Kit and method for determining whether patient suffers from dementia

Abstract

The purpose of the present invention is to provide a biomarker with which it is possible to determine the presence or absence of dementia with high sensitivity and specificity by means of a simple operation, and to provide a method, a kit, and a device for the determination. Provided is a method for determining the presence or absence of a risk of dementia in a subject, said method comprising a step for calculating the expression level of connexin 43 gene and / or the ratio of the expression level of prolyl hydroxylase domain protein 3 gene to the expression level of connexin 37 gene.

Description

Technical Field

[0001] The present invention relates to a biomarker for determining whether a subject has dementia, a method for determining the presence or absence of the risk of developing or onset of dementia based on the biomarker, a kit, and a device. Background Art

[0002] With the progress of the aging process, the number of cases of dementia is increasing, and attempts are being made to treat dementia through early detection and lifestyle improvement.

[0003] As an imaging examination method for diagnosing dementia, amyloid PET (positron emission tomography) and Tau PET are known. However, these imaging examination methods are not suitable for early determination of dementia due to high cost, few facilities, and long restraint time during the examination.

[0004] In addition, cognitive function tests such as the Mini Mental State Examination (MMSE) and the Hasegawa's Dementia Scale for screening dementia are known. However, these cognitive function tests are used after the patient himself / herself recognizes the progression of symptoms and cannot be used to determine dementia at the initial stage of onset. In addition, there is also a problem that the results of the cognitive function tests are unclear.

[0005] In addition to the above, a method for determining dementia by examining proteins in cerebrospinal fluid is also known, but it is highly invasive and not suitable for early diagnosis of dementia.

[0006] As patent documents disclosing a method for determining whether a subject has dementia based on the gene expression level (biomarker) of a specific protein, patent documents 1 to 5 can be exemplified.

[0007] Patent Document 1 describes a method for examining Alzheimer's type dementia, including the steps of detecting in vitro a decrease in the level or function of at least one factor in the insulin / IGF signaling pathway such as insulin in a central nervous system tissue derived from a subject.

[0008] Patent Document 2 describes a method of detecting phosphorylation of at least one substrate protein such as MARCKS in a subject, and determining that the subject has a risk of developing or having Alzheimer's type dementia when the degree of phosphorylation is higher than that of a normal specimen.

[0009] Patent Document 3 describes a biomarker for detecting a cognitive dysfunction disease. The biomarker described in Patent Document 3 is described as a protein dependent on complement C4, prothrombin, complement C3, gelsolin, etc.

[0010] Patent Document 4 discloses that the expression levels of genes related to metabolism represented by the respiratory chain, such as the prolyl hydroxylase domain-containing protein 3 gene, are useful as biomarkers for dementia and the like.

[0011] Patent Document 5 discloses the following method: using the biomarkers described in Patent Document 4 to screen for agents useful for the treatment of dementia and the like.

[0012] Prior Art Documents

[0013] Patent Documents

[0014] Patent Document 1: Japanese Patent Application Laid-Open No. 2008-522199

[0015] Patent Document 2: International Publication WO2015 / 099094

[0016] Patent Document 3: International Publication WO2019 / 012671

[0017] Patent Document 4: International Publication WO2021 / 177447

[0018] Patent Document 5: International Publication WO2021 / 177448 Summary of the Invention

[0019] As described above, biomarkers for dementia have been widely studied. However, many biomarkers useful for definitive diagnosis require complex operations in their detection. On the other hand, biomarkers that include the prolyl hydroxylase domain-containing protein 3 gene and can be detected by simple operations often do not have sufficient sensitivity and specificity to be used alone for definitive diagnosis.

[0020] Therefore, an object of the present invention is to provide a biomarker that can determine whether a subject has dementia with high sensitivity and specificity by simple operations, and at the same time provide a method, a kit, and a device for performing such determination.

[0021] The present inventors conducted intensive studies and as a result, completed the following invention.

[0022] [1] A method for determining the presence or absence of the risk of developing or having dementia in a subject, comprising: a step of measuring the expression levels of connexin 37 gene, and connexin 43 gene and / or prolyl hydroxylase domain protein 3 gene in a sample from the subject; a step of calculating the ratio of the expression level of the connexin 43 gene and / or the expression level of the prolyl hydroxylase domain protein 3 gene to the expression level of the connexin 37 gene; and indicating that the subject has dementia or has a risk of developing dementia based on the comparison of the ratio with a preset cutoff value or a control value calculated in the same manner in a sample from a healthy individual.

[0023] [2] The method according to [1], wherein the sample is blood.

[0024] [3] The method according to [1] or [2], wherein the dementia is Alzheimer's dementia.

[0025] [4] A kit for determining the presence or absence of the risk of developing or having dementia, comprising: a reagent for measuring the expression level of connexin 37 gene in a sample from a subject, and a reagent for measuring the expression level of connexin 43 gene and / or prolyl hydroxylase domain protein 3 gene in the sample, wherein the reagent comprises a primer that can amplify all or a part of the base sequence of the gene or a base sequence complementary thereto and / or a probe that can hybridize with all or a part of the base sequence of the gene or a base sequence complementary thereto.

[0026] [5] The kit according to [4], wherein the primer and the probe are a polynucleotide or a fragment thereof described in any one of the following (1) to (6). (1) A polynucleotide fragment consisting of 15 or more consecutive bases selected from the polynucleotides having the base sequences shown in SEQ ID NO: 1, 3 or 5; (2) A polynucleotide fragment having 1 or 2 base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment of (1); (3) A polynucleotide having 15 or more consecutive bases selected from the polynucleotides having the base sequences shown in SEQ ID NO: 1, 3 or 5; (4) A polynucleotide fragment having 1 or 2 base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment of (3); (5) A polynucleotide that hybridizes with a polynucleotide fragment having 15 or more consecutive bases selected from the base sequences shown in SEQ ID NO: 1, 3 or 5 under highly stringent conditions; and (6) A polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotide or its fragment of (1) to (5).

[0027] [6]A kit is a kit for determining the presence or absence of the risk of developing or having dementia, comprising: a connexin 37 binding molecule in a sample derived from a subject, and a connexin 43 binding molecule and / or a prolyl hydroxylase domain protein 3 binding molecule in the above sample, and the above binding molecule comprises an antibody or an active fragment thereof capable of binding to a target protein, or an aptamer.

[0028] [7]An apparatus is an apparatus for determining the presence or absence of the risk of developing or having dementia, comprising: a reagent for measuring the expression level of the connexin 37 gene in a sample derived from a subject, and a reagent for measuring the expression level of the connexin 43 gene and / or the prolyl hydroxylase domain protein 3 gene in the above sample, and the above reagent comprises a primer capable of amplifying all or a part of the base sequence of the above gene or a base sequence complementary thereto and / or a probe capable of hybridizing with all or a part of the base sequence of the above gene or a base sequence complementary thereto.

[0029] [8]The apparatus according to [7], wherein the above primer and the above probe are polynucleotides or fragments thereof described in any one of the following (1) to (6). (1) A fragment consisting of 15 or more consecutive bases of a polynucleotide selected from the polynucleotides consisting of the base sequences shown in SEQ ID NO: 1, 3 or 5; (2) A polynucleotide fragment containing one or two base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment of (1); (3) A polynucleotide fragment of a polynucleotide selected from the polynucleotides consisting of the base sequences shown in SEQ ID NO: 1, 3 or 5 and containing 15 or more consecutive bases; (4) A polynucleotide fragment containing one or two base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment of (3); (5) A polynucleotide that hybridizes under high stringency conditions with a polynucleotide fragment sequence containing 15 or more consecutive bases selected from the base sequences shown in SEQ ID NO: 1, 3 or 5; and (6) A polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotide or its fragment of (1) to (5).

[0030] [9]An apparatus is an apparatus for determining the presence or absence of the risk of developing or having dementia, comprising: a connexin 37 binding molecule in a sample derived from a subject, and a connexin 43 binding molecule and / or a prolyl hydroxylase domain protein 3 binding molecule in the above sample, and the above binding molecule comprises an antibody or an active fragment thereof capable of binding to a target protein, or an aptamer.

[0031]

[10] A biomarker, which is any one of (a) to (d) selected from the following, is used for determining the presence or absence of dementia or the risk of onset. (a) A polynucleotide composed of the connexin 37 gene or a fragment thereof and a polynucleotide composed of the connexin 43 gene or a fragment thereof; (b) A polynucleotide composed of the connexin 37 gene or a fragment thereof and a polynucleotide composed of the prolyl hydroxylase domain protein 3 gene or a fragment thereof; (c) A polypeptide composed of connexin 37 or a fragment thereof and a polypeptide composed of connexin 43 or a fragment thereof; and (d) A polypeptide composed of connexin 37 or a fragment thereof and a polypeptide composed of prolyl hydroxylase domain protein 3 or a fragment thereof

[0032] This specification includes the disclosure of Japanese Patent Application No. 2022-169057, which is the basis of the priority of this application.

[0033] The biomarker according to the present invention can provide a biomarker capable of determining whether a subject has dementia with high sensitivity and specificity.

[0034] The method for determination according to the present invention can determine whether a subject has dementia by a simple method.

[0035] The kit and device according to the present invention can easily measure the biomarker of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a graph showing the proportion of monocytes in samples from healthy subjects and dementia patients.

[0037] Figure 2 It is a graph showing the expression levels of connexin genes in samples containing monocytes from healthy subjects and dementia patients.

[0038] Figure 3 It is a graph showing the expression levels of connexin genes in whole blood samples from healthy subjects and dementia patients.

[0039] Figure 4 It is a graph showing the relative values of the expression level of the connexin 37 gene relative to the expression levels of each gene in samples containing monocytes from healthy subjects and dementia patients. In the figure, * indicates p < 0.05.

[0040] Figure 5 It is a graph showing the relative value of the expression level of the connexin 37 gene relative to the expression level of the prolyl hydroxylase domain protein 3 (PHD3) gene in whole blood samples from healthy subjects and dementia patients. In the figure, * indicates p < 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0041] 1. Biomarker

[0042] 1-1. General Introduction

[0043] The first aspect of the present invention is a biomarker for determining the presence or absence of the risk of dementia onset or development. As a biomarker for determining the presence or absence of the risk of dementia onset or development in a subject, the biomarker of the present invention can be used in the determination method of the second aspect.

[0044] 1-2. Definitions

[0045] The terms used in this specification are defined as follows.

