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WO2021071219A1 - Biomarqueur pour le diagnostic de maladies neurodégénératives - Google Patents

Biomarqueur pour le diagnostic de maladies neurodégénératives Download PDF

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WO2021071219A1
WO2021071219A1 PCT/KR2020/013614 KR2020013614W WO2021071219A1 WO 2021071219 A1 WO2021071219 A1 WO 2021071219A1 KR 2020013614 W KR2020013614 W KR 2020013614W WO 2021071219 A1 WO2021071219 A1 WO 2021071219A1
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group
disease
neurodegenerative diseases
diagnosis
expression level
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조한나
이형근
류철형
함승주
정재훈
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to a biomarker for diagnosis of neurodegenerative diseases.
  • each marker has a limitation in that it is very difficult to distinguish between people with normal cognitive function and patients with weak cognitive impairment.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • APOE-e4 gene analysis is used in the diagnosis method of neurodegenerative diseases such as Alzheimer's disease, but there is still a limitation in that it is not reliable compared to clinical results in early diagnosis of neurodegenerative diseases.
  • the molecular level markers that have been identified to date are not suitable for regular diagnosis in that they are measured in samples that can be collected by invasive methods such as brain tissue, rectal tissue, bone marrow or spinal fluid.
  • An object of the present invention is a biomarker for diagnosis of neurodegenerative diseases; It is to provide a composition.
  • Another object of the present invention is to provide a kit for diagnosing neurodegenerative diseases including the composition for diagnosing neurodegenerative diseases.
  • Another object of the present invention is to provide a method of providing information for diagnosis of neurodegenerative diseases.
  • a biomarker for diagnosing neurodegenerative diseases is provided.
  • the biomarker of the present invention includes at least one or more genes selected from the following groups 1 to 3; Or a protein encoded thereby:
  • CAP1 Addenylyl Cyclase-Associated Protein 1
  • GLUL Glutamate-Ammonia Ligase
  • SERPINC1 Sepin Family C Member 1
  • SCGB1D1 Secretoglobin Family 1D Member 1
  • AZGP1 Zinc-alpha-2-glycoprotein
  • SCGB2A1 Secretoglobin Family 2A Member 1
  • A2M Alpha-2-macroglobulin
  • IGHM Immunoglobulin heavy constant mu
  • CST4 Cystatin-S
  • PIP Prolactin Induced Protein
  • LCN1 Lipocalin 1
  • LCP1 Lymphocyte Cytosolic Protein 1
  • LTF Lactotransferrin
  • APOA1 Apolipoprotein A-I
  • TF Serotransferrin
  • PKM Pyruvate kinase PKM
  • GC Vitamin D-binding protein
  • PRTN3 Myeloblastin
  • ANXA2 Annexin A2
  • IGHA1 Immunoglobulin heavy constant alpha 1
  • ACTN4 Alpha-actinin- 4
  • the biomarker of the present invention may be measured in a biological sample of a target individual, for example, tear fluid, but is not limited thereto.
  • the object of the present invention is the occurrence of neurodegenerative diseases;
  • the individual may be highly likely to be diagnosed as a neurodegenerative disease, but is not limited thereto.
  • the biological sample of the present invention is any material obtained from or derived from the object of interest, and may be a tear fluid for the purposes of the present invention, but is not limited thereto.
  • a tear fluid for the purposes of the present invention, but is not limited thereto.
  • biomarker refers to whether a specific disease has occurred in a biological sample isolated from a target individual; Likelihood of onset; It refers to an index such as a gene or protein capable of predicting the course of a disease, and for the purposes of the present invention, the biomarker refers to a biomarker capable of diagnosing neurodegenerative diseases.
  • the biomarkers included in the first group of the present invention are patients with neurodegenerative diseases based on a normal control group (no cognitive impairment; no beta amyloid accumulation and no cognitive impairment; and beta amyloid accumulation, but no cognitive impairment).
  • the expression level may be increased in, but is not limited thereto.
