FOXP1 gene, more specifically, the CpG region appearing in the 71063226 to 71063347 nucleotide sequences (SEQ ID NO: 1) of chromosome 3 It may include measuring the methylation level of cytosine of More specifically, it may include measuring the methylation of cytosine located at the 71063286th base (61st base of SEQ ID NO: 1) of chromosome 3.
  • Measuring the methylation level of the CpG region of the NAA60 gene promoter in the present invention is to measure the methylation level of the NAA60 gene promoter CpG region, more specifically, the cytosine of the CpG region appearing in the nucleotide sequence 3457850 to 3457971 of chromosome 16 (SEQ ID NO: 2). measuring the methylation level. More specifically, the method may include measuring methylation of cytosine located at base 3457910 of chromosome 16 (base 61 of SEQ ID NO: 2).
  • Measuring the methylation level of the CpG region of the SLC38A2 gene promoter in the present invention is to measure the methylation level of the CpG region of the SLC38A2 gene promoter, more specifically, the cytosine of the CpG region in the 46368865 to 46368986 nucleotide sequences of chromosome 12 (SEQ ID NO: 3). measuring the methylation level. More specifically, the method may include measuring the methylation of cytosine located at the 46368925th base (61st base of SEQ ID NO: 3) of chromosome 12.
  • the promoter CpG region of the LOC391322 gene more specifically, the cytosine of the CpG region appearing in the 267036 to 267157 nucleotide sequences (SEQ ID NO: 4) of chromosome 22 measuring the methylation level. More specifically, the method may include measuring the methylation of cytosine located at the 267096th base of chromosome 22 (the 61st base of SEQ ID NO: 4).
  • Measuring the methylation level of the CpG region of the CD96 gene promoter in the present invention is to measure the methylation level of the CpG region of the CD96 gene promoter, more specifically, the cytosine of the CpG region appearing in the nucleotide sequences 111541849 to 111541970 of chromosome 3 (SEQ ID NO: 5). measuring the methylation level. More specifically, it may include measuring the methylation of cytosine located at the 111541909 th base of chromosome 3 (61 st base of SEQ ID NO: 5).
  • Measuring the methylation level of the CpG region of the PLEC1 gene promoter in the present invention is to measure the methylation level of the CpG region of the PLEC1 gene promoter, more specifically, the cytosine of the CpG region appearing in nucleotide sequences 143940809 to 143940930 of chromosome 8 (SEQ ID NO: 6). measuring the methylation level. More specifically, the method may include measuring the methylation of cytosine located at the 143940869 base of chromosome 8 (the 61st base of SEQ ID NO: 6).
  • Measuring the methylation level of the CpG region of the NHLRC4 gene promoter in the present invention is to measure the methylation level of the CpG region of the NHLRC4 gene promoter, more specifically, the cytosine of the CpG region appearing in the 566797 to 566918 nucleotide sequences of chromosome 16 (SEQ ID NO: 7). measuring the methylation level. More specifically, it may include measuring the methylation of cytosine located at the 566857th base (61st base of SEQ ID NO: 7) of chromosome 16.
  • the nucleotide sequence of the human genome chromosomal region is expressed according to the latest version of the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38), but the specific sequence of the human genome chromosomal region is expressed as the genome sequence study results are updated. This may be slightly altered, and the expression of the human genome chromosomal region of the present invention may be different according to such alteration. Therefore, the human genome chromosomal region expressed according to the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38) of the present invention has been updated with the human reference sequence since the filing date of the present invention to express the human genome chromosomal region. Even if this is changed differently from now, it will be apparent that the scope of the present invention extends to the altered human genome chromosomal region. These changes can be easily recognized by anyone with ordinary skill in the art to which the present invention pertains.
  • Alzheimer's disease is a representative disease of neurodegenerative brain disease, with memory loss as an initial symptom, and then overall cognitive function decline.
  • AD Alzheimer's disease
  • the diagnosis of Alzheimer's disease is a post-mortem autopsy of the brain, the pathological accumulation of amyloid protein and tau protein, which are known as representative features of autopsy findings in the brain, can be indirectly confirmed by PET image or cerebrospinal fluid test. Compared to the era when diagnosis was based only on modalities, the accuracy of diagnosis has dramatically improved.