[0046] "Dementia" refers to a disease with core symptoms such as memory impairment, orientation disorder, motor disorder, understanding / judgment disorder, and executive function disorder caused by damage to brain cells. Examples of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, etc., according to the diseases or disorders that act as inducements. Dementia in the present invention includes all of these diseases, and the diseases or disorders that act as inducements are not particularly limited.

[0047] "Alzheimer's disease" refers to a type of dementia that shows atrophy of the medial temporal lobe (hippocampus) on CT and MRI and is accompanied by memory impairment, which is the core symptom of cognitive memory impairment. It may be accompanied by other cognitive disorders such as visuospatial cognitive disorders such as structural disorders and topographical orientation disorders, and recent memory impairment. It is a general term for dementia caused by Alzheimer's disease characterized by the aggregation of amyloid-β and dementia with symptoms similar to Alzheimer's disease.

[0048] "Vascular dementia" refers to a type of dementia that appears in association with cerebrovascular diseases and is accompanied by complex attention including information processing speed and frontal lobe executive function disorder. According to the type of vascular lesion, it can be classified into multi-infarct dementia, small vessel dementia, low perfusion vascular dementia, hemorrhagic vascular dementia, etc. It coexists with Alzheimer's disease at a high frequency.

[0049] "Lewy body dementia" refers to a type of dementia with the appearance of inclusions (Lewy bodies) caused by abnormal accumulation of α-synuclein in nerve cells as the main lesion and accompanied by symptoms such as cognitive blurring, visual hallucinations, and / or idiopathic Parkinson's disease. It should be noted that it may be accompanied by structural disorders, severe allergy to antipsychotics, and / or REM sleep behavior disorder, etc. There are cases where Lewy bodies can also be observed outside the central nervous system.

[0050] "Frontotemporal dementia" refers to a dementia in which brain atrophy is observed in the frontal lobe and / or temporal lobe and is accompanied by progressive aphasia. It should be noted that there are cases accompanied by social cognitive disorders such as personality changes and executive function disorders, and memory disorders are often relatively mild. It can be roughly divided into symptoms of Tau accumulation and symptoms of TDP-43, but symptoms of FUS (fused in sarcoma) accumulation are also known.

[0051] In this specification, a "subject" refers to a human or non-human animal that is the subject for determining the presence or absence of the risk of developing or having dementia. As non-human animals, for example, non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats can be cited. Subjects include, for example, individuals with dementia, individuals who may develop dementia in the future, or healthy individuals.

[0052] In this specification, "determining whether having" means determining that a subject has or does not have a specific disease. It also includes differentiating from other diseases with similar lesions or symptoms.

[0053] In this specification, the "risk of onset" refers to the degree of risk of developing from the current state to a specific disease state in the future and presenting the symptoms of that specific disease. As a specific way of expressing the risk of onset, for example, there can be expressions using probability when setting the exact onset situation as 100%, expressions using stages, or expressions within a rough period until onset. In addition, "having a risk of onset" means that the degree of risk is a certain level or above.

[0054] In this specification, an "individual with a possibility of developing dementia in the future" refers to an individual with a high risk of developing dementia. Specifically, for example, there can be cited individuals showing symptoms such as reduced cognitive function that appear in dementia, individuals determined to have a risk of developing dementia based on the discovery of biomarkers, etc., and individuals with risk factors for dementia such as advanced age, hearing loss, smoking, depression, diabetes, and hypertension.

[0055] A "healthy individual" refers to an individual in a healthy state. In this specification, the "healthy state" refers to a state of at least not having the disease of the comparison object. Therefore, an individual who at least does not have dementia is understood to be a healthy individual even if they have other diseases. A healthy individual is preferably a healthy individual who does not have any diseases. In this specification, healthy individuals are mainly used as control individuals that serve as a comparison benchmark for subjects. Therefore, healthy individuals in this specification are of the same species as subjects. Furthermore, it is preferred that conditions such as subspecies (including race), age (including months, weeks), gender, body length, and weight are the same.

[0056] "Subject-derived sample" refers to tissues, cells, and body fluids taken from a subject and used in various aspects of the present invention. As samples that can be used in this specification, for example, body fluid samples such as blood (including peripheral blood), cerebrospinal fluid, saliva, urine, sputum, sweat, throat swabs, nasal swabs, etc., central nervous tissues such as bone marrow and brain tissue, vascular tissue, skin tissue, or adipose tissue can be mentioned. As blood, for example, various forms such as serum, plasma, and whole blood can be mentioned. As a sample derived from bone marrow, bone marrow fluid can be mentioned. In addition, as a body fluid sample or bone marrow sample derived from a subject, for example, a sample containing cells with one nucleus isolated from the body fluid or bone marrow of a subject can be used.

[0057] "White blood cell" is a general term for nucleated cells among the cells contained in blood. Red blood cells and platelets are not included in white blood cells. White blood cells include polymorphonuclear granulocytes and monocytes. Polymorphonuclear granulocytes include eosinophils, neutrophils, and basophils, and are characterized by having a multilobed nucleus and many cytoplasmic granules. Therefore, polymorphonuclear granulocytes are also called granulocytes.

[0058] "Mononuclear cells (Peripheral Blood Mononuclear Cells: PBMC)" refers to cells in white blood cells that have a single round nucleus. Mononuclear cells include lymphocytes and monocytes, etc. The term "mononuclear cell" generally only refers to cells present in peripheral blood. Therefore, mononuclear cells in this specification also include mononuclear cells that migrate to other sites through migration, etc., and broadly include lymphocytes and monocytes.

[0059] In this specification, the "cut-off value" refers to a value that can be used as a reference to determine the risk of disease prevalence or onset. Generally, the ROC curve drawn based on a direct comparison between a control group and a disease group for comparison is derived using a known method, but the method for determining the cut-off value is not limited to this. For example, the cut-off value can be set by a method that does not use a direct comparison between a control group and a disease group for comparison or a method that does not use an ROC curve. As a method for determining the cut-off value using an ROC curve, any known method can be used. As a specific method, for example, a method using the Youden index, a method using the distance from the upper left corner (the point where both sensitivity and specificity are 100%), etc. can be mentioned.

[0060] "ROC curve (Receiver Operating Characteristic curve) is made as follows: the vertical axis is the true positive rate (TPF: True Position Fraction), i.e., sensitivity, and the horizontal axis is the false positive rate (FPF: False Position Fraction), i.e., (1 - specificity). The value of the judgment result that is used as the threshold for a certain finding, i.e., the cutoff point, is used as a mediating variable and varied to draw the curve. Here, sensitivity refers to the ratio of accurately judging positive cases as positive, and specificity refers to the ratio of accurately judging negative cases as negative."

[0061] "AUC (Area Under the Curve) refers to the value of the area under the ROC curve and is a numerical value representing the discrimination ability of the index of this ROC curve. Generally speaking, the closer the AUC is to 1, the higher the discrimination ability, and the closer it is to 0.5, the lower the discrimination ability."

[0062] In this specification, "significant" means statistically significant. Statistically significant means that there is a significant difference between the measured value of the test object and the control value when the difference is statistically processed. For example, when the hazard ratio (significance level) of the obtained value is small, specifically, cases less than 5% (p < 0.05), less than 1% (p < 0.01), and less than 0.1% (p < 0.001) can be cited. The "p (value)" shown here represents the probability that the test statistic accidentally takes this value in the distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic takes this value, meaning that the null hypothesis is more likely to be rejected. The test method for statistical processing can be any well-known test method that can judge significance as long as it is appropriately used, and there is no particular limitation. For example, the student t-test method, paired student t-test method, Welch t-test method, Wilcoxon rank sum test, analysis of variance, Tukey post hoc test, etc. can be used, and there is no particular limitation."

[0063] In this specification, "biomarker" refers to a biological molecule (e.g., nucleic acid molecule, protein molecule) that is used as an index for determining the risk of disease onset or prevalence."

[0064] "One or more" means 1 to 10, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, and particularly preferably 1 or 2."

[0065] 1 - 3. Composition

[0066] The biomarker of the present invention is composed of a combination of two or more proteins or genes, or fragments thereof."

[0067] 1-3-1. Factors Constituting Biomarkers

[0068] The biomarker of the present invention is composed of a combination of connexin 37 and connexin 43 or prolyl hydroxylase domain protein 3, a protein or a gene or a fragment thereof. Each factor will be described below.

[0069] (1) Connexin 37

[0070] "Connexin 37 (Cx37)" is one of the proteins belonging to the connexin family and is a cell membrane protein with a molecular weight of about 37 kDa that constitutes gap junctions. In addition, "connexin 37 gene (Cx37 gene)" refers to the gene encoding connexin 37. Connexin 37 is also known by other names such as GJA4, and it has been reported that mutations in this gene are associated with an increased risk of arteriosclerosis and myocardial infarction.

[0071] The specific amino acid sequence of connexin 37 and the base sequence of its gene are not particularly limited. For example, as the amino acid sequence of the protein, the amino acid sequence of human connexin 37 shown in SEQ ID NO: 2 (333 amino acids) can be cited. In addition, as the base sequence of the gene, the base sequence shown in SEQ ID NO: 1 (1621 bases) encoding human connexin 37 shown in SEQ ID NO: 2 can be cited.

[0072] (2) Connexin 43

[0073] "Connexin 43 (Cx43)" is one of the proteins belonging to the connexin family and is a cell membrane protein with a molecular weight of about 43 kDa that constitutes gap junctions. In addition, "connexin 43 gene (Cx43 gene)" refers to the gene encoding connexin 43. Connexin 37 is also known by other names such as GJA1 and is considered to play an important role particularly in the coordinated contraction of the myocardium. It has been reported that mutations in this gene are associated with abnormal formation of the eyes, teeth, fingers, toes, skull, and heart.

[0074] The specific amino acid sequence of connexin 43 and the base sequence of its gene are not particularly limited. For example, as the amino acid sequence of the protein, the amino acid sequence of human connexin 43 shown in SEQ ID NO: 4 (382 amino acids) can be cited. In addition, as the base sequence of the gene, the base sequence shown in SEQ ID NO: 3 (3083 bases) encoding human connexin 43 shown in SEQ ID NO: 4 can be cited.

[0075] (3) Prolyl Hydroxylase Domain Protein 3

[0076] "Prolyl hydroxylase domain protein 3 (PHD3)" is one of the proteins belonging to the PHD (prolyl hydroxylase domain-containing protein) family. Additionally, "prolyl hydroxylase domain protein 3 gene (PHD3 gene)" refers to the gene encoding PHD3. PHD3 is a protein present in the cytoplasm or nucleus, also known by names such as EGLN3 and HPH1, and is known to be involved in apoptosis and the hypoxic response.