  • the CAP1 may be composed of an amino acid sequence represented by SEQ ID NO: 1
  • the GLUL may be composed of an amino acid sequence represented by SEQ ID NO: 2
  • the SERPINC1 may consist of an amino acid sequence represented by SEQ ID NO: 3
  • SCGB1D1 may consist of an amino acid sequence represented by SEQ ID NO: 4
  • AZGP1 may consist of an amino acid sequence represented by SEQ ID NO: 5
  • SCGB2A1 may be composed of an amino acid sequence represented by SEQ ID NO: 6
  • A2M may be composed of an amino acid sequence represented by SEQ ID NO: 7
  • IGHM may be composed of an amino acid sequence represented by SEQ ID NO: 8
  • CST4 is It may consist of an amino acid sequence represented by SEQ ID NO: 9, but is not limited thereto.
  • the biomarkers included in the second group of the present invention may be those whose expression levels are reduced in patients with neurodegenerative diseases based on the normal control group, but are not limited thereto.
  • the PIP may consist of an amino acid sequence represented by SEQ ID NO: 10
  • the LCN1 may consist of an amino acid sequence represented by SEQ ID NO: 11
  • the LCP1 may consist of an amino acid sequence represented by SEQ ID NO: 12
  • the LTF may consist of an amino acid sequence represented by SEQ ID NO: 13
  • the APOA1 may consist of an amino acid sequence represented by SEQ ID NO: 14.
  • the biomarkers included in the 3 groups of the present invention have increased expression in patients with mild cognitive impairment based on the normal control group, but may have decreased expression in patients with neurodegenerative diseases, for example, in Alzheimer's patients, but are limited thereto. no. That is, when the biomarkers included in the 3 groups are increased based on the normal control group, it may be a mild cognitive impairment or not Alzheimer's disease.
  • the TF may be composed of an amino acid sequence represented by SEQ ID NO: 15, and the PKM may be composed of an amino acid sequence represented by SEQ ID NO: 16, and ,
  • the GC may consist of an amino acid sequence represented by SEQ ID NO: 17,
  • the PRTN3 may consist of an amino acid sequence represented by SEQ ID NO: 18,
  • the ANXA2 may consist of an amino acid sequence represented by SEQ ID NO: 19
  • the IGHA1 may be composed of the amino acid sequence represented by SEQ ID NO: 20
  • the ACTN4 may be composed of the amino acid sequence represented by SEQ ID NO: 21, but is not limited thereto.
  • the "protein” of the present invention is not only the protein itself, but also a protein isomer (Isoform) or protein variant (Variant) that can be produced by splicing and variable promoters, or due to genetic changes such as mutations or polymorphisms. Includes.
  • the diagnostic biomarker includes at least one or more genes selected from the group consisting of SERPINC1, PIP, LCP1, and GLUL; Or it may include a protein encoded thereby.
  • the diagnostic biomarker includes at least one or more genes selected from the group consisting of LCP1 and CAP1; Or it may include a protein encoded thereby.
  • the sensitivity and selectivity may be very remarkably increased compared to the case of using the protein encoded thereby.
  • the expression level of the biomarker of the present invention can be changed according to changes in the degree of cognition and accumulation of beta amyloid compared to the normal control group, among degenerative neurological diseases, especially patients with mild cognitive impairment and examples having severe cognitive impairment For example, Alzheimer's patients can be effectively identified and the severity of the disease can be predicted as symptoms progress in mild cognitive impairment.
  • the neurodegenerative disease of the present invention is a disease in which the function of the nervous system is lost or lost due to degenerative changes in neurons, and is mild cognitive impairment, Alzheimer's disease, and frontotemporal dementia.
  • the "diagnosis" of the present invention is to confirm the presence or characteristics of a pathological condition, and for the purposes of the present invention, the diagnosis may include the onset of neurodegenerative diseases; Likelihood of onset; Course of the disease; And predicting the prognosis or effect due to treatment.
  • Another embodiment of the present invention provides a composition for diagnosing neurodegenerative diseases.
  • the diagnostic composition of the present invention comprises at least one or more genes selected from the following groups 1 to 3; Or an agent for measuring the expression level of the protein encoded thereby:
  • Group 1 CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4
  • Group 3 TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4
  • composition for diagnosis of neurodegenerative diseases of the present invention the expression levels of groups 1 to 3, proteins, objects of interest, biological samples, tears, neurodegenerative diseases and diagnosis, etc. are the same as described in the biomarker. Is omitted to avoid excessive repetition.