  • Biomarkers for defining Alzheimer's disease include beta-amyloid-related markers (e.g., cortical amyloid-PET ligand binding or decreased A ⁇ 42 in cerebrospinal fluid), neurofibrillary tangible tau-related markers (e.g., For example, an increase in phosphorylated tau in the cerebrospinal fluid or binding of tau-PET ligand in the cerebral cortex), or an indicator of neurodegeneration (e.g., an increase in cerebrospinal fluid total tau or brain atrophy in MRI and decreased brain metabolism in FDG-PET) ) and the like.
  • beta-amyloid-related markers e.g., cortical amyloid-PET ligand binding or decreased A ⁇ 42 in cerebrospinal fluid
  • neurofibrillary tangible tau-related markers e.g., For example, an increase in phosphorylated tau in the cerebrospinal fluid or binding of tau-PET ligand in the cerebral cortex
  • an indicator of neurodegeneration e.
  • Alzheimer's Association AA
  • NIA-AA Alzheimer's Association
  • AD preclinical Alzheimer's disease
  • prodromal Alzheimer's disease Symptomatic Alzheimer's disease through a combination of biomarkers and cognitive stages.
  • AD preclinical Alzheimer's disease
  • Symptomatic Alzheimer's disease through a combination of biomarkers and cognitive stages.
  • Alzheimer's disease Normal cognitive function shows pathological findings of Alzheimer's disease in biological marker tests, but corresponds to the preclinical stage without clinical symptoms. The next stage, Alzheimer's disease mild cognitive impairment, shows pathological findings of Alzheimer's disease in the biological marker test, but there is a mild objective cognitive decline and independent daily life performance is maintained.
  • the term "AD with mild cognitive impairment” or “prodromal AD” is also used.
  • Alzheimer's disease dementia shows the pathological findings of Alzheimer's disease in biomarker tests, develops dementia symptoms, and decreases objective cognitive function As a result, the ability to live independently is impaired.
  • Alzheimer's disease mild cognitive impairment is premised on representing the pathological characteristics (biomarkers) of Alzheimer's disease, but there is objective cognitive decline, but the ability to perform daily life is preserved, so it is not dementia It refers to the stage before the onset of Alzheimer's disease dementia.
  • MCI cognitive impairment
  • the general "mild cognitive impairment (MCI)” includes Alzheimer's disease, as well as other neurodegenerative diseases or clinically determined cognitive decline due to various factors. Compared to 1 ⁇ 2% of the general elderly population progressing to dementia every year, it is known that 5-20% of the elderly with mild cognitive impairment progress to dementia per year, so mild cognitive impairment is considered a high-risk group for dementia. However, mild cognitive impairment does not progress to Alzheimer's disease, but to other dementia diseases such as frontal temporal dementia or vascular dementia. known to be maintained.
  • Alzheimer's disease mild cognitive impairment refers to mild cognitive impairment due to Alzheimer's disease, that is, mild cognitive impairment showing the pathological characteristics (biomarkers) of Alzheimer's disease, compared to general mild cognitive impairment. Alzheimer's disease is more likely to progress to dementia.
  • 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 Alzheimer's mild cognitive impairment according to the NIA-AA diagnostic criteria, Alzheimer's within 3 years The rate of progression to disease dementia was very high, about 59% (Brain 2015: 138; 1327-1338).
  • the methylation marker LOC391322 of the present invention is specifically hypomethylated in the Alzheimer's disease mild cognitive impairment patient group and the Alzheimer's disease dementia patient group compared to the normal control group, and the effectiveness of distinguishing the normal group from the patient group through ROC curve analysis was confirmed, It can be used as a marker to diagnose Alzheimer's disease mild cognitive impairment and early diagnosis of Alzheimer's dementia from mild cognitive impairment by accurately predicting patients who progress from Alzheimer's disease mild cognitive impairment to Alzheimer's disease dementia. . Therefore, the compositions, kits and methods of the present invention can delay the exacerbation of the disease through active preventive management and early treatment by selecting patients with a high probability of progressing to Alzheimer's disease dementia by diagnosing Alzheimer's dementia early. In addition, if clinically used in the future, early detection can delay the onset of dementia and reduce the prevalence of dementia, ultimately improving the quality of life of patients and their families, and further reducing the socioeconomic cost of the state. can
  • the methylation markers CD96 and NHLRC4 of the present invention are specifically hypomethylated in the Alzheimer's disease mild cognitive impairment patient group compared to the normal control group, and PLEC1 is specifically highly methylated in the Alzheimer's disease mild cognitive impairment patient group compared to the normal control group, all of which are ROC Since the effectiveness of discriminating between the normal group and the patient group was confirmed through curve analysis, it is possible to diagnose Alzheimer's disease mild cognitive impairment as well as predict the risk of progression from Alzheimer's disease mild cognitive impairment to Alzheimer's disease dementia, Alzheimer's disease It can be used as a predictive diagnostic marker for dementia.
