Method for identifying alzheimer's disease patient

Abstract

To provide a technique for testing Alzheimer's disease that is suitable for repeated measurements and mass screening.SOLUTION: A method for identifying an Alzheimer's disease patient includes the step of measuring the level of miR203 present in urine-derived exosomes of a subject. A significantly elevated level of miR203 relative to that in urine-derived exosomes of healthy persons indicates that the subject might be suffering from Alzheimer's disease.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting a patient with Alzheimer's disease, more particularly, to a method for detecting a patient with Alzheimer's disease, a method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, a kit for determining whether or not a subject may be affected by Alzheimer's disease, and a kit for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease (AD) is a typical dementia disease that shows progressive cognitive decline due to accumulation of amyloid beta (Aβ) and tau protein in the brain and damage to neurons in the hippocampus. AD is rapidly increasing in aging societies, and it is estimated that there are 8 million patients in Japan. As there is no cure for AD, the increase in AD patients has become a major social problem.

[0003] In recent years, a monoclonal antibody therapy (lecanemab) has been developed to remove accumulated Aβ. As monoclonal antibody therapy is expected to be more effective in patients at an early stage of the disease, there has been a great demand for early diagnosis of AD.

[0004] Diagnostic imaging methods for AD include VSRAD, which uses magnetic resonance imaging (MRI) to measure hippocampal atrophy, cerebral blood flow scintigraphy, and amyloid PET, which detects accumulation of Aβ in the brain. However, all of these methods are expensive and not suitable for repeated testing. In addition, amyloid PET can only be performed at a limited number of facilities.

[0005] As a method for diagnosing AD by body fluid testing, it has been reported that the method of measuring p-tau181 in cerebrospinal fluid using the single molecule array (SIMOA) method can diagnose AD with a specificity of 73.1% and a sensitivity of 67.9% (see Non-Patent Document 1). 1-42It has been reported that the measurement of peptides using the SIMOA method was able to diagnose AD with a specificity of 76.9% and a sensitivity of 96.4% (see Non-Patent Document 1). However, because cerebrospinal fluid testing is highly invasive, it is difficult to perform repeated measurements.

[0006] Therefore, the development of a test method using blood is being promoted. For example, Non-Patent Document 2 reports that a method of detecting Aβ peptides in blood using a high-precision mass spectrometer (MALDI-MS) was able to diagnose AD with a specificity of 91.5% and a sensitivity of 92.0%. However, this device is extremely expensive, is not very versatile, and is not suitable for repeated measurements or mass screening. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Shaw LM, et al., Cerebrospinal Fluid Biomarker Signature in Alzheimer's Disease Neuroimaging Initiative Subjects, Ann Neurol., 65 (4), 403-413, 2009. [Non-Patent Document 2] Nakamura A., et al., High performance plasma amyloid-β biomarkers for Alzheimer's disease, Nature, 554 (7691), 249-254, 2018. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a testing technique for Alzheimer's disease that is suitable for repeated measurements and mass screening. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A method for detecting Alzheimer's disease patients, comprising the step of measuring the amount of miR203 present in urinary exosomes of a subject, wherein the amount of miR203 present is significantly higher than the amount of miR203 present in urinary exosomes of healthy subjects, indicating that the subject may be suffering from Alzheimer's disease. [2] The method for detecting Alzheimer's disease patients according to [1], further comprising a step of measuring the abundance of miR203 and miR30c-2 in blood-derived exosomes of a subject shown by the primary screening to be likely to be affected by Alzheimer's disease, wherein a ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), is significantly higher than the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject, indicating that the subject is likely to be affected by Alzheimer's disease. miR203 / miR30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1) [3] A method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, comprising a step of measuring the amount of miR203 present in exosomes derived from the blood of the patient, wherein the amount of miR203 present corresponds to the degree of progression of Alzheimer's disease in the patient. [4] A method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease according to claim 3, further comprising a step of measuring the amount of miR30c-2 present in blood-derived exosomes of the patient with Alzheimer's disease, wherein the magnitude of the ratio of the amounts of miR203 and miR30c-2 present (miR203 / miR30c-2 ratio), calculated by the following formula (1), corresponds to the degree of progression of Alzheimer's disease in the patient. miR203 / miR30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1) [5] A kit for determining whether or not a subject is likely to be affected by Alzheimer's disease, the kit comprising a primer set for amplifying miR203, the kit being used to measure the amount of miR203 present in urinary exosomes of a subject, the kit indicating that the subject is likely to be affected by Alzheimer's disease if the amount of miR203 present is significantly higher than the amount of miR203 present in urinary exosomes of healthy subjects. [6] The kit for determining whether or not a subject is likely to be affected by Alzheimer's disease according to [5], further comprising a primer set for amplifying miR30c-2, and used to measure the abundance of miR203 and miR30c-2 in blood-derived exosomes of the subject, wherein a ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), is significantly higher than the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject, indicating the subject is likely to be affected by Alzheimer's disease. miR203 / miR30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1) [7] The kit described in [5] or [6], further comprising a reagent for extracting exosomes from a biological sample. [8] A kit for assessing the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, the kit comprising a primer set for amplifying miR203 and used to measure the amount of miR203 present in exosomes derived from the blood of a patient with Alzheimer's disease, the amount of miR203 present corresponding to the degree of progression of Alzheimer's disease in the patient. [9] The kit for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease according to [8], further comprising a primer set for amplifying miR30c-2, and used to measure the abundance of miR203 and miR30c-2 in blood-derived exosomes of a patient with Alzheimer's disease, wherein the magnitude of the ratio of the abundance of miR203 to miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), corresponds to the degree of progression of Alzheimer's disease in the patient.