[0077] The specific amino acid sequence of PHD3 and the base sequence of its gene are not particularly limited. For example, as the amino acid sequence of the protein, the amino acid sequence of human PHD3 shown in SEQ ID NO: 6 (239 amino acids) can be cited. Additionally, as the base sequence of the gene, the base sequence of the human PHD3 gene shown in SEQ ID NO: 5 (2706 bases) encoding human prolyl hydroxylase domain protein 3 shown in SEQ ID NO: 6 can be cited.

[0078] It should be noted that the base sequences of SEQ ID NOs: 1, 3, and 5 all contain the base sequence (CDS) encoding the amino acid sequence of the protein, as well as the base sequences of the untranslated regions (UTRs) located upstream and downstream of the CDS.

[0079] 1 - 3 - 2. Composition of the biomarker

[0080] The biomarker of the present invention is composed of a combination of the following three factors.

[0081] (a) A polynucleotide composed of the connexin 37 gene or a fragment thereof and the connexin 43 gene or a fragment thereof (in this specification, often expressed as "Cx37 gene etc. / Cx43 gene etc.");

[0082] (b) A polynucleotide composed of the connexin 37 gene or a fragment thereof and the prolyl hydroxylase domain protein 3 gene or a fragment thereof (in this specification, often expressed as "Cx37 gene etc. / PHD3 gene etc.");

[0083] (c) A polypeptide composed of connexin 37 or a fragment thereof and connexin 43 or a fragment thereof (in this specification, often expressed as "Cx37 etc. / Cx43 etc."); and

[0084] (d) A polypeptide composed of connexin 37 or a fragment thereof and prolyl hydroxylase domain protein 3 or a fragment thereof (in this specification, often expressed as "Cx37 etc. / PHD3 etc.").

[0085] For the fragment in the biomarker of the present invention, the presence of the fragment does not need to show the expression of other proteins or genes, but only needs to show the expression of the target protein or gene, and its length is not particularly limited. Specifically, in the case of polynucleotides, for example, it is 15 bases or more, 17 bases or more, 19 bases or more, 20 bases or more, 25 bases or more, 30 bases or more, 40 bases or more, 50 bases or more, 100 bases or more, 200 bases or more, 300 bases or more, 500 bases or more, 1000 bases or more, 1500 bases or more, 2000 bases or more. In addition, in the case of polypeptides, for example, it is 10 amino acids or more, 20 amino acids or more, 30 amino acids or more, 40 amino acids or more, 50 amino acids or more, 100 amino acids or more, 150 amino acids or more, 200 amino acids or more, 300 amino acids or more.

[0086] 2. Method for determination

[0087] 2-1. Summary

[0088] The second aspect of the present invention is a method for determining the presence or absence of the risk of developing or onset of dementia. According to the method of this aspect, it is possible to determine whether a subject has the risk of developing or onset of dementia based on the biomarker described in the first aspect.

[0089] 2-2. Method

[0090] The method for determination of the present invention includes, as essential steps, a step of measuring the amount of the biomarker and a step of calculating the ratio of the expression level, and shows the presence or absence of the risk of developing or onset of dementia based on the calculated ratio. Hereinafter, each step will be specifically described.

[0091] 2-2-1. Step of measuring the amount of the biomarker

[0092] This step is a step of measuring the amount of the biomarker described in the first aspect in a sample derived from a subject.

[0093] In the case of using a biomarker composed of polynucleotides, this step is a step of measuring the expression levels of the Cx37 gene, and the Cx43 gene and / or the PHD3 gene in a sample derived from a subject.

[0094] On the other hand, in the case of using a biomarker composed of polypeptides, this step is a step of measuring the protein amounts of Cx37, and Cx43 and / or PHD3 in a sample derived from a subject.

[0095] A specified amount of a sample derived from a subject is used in this step.

[0096] The biological species of the subject in the method of this mode is not particularly limited. For example, it can be any mammal shown in the defined items, preferably a non-human primate or a human.

[0097] The state of the subject in the method of this mode is not particularly limited. For example, it can be any one of an individual with dementia, an individual who may develop dementia in the future, an individual suspected of having dementia, or an apparently healthy individual.

[0098] Examples of the specimen used in this step include body fluids, bone marrow, or central nervous tissue, etc. From the perspective of invasiveness during specimen collection, blood, saliva, urine, sputum, sweat, throat swabs, or nasal swabs, etc. are preferred. It should be noted that when comparing with the control value later, the specimens of the subject and the healthy individual used to obtain the control value are, in principle, specimens of the same type as each other. For example, if one is blood, the other is also blood.

[0099] The "specified amount" refers to an amount predetermined by volume or weight. The specified amount is not particularly limited, and it is required that at least the biomarker described in the first mode contained in the specimen is an amount that can be measured. For example, when using blood as the specimen, it can be 100 μL to 20 mL, etc.

[0100] The method for collecting the specimen can be any known method and is not particularly limited. For example, if it is cerebrospinal fluid, it can be collected by lumbar puncture. If it is blood, saliva, urine, sputum, sweat, throat swab, or nasal swab, it can be collected according to a known method.

[0101] The specimen used in the present invention is preferably a specimen containing cells with cell nuclei. The type of cells contained in the specimen is not particularly limited. For example, if the specimen is blood, monocytes are preferred.

[0102] For example, when the specimen contains monocytes, the ratio of monocytes in the specimen is not particularly limited. For example, it can be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%. In this specification, the "ratio of monocytes in the specimen" refers to the proportion of the number of monocytes contained in the specimen in the number of all white blood cells. The ratio of monocytes in the specimen can be measured, for example, by a hematology analyzer, a hemocytometer, a flow cytometer, etc., but is not limited thereto.

[0103] In addition, as a sample, a cell-containing sample isolated from a subject can also be used. The cells can be non-specifically isolated regardless of their types, or specific cells can be specifically isolated. When the sample contains cells, it preferably contains monocytes. In the present specification, a sample containing isolated monocytes is specifically referred to as a monocyte-containing sample.

[0104] The ratio of monocytes in the above-mentioned monocyte-containing sample is not particularly limited. For example, it can be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%. In addition, the ratio of granulocytes in the above-mentioned monocyte-containing sample is preferably less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, or 0%. In the present specification, the "ratio of monocytes or granulocytes in the monocyte-containing sample" refers to the proportion of the number of monocytes or granulocytes contained in the monocyte-containing sample in the total number of all cells. The ratio of monocytes or granulocytes in the monocyte-containing sample can be measured, for example, by a hematology analyzer, a hemocytometer, a flow cytometer, etc., but is not limited thereto.

[0105] The sample derived from a subject sometimes contains granulocytes, and the granulocytes in the sample can sometimes be an important factor reducing the measurement accuracy of the amount of a biomarker. Therefore, a monocyte-containing sample with an increased ratio of monocytes can be used by separating monocytes from the above-mentioned sample or removing granulocytes. By supplying the monocyte-containing sample in the measurement, a highly accurate measurement value can be obtained.

[0106] A method for obtaining a sample containing monocytes from body fluids as a subject source, particularly from blood or bone marrow, preferably uses a reagent and / or a chromatography column for separating monocytes. Examples of the above-mentioned reagent and / or chromatography column include Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Diagnostics Technologies), Human Peripheral Blood Mononuclear Cell Isolation and Viability Kit (manufactured by BioVision), BD Vacutainer (registered trademark) CPT monocyte isolation blood collection tube (manufactured by Becton Dickinson), pluriMate (manufactured by pluriSelect), SepMate (manufactured by STEMCELL Technologies), etc. By using the above-mentioned reagent and / or chromatography column, monocytes in the above-mentioned sample can be separated from granulocytes, and a sample containing monocytes for supply to the assay can be effectively obtained.

[0107] In this step, the amount of the biomarker described in the first mode is measured in the above-mentioned sample. In this specification, the "amount of the biomarker" refers to a value indicating the amount of each factor in the biomarker measured in this step. When the biomarker is composed of polynucleotides, the "amount of the biomarker" is the "expression level", that is, the "amount of transcript (including cDNA amount)". On the other hand, when the biomarker is composed of polypeptides, the "amount of the biomarker" is the "amount of protein".

[0108] The amount of the biomarker can be an absolute value such as volume or weight, or can also be a relative value such as concentration, ionic strength, absorbance or fluorescence intensity. In addition, it can also be a value normalized by the amount of an endogenous control such as a housekeeping gene, its transcript protein, etc. However, in the method of the present invention, since the quotient of the gene expression level or the amount of protein is calculated in the subsequent step of calculating the relative value, normalization based on an endogenous control is not necessarily required.

[0109] As an endogenous control, housekeeping genes such as TBP (TATA-binding protein), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2M (β2 microglobulin), HPRT1 (hypoxanthine phosphoribosyltransferase 1), and 18S ribosomal RNA can be used.

[0110] The amount of the biomarker can be determined by detecting the amount of mRNA (including the coding region and the untranslated region) of the target gene in the test sample. Alternatively, it can also be determined by detecting the amount of the target protein in the test sample.

[0111] In the detection of mRNA, Northern blotting, RT-PCR, real-time RT-PCR, nucleic acid array method (method using a nucleic acid chip), dot blotting, RNase protection assay method, in situ hybridization method, etc. can be used. These methods can be carried out according to known procedures. In addition, the measurement in this step can be performed using the primers and / or probes described in the third mode.

[0112] In the case of using Northern blotting, the presence or absence and the expression level of each gene expression in RNA can be detected and measured by using the probe described in the third mode. Specifically, for example, the following steps can be carried out. First, the probe (complementary strand) is labeled with a radioisotope ( 32 P, 33 P, 35 S, etc.), a fluorescent substance, etc. Next, the RNA in the test sample from the subject is transferred onto a nylon membrane or the like. The labeled probe is applied to this membrane to hybridize the labeled probe with the RNA. Finally, the signal of the label from the formed DNA / RNA double strand is detected and measured using a radiation detector, a fluorescence detector, or the like.

[0113] In the case of using quantitative RT-PCR, the presence or absence of gene expression in RNA and its expression level can be detected and measured by using the primers described in the third method. Specifically, for example, the following steps can be carried out. First, cDNA is prepared from RNA in a sample derived from a subject. Next, using this as a template, PCR is performed using a pair of primers that can amplify the target nucleic acid region, and the resulting double-stranded DNA is detected. It should be noted that as a method for detecting double-stranded DNA, examples include: a method of performing the above PCR using primers pre-labeled with a radioisotope or a fluorescent substance; a method of subjecting the PCR product to electrophoresis on an agarose gel and staining the double-stranded DNA with ethidium bromide or the like for detection; a method of transferring the generated double-stranded DNA onto a nylon membrane or the like by a conventional method and hybridizing it with a labeled probe for detection, etc.

