WO2021071219A1 - Biomarker for diagnosis of neurodegenerative disease - Google Patents

Abstract

The present invention relates to a biomarker for diagnosis of neurodegenerative disease. The present invention can predict neurological diseases very easily, without using invasive methods or contrast media. Furthermore, the biomarker can vary in expression level depending on a change in cognition degree and amyloid beta accumulation relative to a normal control group and thus can effectively discriminate between patients with mild cognitive impairment and patients with severe cognitive impairment, for example, Alzheimer's disease, among neurodegenerative diseases and can predict if mild cognitive impairment deteriorates to severity with the progression of the disease.

Description

Biomarker for diagnosis of neurodegenerative diseases

The present invention relates to a biomarker for diagnosis of neurodegenerative diseases.

With the rapid aging of the global population, patients with neurodegenerative diseases such as cognitive impairment, Alzheimer's disease, Parkinson's disease, and stroke are increasing rapidly. In the case of Korea, it was estimated that 12.2% of the population aged 65 or older accounted for 12.2% of the total population in 2013, and it has already entered an aging country, and is expected to become a super-aged country by 2030. As described above, as the population generally ages, the prevalence of patients with neurodegenerative diseases increases sharply, and social costs accordingly increase continuously, leading to a social problem.

There is currently no cure for neurodegenerative disease, so it is currently the best method to diagnose the disease early and slow the progression of symptoms. However, although studies of various methods for early diagnosis of neurodegenerative diseases have been conducted to date, no markers used in a method capable of very accurately diagnosing degenerative neurological diseases have yet been found.

In the case of Alzheimer, which is the most researched among neurodegenerative diseases, despite the development of various types of neuroimaging techniques, 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. Exists. Specifically, a method for diagnosing brain imaging using magnetic resonance imaging (MRI) or computed tomography (CT); A diagnostic method for measuring the amount of tau protein or beta-amyloid in cerebrospinal fluid (CSF) or blood; Or, as a genetic risk factor, 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. exist. In addition, 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.

Thus, the basic characteristics of neuropathology are well reflected, and the situation in which symptoms gradually worsen from mild cognitive impairment to degenerative neurological disease can be well reflected, and the molecular level of neurodegenerative diseases having advantages such as low cost and non-invasiveness. There is a need for research on markers.

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.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

In one embodiment of the present invention, 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:

Group 1: CAP1 (Adenylyl Cyclase-Associated Protein 1), GLUL (Glutamate-Ammonia Ligase), SERPINC1 (Serpin 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) and CST4 (Cystatin-S)

Group 2: PIP (Prolactin Induced Protein), LCN1 (Lipocalin 1), LCP1 (Lymphocyte Cytosolic Protein 1), LTF (Lactotransferrin) and APOA1 (Apolipoprotein A-I)

Group 3: TF (Serotransferrin), PKM (Pyruvate kinase PKM), GC (Vitamin D-binding protein), PRTN3 (Myeloblastin), ANXA2 (Annexin A2), IGHA1 (Immunoglobulin heavy constant alpha 1) and 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; Alternatively, as an individual whose symptoms such as progression are unclear, 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. As described above, in the case of measuring the biomarker in the tear fluid, there is an advantage in that the disease can be diagnosed very easily without the use of an invasive method used for diagnosing a neurodegenerative disease or a contrast agent.

The "biomarker" of the present invention 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.

In the protein encoded by the gene included in the group 1 of the present invention, the CAP1 may be composed of an amino acid sequence represented by SEQ ID NO: 1, and 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, and 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.

In the protein encoded by the gene included in the group 2 of the present invention, the PIP may consist of an amino acid sequence represented by SEQ ID NO: 10, and the LCN1 may consist of an amino acid sequence represented by SEQ ID NO: 11, and , 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, and the APOA1 may consist of an amino acid sequence represented by SEQ ID NO: 14. However, it is not limited thereto.

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.

In the protein encoded by the gene included in the group 3 of the present invention, 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, and the ANXA2 may consist of an amino acid sequence represented by SEQ ID NO: 19 In addition, the IGHA1 may be composed of the amino acid sequence represented by SEQ ID NO: 20, and 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.

In one embodiment of the present invention, 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.

In another embodiment of the present invention, 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.

In the case of diagnosing neurodegenerative diseases through a specific combination as described above in the diagnostic biomarker of the present invention, a single gene; Alternatively, the sensitivity and selectivity may be very remarkably increased compared to the case of using the protein encoded thereby.