[10] The kit described in [8] or [9], further comprising a reagent for extracting exosomes from a biological sample.

[0010] It can also be said that the present invention includes the following aspects. [P1] A method for detecting a patient with Alzheimer's disease, comprising the step of measuring the amount of miR203 present in urinary exosomes of a subject, wherein the amount of miR203 present is significantly higher than the amount of miR203 present in urinary exosomes of healthy subjects, indicating that the subject may be suffering from Alzheimer's disease. [P2] [P1] A method for detecting a patient with Alzheimer's disease, comprising the steps of: measuring the abundance of miR203 and miR30c-2 in blood-derived exosomes of a subject shown to be possibly suffering from Alzheimer's disease by the detection method as a first screening; and calculating the abundance ratio of the miR203 and miR30c-2 (miR203 / 30c-2 ratio) by the following formula (1), wherein a significantly higher miR203 / 30c-2 ratio than the miR203 / 30c-2 ratio in blood-derived exosomes of a healthy subject indicates a higher likelihood that the subject is suffering from Alzheimer's disease. miR203 / 30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1) [P3] A method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, wherein the ratio of the abundance of miR203 and miR30c-2 in blood-derived exosomes of the patient with Alzheimer's disease (miR203 / 30c-2 ratio), calculated by the following formula (1), corresponds to the degree of progression of Alzheimer's disease in the patient: miR203 / 30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1) Effect of the Invention

[0011] According to the present invention, a testing technique for Alzheimer's disease that is suitable for repeated measurements and mass screening can be provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of heat map analysis of microRNA expression in Experimental Example 1. [Diagram 2] FIG. 2 is a graph showing the results of quantitative PCR of miR203 and miR30c-2 in serum exosomes in Experimental Example 2. [Diagram 3] FIG. 3 is a graph showing the results of quantitative PCR in Experimental Example 2, in which AD patients were divided into mild patients and intermediate to advanced stage patients. [Figure 4] FIG. 4 is a graph showing the results of quantitative PCR in Experimental Example 2, with the subjects divided by sex and age at the time of testing. [Diagram 5] FIG. 5 shows a graph and an ROC curve based on the calculated miR203 / miR30c-2 ratio in Experimental Example 2. [Figure 6] FIG. 6 is a graph based on the miR203 / miR30c-2 ratio in serum exosomes in Experimental Example 3. [Figure 7] FIG. 7 is a graph showing the results of quantitative PCR of miR203 and miR30c-2 in urinary exosomes in Experimental Example 3. [Figure 8] FIG. 8 is a graph showing the results of quantitative PCR in Experimental Example 3, where AD patients were divided into mild patients and intermediate to advanced stage patients. [Figure 9] FIG. 9 is a graph showing the correlation between the expression level of miR203 in serum exosomes and the expression level of miR203 in urinary exosomes in Experimental Example 3. [Figure 10] FIG. 10 is an ROC curve based on the expression level of miR203 in urinary exosomes in Experimental Example 3. [Figure 11] FIG. 11 shows a graph and an ROC curve based on the calculated miR203 / miR30c-2 ratio in Experimental Example 4. [Figure 12]Figure 12 is a graph showing the correlation between the miR203 / miR30c-2 ratio in serum exosomes and the Hasegawa Dementia Rating Scale (HDS-R) in Experimental Example 4, and a graph showing the correlation between the miR203 / miR30c-2 ratio in serum exosomes and the results of the Mini-Mental State Examination (MMSE). [Figure 13] FIG. 13 is a graph showing the analysis results of miR203 in Experimental Example 5. [Figure 14] FIG. 14 is a graph showing the analysis results of miR30c-2 in Experimental Example 5. [Figure 15] FIG. 15 is a flow chart showing the method for determining whether or not a patient is an AD patient in Experimental Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] [Methods for detecting Alzheimer's disease patients] In one embodiment, the present invention provides a method for detecting a patient with Alzheimer's disease, comprising a step of measuring the abundance of miR203 in urinary exosomes of a subject, wherein the abundance of miR203 is significantly higher than the abundance of miR203 in urinary exosomes of healthy subjects, indicating that the subject may be suffering from Alzheimer's disease.

[0014] As described later in the Examples, the inventors have clarified that when the abundance (expression amount) of miR203 in urinary exosomes is significantly higher than the abundance (expression amount) of miR203 in urinary exosomes from healthy individuals, this indicates that the subject may be suffering from Alzheimer's disease. The method of the present embodiment can also be said to be a method for determining whether or not the subject may be suffering from Alzheimer's disease.

[0015] The urine may be a random urine sample. Since urine can be collected non-invasively, it is easy to perform repeated measurements. The method of this embodiment is suitable for mass screening at health checkup centers, etc.