  • the diagnostic composition includes at least one or more genes selected from the group consisting of SERPINC1, PIP, LCP1, and GLUL; Or it may include an agent for measuring the expression level of the protein encoded thereby.
  • the diagnostic composition comprises at least one or more genes selected from the group consisting of LCP1 and CAP1; Or it may include an agent for measuring the expression level of the protein encoded thereby.
  • the agent for measuring the expression level of the gene of the present invention may be included as long as it is capable of measuring the expression level of DNA present in a biological sample or mRNA transcribed by it, for example, it specifically binds to the gene. It may be at least one selected from the group consisting of primers, probes, and antisense nucleotides, but is not limited thereto.
  • the "primer” of the present invention is a fragment that recognizes a target gene sequence, and includes forward and reverse primer pairs, preferably, a primer pair that provides an analysis result having specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence inconsistent with the non-target sequence present in the sample, a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification can give high specificity. .
  • the "probe” of the present invention means a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding.
  • the type of probe is not limited as a material commonly used in the art, but preferably may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, RNA or DNA, and most preferably Hagi is PNA.
  • the probe is a biomaterial that includes an organism-derived or similar thing or a thing produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
  • LNA locked nucleic acids
  • the "locked nucleic acids (LNA)" of the present invention means a nucleic acid analog containing 2'-O, 4'-C methylene bridges [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502 ].
  • LNA nucleosides contain common nucleic acid bases of DNA and RNA, and can form base pairs according to the Watson-Crick base pairing rules. However, due to the'locking' of the molecule due to the methylene bridge, the LNA cannot form an ideal shape in the Watson-Crick bond.
  • the LNA can more quickly pair with a complementary nucleotide chain to increase the stability of the double helix.
  • the "antisense” of the present invention is a sequence of nucleotide bases in which the antisense oligomer is hybridized with the target sequence in RNA by Watson-Crick base pairing, allowing the formation of typically mRNA and RNA: oligomer heterodimer within the target sequence. And an oligomer having a backbone between subunits. Oligomers may have exact sequence complementarity or approximate complementarity to the target sequence.
  • the agent for measuring the expression level of the protein of the present invention may be included as long as it can measure the amount of protein present in a biological sample, for example, an antibody, an oligopeptide, a ligand that specifically binds to the protein, It may be at least one selected from the group consisting of peptide nucleic acid (PNA) and aptamer, but is not limited thereto.
  • PNA peptide nucleic acid
  • the "antibody” of the present invention refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
  • an antibody refers to an antibody that specifically binds to the biomarker protein.
  • the antibody of the present invention includes all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • the antibody can be easily prepared using techniques well known in the art.
  • polyclonal antibodies can be produced by methods well known in the art, including the process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain a serum containing the antibody.
  • Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog.
  • the monoclonal antibody is a hybridoma method well known in the art (hybridoma method; see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or phage antibody library technology (Clackson et al, Nature, 352 :624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991).
  • the antibody prepared by the above method may be separated and purified using a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
  • the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules.
  • the functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and means including all of Fab, F(ab'), F(ab')2, Fv, and the like.
  • the "PNA” of the present invention refers to a polymer similar to artificially synthesized DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by a peptide bond, which greatly increases the binding capacity and stability to DNA or RNA, resulting in molecular biology. , Diagnostic analysis and antisense therapy. The PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500].
  • the "aptamer” of the present invention is an oligonucleotide or a peptide molecule, and the general information of the aptamer is described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "Anartificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24): 142727(1998)].
  • the antibodies, oligopeptides, ligands, PNAs and aptamers of the present invention can be easily prepared by a person skilled in the art based on the amino acid sequences represented by SEQ ID NOs: 1 to 21.
  • the diagnostic composition of the present invention measures the expression level of a gene or protein present in tear fluid, and has the advantage of being able to diagnose a disease very easily without using an invasive method or a contrast medium.
  • Another embodiment of the present invention provides a diagnostic kit for neurodegenerative diseases comprising the diagnostic composition according to the present invention.