  • compositions, kits and methods of the present invention predict the risk of Alzheimer's disease in advance, and by selecting patients with a high probability of progressing to Alzheimer's disease dementia, it is possible to delay the deterioration of the disease through active preventive management and early treatment.
  • early detection can delay the onset of dementia and reduce the prevalence of dementia, ultimately improving the quality of life of patients and their families, and further reducing the socioeconomic cost of the state.
  • the methylation markers FOXP1, NAA60 and SLC38A2 of the present invention are specifically hypomethylated in the Alzheimer's disease dementia patient group compared to the normal control group, and the effectiveness of distinguishing the normal group from the patient group through ROC curve analysis was confirmed, so Alzheimer's disease dementia can be used as a marker for diagnosing
  • diagnosis means to confirm the presence of a disease, the presence or characteristics of a disease pathology.
  • diagnosis includes determining whether a person has a specific disease, measuring the condition before symptoms appear, determining the prognosis of the disease, and further determining whether the disease has progressed or worsened. can do.
  • “early diagnosis” refers to confirming the presence or characteristics of a disease, a disease pathology, or predicting whether a disease occurs, progresses, or worsens from a state before symptoms appear.
  • predicting the risk of progression to Alzheimer's disease dementia means pre-assessing and predicting the risk of developing Alzheimer's disease dementia in the future or progressing to Alzheimer's disease dementia at a stage prior to the appearance of symptoms of Alzheimer's disease dementia.
  • the agent (reagent) for measuring the methylation level of the CpG site is a compound that modifies an unmethylated cytosine base or a methylation sensitive restriction enzyme, a primer specific for a methylated allele sequence of a gene, an unmethylated allele sequence It may include a primer specific for , a methylated CpG binding domain, or a methylated DNA antibody that specifically binds to the methylated DNA (eg, an antibody that specifically binds to methylcytosine).
  • the compound that modifies the unmethylated cytosine base may be, but is not limited to, bisulfite or a salt thereof, and preferably sodium bisulfite.
  • Bisulfite-modified DNA can detect methylation through various methods, such as sequencing or methylation-specific PCR. It is well known in the art (eg WO01/26536; US2003/0148326A1).
  • the methylation-sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting methylation of a CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. Examples include, but are not limited to, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like. Depending on the methylation or unmethylation at C of the restriction enzyme recognition site, whether or not cleavage by the restriction enzyme is changed, which can be detected through PCR or Southern blot analysis. 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. Thereafter, the processed DNA can be amplified by PCR using primers and measured by confirming the presence or absence of the amplified product.
  • the formulation of the present invention may include a primer specific for a methylated allele sequence of a gene and a primer specific for an unmethylated allele sequence.
  • the term "primer” refers to a short nucleic acid sequence that is capable of base pairing with a complementary template with a nucleic acid sequence having a short free three-terminal hydroxyl group and serves as a starting point for template strand copying. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.
  • the primers may incorporate additional features that do not change the basic properties of the primers, which are sense and antisense nucleic acids with a sequence of 7 to 50 nucleotides, which serve as the starting point of DNA synthesis.
  • the primers of the present invention can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and each methylated primer pair capable of specifically amplifying cytosine that has not been modified by bisulfite; and a primer pair capable of specifically amplifying a cytosine modified by bisulfite because it is not methylated.
  • a methylated DNA antibody refers to an antibody that specifically binds to a methylated base in DNA.
  • an antibody having a property of recognizing and binding to methylated cytosine in a DNA chain, such as an antibody against methylcytosine is mentioned.
  • it may be an antibody capable of specifically recognizing and specifically binding to the methylated DNA described herein.
  • a methylated DNA antibody can be prepared by a conventional method using a methylated base, methylated DNA, or the like as an antigen.
  • a methylated base methylated DNA
  • an antigen for example, in order to produce a methylcytosine antibody, 5-methylcytosine, 5-methylcytosine, or DNA containing 5-methylcytosine is used as an antigen to prepare an antibody, and then the antibody is prepared using methylcytosine in DNA. of specific binding can be selected as an indicator.
  • methylated DNA is immunoprecipitated using these, and then Southern blot, PCR, microarray, or sequence A specific CpG site may be identified through sequencing or the like.