[0114] In the case of using nucleic acid array analysis, an RNA chip or a DNA chip in which the probes described in the third method are immobilized on a solid phase such as a substrate is used. In this method, by applying a nucleic acid molecule obtained from a sample derived from a subject to the chip and detecting the signal of the chip, it is detected whether the target gene is expressed.

[0115] The technique of immobilizing nucleic acids on a substrate is generally referred to by names such as nucleic acid chip, nucleic acid array, and microarray. In addition, DNA or RNA arrays include DNA or RNA microarrays and DNA or RNA microarrays. In this specification, when referred to as a chip, all of these techniques are included.

[0116] As a method for measuring the signal of the chip, there is no limitation. For example, a method of detecting and measuring the signal of a marker from a detection composition using an image detector (examples include Typhoon 9410 (GE Healthcare), 3D-Gene (registered trademark) scanner (Toray Industries, Inc.), etc.) can be cited.

[0117] "Surface Plasmon Resonance (SPR) method" refers to a method for highly sensitive detection and quantification of adsorbates on the surface of a metal thin film by utilizing the surface plasmon resonance phenomenon. The surface plasmon resonance phenomenon refers to the phenomenon that when the incident angle of the laser irradiated on the metal thin film is changed, the intensity of the reflected light significantly attenuates at a specific incident angle (resonance angle). In the present invention, a nucleic acid probe, which is a biomarker and has a sequence complementary to the base sequence of the target nucleic acid molecule, is immobilized on the surface of the metal thin film. After the surface of other parts of the metal thin film is blocked, a liquid sample such as body fluid collected from a subject is allowed to flow on the surface of the metal thin film, thereby forming a base pair between the target nucleic acid molecule and the nucleic acid probe. The target nucleic acid molecule is detected and quantified based on the difference in the measured values before and after the sample flows. Detection and quantification based on the surface plasmon resonance method can be performed, for example, using an SPR sensor commercially available from Biacore. This technology is well-known in this field. For example, refer to Kazuhiro Nagata and Hiroshi Handa, Real-Time Analysis Experimental Method for Biomolecular Interactions, Springer-Verlag Tokyo, Tokyo, 2000.

[0118] "Quartz Crystal Microbalance (QCM) method" refers to a mass measurement method for quantitatively capturing an extremely small amount of adsorbates based on the change in the resonance frequency according to the phenomenon that when a substance adsorbs on the surface of an electrode mounted on a quartz crystal oscillator, the resonance frequency of the quartz crystal oscillator decreases corresponding to its mass. Detection and quantification based on this method also utilize a commercially available QCM sensor in the same way as the SPR method. For example, the target nucleic acid molecule can be detected and quantified by the base pairing between a nucleic acid probe having a sequence complementary to the base sequence of the target nucleic acid molecule immobilized on the electrode surface and the target nucleic acid molecule in a sample collected from a subject. This technology is well-known in this field. For example, refer to J. Christopher Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo, G. M. Whitesides (2005) Self-Assembled Monolayers of a Form of Nanotechnology, Chemical Review, 105: 1103-1170; Yutaka Morizumi, Takamichi Nakamoto (1997) Sensor Engineering, Shoko-do.

[0119] There is no limitation on the calculation of gene expression levels. For example, in the present invention, statistical processing described in Statistical analysis of gene expression microarray data (written by Speed T., Chapman and Hall / CRC), and A beginner's guide Microarray gene expression data analysis (written by Causton H.C. et al., Blackwell publishing), etc. can be used. For example, the average value of the measured values of the blank spots on the DNA chip can be added to 2 times, 3 times, or 6 times the standard deviation of the measured values of the blank spots, and the probe spots with signal values equal to or greater than this value can be regarded as detection spots. The average value of the measured values of the blank spots can be further regarded as the background, and the value subtracted from the measured values of the probe spots can be used as the gene expression level.

[0120] Missing values of gene expression levels can be excluded from the analysis object, preferably replaced by the minimum value of the gene expression levels in each DNA chip, and more preferably replaced by the value obtained by subtracting 0.1 from the logarithm of the minimum value of the gene expression levels. Furthermore, in order to remove genes with low signals, only genes with gene expression levels of 2 to the power of 6, 2 to the power of 8, or 2 to the power of 10 or more in 20% or more, 50% or more, or 80% or more of the number of measured samples can be selected as the analysis object.

[0121] The detection of the amount of protein can be carried out by immunoassay methods using antibodies that specifically recognize and bind to the target protein. Antibodies can be prepared by known methods. As immunoassay methods, methods using solid-phase carriers immobilized with antibodies that specifically bind to the target protein to be detected, flow cytometry, Western blotting, etc. can be mentioned. As the method using a solid-phase carrier, for example, enzyme immunoassay (ELISA) using an immobilized microtiter plate, agglutination method using immobilized particles (immunoprecipitation method), etc. can be mentioned, and it is not limited thereto. Known immunological assay methods can be used to detect the amount of the target protein in the sample. In addition, the detection of the amount of the target protein can also be carried out by methods using protein amount analysis techniques that do not use antibodies, such as LC-MS / MS MRM, etc. These detection methods can also be implemented according to the protocols of conventional methods. In addition, the measurement of this step can be carried out using the peptide-binding molecule described in the third mode.

[0122] Before measuring the amount of the biomarker through this step, any treatment can be carried out as needed. For example, extraction treatment of nucleic acids and proteins from the sample can be carried out.

[0123] The method for extracting nucleic acid from a sample is not particularly limited. Under the condition that the target polynucleotide is not decomposed, conventionally known buffers, devices, kits, etc. can be used to extract nucleic acid. For example, the acidic phenol method, phenol / chloroform extraction method, etc. can be used to extract nucleic acid. As a method for recovering nucleic acid from these extraction solutions, for example, the ethanol precipitation method can be used. In addition, commercially available nucleic acid extraction kits can be used. As the nucleic acid extraction kit, DNeasy Blood&Tissue Kit (QIAGEN), Quick-DNA kit (Zymoresearch), etc. can be used. In addition, reagents or kits specifically for extracting RNA can also be used.

[0124] In the method of the present invention, as a method for extracting nucleic acid from a specimen, for example, 3D-Gene (registered trademark) RNA extraction reagent from liquid sample kit (Toray Industries, Inc.) etc. can be used.

[0125] In addition, the method for extracting protein from a sample is not particularly limited. Under the condition that the target protein is not decomposed, conventionally known buffers, devices, kits, etc. can be used to extract protein. For example, extraction can be carried out by using a protein denaturant and / or performing mechanical pulverization treatment. As the protein denaturant, for example, urea, guanidine salt, trichloroacetic acid (TCA), surfactants, etc. can be cited. In addition, as the surfactant, for example, SDS (sodium dodecyl sulfate), CHAPS, Triton X-100, Tween 20, Nonidet P-40, etc. can be cited. As the mechanical pulverization treatment, for example, pulverization based on a homogenizer, ultrasonic treatment, French press treatment, pulverization using glass beads, etc. can be cited.

[0126] 2-2-2. Step of calculating the ratio

[0127] This step is a step of calculating the ratio of the expression level of the Cx37 gene or the protein amount of Cx37 to the expression level of other genes or the protein amount of other genes. This step can be carried out simultaneously with or after the step of measuring the amount of the biomarker.

[0128] When the measured biomarker is composed of polynucleotide, in this step, the ratio of the expression level of the Cx43 gene and / or the expression level of the PHD3 gene to the expression level of the Cx37 gene is calculated.

[0129] The ratio calculated in this case is the relative value of the expression level of the Cx37 gene to the expression level of the Cx43 gene and / or the expression level of the PHD3 gene (hereinafter, often simply referred to as "the relative value of the expression level of the Cx37 gene" in this specification) or its reciprocal (the relative value of the expression level of the Cx43 gene and / or the expression level of the PHD3 gene to the expression level of the Cx37 gene (hereinafter, often simply referred to as "the relative value relative to the expression level of the Cx37 gene" in this specification)).

[0130] When the measured biomarker is composed of a polypeptide, in this step, calculate the ratio of the protein amount of Cx43 and / or the protein amount of PHD3 to the protein amount of Cx37.

[0131] The ratio calculated in this case is the relative value of the protein amount of Cx37 to the protein amount of Cx43 and / or the protein amount of PHD3 (hereinafter, often simply referred to as "the relative value of the protein amount of Cx37" in this specification) or its reciprocal (the relative value of the protein amount of Cx43 and / or the protein amount of PHD3 to the protein amount of Cx37 (hereinafter, often simply referred to as "the relative value relative to the protein amount of Cx37" in this specification)).

[0132] "Ratio" refers to the quotient value of the values of multiple indicators with respect to each other.

[0133] The "relative value" of the first value and the second value refers to the value obtained as a result of comparing the first value and the second value, and usually refers to the value obtained by dividing the first value by the second value.

[0134] There is no particular limitation on the calculation method of the relative value in this step. For example, it can be performed by manual calculation or using an arithmetic device.

[0135] Based on a preset cut-off value, or a comparison of the control value calculated in the same way in a sample from a healthy individual with the ratio calculated in this step, it is shown that the subject has dementia or has a risk of developing dementia. This judgment can be carried out as a step for making a determination. The detailed content of the judgment is described in detail in the step for making a determination.

[0136] 2-2-3. Step for making a determination

[0137] This step is a step for determining that the subject has dementia or has a risk of developing dementia based on the ratio. This step can be carried out simultaneously with or after the step of calculating the ratio.

[0138] The determination can be made by comparing the calculated ratio with the cut-off value or with the control value.

[0139] When a cut-off value is used in this step, if the relative value of the expression level of the Cx37 gene or the protein amount of Cx37 is lower than a pre-set cut-off value, or if the relative value of the expression level of the Cx37 gene or the protein amount of Cx37 is higher than a pre-set cut-off value, it is determined that the subject has dementia or is at risk of developing dementia.