  • the contents related to genes, proteins, neurodegenerative diseases, agents for measuring the expression level of genes or proteins, objects of interest, biological samples, tears, diagnosis, etc. are as described in the biomarkers and diagnostic compositions. The same is omitted to avoid excessive repetition of the specification.
  • the kit of the present invention measures the expression level of the gene or the protein encoded by it in the naturally released tear fluid that does not depend on an invasive method, it is very easy to diagnose a disease without using an invasive method or a contrast agent. In addition, it is possible to distinguish and diagnose mild cognitive impairment and neurodegenerative diseases that are similar in initial state and symptoms of neurodegenerative disease very effectively.
  • the kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit, but is not limited thereto.
  • MRM multiple reaction monitoring
  • the kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method.
  • the kit may further include essential elements necessary to perform a reverse transcription polymerase reaction.
  • the reverse transcription polymerase reaction kit contains a primer pair specific for the gene.
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp.
  • a primer specific to the nucleic acid sequence of a control gene eg, a housekeeping gene
  • reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (variable in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors. DEPC-water, sterilized water, and the like may be included, but are not limited thereto.
  • the diagnostic kit of the present invention may include essential elements necessary to perform a DNA chip.
  • the DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe.
  • the substrate may include a control gene (eg, a housekeeping gene) or a cDNA or oligonucleotide corresponding to a fragment thereof, but is not limited thereto.
  • the diagnostic kit of the present invention may contain essential elements necessary to perform ELISA.
  • the ELISA kit contains an antibody specific for the protein.
  • Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal, polyclonal, or recombinant antibodies.
  • the ELISA kit may include an antibody specific for a control (eg, housekeeping protein) protein.
  • Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or antibodies capable of binding. Other materials may be included.
  • Another embodiment of the present invention provides a method of providing information for diagnosis of neurodegenerative diseases.
  • the method of providing the information of the present invention includes at least one gene selected from the following groups 1 to 3 from a biological sample isolated from a target individual; Or measuring the expression level of the protein encoded thereby; includes:
  • Group 1 CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4
  • Group 3 TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4.
  • the method of the present invention comprises the gene in the naturally released tear fluid that does not depend on the invasive method; Alternatively, since the expression level of the protein encoded by this is measured, the disease can be diagnosed very easily without using an invasive method or a contrast medium. Furthermore, genes included in the three groups; Alternatively, in the case of measuring the expression level of the protein encoded by this, it is possible to specifically select only whether or not Alzheimer's disease occurs among mild cognitive impairment or neurodegenerative diseases.
  • the subject matter biological sample, gene, protein, neurodegenerative disease, agent for measuring the expression level of a gene or protein, diagnosis, etc. are described in the biomarker and diagnostic composition. As described above, it is omitted to avoid excessive repetition of the specification.
  • the method of providing the information includes at least one or more genes selected from the group consisting of SERPINC1, PIP, LCP1, and GLUL; Or it may include the step of measuring the expression level of the protein encoded thereby.
  • the method of providing the information includes at least one or more genes selected from the group consisting of LCP1 and CAP1; Or it may include the step of measuring the expression level of the protein encoded thereby.
  • the step of measuring the expression level of the gene of the present invention is reverse transcription using an agent for measuring the expression level of at least one gene selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene.
  • Polymerase reaction RT-PCR
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Real-time RT-PCR real-time RT-PCR
  • RNase protection assay RNase protection assay
  • Northern blotting Northern blotting
  • DNA chip but is not limited thereto.
  • the step of measuring the expression level of the protein of the present invention is at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNA), and aptamers that specifically bind to the protein.
  • the expression level of a gene included in group 1 or a protein encoded by it when the expression level of a gene included in group 1 or a protein encoded by it is increased compared to a normal control group; Or when the expression level of the gene included in the group 2 or the protein encoded by it is decreased compared to the normal control group; It may further include predicting a neurodegenerative disease.
  • the expression level of the gene or the protein encoded by the gene included in the group 3 is increased compared to the normal control group, among degenerative neurological diseases, in particular, predicting a mild cognitive impairment is further performed. It may be included, but is not limited thereto.