Since 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. ), dementia with Lewy bodies, corticobasal degeneration, Parkinson's disease, multiple system atrophy, progressive supranuclear palsy, Huntington's disease), dental-rubro-pallido-luysian atrophy, spinocerebellar ataxia, amyotrophic lateral sclerosis, primary lateral sclerosis and spinal cord It may be at least one or more selected from the group consisting of spinal muscular atrophy, and may be, for example, mild cognitive impairment or Alzheimer's disease, but is not limited thereto.

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 2: PIP, LCN1, LCP1, LTF and APOA1

Group 3: TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4

In the 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.

In one embodiment of the present invention, 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.

In another embodiment of the present invention, 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. More specifically, 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.

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. When LNA is included in a DNA or RNA oligonucleotide, 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.

Since the information on the gene of the present invention can be easily derived by a person skilled in the art based on the amino acid sequence shown in SEQ ID NOs: 1 to 21, a primer that specifically binds to the gene based on the gene sequence derived by the skilled person Probes or antisense nucleotides can be easily prepared.

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.

The "antibody" of the present invention refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, 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. For example, 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. In addition, 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. In addition, 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.

In the kit of 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.

Since 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.

The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method.

In one embodiment of the present invention, 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. In addition, a primer specific to the nucleic acid sequence of a control gene (eg, a housekeeping gene) may be included. Other 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.

In another embodiment of the present invention, 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. In addition, 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.

In another embodiment of the present invention, 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. In addition, 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 2: PIP, LCN1, LCP1, LTF and APOA1

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.

In the method of providing the information of the present invention, 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.

In one embodiment of the present invention, 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.

In another embodiment of the present invention, 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 reverse transcription polymerase reaction (Competitive RT-PCR), real-time RT-PCR, RNase protection assay (RPA), Northern blotting ( Northern blotting) or through a 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. Protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption) using an agent that measures the expression level of protein /Ionization Time of Flight Mass Spectrometry) analysis, radiation immunity analysis, radioimmune diffusion method, octeroni immunity diffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis method, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (liquid chromatographyMass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), Western blotting, or ELISA (enzyme linked immunosorbentassay), etc., but is not limited thereto.

In the method of providing the information of the present invention, 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.

In the method of providing the information of the present invention, when 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.

1 is an AUC for diagnosing a second group (mild cognitive impairment; MCI) based on a first group (normal control without beta amyloid accumulation; CU) using a biomarker panel according to an embodiment of the present invention. It shows the result of measuring the value.

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.

In an embodiment of the present invention, at least one or more genes selected from the following groups 1 to 3; Or it provides a biomarker for diagnosing neurodegenerative diseases comprising a protein encoded thereby:

Group 1: CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4

Group 2: PIP, LCN1, LCP1, LTF and APOA1

Group 3: TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4

In another embodiment of the present invention, at least one or more genes selected from groups 1 to 3; Or it provides a composition for diagnosing neurodegenerative diseases comprising an agent for measuring the expression level of the protein encoded thereby.

In another embodiment of the present invention, a kit for diagnosing neurodegenerative diseases including the composition for diagnosing neurodegenerative diseases according to the present invention is provided.

In another embodiment of the present invention, at least one or more genes selected from groups 1 to 3 from a biological sample isolated from a target individual; Or it provides a method of providing information for diagnosis of neurodegenerative diseases comprising; measuring the expression level of the protein encoded thereby.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

[Example 1] In-solution digestion for protein sample preparation

As shown in Table 1 below, samples included in each group were subjected to sample preparation for individual analysis of tear fluid obtained from patients belonging to each group. Then, in-solution digestion was performed to digest each of the three samples containing the 100 μg protein into peptides. Thereafter, 8 M of urea dissolved in 100 mM ammonium bicarbonate was mixed with each of the digested samples at a volume ratio of 1:1, and incubated for 20 minutes at room temperature (RT). After the peptide was reduced by adding 10 mM DTT (Dithiothreitol) to the cultured samples, 30 mM IAA (Iodoacetamide) was added to induce alkylation of the peptide. Then, trypsin equivalent to 1/50 of the sample volume was added to the alkylation-induced samples and incubated overnight at 37°C, 0.4% TFA was added to terminate the trypsin reaction, and a C18 Harvard macro spin column The salt of the peptide was removed using (C18 Havard macro spin column). The salt-free peptide was completely dried and then stored at -80°C until used in experiments.