[0140] The specific value of the cut-off value may vary depending on the biomarker used in the method of the present invention, the sample used, and the method for measuring the biomarker. For example, when the cut-off value for the relative value of the expression level of the Cx37 gene and the expression level of the PHD3 gene is measured by nucleic acid detection in a sample containing monocytes, according to the results of Example 3, it is 0.893, and when the same measurement is performed in whole blood, according to the results of Example 4, it is 0.744. In addition, when the cut-off value for the relative value of the expression level of the Cx37 gene and the expression level of the Cx43 gene is measured by nucleic acid detection in a sample containing monocytes, according to the results of Example 3, it is 0.651. Similarly, for example, when the cut-off value for the relative value of the expression level of the PHD3 gene relative to the expression level of the Cx37 gene is measured by nucleic acid detection in a sample containing monocytes, it is 1.120, and when the same measurement is performed in whole blood, it is 1.344. In addition, when the cut-off value for the relative value of the expression level of the Cx43 gene relative to the expression level of the Cx37 gene is measured by nucleic acid detection in a sample containing monocytes, it is 1.536.

[0141] As the cut-off value used in the present invention, the sensitivity and specificity are sufficiently high. For example, it is preferably a cut-off value with a sensitivity and specificity of 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. In addition, it is preferably a cut-off value with an AUC representing the discrimination ability of the biomarker of 0.75 or more, 0.78 or more, 0.79 or more, 0.8 or more, 0.85 or more, 0.88 or more, 0.89 or more, or 0.9 or more.

[0142] When a control value is used in this step, generally, if the relative value of the expression level of the Cx37 gene or the protein amount of Cx37 is significantly lower than the control value, or if the relative value of the expression level of the Cx37 gene or the protein amount of Cx37 is significantly higher than the control value, it is judged that the subject has dementia or is at risk of developing dementia.

[0143] In the present invention, the "control value" refers to the relative value calculated in the same way as that of the subject in a sample derived from a healthy individual. Therefore, when the relative value is significantly lower or higher than the control value calculated in the same way in a sample derived from a healthy individual, it can be determined that the subject has dementia or is at risk of developing dementia.

[0144] There is no particular limitation on the specific method for determining the control value. For example, the average value of the relative values measured and calculated in advance for healthy individuals using the same method can be used as the control value, or the average value of the relative values obtained from healthy individuals at the same time as the subject can be used as the control value. When using a pre-calculated control value, it is preferable to provide a confidence interval corresponding to the desired significance level together with the control value. In this case, when the relative value of the subject exceeds the confidence interval and is lower or higher, it can be determined that the subject has dementia or is at risk of developing dementia.

[0145] There is no particular limitation on the significance level when using the control value. Specifically, for example, any significance level described in the defined terms can be used. In addition, any other commonly used significance level can be used according to the purpose, etc. In addition, multiple significance levels can be used in the determination according to the purpose.

[0146] In addition, when the relative value of the subject is lower or higher than the control value by a certain degree or more, it can also be determined that the subject has dementia or is at risk of developing dementia. When using the relative value of the expression level of the Cx37 gene or the protein amount of Cx37, the ratio of the relative value to the control value can be, for example, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less. In addition, when using the relative value of the expression level of the Cx37 gene or the protein amount of Cx37, the ratio of the relative value to the control value can be, for example, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more, 3.0 or more, 5.0 or more, or 10 or more.

[0147] In this method, multiple values can be used as the cut-off value and the control value. For example, the cut-off value or the control value for determining the risk of developing dementia and the cut-off value or the control value for determining the presence of dementia can be determined separately. When using the relative value of the expression level of the Cx37 gene or the protein amount of Cx37, for example, if the relative value of the subject is lower than the cut-off value for determining the risk of developing dementia but not higher than the cut-off value for determining whether the subject has dementia, it can be determined that the subject has a risk of developing dementia. Or, for example, the degree of the risk of onset can also be divided into multiple stages, and the cut-off value or the control value can be set separately.

[0148] It should be noted that the method of this mode includes not only the case of using only either the cut-off value or the control value, but also the case of using a combination of both.

[0149] The type of the determination result in this step is not particularly limited. For example, in addition to determining whether the subject has dementia or the risk of developing dementia as described above, it can also be used to determine the severity of dementia in the subject. In addition, the type of dementia as the determination object is not particularly limited. For example, Alzheimer's dementia, Lewy body dementia, vascular dementia, etc. can be cited.

[0150] The method of the present invention can also be used in combination with known biomarkers for dementia examination such as amyloid-β protein and Tau protein, and image detection such as PET for visualizing them. When this step is included, the method of this mode can be used as a determination method and a method for assisting a physician's determination.

[0151] 2 - 3. Effects

[0152] According to the method of this mode, it is possible to determine the presence or absence of dementia and the risk of onset with high sensitivity and specificity through a simple operation with low invasiveness.

[0153] In addition, according to the method of this mode, it is possible to determine whether it is necessary to take intervention measures for preventing, improving, or treating dementia. In this case, the method of this mode can be used as a method for selecting an animal individual to be treated with an active substance for improving, treating, and / or preventing dementia.

[0154] 3. Kit

[0155] 3 - 1. Overview

[0156] The third mode of the present invention is a kit for determining the presence or absence of dementia or the risk of onset. According to the kit of this mode, the biomarker described in the first mode can be detected and / or its amount can be measured.

[0157] 3 - 2. Composition

[0158] The kit of the present invention contains a reagent for measuring the expression level of a gene or a peptide-binding molecule as an essential component.

[0159] When the kit of the present invention contains a reagent for measuring the expression level of a gene, the kit of the present invention contains: a reagent for measuring the expression level of the Cx37 gene in a sample derived from a subject, and a reagent for measuring the expression level of the Cx43 gene and / or the PHD3 gene in the above sample.

[0160] On the other hand, when the kit of the present invention contains a peptide-binding molecule, the kit of the present invention contains: a Cx37-binding molecule in a sample derived from a subject, and a Cx43-binding molecule and / or a PHD3-binding molecule in the above sample.

[0161] Hereinafter, the reagent and the binding molecule will be specifically described.

[0162] 3-2-1. Reagent for measuring the expression level of a gene

[0163] The reagent for measuring the expression level of the gene used in the kit of the present invention refers to a molecule that specifically binds to a polynucleotide or a fragment thereof among the biomarkers described in the first aspect. Specifically, it refers to a primer that can amplify all or a part of the base sequence of the target gene or a base sequence complementary thereto, and / or a probe that can hybridize with all or a part of the base sequence of the target gene or a base sequence complementary thereto.

[0164] The polynucleotide of the biomarker mentioned here, that is, the target nucleotide, refers to Cx37 gene, etc. / Cx43 gene, etc. and Cx37 gene, etc. / PHD3 gene, etc.

[0165] The base sequences of the primer and the probe in the kit of the present invention are not particularly limited as long as they perform the above functions. For example, the following polynucleotides can be mentioned:

[0166] (1) A polynucleotide fragment composed of 15 or more consecutive bases selected from the polynucleotides having the base sequences shown in SEQ ID NO: 1, 3, or 5;

[0167] (2) A polynucleotide fragment containing one or two base deletions, substitutions, additions, or insertions in the base sequence of the polynucleotide fragment in (1);

[0168] (3) A polynucleotide fragment containing 15 or more consecutive bases of the polynucleotide having the base sequences shown in SEQ ID NO: 1, 3, or 5;

[0169] (4) A polynucleotide fragment containing one or two base deletions, substitutions, additions, or insertions in the base sequence of the polynucleotide fragment in (3);

[0170] (5) A polynucleotide that hybridizes under high stringency conditions with a polynucleotide fragment sequence of 15 or more consecutive bases selected from the base sequences shown in SEQ ID NO: 1, 3, or 5; and

[0171] (6) A polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotide of (1) to (5) or a fragment thereof.

[0172] The "probe" in this specification includes a polynucleotide, a polynucleotide complementary thereto, and an aptamer that can specifically recognize and detect RNA produced by gene expression or a polynucleotide derived from the RNA.

[0173] The "primer" in this specification includes a polynucleotide that can specifically recognize and amplify RNA produced by gene expression or a polynucleotide derived from the RNA, and a polynucleotide complementary thereto.

[0174] "Complementary" means that nucleic acid bases can form a base-pairing relationship with each other via hydrogen bonds. It corresponds to the so-called Watson-Crick base pairs (natural base pairs) or Hoogsteen-type base pairs.

[0175] "Hybridization" or "hybridizable" in this specification means that polynucleotides having mutually complementary base sequences form completely or partially complementary double strands by base pairing.

[0176] "Stringent conditions" in this specification mean conditions under which a nucleic acid probe hybridizes with its target sequence to a greater extent (for example, a measurement value of the average of the background measurement value + 2 times the standard error of the background measurement value or more) compared to other sequences. Stringent conditions depend on the sequence and vary depending on the environment in which hybridization is performed.

[0177] In this specification, "high stringent conditions" refer to environmental conditions in which non-specific hybridization is not likely to occur. Under high stringent conditions, nucleic acids with a target base sequence can hybridize, but nucleic acids with non-specific base sequences substantially cannot form hybrids. Generally speaking, high stringent conditions refer to low salt concentration and high temperature conditions. Low salt concentration means, for example, 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM, or 15 to 200 mM. For example, it includes 1.5 to 3.5×SSC, 2 to 3×SSC, 2 to 2.5×SSC. In addition, high temperature means, for example, 50 to 68°C or 55 to 70°C. As another condition for defining stringent conditions, namely the washing conditions after hybridization, for example, the conditions of washing at 65°C with 0.1×SSC and 0.1% SDS in the washing after hybridization can be cited. Preferably, the polynucleotides contained in the kit of the present invention remain hybridized to the sense strand as the object even when washed under the above conditions. As specific polynucleotides, polynucleotides composed of a base sequence that is in a completely complementary relationship with the base sequence of the sense strand as the object, and polynucleotides composed of a base sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the polynucleotide can be exemplified.

[0178] Other examples of "stringent conditions" in these hybridizations are described, for example, in Sambrook, J. & Russel, D., Molecular Cloning, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, issued on January 15, 2001, Volume 1, 7.42 - 7.45, Volume 2, 8.9 - 8.17, etc., and can be utilized in the present invention.

[0179] The value of homology represents the value calculated under default settings using software for calculating the homology between multiple base sequences (for example, FASTA, DNASIS, and BLAST). The value of homology of a base sequence is calculated by counting the number of bases that are consistent when aligning a pair of base sequences in the most consistent manner, and taking it as the ratio of the number of the consistent bases to all the bases of the compared base sequences. Here, in the case of the presence of a gap, the above all the bases are the number of bases obtained by counting 1 gap as 1 base. For the detailed content of the method for determining homology, please refer to, for example, Altschul et al, Nuc. Acids Res. 25, 3389 - 3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403 - 410, 1990.

[0180] The length of the consecutive bases of these polynucleotides is not particularly limited as long as it is 15 bases or more. For example, it can be 17 bases or more, 18 bases or more, 19 bases or more, 20 bases or more, 21 bases or more, etc.