  • the present invention relates to a biomarker for diagnosing neurodegenerative diseases, and the present invention can predict neurological diseases very easily without using an invasive method or contrast medium. Furthermore, since the expression level of the biomarker can be changed according to the change in the degree of cognition and accumulation of beta amyloid compared to the normal control group, among degenerative neurological diseases, especially patients with mild cognitive impairment and severe cognitive impairment are described. For example, Alzheimer's patients can be effectively distinguished.
  • MCI cognitive impairment
  • CU normal control without beta amyloid accumulation
  • FIG. 2 shows the results of measuring AUC values for diagnosing a third group (Alzheimer's; AC) based on the first group using the biomarker panel according to an embodiment of the present invention.
  • Group 1 CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4
  • Group 3 TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4
  • kits for diagnosing neurodegenerative diseases including the composition for diagnosing neurodegenerative diseases according to the present invention is provided.
  • CU group Characteristic Number of patients First group Normal (CU * ) 30 2nd group Mild cognitive impairment (MCI) 32 3rd group Alzheimer's (AD) 41 * CU (Cognitively impaired): Normal person with no cognitive dysfunction
  • the PRM technique is a quantitative analysis method that increases the quantitative sensitivity and selectivity by selectively quantitatively analyzing only the desired peptide by entering the molecular weight and liquid chromatography elution time of the target peptide to be quantitatively analyzed.
  • Example 2 the sample of Example 2 was loaded onto a column, and 5 to 40% of D solvent for 60 minutes, 40 to 80% of D solvent for 5 minutes, and 80% D solvent for 10 minutes.
  • the mobile phase of the column was set under the conditions of the concentration gradient, and finally, it was allowed to equilibrate for 10 minutes in 1% D solvent.
  • the C solvent is 0.1% formic acid dissolved in water
  • the D solvent is acetonitrile dissolved using 0.1% formic acid.
  • the peptide was eluted from the column using a trap column, and then the peptide was ionized using an EASY-spray column (50 cm ⁇ 75 ⁇ m ID) filled with 2 ⁇ m C18 particles at a potential of 1.8 kV.
  • Total mass spectrometry data was collected at m/z 200, resolution 70,000, and scan range 400 to 2,000 Th, and the target value of AGC (Automatic Gain Control) was 1.0 ⁇ 10 6 , and the maximum ion implantation was 120 ms.
  • MS/MS maximum ion implantation time was set to 60 ms at a resolution of 17,500, and dynamic exclusion time was set to 30 seconds.
  • quantification was performed by extracting the amount of fragment ions of each peptide using a Skyline search engine.
  • normalization quantitative standardization
  • the proportion of protein was expressed as the median of the proportion of all quantifiable spectra of the protein-related peptide, compared to the first group (normal, CU), and the second group (mild cognitive impairment) and In the third group (Alzheimer's), the condition was set so that the protein whose expression level was increased or decreased was selected when the p value was changed by a fold greater than 2, which is less than 0. Furthermore, proteins showing differences in expression in the second group or the third group were selected based on the normal group without cognitive impairment (CU), and 21 proteins of statistically significant proteins obtained through the TMT qualitative/quantitative method were selected. Fold change and AUC values were derived, and the results are shown in Table 2 below.
  • Group 1 CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4
  • Group 3 TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4
  • neurodegenerative diseases including mild cognitive impairment and Alzheimer's
  • genes included in group 3 when genes included in group 3 are used, degenerative neurons Among the diseases, it can be seen that a disease such as Alzheimer's does not exist, but only mild cognitive impairment can be specifically selected.
  • a biomarker panel was constructed as follows using a protein significantly changed in the mild cognitive impairment or Alzheimer group, and accordingly, the process of deriving the ROC curve, AUC and P-value was performed. By comparing the selectivity and sensitivity, it is shown in Figures 1 and 2:
  • the AUC values were calculated using the first panel and the second panel for the second group (mild cognitive impairment; MCI) based on the first group (normal control without beta amyloid accumulation; CU). When measured, the values corresponded to 0.963 (first panel) and 0.951 (second panel), indicating very high selectivity and sensitivity.
  • the present invention relates to a biomarker for diagnosing neurodegenerative diseases, and the present invention can predict neurological diseases very easily without using an invasive method or contrast medium. Furthermore, since the expression level of the biomarker can be changed according to the change in the degree of cognition and accumulation of beta amyloid compared to the normal control group, among degenerative neurological diseases, especially patients with mild cognitive impairment and severe cognitive impairment are described. For example, Alzheimer's patients can be effectively identified and the severity of the disease can be predicted as symptoms progress in mild cognitive impairment.