  • a substrate an appropriate buffer solution, a chromogenic enzyme or fluorescent substance label, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, and a chromogenic substrate may be used.
  • a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass may be used, and the chromogenic enzyme is peroxidase, alkaline phosphorus Alkaline phosphatase, etc. may be used, the fluorescent material may be FITC, RITC, etc., and the color developing substrate solution may be ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfur).
  • radioactive isotope labels examples include radioactive isotope labels, latex bead labels, colloidal labels, biotin labels, etc. may be used, but are limited thereto no.
  • composition and kit may further include a polymerase agarose, a buffer solution required for electrophoresis, and the like, in addition to the preparation.
  • kit may be implemented in the form of a DNA methylation microarray.
  • sample includes samples such as cells, tissues, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum or urine with different methylation levels of genes due to Alzheimer's disease. Any sample that can be extracted can be used. Preferred embodiments include, but are not limited to, skin tissue or skin cells, for example, skin fibroblasts, and the like.
  • the measurement of the methylation level of the CpG region of the gene promoter is a compound or methylation sensitive restriction enzyme that modifies an unmethylated cytosine base, a primer specific for the methylated sequence of the CpG region of the gene promoter, and a non-methylated sequence specific It can be measured using a primer, a methylated CpG binding domain, or an antibody that specifically binds to methylcytosine.
  • a method using a restriction enzyme or bisulfite using the difference between a methylated base and a non-methylated base an immunoprecipitation method using a methylated CpG binding domain or an anti-methylcytosine antibody (e.g., MIRA, MeDIP) ), a method using a DNA methylation microarray, and the like.
  • 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 may be measured or detected using pyrosequencing, bisulfite sequencing, DNA methylation microarray, or immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.
  • the step of measuring the methylation level of the CpG region of the gene promoter in the method of the present application comprises the steps of (a) treating the genomic DNA in the obtained sample with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme; and (b) amplifying the processed DNA by PCR using a primer 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 whether a gene is methylated by modifying an unmethylated cytosine residue using such a bisulfite is well known in the art.
  • the methylation-sensitive restriction enzyme in step (a) is a restriction enzyme capable of specifically detecting methylation of a specific CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme.
  • a restriction enzyme capable of specifically detecting methylation of a specific 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.
  • the amplification in step (b) may be performed by a conventional PCR method.
  • the primer used at this time can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and specifically amplifies cytosine that has been methylated and has not been modified by bisulfite. and a primer pair capable of specifically amplifying a cytosine modified by bisulfite because it is not methylated.
  • the step of measuring the methylation level of the specific CpG region of the gene promoter may further include (c) confirming the presence or absence of the amplified product in step (b).
  • the presence or absence of the amplified product in step (c) may be performed according to whether a band at a desired position is detected by a method known in the art, for example, electrophoresis.
  • a primer capable of specifically amplifying methylated cytosine that has not been modified by bisulfite The degree of methylation can be determined according to the presence or absence of a PCR product amplified by a pair and a primer pair capable of specifically amplifying a cytosine modified by bisulfite because it is not methylated.
  • 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 corresponding gene is amplified by PCR, and the nucleotide sequence of the amplified region is analyzed. .
  • CpG is methylated by a method known in the art, for example, if there is a PCR product in the DNA treated with a restriction enzyme in the state where the PCR product is shown in mock DNA, If there is no PCR product in the DNA treated with the enzyme, whether CpG is methylated can be determined according to the determination that it is unmethylated, which is apparent to those skilled in the art.
  • mock DNA refers to sample DNA separated from the sample and untreated.
  • an immunoprecipitation method using a methylated DNA antibody for example, an antibody against a methylated CpG binding domain (MBD) or methylcytosine
  • a specific CpG site can be identified through Southern blot, PCR, microarray, or sequencing.
  • the method of the present application comprises a step of comparing the result of measuring the methylation level of the CpG region of one or more gene promoters selected from the group consisting of FOXP1, NAA60, SLC38A2, LOC391322, CD96, PLEC1, and NHLRC4 with a reference value,
  • the reference value may be measured from a sample of a healthy individual or a sample of an individual not diagnosed with Alzheimer's disease, or may be a predetermined cut-off value.
  • Alzheimer's disease dementia is diagnosed; If the CpG region of the LOC391322 gene promoter in the sample of the subject is hypomethylated than the reference value, it is diagnosed as Alzheimer's disease mild cognitive impairment or Alzheimer's disease dementia; If the CpG region of one or more gene promoters selected from the group consisting of CD96 and NHLRC4 in the subject's sample is hypomethylated than the reference value or the CpG region of the PLEC1 gene promoter is hypermethylated than the reference value, Alzheimer's disease mild cognitive impairment is diagnosed. or a risk of progression to Alzheimer's disease or dementia.