[0016] Exosomes are small vesicles with a diameter of about 40 to 100 nm that are released from cells. Exosomes contain cell-derived proteins, mRNA, microRNA (miRNA), DNA, etc. Since exosomes can pass through the blood-brain barrier, it is thought that exosomes derived from brain cells are also contained in blood and urine.

[0017] Extraction of exosomes from urine may be performed by a commonly used method, or may be performed using a commercially available kit. Extraction of microRNA from exosomes may also be performed by a commonly used method, or may be performed using a commercially available kit.

[0018] Human miR203 is generated from a precursor, hsa-mir-203a. The accession number of hsa-mir-203a in the public microRNA database miRBase is MI0000283. The base sequence of hsa-mir-203a is 5'-guguuggggacucgcgcgcuggguccAGUGGUUCUUAACAGUUCAACAGUUcuguagcgcaauuGUGAAAUGUUUAGGACCACUAGacccggcgggcgcggcgacagcga-3' (SEQ ID NO: 1).

[0019] From hsa-mir-203a, mature miRNAs hsa-miR-203a-5p and hsa-miR-203a-3p are generated. The base sequence of hsa-miR-203a-5p is 5'-AGUGGUUCUUAACAGUUCAACAGUU-3' (SEQ ID NO: 2), and the base sequence of hsa-miR-203a-3p is 5'-GUGAAAUGUUUAGGACCACUAG-3' (SEQ ID NO: 3).

[0020] hsa-miR-203a-5p corresponds to the 27th to 51st bases of the nucleotide sequence of hsa-mir-203a, and hsa-miR-203a-3p corresponds to the 65th to 86th bases of the nucleotide sequence of hsa-mir-150.

[0021] Mouse miR203 is generated from a precursor, mmu-mir-203. The accession number of mmu-mir-203 in miRBase is MI0000246. The nucleotide sequence of mmu-mir-203 is 5'-gccugguccAGUGGUUCUUGACAGUUCAACAguucuguagcacaauuGUGAAAUGUUUAGGACCACUAGacccggc-3' (SEQ ID NO: 4).

[0022] From mmu-mir-203, mature miRNAs mmu-miR-203-5p and mmu-miR-203-3p are generated. The base sequence of mmu-miR-203-5p is 5'-AGUGGUUCUUGACAGUUCAACA-3' (SEQ ID NO: 5), and the base sequence of mmu-miR-203-3p is 5'-AGUGGUUCUUGACAGUUCAACA-3' (SEQ ID NO: 6).

[0023] mmu-miR-203-5p corresponds to the 10th to 31st bases of the mmu-mir-203 nucleotide sequence, and mmu-miR-203-3p corresponds to the 48th to 69th bases of the mmu-mir-203 nucleotide sequence.

[0024] The miR203 detected in the method of this embodiment may be hsa-miR-203a-5p or hsa-miR-203a-3p.

[0025] The amount of miR203 present in urinary exosomes from a subject can be measured by a commonly used method, for example, quantitative real-time PCR.

[0026] When the abundance of miR203 in urinary exosomes is significantly higher than that in urinary exosomes from healthy individuals, it can be determined that the subject may be suffering from Alzheimer's disease.

[0027] In the method of this embodiment, the presence of miR203 in urinary exosomes of a subject being significantly higher than the presence of miR203 in urinary exosomes of a healthy subject may mean that the presence of miR203 in urinary exosomes of the subject is higher than a predetermined threshold.

[0028] Here, the predetermined threshold may be empirically set by accumulating data of the evaluation results. It is preferable to set a value that can distinguish with high accuracy the possibility that the subject is suffering from Alzheimer's disease as the predetermined threshold. Here, a discriminant formula may be created by a statistical analysis method known in the art, and the threshold may be calculated from these accumulated measured values ​​using this formula. Alternatively, the accumulated measured values ​​may be machine-learned by AI to automatically determine the possibility that the subject is suffering from Alzheimer's disease.

[0029] As described later in the Examples, the predetermined threshold may be a cutoff value determined as a result of ROC analysis. In the method of this embodiment, the cutoff value may be about 1.275, more preferably 1.275.

[0030] In this specification, "about" means a value including ±10%. For example, about 10 means 9 to 11. By adopting about 1.275 as the cutoff value and determining that a subject having an abundance of miR203 in urinary exosomes of about 1.275 or more is likely to suffer from Alzheimer's disease, it is possible to pick up a wider range of subjects who may suffer from Alzheimer's disease.

[0031] Alternatively, the cutoff value may be about 2.370, more preferably 2.370. As described later in the Examples, by adopting a cutoff value of about 2.370 and determining that a subject having an abundance of miR203 in urinary exosomes of about 2.370 or more is likely to be affected by Alzheimer's disease, it is possible to determine whether a subject is affected by Alzheimer's disease with a specificity of 100% and a sensitivity of 60%.