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

[Example 2] Global profiling using LC MS/MS

Among the mild cognitive impairment and Alzheimer-specific expression difference proteins obtained through global profiling, 72 proteins that were able to be analyzed by the parallel reaction monitoring (PRM) technique were individually analyzed. 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.

After resuspending 0.1% formic acid dissolved in water into the peptide sample, a ^{quantitative PRM analysis was performed using a Q Exactive TM} Orbitrap hybrid mass spectrometer (Waters, USA) coupled with a nanoAQUITY UPLC system.

In addition, for PRM quantitative analysis, 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. Here, the C solvent is 0.1% formic acid dissolved in water, and the D solvent is acetonitrile dissolved using 0.1% formic acid. Thereafter, 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 μm ID) filled with 2 μm 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.

In order to obtain quantitative information from the MS/MS spectrum of each target peptide obtained as described above, quantification was performed by extracting the amount of fragment ions of each peptide using a Skyline search engine. For normalization (quantitative standardization), an equal amount of e-coli beta-galactosidase peptide, which is not present in human tear samples, was spiked in to each sample, and then standardized using the quantitative information value.

In the mass spectrometry results, 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.

Uniprot ID	gene	Fold change			AUC
		CI/CU	MCI/CU	AD/CU	CU vs CI	CU vs MCI	MCI vs AD
P12273	PIP	0.33	0.19	0.43	0.874 **	0.876 **	0.689 *
P31025	LCN1	0.14	0.14	0.14	0.853 **	0.856 **	0.547
P02787	TF	1.94	3.08	0.34	0.838 **	0.840 **	0.813 **
Q01518	CAP1	2.24	3.30	1.42	0.825 **	0.828 **	0.757 **
P15104	GLUL	2.86	3.48	2.37	0.806 **	0.807 **	0.574
P13796	LCP1	0.42	0.51	0.36	0.774 **	0.774 **	0.627 *
P01008	SERPINC1	3.02	4.88	1.56	0.716 **	0.715 **	0.608
O95968	SCGB1D1	3.59	4.45	2.92	0.704 **	0.703 **	0.567
P25311	AZGP1	2.00	2.30	1.76	0.694 **	0.693 **	0.570
P14618	PKM	1.01	1.78	0.40	0.683 **	0.685 *	0.858
O75556	SCGB2A1	2.40	2.32	2.46	0.654 *	0.655 *	0.541
P02774	GC	0.94	1.60	0.43	0.648 *	0.649 *	0.773
P01023	A2M	1.87	2.46	1.41	0.645 *	0.648 *	0.583
P24158	PRTN3	3.17	5.88	1.05	0.627	0.632	0.584
P02788	LTF	0.78	0.77	0.78	0.614	0.617	0.563
P07355_2	ANXA2	1.46	2.06	1.00	0.613	0.604	0.609
P01876	IGHA1	1.28	2.01	0.72	0.600	0.573	0.660
P02647	APOA1	0.76	0.63	0.86	0.581	0.584	0.591
P01871	IGHM	1.34	1.34	1.33	0.554	0.568	0.633 *
P01036	CST4	1.56	1.30	1.76	0.540	0.555	0.571
O43707	ACTN4	0.85	1.05	0.71	0.465	0.556	0.579
* P <0.05, ** P <0.01, AUC: Area under curves1. CU (Cognitively unimpaired): When recognition is not impaired 2. CI (Cognitively impaired): Mild cognitive impairment and Alzheimer's 3. Mild cognitive impairment (MCI): mild cognitive impairment 4. AD: Alzheimer's

As shown in Table 2, on the basis of normal without cognitive impairment (CU), group 1 with increased expression in both the second group and the third group; Group 2 with reduced expression in both the second group (mild cognitive impairment; MCI) and the third group (Alzheimer's; AD); And 21 genes were classified into 3 groups with increased expression in the second group (MCI), but decreased expression in the third group (AD):

Group 1: CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4

Group 2: PIP, LCN1, LCP1, LTF and APOA1

Group 3: TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4

Through the above results, when the genes included in groups 1 and 2 are used, neurodegenerative diseases (including mild cognitive impairment and Alzheimer's) can be effectively screened, and 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.

[Example 3] Selectivity and sensitivity check according to biomarker panel composition

Compared with the normal group in Example 2, 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:

First Panel: SERPINC1, PIP, LCP1 and GLUL

Second Panel: LCP1 and CAP1

As shown in FIG. 1, 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.