  • the change in methylation of a specific CpG region of a gene can be used as a biomarker to be usefully used for diagnosing Alzheimer's disease.
  • hypomethylation of a specific CpG region of FOXP1, NAA60 and/or SLC38A2 genes can be used as a biomarker to be usefully used for diagnosing Alzheimer's disease dementia.
  • hypomethylation of a specific CpG region of the LOC391322 gene can be used as a biomarker to be usefully used in the diagnosis of mild cognitive impairment in Alzheimer's disease or early diagnosis of dementia in Alzheimer's disease.
  • the low methylation of the specific CpG region of the CD96 and/or NHLRC4 gene or the high methylation of the specific CpG region of the PLEC1 gene are used as biomarkers to diagnose Alzheimer's disease mild cognitive impairment or predict the risk of progression to Alzheimer's disease dementia. can be used
  • the Alzheimer's disease patient group includes three groups: Alzheimer's disease preclinical Alzheimer's disease (AD), Alzheimer's disease mild cognitive impairment (Alzheimer's disease), and Symptomatic Alzheimer's disease.
  • Controls include a normal control group (a group with normal cognitive function and no amyloid pathology), mild cognitive impairment due to non-AD, and dementia not caused by Alzheimer's disease (dementia). It is composed of three groups: due to non-AD).
  • the degree of current cognitive decline and daily living ability were judged through a neuropsychological test.
  • three stages of cognitive decline ie, normal cognitive function, mild cognitive impairment, and dementia, were determined.
  • the normal cognitive function group apart from complaints of subjective cognitive decline, all items of the objective neuropsychological test showed test results higher than the standard score (z score ⁇ -1.5), and there were no objective findings of cognitive decline and daily living ability. is undamaged.
  • the neuropsychological test shows a result of 1.5 or less (z score ⁇ -1.5) in the standard score, and there is a decrease in objective memory, but overall cognitive function and It is a group in which daily living ability is maintained.
  • the dementia group shows objective deterioration (standard score of 1.5 or less) in two or more cognitive domains, and is a group that requires the care of others due to impairment of daily living ability, along with a slowly progressing clinical picture.
  • a third step blood tests, structural imaging of the brain (computed tomography, CT or magnetic resonance imaging, MRI), and functional imaging of the brain (functional MRI, amyloid positron emission tomography, amyloid PET) are performed to reduce cognitive decline. Other possible causes were excluded, and the accumulation of amyloid pathological findings in the brain was checked. Excluding other causes that may cause cognitive decline is an important step in the diagnosis of Alzheimer's disease. In addition, alcoholism, drug addiction, major depressive disorder, bipolar disorder, schizophrenia, and convulsive disease were excluded because they may be associated with cognitive decline. In addition, on brain imaging, structural brain abnormalities such as normostatic hydrocephalus, stroke, brain tumors, etc.
  • Alzheimer's disease research diagnostic criteria positive amyloid PET (Brain amyloid Plaque load, BAPL 2 or 3) was judged to have Alzheimer's disease, and one of preclinical, prodromal, and symptomatic AD was determined according to clinical symptoms. group was judged.
  • Negative amyloid PET (BAPL 1) is judged as a control group, and according to clinical symptoms, normal control group (group with normal cognitive function and no amyloid pathology), mild cognitive impairment due to not Alzheimer's disease non-AD), it was decided as one of the dementia due to non-AD groups.
  • a final judgment was made into six groups.
  • a skin biopsy was collected from the inside of the patient's thigh using a cylindrical blade with a diameter of 2 mm. Divide the collected skin biopsy into 6 equal parts and place medium (DMEM/20% FBS) sufficient to submerge the skin biopsy pieces in a 24-well cell culture plate coated with 0.1% gelatin, and wait for 7 days to prevent the cells from protruding from the edges of the skin biopsy pieces. observed. Medium was replenished every 2-3 days to prevent the skin biopsy pieces from drying out. 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 filled to the outside of the culture well, it was transferred to a 35 mm culture dish and the right side of the medium was removed.
  • DMEM/20% FBS medium
  • fetal serum FBS
  • FBS fetal serum
  • Genomic DNA from the skin fibroblasts cultured in Example 2 was extracted 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 DNA state was checked for degradation by electrophoresis on a 1% agarose gel.