  • SEQ ID NO: 1 CAP1 amino acid sequence
  • SEQ ID NO: 2 GLUL amino acid sequence
  • SEQ ID NO: 3 SERPINC1 amino acid sequence
  • SEQ ID NO: 4 SCGB1D1 amino acid sequence
  • SEQ ID NO: 5 AZGP1 amino acid sequence
  • SEQ ID NO: 6 SCGB2A1 amino acid sequence
  • SEQ ID NO: 7 A2M amino acid sequence
  • MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR QGIPFFGQVR LVDGKGVPIP NKVI
  • GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF SWKYKNNSDI SSTRGFPSVL RGGKYAATSQ VLLPSKDVMQ GTDEHVVCKV QHPNGNKEKN VPLPVIAELP PKVSVFVPPR DGFFGNPRKS KLICQATGFS PRQIQVSWLR EGKQVGSGVT TDQVQAEAKE SGPTTYKVTS TLTIKESDWL GQSMFTCRVD HRGLTFQQNA SSMCVPDQDT AIRVFAIPPS FASIFLTKST KLTCLVTDLT TYDSVTISWT RQNGEAVKTH TNISESHPNA TFSAVGEASI CEDDWNSGER FTCTVTHTDL PSPLKQTISR PKGVALHRPD VYLLPPAREQ LNLRESATIT CLVTGFSPAD VFVQWMQRGQ PLSPEKYVTS APMPEPQAPG RYFAHSILTV SEEEWNTGET Y
  • SEQ ID NO: 10 PIP amino acid sequence
  • SEQ ID NO: 11 LCN1 amino acid sequence
  • SEQ ID NO: 12 LCP1 amino acid sequence
  • SEQ ID NO: 13 LTF amino acid sequence
  • SEQ ID NO: 14 APOA1 amino acid sequence
  • SEQ ID NO: 15 TF amino acid sequence
  • SEQ ID NO: 16 PKM amino acid sequence
  • SEQ ID NO: 17 GC amino acid sequence
  • SEQ ID NO: 18 PRTN3 amino acid sequence
  • SEQ ID NO: 20 IGHA1 amino acid sequence
  • SEQ ID NO: 21 ACTN4 amino acid sequence

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Neurology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Biomarqueur pour le diagnostic de maladies neurodégénératives. La présente invention peut prédire très facilement des maladies neurologiques, sans utiliser de procédés invasifs ou de produits de contraste. En outre, le biomarqueur peut varier dans le niveau d'expression en fonction d'un changement du niveau de cognition et de l'accumulation de bêta-amyloïde par rapport à un groupe témoin normal, et peut ainsi faire la distinction efficace entre des patients présentant un trouble cognitif léger et des patients présentant une déficience cognitive grave, par exemple, la maladie d'Alzheimer, parmi les maladies neurodégénératives et peut prédire si une déficience cognitive légère se détériore en devenant sévère avec la progression de la maladie.
PCT/KR2020/013614 2019-10-07 2020-10-07 Biomarqueur pour le diagnostic de maladies neurodégénératives Ceased WO2021071219A1 (fr)

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WO2024014834A1 (fr) * 2022-07-12 2024-01-18 재단법인대구경북과학기술원 Biomarqueur pour le diagnostic précoce de la maladie d'alzheimer et son utilisation
CN119724538A (zh) * 2024-11-04 2025-03-28 湖北中医药大学 一种基于生信大数据的智能双疾病诊断方法

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WO2024014834A1 (fr) * 2022-07-12 2024-01-18 재단법인대구경북과학기술원 Biomarqueur pour le diagnostic précoce de la maladie d'alzheimer et son utilisation
CN115494241A (zh) * 2022-09-29 2022-12-20 浙江大学 脑脊液中蛋白标志物在制备诊断轻度认知功能损害的产品中的应用
CN119724538A (zh) * 2024-11-04 2025-03-28 湖北中医药大学 一种基于生信大数据的智能双疾病诊断方法

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