[0181] The level of homology is preferably 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

[0182] In addition, these polynucleotides may include polynucleotides containing a base sequence having a certain level or more of homology with a consecutive partial base sequence or its complementary base sequence contained in the base sequence of the target gene.

[0183] When used as a primer, it is preferably included the above consecutive bases on the 3'-terminal side as the extension direction. In addition, it preferably includes a base sequence region composed of a base sequence having 100% homology with the target base sequence starting from the 3'-terminal, and a base sequence region composed of a base sequence having a certain level or more of homology with the target base sequence. The length of the base sequence region composed of a base sequence homologous to the target base sequence is not particularly limited. For example, it is 2 bases or more, 3 bases or more, 5 bases or more, 10 bases or more, 15 bases or more, 17 bases or more, 19 bases or more.

[0184] In this case, the 5'-terminal side may include any additional base sequence. The total number of bases of the entire primer is not particularly limited. For example, it can be 50 bases or less, 40 bases or less, or 30 bases or less, etc.

[0185] For the primer pair included in the kit of the present invention, specifically, for example, a pair of polynucleotides having the base sequences of SEQ ID NOs: 9 and 10 or composed thereof for the Cx37 gene; a pair of polynucleotides having the base sequences of SEQ ID NOs: 11 and 12 or composed thereof for the Cx43 gene; a pair of polynucleotides having the base sequences of SEQ ID NOs: 17 and 18 or composed thereof for the PHD3 gene, etc.

[0186] As an example of the conditions when performing PCR using the primer in the kit of the present invention, for example, it can be mentioned using a PCR buffer composed of 10 mM Tris-HCL (pH 8.3), 50 mM KCL, 1 - 2 mM MgCl 2 etc., and treating at a temperature of Tm value + 5 - 10 °C calculated from the sequence of this primer for about 15 seconds to 1 minute. As a method for calculating the above Tm value, it can be mentioned that Tm value = 2 × (number of adenine residues + number of thymine residues) + 4 × (number of guanine residues + number of cytosine residues), etc.

[0187] The nucleic acid of the probe contained in the kit of the present invention for use in the above hybridization method is usually DNA that can be synthesized at low cost and has high stability. It may also contain all or part of PNA (peptide nucleic acid), BNA (bridged nucleic acid) / LNA (locked nucleic acid), methylphosphonate-type DNA, phosphorothioate-type DNA, 2'-O-methyl-type RNA and other chemically modified nucleic acids, mimetic nucleic acids, or combinations thereof as needed. In addition, the probe contained in the kit of the present invention can be modified or labeled with fluorescent dyes (e.g., fluorescamine and its derivatives, rhodamine and its derivatives, DIG, FITC, Cy3, Cy5, FAM, HEX, VIC), quencher substances (TAMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3), biotin or (strept)avidin, or magnetic beads and other modifying substances, or radioisotopes (e.g., 32 P, 33 P, 35 S), etc. Hybridization is carried out under stringent conditions at low salt concentration and high temperature in order to exclude nucleic acids other than those for the purpose of non-specific hybridization.

[0188] The polynucleotide or its fragment used in the kit of the present invention can be either DNA or RNA.

[0189] The polynucleotide used in the kit of the present invention can be prepared using general techniques such as DNA recombination techniques, PCR methods, and methods based on DNA / RNA automatic synthesizers.

[0190] For DNA recombination techniques and PCR methods, the techniques described in, for example, Ausubel et al., Current Protocols in Molecular Biology, John Willey & Sons, US (1993); Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, US (1989), etc. can be used.

[0191] The base sequence of interest is publicly known, and the method for obtaining it as described above is also known. Therefore, by cloning this gene, a polynucleotide that can be used as a probe or primer in the kit of the present invention can be prepared.

[0192] Such probes or primers can be chemically synthesized using a DNA automatic synthesizer. In this synthesis, the phosphoramidite method is generally used, and by using this method, single-stranded DNA of up to about 100 bases can be automatically synthesized. DNA automatic synthesizers are commercially available from, for example, Polygen Corporation, ABI, Thermo Fisher Scientific, etc. Alternatively, they can also be prepared using the cDNA cloning method.

[0193] The kit of the present invention can contain probes and / or primers in any combination. Generally, the primers are contained in the form of a primer pair of a forward primer and a reverse primer. In addition, it preferably contains at least one set of primer sets for detecting a set of biomarkers described in the first aspect or a probe set for detecting a set of biomarkers described in the first aspect.

[0194] The kit of the present invention can further additionally contain probes and / or primers for any known gene that can be used as a biomarker for determining the presence or absence of the risk of dementia onset or disease.

[0195] 3-2-2. Peptide-binding molecule

[0196] In this specification, the "peptide-binding molecule" refers to a molecule that specifically binds to a polypeptide or a peptide fragment thereof among the biomarkers described in the first aspect. Specifically, it refers to an antibody or an active fragment thereof that can bind to the target protein, or an aptamer.

[0197] The polypeptide of the biomarker mentioned here, that is, the target polypeptide, refers to Cx37 etc. / Cx43 etc. and Cx37 etc. / PHD3 etc.

[0198] The kit of the present invention contains more than one peptide-binding molecule that binds to these polypeptides. The peptide-binding molecule can be any one of a peptide, a nucleic acid, a low-molecular compound, or a combination thereof.

[0199] 3-2-3. Other components

[0200] The kit of the present invention can further contain: reagents for extracting nucleic acids (such as total RNA) or polypeptides from body fluids, cells, or tissues, fluorescent substances for labeling, enzymes for nucleic acid amplification and culture media, instruction manuals, dilution or reaction buffers containing components required for measurement, washing solutions, color-developing reagents, reaction containers, etc.

[0201] The kit of the present invention may further comprise a reagent and / or a chromatographic column for separating monocytes from the above-mentioned specimen. As the above-mentioned reagent and / or chromatographic column, for example, Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Diagnostics Technologies), Human Peripheral Blood Mononuclear Cell Isolation and Viability Kit (manufactured by BioVision), BD Vacutainer (registered trademark) CPT monocyte isolation blood collection tube (manufactured by Becton Dickinson), pluriMate (manufactured by pluriSelect), SepMate (manufactured by STEMCELL Technologies), etc. Monocytes in the above-mentioned specimen can be separated from granulocytes to effectively obtain a specimen containing monocytes for measurement.

[0202] 4. Device

[0203] 4-1. Overview

[0204] The fourth aspect of the present invention is a device for determining the presence or absence of the risk of developing or having dementia. The device according to this aspect can detect the biomarker described in the first aspect and / or measure its amount.

[0205] 4-2. Configuration

[0206] The device of the present invention includes a reagent or a peptide-binding molecule for measuring the expression level of a gene as an essential component.

[0207] When the device of the present invention includes a reagent for measuring the expression level of a gene, the device of the present invention includes: a reagent for measuring the expression level of the Cx37 gene in a specimen derived from a subject, and a reagent for measuring the expression level of the Cx43 gene and / or the PHD3 gene in the above-mentioned specimen.

[0208] On the other hand, when the device of the present invention includes a peptide-binding molecule, the kit of the present invention includes: a Cx37-binding molecule in a specimen derived from a subject, and a Cx43-binding molecule and / or a PHD3-binding molecule in the above-mentioned specimen.

[0209] The reagent for measuring the expression level and the peptide-binding molecule have been described in detail in the third aspect, so the description is omitted here.

[0210] Other configurations are basically based on the description in the third aspect.

[0211] The device of the present invention may additionally include, as needed: means for collecting a sample from a subject, means for measuring the amount of a biomarker, and means for calculating a ratio, etc.

[0212] The device of the present invention is, for example, a device for measuring a biomarker in which a polynucleotide, a mutant thereof, a derivative thereof, or a fragment thereof, etc. in the present invention described above binds or adheres to a solid phase. Examples of the material of the solid phase are plastic, paper, glass, silicon, etc., and due to easy processing, the preferred material of the solid phase is plastic. The shape of the solid phase is arbitrary, for example, square, circular, short strip-shaped, film-shaped, etc. The device of the present invention includes, for example, a device for measurement by a hybridization technique. Specifically, a blotting device, a nucleic acid array (for example, a microarray, a DNA chip, an RNA chip), etc. can be exemplified.

[0213] The nucleic acid array technology is a technology for producing an array such as a chip by binding or adhering each of the above nucleic acids to the surface of a solid phase that has been subjected to surface treatment such as L-lysine coating, introduction of functional groups such as amino and carboxyl groups, etc. as needed, and using this array to perform measurement of nucleic acids during hybridization. As a method for binding or adhering nucleic acids, for example, a method of spotting nucleic acids using a high-density dispenser called a spotter or an array spotter, a method of blowing nucleic acids onto a solid phase using an inkjet device that ejects minute droplets using a piezoelectric element or the like by a nozzle, or a method of sequentially performing nucleotide synthesis on a solid phase, etc. can be cited.

[0214] 5. Method for screening candidate substances or treatment conditions that may be effective for improvement, treatment, and / or prevention of dementia

[0215] 5-1. Overview

[0216] The fifth aspect of the present invention is a method for screening candidate substances or treatment conditions that may be effective for improvement, treatment, and / or prevention of dementia. According to the method of this aspect, candidate substances or treatment conditions of a medicament useful for the treatment of dementia, etc. can be screened based on the biomarker described in the first aspect.

[0217] 5-2. Method

[0218] The method of the present invention includes, as essential steps, a step of exposing to a test substance or a test condition, a step of measuring the amount of a biomarker, a step of calculating a ratio, and a step of making a determination.

[0219] 5-2-1. Step of exposing to a test substance or a test condition

[0220] This step is a step of exposing cells to a test substance or a test condition.

[0221] As used in this specification, "treatment conditions" refer to the treatment conditions for the improvement, treatment, and / or prevention of dementia. "Test conditions" as used in this specification refer to treatment conditions that may be used for the improvement, treatment, and / or prevention of dementia. Treatment conditions, for example, in the case of administering a device such as a medical device or implementing a treatment such as exercise therapy, refer to the administration conditions and implementation conditions thereof. "Exposure to test conditions" means applying the test conditions to the exposure subject.

[0222] The type of cells is not particularly limited and is preferably the cells contained in the sample in this specification. Specifically, for example, cells contained in body fluids, cells contained in bone marrow, monocytes, etc. can be cited. The cells can be cells in a living organism or isolated cells. When using cells in a living organism as the cells, the cells are preferably cells in a living organism of a non-human animal. In addition, when using isolated cells, the method of this embodiment is an in vitro method. The cells used in the method of this embodiment are cells in which changes in the cells accompanying the exposure can be used as an index for determining whether a test substance or test conditions are effective for the treatment and / or prevention of dementia. Specifically, for example, cells from an individual suffering from dementia can be used in the method of this embodiment.