[0032] The method of this embodiment may comprise the step of detecting Alzheimer's disease patients based on the amount of miR203 present in urine-derived exosomes as a first screening step, and further comprising the step of measuring the amounts of miR203 and miR30c-2 present in blood-derived exosomes of a subject shown by the first screening to be likely to be affected by Alzheimer's disease, wherein a ratio of the amounts of miR203 and miR30c-2 present (miR203 / miR30c-2 ratio), calculated by the following formula (1), being significantly higher than the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject, indicates that the subject is likely to be affected by Alzheimer's disease. miR203 / miR30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1)

[0033] As described later in the Examples, the inventors revealed that when the miR203 / miR30c-2 ratio in blood-derived exosomes is significantly higher than the miR203 / miR30c-2 ratio in blood-derived exosomes from healthy individuals, the subject is more likely to be suffering from Alzheimer's disease.

[0034] By conducting mass screening (group testing) using urine in the first screening and testing using the miR203 / miR30c-2 ratio in blood-derived exosomes as an indicator in the second screening, it will be possible to meet the demand for early diagnosis of Alzheimer's disease.

[0035] Examples of blood include serum and plasma. Extraction of exosomes from blood may be performed by a commonly used method, or may be performed using a commercially available kit. Extraction of microRNA from exosomes may also be performed by a commonly used method, or may be performed using a commercially available kit.

[0036] Human miR30c-2 is generated from the precursor hsa-mir-30c-2. The accession number of hsa-mir-30c-2 in the public microRNA database miRBase is MI0000254. The base sequence of hsa-mir-30c-2 is 5'-agauacUGUAAACAUCCUACACUCUCAGCuguggaaaguaagaaagCUGGGAGAAGGCUGUUUACUCUuucu-3' (SEQ ID NO: 7).

[0037] From hsa-mir-30c-2, mature miRNAs hsa-miR-30c-2-5p (hsa-miR-30c-5p) and hsa-miR-30c-2-3p are generated. The base sequence of hsa-miR-30c-2-5p is 5'-UGUAAACAUCCUACACUCUCAGC-3' (SEQ ID NO: 8), and the base sequence of hsa-miR-30c-2-3p is 5'-CUGGGAGAAGGCUGUUUACUCU-3' (SEQ ID NO: 9).

[0038] hsa-miR-30c-2-5p corresponds to the 7th to 29th bases of the nucleotide sequence of hsa-mir-30c-2. Also, hsa-miR-30c-2-3p corresponds to the 47th to 68th bases of the nucleotide sequence of hsa-mir-30c-2.

[0039] Mouse miR203 is generated from the precursor mmu-mir-30c-2. The accession number of mmu-mir-30c-2 in miRBase is MI0000548. The nucleotide sequence of mmu-mir-30c-2 is 5'-gagugacagauauUGUAAACAUCCUACACUCUCAGCugugaaaaguaagaaagCUGGGAGAAGGCUGUUUACUCUcucugccuu-3' (SEQ ID NO: 10).

[0040] From mmu-mir-30c-2, the mature miRNAs mmu-miR-30c-2-5p (mmu-miR-30c-5p) and mmu-miR-30c-2-3p are generated. The base sequence of mmu-miR-30c-2-5p is 5'-UGUAAACAUCCUACACUCUCAGC-3' (SEQ ID NO: 11), and the base sequence of mmu-miR-30c-2-3p is 5'-CUGGGAGAAGGCUGUUUACUCU-3' (SEQ ID NO: 12).

[0041] mmu-miR-30c-2-5p corresponds to the 14th to 36th bases of the mmu-mir-30c-2 nucleotide sequence, and mmu-miR-30c-2-3p corresponds to the 54th to 75th bases of the mmu-mir-30c-2 nucleotide sequence.

[0042] The miR30c-2 detected in the method of this embodiment may be hsa-miR-30c-2-5p or hsa-miR-30c-2-3p.

[0043] In the method of this embodiment, the miR203 / miR30c-2 ratio in the blood-derived exosomes of a subject being significantly higher than the miR203 / miR30c-2 ratio in the blood-derived exosomes of a healthy subject may mean that the miR203 / miR30c-2 ratio in the blood-derived exosomes of the subject is higher than a predetermined threshold value.

[0044] Here, the predetermined threshold may be empirically set by accumulating data of the evaluation results. It is preferable to set a value that can distinguish with high accuracy the possibility that the subject is suffering from Alzheimer's disease as the predetermined threshold. Here, a discriminant formula may be created by a statistical analysis method known in the art, and the threshold may be calculated from these accumulated measured values ​​using this formula. Alternatively, the accumulated measured values ​​may be machine-learned by AI to automatically determine the possibility that the subject is suffering from Alzheimer's disease.

[0045] As described later in the Examples, the predetermined threshold value may be a cutoff value determined as a result of ROC analysis. In the method of this embodiment, the cutoff value may be about 3.076, more preferably 3.076. By adopting about 3.076 as the cutoff value and determining that a subject with a miR203 / miR30c-2 ratio of about 3.076 or more in blood-derived exosomes may be affected by Alzheimer's disease, it is possible to determine that a subject is affected by Alzheimer's disease with a specificity of 100% and a sensitivity of 90%.

[0046] [Methods for assessing the degree of progression of Alzheimer's disease in patients with Alzheimer's disease] In one embodiment, the present invention provides a method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, comprising a step of measuring the abundance of miR203 in blood-derived exosomes of the patient, wherein the abundance of miR203 corresponds to the degree of progression of Alzheimer's disease in the patient.