In addition, as shown in FIG. 2, when the AUC value is measured using the first panel and the second panel for the third group (Alzheimer's; AD) based on the first group, the value is 0.946 (the first Panel) and 0.925 (second panel) showed very high selectivity and sensitivity.

Through the above results, it is possible to diagnose mild cognitive impairment and Alzheimer's with a very high level of selectivity and sensitivity compared to the normal control group by using the biomarker panel corresponding to the combination of the genes corresponding to the biomarker according to the present invention. Able to know.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

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

MADMQNLVER LERAVGRLEA VSHTSDMHRG YADSPSKAGA APYVQAFDSL LAGPVAEYLK ISKEIGGDVQ KHAEMVHTGL KLERALLVTA SQCQQPAENK LSDLLAPISE QIKEVITFRE KNRGSKLFNH LSAVSESIQA LGWVAMAPKP GPYVKEMNDA AMFYTNRVLK EYKDVDKKHV DWVKAYLSIW TELQAYIKEF HTTGLAWSKT GPVAKELSGL PSGPSAGSCP PPPPPCPPPP PVSTISCSYE SASRSSLFAQ INQGESITHA LKHVSDDMKT HKNPALKAQS GPVRSGPKPF SAPKPQTSPS PKRATKKEPA VLELEGKKWR VENQENVSNL VIEDTELKQV AYIYKCVNTT LQIKGKINSI TVDNCKKLGL VFDDVVGIVE IINSKDVKVQ VMGKVPTISI NKTDGCHAYL SKNSLDCEIV SAKSSEMNVL IPTEGGDFNE FPVPEQFKTL WNGQKLVTTV TEIAG

SEQ ID NO: 2: GLUL amino acid sequence

MTTSASSHLN KGIKQVYMSL PQGEKVQAMY IWIDGTGEGL RCKTRTLDSE PKCVEELPEW NFDGSSTLQS EGSNSDMYLV PAAMFRDPFR KDPNKLVLCE VFKYNRRPAE TNLRHTCKRI MDMVSNQHPW FGMEQEYTLM GTDGHPFGWP SNGFPGPQGP YYCGVGADRA YGRDIVEAHY RACLYAGVKI AGTNAEVMPA QWEFQIGPCE GISMGDHLWV ARFILHRVCE DFGVIATFDP KPIPGNWNGA GCHTNFSTKA MREENGLKYI EEAIEKLSKR HQYHIRAYDP KGGLDNARRL TGFHETSNIN DFSAGVANRS ASIRIPRTVG QEKKGYFEDR RPSANCDPFS VTEALIRTCL LNETGDEPFQ YKN

SEQ ID NO: 3: SERPINC1 amino acid sequence

MYSNVIGTVT SGKRKVYLLS LLLIGFWDCV TCHGSPVDIC TAKPRDIPMN PMCIYRSPEK KATEDEGSEQ KIPEATNRRV WELSKANSRF ATTFYQHLAD SKNDNDNIFL SPLSISTAFA MTKLGACNDT LQQLMEVFKF DTISEKTSDQ IHFFFAKLNC RLYRKANKSS KLVSANRLFG DKSLTFNETY QDISELVYGA KLQPLDFKEN AEQSRAAINK WVSNKTEGRI TDVIPSEAIN ELTVLVLVNT IYFKGLWKSK FSPENTRKEL FYKADGESCS ASMMYQEGKF RYRRVAEGTQ VLELPFKGDD ITMVLILPKP EKSLAKVEKE LTPEVLQEWL DELEEMMLVV HMPRFRIEDG FSLKEQLQDM GLVDLFSPEK SKLPGIVAEG RDDLYVSDAF HKAFLEVNEE GSEAAASTAV VIAGRSLNPN RVTFKANRPF LVFIREVPLN TIIFMGRVAN PCVK

SEQ ID NO: 4: SCGB1D1 amino acid sequence

MRLSVCLLLL TLALCCYRAN AVVCQALGSE ITGFLLAGKP VFKFQLAKFK APLEAVAAKM EVKKCVDTMA YEKRVLITKT LGKIAEKCDR

SEQ ID NO: 5: AZGP1 amino acid sequence

MVRMVPVLLS LLLLLGPAVP QENQDGRYSL TYIYTGLSKH VEDVPAFQAL GSLNDLQFFR YNSKDRKSQP MGLWRQVEGM EDWKQDSQLQ KAREDIFMET LKDIVEYYND SNGSHVLQGR FGCEIENNRS SGAFWKYYYD GKDYIEFNKE IPAWVPFDPA AQITKQKWEA EPVYVQRAKA YLEEECPATL RKYLKYSKNI LDRQDPPSVV VTSHQAPGEK KKLKCLAYDF YPGKIDVHWT RAGEVQEPEL RGDVLHNGNG TYQSWVVVAV PPQDTAPYSC HVQHSSLAQP LVVPWEAS