[0223] The method of exposing to the test substance in this step is not particularly limited and can be appropriately determined according to the properties of the test substance, the type of cells, etc. The method of exposing to the test conditions in this step is not particularly limited and is appropriately determined according to the type and nature of the phenomenon utilized, the type of device and instrument used, the type of treatment means (for example, an increase in the amount of exercise), etc.

[0224] For example, when the exposure subject is isolated cells, the exposure can be carried out by incubating the cells in the presence of the test substance or test conditions. In addition, when the exposure subject is cells in a living organism of a non-human animal, the test substance or test conditions are administered to the non-human animal.

[0225] The administration method of the test substance is not particularly limited and can be either oral administration or parenteral administration.

[0226] The specific administration method or implementation method of the test conditions is not particularly limited. For example, in the case of using a device such as a medical device, various parameters such as the administration target site, the administered energy (output), time, frequency, period, etc. are appropriately determined according to the subject individual, the type of cells, etc. In addition, the implementation method in the case of a treatment including a treatment without using a device such as exercise therapy is not particularly limited. For example, in the case of exercise therapy, various parameters such as the site of exercise, speed, range, load, difficulty, time, frequency, period, etc. are appropriately determined according to the specific means such as the type of exercise and the health status of the subject individual.

[0227] Step 5-2-2. Procedure for Measuring the Amount of Biomarker

[0228] This step is a procedure for measuring the amount of the biomarker described in the first mode in cells. This step can be carried out simultaneously with or after the step of exposing to the test substance or test conditions.

[0229] The content of this step is based on the "procedure for measuring the amount of biomarker" in the second mode. Therefore, only the differences are described here.

[0230] When the cells to be exposed in the step of exposing to the test substance or test conditions are cells in the body of a non-human animal, in this step, a sample can be isolated from the non-human animal, and the amount of the biomarker in the isolated sample can be measured. The method for isolating the sample is not particularly limited.

[0231] Step 5-2-3. Procedure for Calculating the Ratio

[0232] This step is a procedure for calculating the ratio of the expression level of the Cx37 gene or the protein amount of Cx37 to the expression level of other genes or the protein amount. This step can be carried out simultaneously with or after the step of measuring the amount of the biomarker.

[0233] The content of this step is based on the "procedure for calculating the ratio" in the second mode. Therefore, detailed description is omitted here.

[0234] Step 5-2-4. Procedure for Making a Judgment

[0235] This step is a procedure for judging whether the test substance or test conditions are effective in improving, treating, and / or preventing dementia based on the relative value. This step can be carried out simultaneously with or after the step of calculating the relative value of the expression level.

[0236] The content of this step is basically based on the "procedure for making a judgment" in the second mode. Therefore, the differences are described here.

[0237] In this step, when the relative value of the expression level of the Cx37 gene or the protein amount of Cx37 is lower than the cut-off value, or when the relative value of the expression level of the Cx37 gene or the protein amount of Cx37 is significantly lower than the control value, it is determined that the test substance or test conditions are candidate substances or treatment conditions effective in improving, treating, and / or preventing dementia.

[0238] Alternatively, when the relative value of the expression level of the Cx37 gene or the amount of Cx37 protein is higher than the cut-off value, or when the relative value of the expression level of the Cx37 gene or the amount of Cx37 protein is significantly higher compared to the control value, it is determined that the test substance or test condition is a candidate substance or treatment condition effective for the improvement, treatment, and / or prevention of dementia.

[0239] Example

[0240] <Example 1. Measurement of the expression level of connexin genes in dementia patients>

[0241] (Purpose)

[0242] To study how the expression level of connexin genes in dementia patients changes compared to healthy individuals.

[0243] (Method)

[0244] 1. Selection of individuals

[0245] From among men aged 65 and above, who are defined as having early-onset dementia, 10 volunteers each of healthy individuals and dementia patients were selected according to the following criteria.

[0246] · Exclude the subject's past medical history, comorbidities, etc.: diabetes, active cancer, during cancer treatment, within 5 years after the end of cancer treatment, thyroid disease, infectious diseases (AIDS, hepatitis B and C), fever at the time of blood collection, chronic subdural hematoma, normal pressure hydrocephalus, during hemodialysis, scheduled for hemodialysis (doctor indicates that hemodialysis is needed in the near future), COPD, during home oxygen therapy, smoking within the past 12 months

[0247] · Exclude the subject's medication status: anti-cancer drugs, steroids, anti-rheumatic drugs, etc., which affect the immune system

[0248] · Suffering from dementia: Dementia patients are individuals who have been diagnosed based on MRI, SPECT examinations, etc. in a medical facility and with the opinion of a radiologist, etc.

[0249] · MMSE examination: For healthy individuals, 28 points or more, and for dementia patients, 23 points or less

[0250] 2. Examinations and blood collection

[0251] Based on the principle of informed consent, the purpose of using the specimens obtained, etc. was explained to each individual, and consent was obtained before the examinations and blood collection. For healthy individuals, the consent of the individual was obtained, and for dementia patients, the consent of the individual and / or the guardian was obtained.

[0252] When the MMSE examination has not been performed within 3 months, the MMSE examination is performed when consent is obtained.

[0253] Fast from the previous evening and collect blood before noon.

[0254] Collect blood from the antecubital vein using a 10 mL syringe with a 21G needle.

[0255] Among a total of 8 mL to 8.5 mL of blood collected, 2.5 mL is aliquoted into a PAXgene (registered trademark) RNA blood collection tube (manufactured by Becton Dickinson) as a specimen for whole blood gene analysis. 0.5 mL to 1 mL is dispensed into a blood collection tube containing EDTA as a specimen for peripheral blood imaging. Additionally, 4 mL is dispensed into a CPT (trademark) mononuclear cell separation blood collection tube (manufactured by Becton Dickinson) as a specimen for preparing mononuclear cell suspension. After the specimen for gene analysis is stored at room temperature for more than 2 hours, it is cryopreserved until use in gene expression analysis.

[0256] The proportion of mononuclear cells is calculated as follows. The numbers of band neutrophils, segmented neutrophils, lymphocytes, mononuclear cells, eosinophils, and basophils in the whole blood specimen are counted by microscopic visual inspection (microscopic examination) of the specimen for peripheral blood imaging. The proportion of mononuclear cells (lymphocytes and mononuclear cells) among the total blood cells counted above is calculated as the proportion of mononuclear cells.

[0257] 3. Preparation of mononuclear cell suspension

[0258] Centrifuge the specimen for preparing mononuclear cell suspension at 1800 × g for 20 minutes at room temperature. After gently inverting and mixing, use a 2.5 mL syringe with a 21G needle to collect 2.5 mL from the supernatant as the mononuclear cell suspension.

[0259] The mononuclear cell suspension and the residual liquid are separately placed into different PAXgene (registered trademark) RNA blood collection tubes (manufactured by Becton Dickinson) and used as specimens for gene analysis. After the specimen for gene analysis is stored at room temperature for more than 2 hours, it is cryopreserved until use in gene expression analysis.

[0260] 4. Gene expression analysis

[0261] First, total RNA is extracted from the specimen for gene expression analysis. The extraction is carried out using an RNA extraction kit (RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific, model: AM1928)) according to the manufacturer's protocol.

[0262] Next, cDNA was synthesized (reverse-transcribed) from 1 μg of total RNA. The cDNA synthesis was carried out using a cDNA synthesis kit (PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Inc., model: 6210B (A×4))) according to the manufacturer's protocol.

[0263] The resulting cDNA was used as a template for real-time PCR reaction. As the nucleic acid amplification reagent, PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific, model: A25777) was used. Additionally, as the real-time PCR system, Agilent AriaMx Real-Time PCR System (Agilent) was used. The real-time PCR reaction conditions were as follows: 1 cycle at 50°C for 3 minutes, 1 cycle at 95°C for 3 minutes, 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle at 95°C for 30 seconds, 1 cycle at 65°C for 30 seconds, and 1 cycle at 95°C for 30 seconds.

[0264] The 18S ribosomal RNA gene, which is a housekeeping gene, was used as an endogenous control, and the relative expression levels of each gene relative to the endogenous control were analyzed using the Pfaffl method. It should be noted that the Pfaffl method is a relative quantification method that is a type of comparative Ct method considering the PCR amplification efficiencies of the housekeeping gene and the gene (Pfaffl MW. Nucleic Acids Res. 2001; 29(9):e45.).

[0265] The base sequences of the primers for each gene are shown in Table 1. 18S represents 18S ribosomal RNA, Glut1 and Glut3 represent glucose transporter 1 and 3 respectively, MCT4 represents monocarboxylate transport protein 4, and PDK1 represents pyruvate dehydrogenase kinase 1. Additionally, Fw represents the forward primer and Rv represents the reverse primer.

[0266] [Table 1]

[0267] Table 1: Primers used in the examples

[0268]

[0269] The determination of the cut-off value and the calculation of the determination accuracy such as sensitivity, specificity, and AUC were performed using the free statistical software EZR (Easy R version 1.54). The cut-off value was determined using the Youden index.

[0270] The calculation of significant differences was performed using the Welch t-test.

[0271] (Results)

[0272] The results are shown in Figures 1 - 3 .

[0273] First, there was no significant difference in the proportion of monocytes between healthy subjects and dementia patients ( Figure 1 ).

[0274] The expression levels of connexin 37 gene and connexin 43 gene did not show significant differences between healthy subjects and dementia patients in the specimens containing monocytes ( Figure 2 ), whole blood specimens ( Figure 3 ). However, in any specimen, a slightly decreasing trend in the expression level of connexin 37 gene was observed in dementia patients compared with healthy subjects. In addition, for connexin 43 gene, conversely, a slightly increasing trend in the expression level was observed in dementia patients. In addition, the determination accuracy of dementia patients when using connexin genes was not high.

[0275] In summary, it can be seen that the expression level of connexin gene in peripheral blood cannot be used alone as a biomarker for dementia.

[0276] <Example 2. Verification of the determination accuracy of dementia using various biomarker monomers>

[0277] (Purpose)

[0278] To study the determination accuracy of dementia using the biomarker monomers found in WO 2021 / 177447.

[0279] (Method)

[0280] Using the analysis results of the gene expression levels in Example 1, the determination accuracy was calculated using the same method as in Example 1.