[0047] As described later in the Examples, the inventors have demonstrated that the abundance (expression amount) of miR203 in blood-derived exosomes is significantly higher in patients with intermediate to advanced stages of Alzheimer's disease than in patients with mild disease. Therefore, the abundance of miR203 in blood-derived exosomes of Alzheimer's disease patients can be used as an indicator to evaluate the degree of progression of Alzheimer's disease in Alzheimer's disease patients.

[0048] As described later in the Examples, when the amount of miR203 present in blood-derived exosomes of a healthy subject or a patient with a neurological disease other than Alzheimer's disease is set to 1, an Alzheimer's disease patient can be evaluated as having mild disease if the amount of miR203 present in blood-derived exosomes is 1.2 or more and less than 5. Furthermore, when the amount of miR203 present in blood-derived exosomes of a healthy subject or a patient with a neurological disease other than Alzheimer's disease is set to 1, an Alzheimer's disease patient can be evaluated as having intermediate to advanced stages.

[0049] In the method of this embodiment, the blood, exosomes, miR203, and the amount of miR203 present are measured in the same manner as described above.

[0050] The method of the present embodiment may be a method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, further comprising a step of measuring the amount of miR30c-2 present in blood-derived exosomes of the patient with Alzheimer's disease, in which the ratio of the amounts of miR203 and miR30c-2 present (miR203 / miR30c-2 ratio), calculated by the following formula (1), corresponds to the degree of progression of Alzheimer's disease in the patient. miR203 / miR30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1)

[0051] As described later in the Examples, the inventors have demonstrated that the miR203 / miR30c-2 ratio in blood-derived exosomes is significantly higher in patients with intermediate to advanced stages of Alzheimer's disease than in patients with mild disease. Therefore, the degree of progression of Alzheimer's disease in Alzheimer's disease patients can be evaluated using the miR203 / miR30c-2 ratio in blood-derived exosomes as an indicator.

[0052] As described later in the Examples, when the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject or a patient with a neurological disease other than Alzheimer's disease is taken as 1, an Alzheimer's disease patient whose blood-derived exosomes have a miR203 / miR30c-2 ratio of 1.2 or more and less than 10 can be evaluated as having mild disease. Furthermore, when the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject or a patient with a neurological disease other than Alzheimer's disease is taken as 1, an Alzheimer's disease patient whose blood-derived exosomes have a miR203 / miR30c-2 ratio of 10 or more and less than 300 can be evaluated as having intermediate to advanced stages.

[0053] In the method of this embodiment, the measurement of miR30c-2 and the amount of miR30c-2 present is similar to that described above.

[0054] [Kit for determining whether or not a subject may have Alzheimer's disease] In one embodiment, the present invention provides a kit for determining whether or not a subject is likely to be affected by Alzheimer's disease, the kit comprising a primer set for amplifying miR203, the kit being used to measure the amount of miR203 present in urinary exosomes of a subject, wherein the amount of miR203 present is significantly higher than the amount of miR203 present in urinary exosomes of healthy subjects, indicating that the subject is likely to be affected by Alzheimer's disease.

[0055] The kit of this embodiment can be used to suitably carry out the above-mentioned method for detecting Alzheimer's disease patients.

[0056] The primer set for amplifying miR203 is not particularly limited as long as it can amplify miR203, and may be a commercially available one. For example, the primer set may be TaqMan TM MicroRNA Assays (Assay ID 478316_mir, Thermo Fisher Scientific) can be used.

[0057] The kit of this embodiment further includes a primer set for amplifying miR30c-2, and is used to measure the abundance of miR203 and miR30c-2 in the blood-derived exosomes of a subject, and may be used for determining whether or not the subject is likely to be affected by Alzheimer's disease, where the ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), is significantly higher than the miR203 / miR30c-2 ratio in the blood-derived exosomes of a healthy subject, indicating the subject is likely to be affected by Alzheimer's disease. miR203 / miR30c-2 ratio = abundance of miR203 / abundance of miR30c-2…(1)

[0058] The kit of this embodiment can be used to suitably carry out the above-mentioned method for detecting Alzheimer's disease patients.

[0059] The primer set for amplifying miR30c-2 is not particularly limited as long as it can amplify miR30c-2, and may be commercially available. For example, the primer set may be TaqMan TM MicroRNA Assays (Assay ID 479401_mir, Thermo Fisher Scientific) can be used.

[0060] The kit of the present embodiment may further include a reagent for extracting exosomes from a biological sample, such as urine, serum, or plasma.

[0061] [A kit for assessing the degree of progression of Alzheimer's disease in patients with Alzheimer's disease] In one embodiment, the present invention provides a kit for assessing the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, the kit comprising a primer set for amplifying miR203, the kit being used to measure the abundance of miR203 in blood-derived exosomes of a patient with Alzheimer's disease, the abundance of miR203 corresponding to the degree of progression of Alzheimer's disease in the patient.

[0062] The kit of the present embodiment can suitably carry out the above-mentioned method for evaluating the degree of progression of Alzheimer's disease in an Alzheimer's disease patient.

[0063] The primer set for amplifying miR203 was the same as that described above.