SEQ ID NO: 6: SCGB2A1 amino acid sequence

MKLLMVLMLA ALLLHCYADS GCKLLEDMVE KTINSDISIP EYKELLQEFI DSDAAAEAMG KFKQCFLNQS HRTLKNFGLM MHTVYDSIWC NMKSN

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 NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDNE DCINRHNVYI NGITYTPVSS TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLQQYEMHGP EGLRVGFYES DVMGRGHARL VHVEEPHTET VRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNA

SEQ ID NO: 8: IGHM amino acid sequence

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 YTCVVAHEAL PNRVTERTVD KSTGKPTLYN VSLVMSDTAG TCY

SEQ ID NO: 9: CST4 amino acid sequence

MARPLCTLLL LMATLAGALA SSSKEENRII PGGIYDADLN DEWVQRALHF AISEYNKATE DEYYRRPLQV LRAREQTFGG VNYFFDVEVG RTICTKSQPN LDTCAFHEQP ELQKKQLCSF EIYEVPWEDR MSLVNSRCQE A

SEQ ID NO: 10: PIP amino acid sequence

MRLLQLLFRA SPATLLLVLC LQLGANKAQD NTRKIIIKNF DIPKSVRPND EVTAVLAVQT ELKECMVVKT YLISSIPLQG AFNYKYTACL CDDNPKTFYW DFYTNRTVQI AAVVDVIREL GICPDDAAVI PIKNNRFYTI EILKVE

SEQ ID NO: 11: LCN1 amino acid sequence

MKPLLLAVSL GLIAALQAHH LLASDEEIQD VSGTWYLKAM TVDREFPEMN LESVTPMTLT TLEGGNLEAK VTMLISGRCQ EVKAVLEKTD EPGKYTADGG KHVAYIIRSH VKDHYIFYCE GELHGKPVRG VKLVGRDPKN NLEDFEKSET AARGKPVRG VKLVGRDPKN NLEDFEK

SEQ ID NO: 12: LCP1 amino acid sequence

MARGSVSDEE MMELREAFAK VDTDGNGYIS FNELNDLFKA ACLPLPGYRV REITENLMAT GDLDQDGRIS FDEFIKIFHG LKSTDVAKTF RKAINKKEGI CAIGGTSEQS SVGTQHSYSE EEKYAFVNWI NKALENDPDC RHVIPMNPNT NDLFNAVGDG IVLCKMINLS VPDTIDERTI NKKKLTPFTI QENLNLALNS ASAIGCHVVN IGAEDLKEGK PYLVLGLLWQ VIKIGLFADI ELSRNEALIA LLREGESLED LMKLSPEELL LRWANYHLEN AGCNKIGNFS TDIKDSKAYY HLLEQVAPKG DEEGVPAVVI DMSGLREKDD IQRAECMLQQ AERLGCRQFV TATDVVRGNP KLNLAFIANL FNRYPALHKP ENQDIDWGAL EGETREERTF RNWMNSLGVN PRVNHLYSDL SDALVIFQLY EKIKVPVDWN RVNKPPYPKL GGNMKKLENC NYAVELGKNQ AKFSLVGIGG QDLNEGNRTL TLALIWQLMR RYTLNILEEI GGGQKVNDDI IVNWVNETLR EAKKSSSISS FKDPKISTSL PVLDLIDAIQ PGSINYDLLK TENLNDDEKL NNAKYAISMA RKIGARVYAL PEDLVEVNPK MVMTVFACLM GKGMKRV