[0281] (Results)

[0282] The results are shown in Tables 2 and 3.

[0283] [Table 2]

[0284] Table 2: Determination accuracy of dementia using each biomarker monomer in the specimen containing monocytes

[0285] PHD3 PDK1 GLUT1 GLUT3 MCT4 Specificity (%) 80 90 50 80 70 Sensitivity (%) 50 30 80 50 50 AUC 0.70 0.55 0.66 0.57 0.49

[0286] [Table 3]

[0287] Table 3: Dementia determination accuracy using each biomarker in whole blood samples

[0288] PHD3 PDK1 GLUT1 GLUT3 MCT4 Specificity (%) 50 60 80 50 80 Sensitivity (%) 90 70 60 80 60 AUC 0.72 0.61 0.68 0.61 0.66

[0289] Table 2 shows the determination accuracy of dementia patients using each biomarker monomer in samples containing monocytes. Table 3 shows the determination accuracy of dementia patients using each biomarker monomer in whole blood samples.

[0290] The sensitivity and specificity of any biomarker did not exceed 70%. In addition, although the value of AUC, which represents the discriminative ability of the index, was the highest for the prolyl hydroxylase domain protein 3 (PHD3) gene, its value also remained at around 0.7.

[0291] In summary, it can be seen that the expression levels of each known biomarker gene cannot achieve a determination accuracy sufficient to diagnose dementia patients as monomers.

[0292] (Example 3. Verification of dementia determination accuracy based on the relative value of the expression level of connexin 37 gene with respect to the expression levels of various biomarker genes)

[0293] (Purpose)

[0294] To study the dementia determination accuracy when using the relative value of the expression level of connexin 37 gene with respect to the expression levels of connexin 43 and the biomarker genes studied in Example 2.

[0295] (Method)

[0296] Using the analysis results of the gene expression levels in Example 1, the determination accuracy was calculated using the same method as in Example 1.

[0297] The relative value was calculated by dividing the expression level of the connexin 37 gene by the expression levels of other genes.

[0298] (Result)

[0299] The results are shown in Figure 4 and Table 4.

[0300] [Table 4]

[0301] Table 4: Dementia determination accuracy based on the relative value of the expression level of connexin 37 gene

[0302]

[0303] Table 4 shows the determination accuracy of dementia patients for each relative value in samples containing monocytes.

[0304] In the case of combination with any gene, the determination accuracy is generally improved compared with the case where the gene expression level is used as a monomer. In particular, when combined with the PHD3 gene (cut-off value: 0.893) and when combined with the connexin 43 gene (cut-off value: 0.651), both the sensitivity and specificity exceed 70%, and the AUC is also much higher than 0.75.

[0305] The result of comparing the values between healthy subjects and dementia patients is the same ( Figure 4 ). As Figure 4 shown, among the combinations studied, only when the expression level of the connexin 37 gene is combined with the expression levels of the PHD3 gene and the connexin 43 gene, the relative value is significantly lower in dementia patients than in healthy subjects.

[0306] In summary, it can be seen that the relative value of the expression level of the connexin 37 gene relative to the expression levels of the PHD3 gene and the connexin 43 gene has a determination accuracy for dementia patients sufficient for a definite diagnosis and is an excellent biomarker.

[0307] <Verification of the determination accuracy of dementia using the relative value of the expression level of the connexin 37 gene relative to the expression level of the PHD3 gene>

[0308] (Objective)

[0309] To study the determination accuracy of dementia when using the relative value of the expression level of the connexin 37 gene relative to the expression level of the PHD3 gene.

[0310] (Method)

[0311] Using the analysis results of the gene expression levels in Example 1, the determination accuracy was calculated by the same method as in Example 1.

[0312] The relative value was calculated by dividing the expression level of the connexin 37 gene by the expression level of the PHD3 gene.

[0313] (Result)

[0314] The results are shown in Figure 5 .

[0315] It can be seen that the sensitivity and specificity of the determination accuracy of dementia patients for the relative value of the expression level of the PHD3 gene in whole blood samples are both 90%, and the AUC is also 0.9 (cut-off value: 0.744), which is an extremely excellent biomarker.

[0316] The result of comparing the values between healthy subjects and dementia patients is the same ( Figure 5 ). As Figure 5As shown, in the whole blood sample as well as in the case of using a sample containing monocytes, the relative value of the expression level of connexin 37 gene relative to the expression level of PHD3 gene was significantly lower in dementia patients compared to healthy subjects.

[0317] In summary, it can be seen that the relative value of the expression level of connexin 37 gene, which is useful in a sample containing monocytes, is also useful in the whole blood sample.

[0318] <Example 5. Verification of the determination accuracy of dementia with respect to the relative value of the expression level of connexin 37 gene>

[0319] (Objective)

[0320] To study the determination accuracy of dementia when using the relative value of the expression level of PHD3 gene divided by the expression level of connexin 37 gene with the numerator and denominator reversed.

[0321] (Method)

[0322] Using the analysis results of the gene expression levels in Example 1, the determination accuracy was calculated by the same method as in Example 1.

[0323] The relative value was calculated by dividing the expression level of PHD3 gene by the expression level of connexin 37 gene.

[0324] (Result)

[0325] When using the relative value of the expression level of connexin 37 gene, the sensitivity, specificity, and AUC of the determination accuracy for dementia patients were exactly the same as when using the relative value of the expression level of connexin 37 gene, both in the sample containing monocytes and in the whole blood sample (cutoff value: for the sample containing monocytes, 1.12; for the whole blood sample, 1.344).

[0326] In summary, it can be confirmed that the ratio of the expression level of connexin 37 gene to the expression levels of other genes such as PHD3 gene is useful for the determination of dementia regardless of which is used as the denominator.

[0327] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference directly into this specification.

Claims

1. A method for determining the presence or absence of the risk of developing or having dementia in a subject, comprising: a step of measuring the expression levels of connexin 37 gene, and connexin 43 gene and / or prolyl hydroxylase domain protein 3 gene in a sample derived from the subject, a step of calculating the ratio of the expression level of the connexin 43 gene and / or the expression level of the prolyl hydroxylase domain protein 3 gene to the expression level of the connexin 37 gene; indicating that the subject has dementia or has a risk of developing dementia based on the comparison of the ratio with a preset cut-off value or the comparison with a control value calculated in the same way in a sample derived from a healthy individual.

2. The method according to claim 1, wherein, the sample is blood.

3. The method according to claim 1 or 2, wherein, the dementia is Alzheimer's dementia.

4. A kit for determining the presence or absence of the risk of developing or having dementia, comprising: a reagent for measuring the expression level of connexin 37 gene in a sample derived from a subject, and a reagent for measuring the expression level of connexin 43 gene and / or prolyl hydroxylase domain protein 3 gene in the sample; the reagent contains primers that can amplify all or part of the base sequence of the gene or a base sequence complementary thereto, and / or probes that can hybridize with all or part of the base sequence of the gene or a base sequence complementary thereto.

5. The kit according to claim 4, wherein, the primers and the probes are polynucleotides or fragments thereof shown in any one of the following (1) to (6), (1) a polynucleotide fragment consisting of 15 or more consecutive bases selected from the polynucleotides consisting of the base sequences shown in SEQ ID NO: 1, 3 or 5; (2) a polynucleotide fragment containing 1 or 2 base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment in (1); (3) a polynucleotide fragment containing 15 or more consecutive bases selected from the polynucleotides consisting of the base sequences shown in SEQ ID NO: 1, 3 or 5; (4) a polynucleotide fragment containing 1 or 2 base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment in (3); (5) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide fragment sequence containing 15 or more consecutive bases selected from the base sequences shown in SEQ ID NO: 1, 3 or 5; and (6) a polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotides or fragments thereof in (1) to (5).

6. A kit for determining the presence or absence of the risk of developing or having dementia, comprising: a connexin 37 binding molecule in a sample derived from a subject, and a connexin 43 binding molecule and / or a prolyl hydroxylase domain protein 3 binding molecule in the sample; the binding molecule contains an antibody or an active fragment thereof that can bind to the target protein, or an aptamer.

7. A device for determining the presence or absence of the risk of developing or having dementia, comprising: A reagent for measuring the expression level of the connexin 37 gene in a sample derived from a subject, and a reagent for measuring the expression level of the connexin 43 gene and / or the prolyl hydroxylase domain protein 3 gene in the sample; The reagent contains: primers that can amplify all or part of the base sequence of the gene or a base sequence complementary thereto, and / or probes that can hybridize with all or part of the base sequence of the gene or a base sequence complementary thereto.

8. The device according to claim 7, wherein, the primers and the probes are polynucleotides or fragments thereof described in any one of the following (1) to (6), (1) A polynucleotide fragment consisting of 15 or more consecutive bases selected from the polynucleotides consisting of the base sequences shown in SEQ ID NO: 1, 3 or 5; (2) A polynucleotide fragment containing 1 or 2 base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment in (1); (3) A polynucleotide fragment containing 15 or more consecutive bases selected from the polynucleotides consisting of the base sequences shown in SEQ ID NO: 1, 3 or 5; (4) A polynucleotide fragment containing 1 or 2 base deletions, substitutions, additions or insertions in the base sequence of the polynucleotide fragment in (3); (5) A polynucleotide that hybridizes with a polynucleotide fragment sequence containing 15 or more consecutive bases selected from the base sequences shown in SEQ ID NO: 1, 3 or 5 under high stringency conditions; and (6) A polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotides or fragments thereof in (1) to (5).

9. A device for determining the presence or absence of the risk of developing or having dementia, comprising: a connexin 37 binding molecule in a sample derived from a subject, and a connexin 43 binding molecule and / or a prolyl hydroxylase domain protein 3 binding molecule in the sample; The binding molecule contains an antibody or an active fragment thereof that can bind to the target protein, or an aptamer.

10. A biomarker, which is any one of the following (a) to (d), for determining the presence or absence of the risk of developing or having dementia, (a) A polynucleotide consisting of the connexin 37 gene or a fragment thereof and a polynucleotide consisting of the connexin 43 gene or a fragment thereof; (b) A polynucleotide consisting of the connexin 37 gene or a fragment thereof and a polynucleotide consisting of the prolyl hydroxylase domain protein 3 gene or a fragment thereof; (c) A polypeptide consisting of connexin 37 or a fragment thereof and a polypeptide consisting of connexin 43 or a fragment thereof; and (d) A polypeptide consisting of connexin 37 or a fragment thereof and a polypeptide consisting of prolyl hydroxylase domain protein 3 or a fragment thereof.

Images