[0064] The kit of this embodiment further includes a primer set for amplifying miR30c-2, and is used to measure the abundance of miR203 and miR30c-2 in blood-derived exosomes of an Alzheimer's disease patient, and the magnitude of the ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), corresponds to the extent of the progression of Alzheimer's disease in the patient, and may be used to evaluate the degree of progression of Alzheimer's disease in an Alzheimer's disease patient.

[0065] The kit of the present embodiment can suitably carry out the above-mentioned method for evaluating the degree of progression of Alzheimer's disease in an Alzheimer's disease patient.

[0066] The primer set for amplifying miR30c-2 was the same as that described above.

[0067] The kit of the present embodiment may further include a reagent for extracting exosomes from a biological sample, such as urine, serum, or plasma, as described above. EXAMPLES

[0068] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0069] [Experimental Example 1] (Searching for microRNAs that are indicators of AD) Exosomes produced by brain cells can freely cross the blood-brain barrier, so the inventors focused on microRNA contained in exosomes.

[0070] We used human beta amyloid precursor protein knock-in mice (APP-KI mice, see Saito T., et al., Single App knock-in mouse models of Alzheimer's disease, Nat Neurosci., 17 (5), 661-663, 2014.), an animal model of AD.

[0071] Exosomes were extracted from the serum of wild-type and APP-KI mice, and microRNAs were extracted from the exosomes. Next, they were analyzed by next-generation sequencing to identify microRNAs whose expression levels were significantly different between wild-type and APP-KI mice.

[0072] FIG. 1 shows the results of heat map analysis of microRNA expression. As a result, the only microRNA whose expression level was significantly increased in APP-KI mice compared to that in wild-type mice was miR203. In addition, five microRNAs whose expression level was significantly decreased in APP-KI mice compared to that in wild-type mice were found, and the one whose expression level was the most decreased was miR30c-2. Thereafter, experiments were conducted focusing on miR203 and miR30c-2.

[0073] [Experimental Example 2] (Analysis of microRNAs derived from AD patients 1) Exosome RNA was purified from serum of the subjects shown in Table 1 below. In Table 1 below, there were six healthy controls (hereinafter sometimes referred to as "HC") and nine patients with neurological diseases other than AD (hereinafter sometimes referred to as "OND"). In addition, of the AD patients, 10 were mildly ill patients and five were in the intermediate to advanced stages. Patients with a score of 18 or more on the Hasegawa Dementia Scale-Report (HDS-R) and a score of 21 or more on the Mini-Mental State Examination (MMSE) were classified as mildly ill patients.

[0074] [Table 1]

[0075] Exosomal RNA was purified using ExoQuick Exosome Isolation and RNA Purification kits (System Biosciences) and exoRNeasy Midi kit (Qiagen). miR203 and miR30c-2 were measured by quantitative PCR using TaqMan microRNA assays (Thermo Fisher Scientific). For miR203, hsa-miR-203a-3p was measured. For miR30c-2, hsa-miR-30c-2-3p was measured.

[0076] The expression levels of microRNAs were calculated using the 2-ΔΔCt method. In addition, the receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff value based on the Youden index, and specificity and sensitivity were calculated.

[0077] Figure 2 is a graph showing the results of quantitative PCR of miR203 and miR30c-2 in serum exosomes. As a result, it was revealed that the expression level of miR203 in serum exosomes of AD patients was significantly increased compared to healthy controls or patients with neurological diseases other than AD. On the other hand, it was revealed that the expression level of miR30c-2 in serum exosomes of AD patients was significantly decreased compared to healthy controls or patients with neurological diseases other than AD.

[0078] Figure 3 shows the results of quantitative PCR for AD patients divided into mild and intermediate to advanced stages. As a result, it was revealed that miR203 was significantly increased in intermediate to advanced stages compared to mild stages. On the other hand, miR30c-2 was decreased in all patients from mild to advanced stages.

[0079] Figure 4 is a graph showing the results of quantitative PCR, with the subjects divided by gender and age at the time of testing. As a result, no significant differences based on gender or age at the time of testing were observed for either miR203 or miR30c-2.

[0080] Next, the miR203 / miR30c-2 ratio was calculated and examined. The miR203 / miR30c-2 ratio was calculated using the following formula (1). miR203 / miR30c-2 ratio = expression level of miR203 / expression level of miR30c-2 … (1)

[0081] Figure 5 shows a graph and ROC curve based on the calculated miR203 / miR30c-2 ratio. As a result, it was revealed that the miR203 / miR30c-2 ratio was significantly higher in mild AD patients than in healthy controls or patients with neurological diseases other than AD, and was further elevated in intermediate to advanced AD patients. In addition, analysis with the ROC curve revealed that, when the cutoff value based on the Youden index was set at 3.984, AD could be diagnosed with 100% specificity and 86% sensitivity based on the miR203 / miR30c-2 ratio in serum exosomes.

[0082] [Experimental Example 3] (Analysis of microRNAs derived from AD patients 2) Exosome RNA was purified from serum and urine of the subjects shown in Table 2 below. In Table 2 below, 4 of the AD patients were mild patients and 11 were intermediate to advanced AD patients. Patients with a score of 18 or more on the Hasegawa Dementia Scale-Report (HDS-R) and a score of 21 or more on the Mini-Mental State Examination (MMSE) were classified as mild AD patients.