SEQ ID NO: 13: LTF amino acid sequence

MKLVFLVLLF LGALGLCLAG RRRSVQWCAV SQPEATKCFQ WQRNMRKVRG PPVSCIKRDS PIQCIQAIAE NRADAVTLDG GFIYEAGLAP YKLRPVAAEV YGTERQPRTH YYAVAVVKKG GSFQLNELQG LKSCHTGLRR TAGWNVPIGT LRPFLNWTGP PEPIEAAVAR FFSASCVPGA DKGQFPNLCR LCAGTGENKC AFSSQEPYFS YSGAFKCLRD GAGDVAFIRE STVFEDLSDE AERDEYELLC PDNTRKPVDK FKDCHLARVP SHAVVARSVN GKEDAIWNLL RQAQEKFGKD KSPKFQLFGS PSGQKDLLFK DSAIGFSRVP PRIDSGLYLG SGYFTAIQNL RKSEEEVAAR RARVVWCAVG EQELRKCNQW SGLSEGSVTC SSASTTEDCI ALVLKGEADA MSLDGGYVYT AGKCGLVPVL AENYKSQQSS DPDPNCVDRP VEGYLAVAVV RRSDTSLTWN SVKGKKSCHT AVDRTAGWNI PMGLLFNQTG SCKFDEYFSQ SCAPGSDPRS NLCALCIGDE QGENKCVPNS NERYYGYTGA FRCLAENAGD VAFVKDVTVL QNTDGNNNEA WAKDLKLADF ALLCLDGKRK PVTEARSCHL AMAPNHAVVS RMDKVERLKQ VLLHQQAKFG RNGLAGPNLKGKFNDLGFKLKTKFLR

SEQ ID NO: 14: APOA1 amino acid sequence

MKAAVLTLAV LFLTGSQARH FWQQDEPPQS PWDRVKDLAT VYVDVLKDSG RDYVSQFEGS ALGKQLNLKL LDNWDSVTST FSKLREQLGP VTQEFWDNLE KETEGLRQEM SKDLEEVKAK VQPYLDDFQK KWQEEMELYR QKVEPLRAEL QEGARQKLHE LQEKLSPLGE EMRDRARAHV DALRTHLAPY SDELRQRLAA RLEALKENGG ARLAEYHAKA TEHLSTLSEK AKPALEDLRQ GLLPVLESFK VSFLSALEEY TKKLNTQ

SEQ ID NO: 15: TF amino acid sequence

MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK KASYLDCIRA IAANEADAVT LDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKP VKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK CGLVPVLAEN YNKSDNCEDT PEAGYFAIAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLAR APNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCEPNNK EGYYGYTGCLVKLSTFRGN

SEQ ID NO: 16: PKM amino acid sequence

MSKPHSEAGT AFIQTQQLHA AMADTFLEHM CRLDIDSPPI TARNTGIICT IGPASRSVET LKEMIKSGMN VARLNFSHGT HEYHAETIKN VRTATESFAS DPILYRPVAV ALDTKGPEIR TGLIKGSGTA EVELKKGATL KITLDNAYME KCDENILWLD YKNICKVVEV GSKIYVDDGL ISLQVKQKGA DFLVTEVENG GSLGSKKGVN LPGAAVDLPA VSEKDIQDLK FGVEQDVDMV FASFIRKASD VHEVRKVLGE KGKNIKIISK IENHEGVRRF DEILEASDGI MVARGDLGIE IPAEKVFLAQ KMMIGRCNRA GKPVICATQM LESMIKKPRP TRAEGSDVAN AVLDGADCIM LSGETAKGDY PLEAVRMQHL IAREAEAAIY HLQLFEELRR LAPITSDPTE ATAVGAVEAS FKCCSGAIIV LTKSGRSAHQ VARYRPRAPI IAVTRNPQTA RQAHLYRGIF PVLCKDPVQE AWAEDVDLRV NFAMNVGKAR GFFKKGDVVI VLTGWRPGSG FTNTMRVVPV P

SEQ ID NO: 17: GC amino acid sequence

MKRVLVLLLA VAFGHALERG RDYEKNKVCK EFSHLGKEDF TSLSLVLYSR KFPSGTFEQV SQLVKEVVSL TEACCAEGAD PDCYDTRTSA LSAKSCESNS PFPVHPGTAE CCTKEGLERK LCMAALKHQP QEFPTYVEPT NDEICEAFRK DPKEYANQFM WEYSTNYGQA PLSLLVSYTK SYLSMVGSCC TSASPTVCFL KERLQLKHLS LLTTLSNRVC SQYAAYGEKK SRLSNLIKLA QKVPTADLED VLPLAEDITN ILSKCCESAS EDCMAKELPE HTVKLCDNLS TKNSKFEDCC QEKTAMDVFV CTYFMPAAQL PELPDVELPT NKDVCDPGNT KVMDKYTFEL SRRTHLPEVF LSKVLEPTLK SLGECCDVED STTCFNAKGP LLKKELSSFI DKGQELCADY SENTFTEYKK KLAERLKAKL PDATPTELAK LVNKHSDFAS NCCSINSPPL YCDSEIDAEL KNIL