  • DNA methylation mutation analysis is performed by extracting DNA from the skin fibroblasts of the patient group and control group, converting unmethylated cytosine to uracil by performing bisulfite conversion, and using 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 with a value of 0 to 1.
  • a ⁇ value of 0 means that the corresponding CpG site is completely unmethylated, and 1 means that the corresponding CpG site is completely methylated.
  • DMGs differentially methylated genes
  • the difference in the DNA methylation degree of the selected gene in the normal control group and the Alzheimer's disease mild cognitive impairment patient group is shown as a scatter dot plot in FIGS. 7, 10, 12 and 14, and the mean ⁇ standard error (mean ⁇ SEM) values are indicated.
  • ROC curve receiver operating characteristics curve
  • receiver-actuated characteristic curves (ROCs) curve receiver operating characteristics curve
  • AUC area under the curve
  • LOC391322 was hypomethylated in both the Alzheimer's disease mild cognitive impairment group and the Alzheimer's disease dementia patient group. was confirmed (FIG. 9). These results show that disease-specific hypomethylation of the LOC391322 gene, which appears in skin fibroblasts of Alzheimer's disease dementia patients, is a non-invasive method for diagnosing Alzheimer's disease mild cognitive impairment, or early diagnosis of Alzheimer's disease dementia from the mild cognitive impairment stage. This indicates that it is effective as an early diagnosis marker for Alzheimer's disease and dementia.
  • PLEC1 was highly methylated in the Alzheimer's disease mild cognitive impairment patient group and the Alzheimer's disease dementia patient group. (Fig. 13). These results show that the disease-specific hypermethylation of the PLEC1 gene in skin fibroblasts of patients with mild cognitive impairment with Alzheimer's disease can be used to diagnose mild cognitive impairment with Alzheimer's disease in a non-invasive way, or to develop Alzheimer's disease dementia among patients with mild cognitive impairment with Alzheimer's disease. It indicates that it is effective as a predictive diagnostic marker for Alzheimer's disease and dementia, which predicts the progression of the disease in advance.

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Abstract

La présente invention concerne une composition, un kit et un procédé de détection ou de mesure du niveau de méthylation d'un site CpG génique, de manière à diagnostiquer un trouble cognitif léger de la maladie d'Alzheimer, diagnostiquer la maladie d'Alzheimer, diagnostiquer la maladie d'Alzheimer à un stade précoce ou prédire le risque de progression de la maladie d'Alzheimer.
PCT/KR2021/002309 2020-02-27 2021-02-24 Diagnostic et prédiction de la maladie d'alzheimer à l'aide d'une modification de méthylation épigénétique de gène Ceased WO2021172864A1 (fr)

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KR10-2020-0024178 2020-02-27
KR10-2020-0024175 2020-02-27
KR10-2020-0024176 2020-02-27
KR1020200024176A KR102313454B1 (ko) 2020-02-27 2020-02-27 유전자 CpG 메틸화 변화를 이용한 알츠하이머병 치매 진단용 조성물 및 이의 이용
KR1020200024180A KR102313455B1 (ko) 2020-02-27 2020-02-27 알츠하이머병 경도인지장애의 진단 또는 알츠하이머병 치매로의 진행 위험성 예측 방법
KR10-2020-0024177 2020-02-27
KR1020200024177A KR102139314B1 (ko) 2020-02-27 2020-02-27 유전자의 후성학적 메틸화 변화를 이용한 알츠하이머병 치매의 조기 진단 및 예측
KR1020200024179A KR102313459B1 (ko) 2020-02-27 2020-02-27 알츠하이머병 치매 특이적 dna 메틸레이션 마커 검출용 조성물 및 검출방법
KR1020200024175A KR102313453B1 (ko) 2020-02-27 2020-02-27 유전자의 dna 메틸레이션 변화를 이용한 알츠하이머병 경도인지장애의 진단 마커
KR10-2020-0024180 2020-02-27
KR10-2020-0024181 2020-02-27
KR10-2020-0024179 2020-02-27
KR1020200024178A KR102313457B1 (ko) 2020-02-27 2020-02-27 알츠하이머병 치매 특이적 유전자 nhlrc4 및 이를 이용한 알츠하이머병 치매 예측
KR1020200024181A KR102313456B1 (ko) 2020-02-27 2020-02-27 Naa60 유전자의 후성유전학적 변화를 이용한 알츠하이머병 치매 진단

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