[0083] [Table 2]

[0084] Exosomal RNA was purified using ExoQuick Exosome Isolation and RNA Purification kits (System Biosciences) and exoRNeasy Midi kit (Qiagen). miR203 and miR30c-2 were measured by quantitative PCR using TaqMan microRNA assays (Thermo Fisher Scientific). For miR203, hsa-miR-203a-3p was measured. For miR30c-2, hsa-miR-30c-2-3p was measured.

[0085] The expression levels of microRNAs were calculated using the 2-ΔΔCt method. The miR203 / miR30c-2 ratio was calculated using the above formula (1). The optimal cutoff value was determined based on the Youden index by analysis using a receiver operating characteristic curve, and the specificity and sensitivity were calculated.

[0086] Figure 6 is a graph based on the miR203 / miR30c-2 ratio in serum exosomes. As a result, it was revealed that the miR203 / miR30c-2 ratio was significantly higher in mild AD patients than in healthy controls, and was further elevated in intermediate to advanced AD patients. In addition, analysis with an ROC curve revealed that AD could be diagnosed with 100% specificity and 87% sensitivity based on the miR203 / miR30c-2 ratio in serum exosomes when the cutoff value was set at 3.984 based on the Youden index.

[0087] These results further support that AD can be reliably diagnosed based on the miR203 / miR30c-2 ratio in serum exosomes.

[0088] Figure 7 is a graph showing the results of quantitative PCR of miR203 and miR30c-2 in urinary exosomes. As a result, it was revealed that the expression level of miR203 in urinary exosomes of AD patients was significantly increased compared to healthy controls. On the other hand, the expression of miR30c-2 in urinary exosomes was not detected in either healthy controls or AD patients.

[0089] Figure 8 is a graph showing the results of quantitative PCR for AD patients divided into mild and intermediate to advanced stages. As a result, it was revealed that miR203 in urinary exosomes was significantly increased in intermediate to advanced stages compared to mild stages.

[0090] Figure 9 is a graph showing the correlation between the expression level of miR203 in serum exosomes and the expression level of miR203 in urinary exosomes. As shown in Figure 9, the expression level of miR203 showed a strong positive correlation between serum and urine. This suggests that most of the miR203 in urine is derived from miR203 in serum exosomes.

[0091] Figure 10 shows the ROC curve based on the expression level of miR203 in urinary exosomes. When the cutoff value was set to 2.370 based on the Youden index, it was found that AD could be diagnosed with 100% specificity and 60% sensitivity. When the cutoff value was set to 1.275, the specificity was 76.9%, but the sensitivity increased to 73.3%.

[0092] [Experimental Example 4] (Analysis of microRNAs derived from AD patients 3) Exosome RNA was purified from serum for the subjects shown in Table 3 below. The subjects shown in Table 3 are a combination of the subjects shown in Table 1 and the subjects shown in Table 2. In Table 3 below, there were 12 healthy controls and 9 patients with neurological diseases other than AD. In addition, of the AD patients, 14 were mild patients and 16 were intermediate to advanced patients. Patients with a score of 18 or more on the Hasegawa Dementia Scale-Report (HDS-R) and a score of 21 or more on the Mini-Mental State Examination (MMSE) were classified as mild patients.

[0093] [Table 3]

[0094] Exosomal RNA was purified using ExoQuick Exosome Isolation and RNA Purification kits (System Biosciences) and exoRNeasy Midi kit (Qiagen). miR203 and miR30c-2 were measured by quantitative PCR using TaqMan microRNA assays (Thermo Fisher Scientific). For miR203, hsa-miR-203a-3p was measured. For miR30c-2, hsa-miR-30c-2-3p was measured.

[0095] The expression levels of microRNAs were calculated using the 2-ΔΔCt method. The miR203 / miR30c-2 ratio was calculated using the above formula (1). The optimal cutoff value was determined based on the Youden index by analysis using a receiver operating characteristic curve, and the specificity and sensitivity were calculated.

[0096] Figure 11 shows a graph and ROC curve based on the calculated miR203 / miR30c-2 ratio. As a result, it was shown that the miR203 / miR30c-2 ratio was significantly higher in AD patients than in healthy controls or patients with neurological diseases other than AD. Furthermore, it was revealed that the miR203 / miR30c-2 ratio was significantly higher in intermediate to advanced AD patients than in mild AD patients.

[0097] In addition, ROC curve analysis revealed that, when a cutoff value of 3.076 was used based on the Youden index, AD could be diagnosed based on the miR203 / miR30c-2 ratio in serum exosomes with 100% specificity and 90% sensitivity.

[0098] These results further support that AD can be reliably diagnosed based on the miR203 / miR30c-2 ratio in serum exosomes.

[0099] Figure 12 is a graph showing the correlation between the miR203 / miR30c-2 ratio in serum exosomes and the Hasegawa Dementia Rating Scale (HDS-R), and a graph showing the correlation between the miR203 / miR30c-2 ratio in serum exosomes and the results of the Mini-Mental State Examination (MMSE). As shown in Figure 12, it was revealed that the miR203 / 30c-2 ratio shows a significant negative correlation with the HDS-R value. It was also revealed that the miR203 / miR30c-2 ratio shows a negative correlation with the MMSE value.