SEQ ID NO: 18: PRTN3 amino acid sequence

MAHRPPSPAL ASVLLALLLS GAARAAEIVG GHEAQPHSRP YMASLQMRGN PGSHFCGGTL IHPSFVLTAA HCLRDIPQRL VNVVLGAHNV RTQEPTQQHF SVAQVFLNNY DAENKLNDVL LIQLSSPANL SASVATVQLP QQDQPVPHGT QCLAMGWGRV GAHDPPAQVL QELNVTVVTF FCRPHNICTF VPRRKAGICF GDSGGPLICD GIIQGIDSFV IWGCATRLFP DFFTRVALYV DWIRSTLRRV EAKGRP

SEQ ID NO: 19: ANXA2 amino acid sequence

MSTVHEILCK LSLEGDHSTP PSAYGSVKAY TNFDAERDAL NIETAIKTKG VDEVTIVNIL TNRSNAQRQD IAFAYQRRTK KELASALKSA LSGHLETVIL GLLKTPAQYD ASELKASMKG LGTDEDSLIE IICSRTNQEL QEINRVYKEM YKTDLEKDII SDTSGDFRKL MVALAKGRRA EDGSVIDYEL IDQDARDLYD AGVKRKGTDV PKWISIMTER SVPHLQKVFD RYKSYSPYDM LESIRKEVKG DLENAFLNLV QCIQNKPLYF ADRLYDSMKG KGTRDKVLIR IMVSRSEVDM LKIRSEFKRK YGKSLYYYIQ QDTKGDYQKA LLYLCGGDD

SEQ ID NO: 20: IGHA1 amino acid sequence

ASPTSPKVFP LSLCSTQPDG NVVIACLVQG FFPQEPLSVT WSESGQGVTA RNFPPSQDAS GDLYTTSSQL TLPATQCLAG KSVTCHVKHY TNPSQDVTVP CPVPSTPPTP SPSTPPTPSP SCCHPRLSLH RPALEDLLLG SEANLTCTLT GLRDASGVTF TWTPSSGKSA VQGPPERDLC GCYSVSSVLP GCAEPWNHGK TFTCTAAYPE SKTPLTATLS KSGNTFRPEV HLLPPPSEEL ALNELVTLTC LARGFSPKDV LVRWLQGSQE LPREKYLTWA SRQEPSQGTT TFAVTSILRV AAEDWKKGDT FSCMVGHEAL PLAFTQKTID RLAGKPTHVN VSVVMAEVDG TCY

SEQ ID NO: 21: ACTN4 amino acid sequence

MVDYHAANQS YQYGPSSAGN GAGGGGSMGD YMAQEDDWDR DLLLDPAWEK QQRKTFTAWC NSHLRKAGTQ IENIDEDFRD GLKLMLLLEV ISGERLPKPE RGKMRVHKIN NVNKALDFIA SKGVKLVSIG AEEIVDGNAK MTLGMIWTII LRFAIQDISV EETSAKEGLL LWCQRKTAPY KNVNVQNFHI SWKDGLAFNA LIHRHRPELI EYDKLRKDDP VTNLNNAFEV AEKYLDIPKM LDAEDIVNTA RPDEKAIMTY VSSFYHAFSG AQKAETAANR ICKVLAVNQE NEHLMEDYEK LASDLLEWIR RTIPWLEDRV PQKTIQEMQQ KLEDFRDYRR VHKPPKVQEK CQLEINFNTL QTKLRLSNRP AFMPSEGKMV SDINNGWQHL EQAEKGYEEW LLNEIRRLER LDHLAEKFRQ KASIHEAWTD GKEAMLKHRD YETATLSDIK ALIRKHEAFE SDLAAHQDRV EQIAAIAQEL NELDYYDSHN VNTRCQKICD QWDALGSLTH SRREALEKTE KQLEAIDQLH LEYAKRAAPF NNWMESAMED LQDMFIVHTI EEIEGLISAH DQFKSTLPDA DREREAILAI HKEAQRIAES NHIKLSGSNP YTTVTPQIIN SKWEKVQQLV PKRDHALLEE QSKQQSNEHL RRQFASQANV VGPWIQTKME EIGRISIEMN GTLEDQLSHL KQYERSIVDY KPNLDLLEQQ HQLIQEALIF DNKHTNYTME HIRVGWEQLL TTIARTINEV ENQILTRDAK GISQEQMQEF RASFNHFDKD HGGALGPEEF KACLISLGYD VENDRQGEAE FNRIMSLVDP NHSGLVTFQA FIDFMSRETT DTDTADQVIA SFKVLAGDKN FITAEELRRE LPPDQAEYCI ARMAPYQGPD AVPGALDYKS FSTALYGESD L