[0100] [Experimental Example 5] (Analysis of microRNAs derived from AD patients 4) Using TargetScanMouse v7.1, we identified target genes of miR203 and miR30c-2, and investigated the biological functions of these target genes by enriched pathway analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID).

[0101] Figure 13 is a graph showing the analysis results of miR203. As a result, miR203 was related to cell proliferation, differentiation, survival, synaptic transmission, insulin signaling, and aging. Figure 14 is a graph showing the analysis results of miR30c-2. As a result, miR30c-2 was related to synaptic transmission, excitotoxicity, insulin signaling, aging, cell proliferation and survival.

[0102] [Experimental Example 6] (Determination of AD patients) Figure 15 is a flow chart of the method for identifying AD patients. First, subjects who may have AD are extracted by a non-invasive screening test using quantitative PCR of miR203 in urinary exosomes. Next, subjects who may have AD are identified with high specificity and sensitivity by a less invasive test measuring the miR203 / miR30c-2 ratio in serum exosomes.

[0103] The urine test uses a lower cutoff value of miR203, 1.275, to more broadly identify subjects with a possibility of AD. Then, a blood test uses the miR203 / miR30c-2 ratio to determine the possibility of AD with high specificity. In this way, subjects who are determined to be possibly suffering from AD by urine and blood tests are given a definitive diagnosis of AD through neurological examinations, cognitive function tests, and imaging tests such as MRI at a hospital.

[0104] Measurement of exosomal miR in urine is non-invasive and can be performed any number of times, allowing detection in urine at any time. This makes it suitable for mass screening at health checkup centers, etc. AD patients can be detected with a specificity of 76.9% and a sensitivity of 73.3%, making it a useful screening method. By combining urine exosomal miR testing with blood exosomal miR testing, AD patients can be identified with a specificity of 100% and a sensitivity of 90%. [Industrial Applicability]

[0105] According to the present invention, a testing technique for Alzheimer's disease that is suitable for repeated measurements and mass screening can be provided.

Claims

1. The method includes measuring the amount of miR203 present in urinary exosomes of a subject, The method for detecting a patient with Alzheimer's disease, wherein the abundance of miR203 is significantly higher than the abundance of miR203 in urinary exosomes of a healthy subject, indicating that the subject may be suffering from Alzheimer's disease.

2. The method according to claim 1 is used as a primary screening, and the method further comprises a step of measuring the abundance of miR203 and miR30c-2 in blood-derived exosomes of a subject who has been shown by the primary screening to be possibly suffering from Alzheimer's disease, 2. The method for detecting a patient with Alzheimer's disease according to claim 1, wherein the ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), is significantly higher than the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject, indicating that the subject may be suffering from Alzheimer's disease. miR203 / miR30c-2 ratio = miR203 abundance / miR30c-2 abundance... (1)

3. Measuring the abundance of miR203 in blood-derived exosomes of Alzheimer's disease patients, A method for assessing the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, wherein the abundance of miR203 corresponds to the degree of progression of Alzheimer's disease in the patient.

4. The method further comprises measuring the abundance of miR30c-2 in blood-derived exosomes of Alzheimer's disease patients; 4. The method for evaluating the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease according to claim 3, wherein the magnitude of the ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), corresponds to the degree of progression of Alzheimer's disease in the patient. miR203 / miR30c-2 ratio = miR203 abundance / miR30c-2 abundance... (1)

5. A primer set for amplifying miR203, A kit for determining whether or not a subject is likely to be affected by Alzheimer's disease, which is used to measure the amount of miR203 present in urinary exosomes of a subject, and indicates that the subject is likely to be affected by Alzheimer's disease if the amount of miR203 present is significantly higher than the amount of miR203 present in urinary exosomes of a healthy subject.

6. Further comprising a primer set for amplifying miR30c-2, The kit according to claim 5, which is used to measure the abundance of miR203 and miR30c-2 in blood-derived exosomes of a subject, and in which a ratio of the abundance of miR203 and miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), is significantly higher than the miR203 / miR30c-2 ratio in blood-derived exosomes of a healthy subject, indicating that the subject is likely to be affected by Alzheimer's disease. miR203 / miR30c-2 ratio = miR203 abundance / miR30c-2 abundance... (1)

7. The kit according to claim 5 or 6, further comprising a reagent for extracting exosomes from a biological sample.

8. A primer set for amplifying miR203, A kit for assessing the degree of progression of Alzheimer's disease in a patient with Alzheimer's disease, the kit being used to measure the amount of miR203 present in blood-derived exosomes of a patient with Alzheimer's disease, the amount of miR203 present corresponding to the degree of progression of Alzheimer's disease in the patient.

9. Further comprising a primer set for amplifying miR30c-2, The kit according to claim 8, which is used to measure the abundance of miR203 and miR30c-2 in blood-derived exosomes of an Alzheimer's disease patient, and the magnitude of the ratio of the abundance of miR203 to miR30c-2 (miR203 / miR30c-2 ratio), calculated by the following formula (1), corresponds to the extent of progression of Alzheimer's disease in the patient.

10. The kit according to claim 8 or 9, further comprising a reagent for extracting exosomes from a biological sample.

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