Claims (16)

1. At least one or more genes selected from the following groups 1 to 3; Or a biomarker for diagnosing neurodegenerative diseases comprising a protein encoded thereby:

   Group 1: CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4

   Group 2: PIP, LCN1, LCP1, LTF and APOA1

   Group 3: TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4
2. The method of claim 1,

   The biomarker is to be measured in tear fluid, a biomarker for diagnosing neurodegenerative diseases.
3. The method of claim 1,

   The neurodegenerative diseases include mild cognitive impairment, Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, corticobasal degeneration, and Parkinson's disease. (Parkinson's disease), multiple system atrophy, progressive supranuclear palsy, Huntington's disease, dental-rubro-pallido-luysian atrophy, spinal cerebellum Ataxia, amyotrophic lateral sclerosis, primary lateral sclerosis, and spinal muscular atrophy. Diagnostic biomarker.
4. At least one or more genes selected from the following groups 1 to 3; Or a composition for diagnosis of neurodegenerative diseases comprising 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 2: PIP, LCN1, LCP1, LTF and APOA1

   Group 3: TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4
5. The method of claim 4,

   The agent for measuring the expression level of the gene is at least one selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene, a composition for diagnosing neurodegenerative diseases.
6. The method of claim 4,

   The agent for measuring the expression level of the protein 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, A composition for diagnosis of neurodegenerative diseases.
7. The method of claim 4,

   The neurodegenerative diseases include mild cognitive impairment, Alzheimer's disease, frontotemporal dementia, Lewy body dementia, cortical basal degeneration, Parkinson's disease, multi-system atrophy, progressive nuclear paralysis, Huntington's disease, tooth red-pale nucleus Luces atrophy, spinal cerebellar ventricle. Manic disease, muscle atrophy lateral sclerosis, primary lateral sclerosis, and spinal muscle atrophy, which is at least one selected from the group consisting of, the diagnostic composition for neurodegenerative diseases.
8. The method of claim 4,

   The expression level of the gene or the protein encoded by it is measured in the tear fluid, the composition for diagnosis of neurodegenerative disease.
9. A kit for diagnosis of neurodegenerative diseases comprising the composition for diagnosis of any one of claims 4 to 8.
10. The method of claim 9,

    The kit is 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.
11. At least one or more genes selected from the following groups 1 to 3 from the biological sample isolated from the subject of interest; Or measuring the expression level of the protein encoded thereby; a method of providing information for diagnosis of neurodegenerative disease comprising:

    Group 1: CAP1, GLUL, SERPINC1, SCGB1D1, AZGP1, SCGB2A1, A2M, IGHM and CST4

    Group 2: PIP, LCN1, LCP1, LTF and APOA1

    Group 3: TF, PKM, GC, PRTN3, ANXA2, IGHA1 and ACTN4
12. The method of claim 11,

    The biological sample is a tear fluid, a method for providing information for diagnosis of neurodegenerative diseases.
13. The method of claim 11,

    The neurodegenerative diseases are mild cognitive impairment, Alzheimer's disease, frontotemporal dementia, Lewy body dementia, cortical basal degeneration, Parkinson's disease, multisystem atrophy, progressive nucleus paralysis, Huntington's disease, tooth red pale nucleus Luce atrophy Disease, spinal cerebellar ataxia, muscle atrophy lateral sclerosis, primary lateral sclerosis, and spinal muscle atrophy.
14. The method of claim 11,

    The expression level of the gene is measured through at least one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene.
15. The method of claim 11,

    The expression level of the protein is measured through at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNAs, and aptamers that specifically bind to the protein, providing information for diagnosis of neurodegenerative diseases. How to.
16. The method of claim 11,

    When the expression level of the gene included in the group 1 or the protein encoded by it is increased compared to the 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; A method of providing information for diagnosis of neurodegenerative disease, further comprising the step of predicting as neurodegenerative disease.

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