Synchronized cell cycle gene expression testing and related therapies for Alzheimer's disease

Abstract

To provide synchronized cell cycle gene expression tests on Alzheimer disease and related therapeutic methods.SOLUTION: The invention provides a gene-based accurate method for determining whether a human subject is afflicted with Alzheimer disease (AD) or with non-Alzheimer type dementia (ADD). The subject method is based on, at least in part, a surprising finding that by synchronizing an appropriate cell population of the patient, then measuring the expression level of a gene expressed differentially between AD cells and non-ADD cells, it is possible to accurately diagnose whether the patient is afflicted with AD or with non-ADD. The invention also provides a method for treating AD by using an agent that affects the expression of a certain gene.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 596,588, filed on 8 December 2017, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced. The disclosures of these publications are incorporated herein by reference to better describe the state of the art in which the present invention pertains. [Background technology]

[0003] Background of the Invention Alzheimer's disease ("AD") has long been the subject of considerable effort to develop accurate diagnostic and treatment methods. Despite these efforts, there remains an unmet need for accurate diagnosis of AD and for differentiating AD from non-Alzheimer's dementia ("non-ADD"). There also remains an unmet need for effective methods to treat AD. [Overview of the project] [Means for solving the problem]

[0004] Summary of the Invention The present invention relates to a method for determining whether a human subject is suffering from AD or not, when the subject is suspected of having AD or not having ADD, (a) A step of synchronizing a suitable population of cells derived from the above subject; and (b) A step of measuring the expression levels of genes known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients in the obtained synchronized cell population. The present invention provides a method that includes (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD.

[0005] The present invention also relates to a method for determining whether a human subject has AD or not has ADD, when the subject is suspected of having AD or not having ADD, (a) A step of synchronizing a population of cultured skin fibroblasts derived from the above subject, wherein the synchronization step includes culturing the fibroblasts until they become overconfluent, and then starving the resulting overconfluent fibroblasts; and (b) A step of measuring the expression levels of each of the following genes in the obtained synchronized fibroblast population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein the step of measuring the expression level of each gene includes measuring the number of RNA transcripts per total number of transcripts. The present invention provides a method that includes (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD.

[0006] The present invention relates to a method for determining whether a human subject is suffering from AD or not, when the subject is suspected of having AD or not having ADD, (a) A step of synchronizing a population of cultured immortalized B lymphocytes derived from the above subject, wherein the synchronization step includes culturing the lymphocytes until they become overconfluent, and then starving the resulting overconfluent lymphocytes; and (b) A step of measuring the expression levels of each of the following genes in the synchronized lymphocyte population obtained above: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein the step of measuring the expression level of each gene includes measuring the number of RNA transcripts per total number of transcripts. The method further includes the following, which (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD.

[0007] The present invention further provides a method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of a drug known to conveniently affect the expression level of one or more genes whose expression levels correlate with Alzheimer's disease.

[0008] Finally, the present invention provides a method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of carfilzomib, bortezomib, bumetanide, furosemide, or torasemide. In certain embodiments, for example, the following are provided: (Item 1) A method for determining whether a human subject has Alzheimer's disease ("AD") or non-Alzheimer's dementia (non-ADD) when the subject is suspected of having AD, (a) A step of synchronizing a suitable population of cells derived from the subject; and (b) A step of measuring the expression levels of genes known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients in the obtained synchronized cell population. This includes, thereby, (i) if the expression level measured in step (b) matches the expression level of the gene in the corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in the corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD. method. (Item 2) The method according to item 1, wherein the appropriate cells derived from the subject are cultured skin cell fibroblasts. (Item 3) The method according to item 1, wherein the appropriate cells derived from the subject are cultured B lymphocytes. (Item 4) The B lymphocytes are immortalized according to the method described in item 3. (Item 5) The method according to item 1, wherein the step of synchronizing the appropriate population of cells includes culturing the cells until they become overconfluent, and then starving the resulting overconfluent cells. (Item 6) The method according to item 1, wherein the gene is known to be expressed at least 50% differentially between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. (Item 7) The method according to item 6, wherein the gene is known to be expressed at least 100% differentially between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. (Item 8) The gene is selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, as described in item 1. (Item 9) The method according to item 1, wherein step (b) comprises measuring the expression levels of several genes, each of which is known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. (Item 10) The method according to item 9, wherein the plurality of genes are selected from the group consisting of at least 2 genes, at least 5 genes, at least 20 genes, at least 100 genes, and at least 1,000 genes. (Item 11) The method according to item 9, wherein each of the aforementioned multiple genes is known to be expressed at least 50% differentially between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. (Item 12) The method according to item 11, wherein each of the aforementioned multiple genes is known to be expressed at least 100% differentially between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. (Item 13) The method according to item 9, wherein the plurality of genes include two or more genes selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. (Item 14) The method according to item 1, wherein the step of measuring the gene expression level includes measuring the number of RNA transcripts of that gene per total number of transcripts. (Item 15) A method for determining whether a human subject has Alzheimer's disease ("AD") or non-Alzheimer's dementia ("non-ADD") when the subject is suspected of having AD, (a) A step of synchronizing a population of cultured skin fibroblasts derived from the subject, wherein the synchronizing step includes culturing the fibroblasts until they become overconfluent, and then starving the resulting overconfluent fibroblasts; and (b) a step of measuring the expression level of each of the following genes in the obtained synchronized fibroblast population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein the step of measuring the expression level of each gene comprises measuring the number of RNA transcripts thereof per the total number of transcripts, comprising: (i) if the expression level measured in step (b) matches the expression level of said gene in corresponding synchronized cells derived from an AD patient, the subject suffers from AD; and (ii) if the expression level measured in step (b) matches the expression level of said gene in corresponding synchronized cells derived from a non-ADD patient, the subject suffers from non-ADD, The method. (Item 16) A method for determining whether a human subject suffers from Alzheimer's disease ("AD") or non-Alzheimer's dementia ("non-ADD") when the subject is suspected of suffering from AD or non-ADD, comprising: (a) synchronizing a population of cultured immortalized B lymphocytes derived from said subject, wherein the synchronizing step comprises culturing said lymphocytes until they become overconfluent, and then starving the obtained overconfluent lymphocytes; and (b) a step of measuring the expression level of each of the following genes in the obtained synchronized lymphocyte population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein the step of measuring the expression level of each gene comprises measuring the number of RNA transcripts thereof per the number of total transcripts, comprising, whereby: (i) the subject suffers from AD when the expression level measured in step (b) matches the expression level of said gene in corresponding synchronized cells derived from an AD patient, and (ii) the subject suffers from non-ADD when the expression level measured in step (b) matches the expression level of said gene in corresponding synchronized cells derived from a non-ADD patient, A method. (Item 17) A method for treating a human subject suffering from Alzheimer's disease, comprising administering to said subject a therapeutically effective amount of an agent known to conveniently affect the expression level of one or more genes whose expression level correlates with Alzheimer's disease. (Item 18) The gene is selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, as described in item 17. (Item 19) A method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of carfilzomib. (Item 20) A method for treating a human subject suffering from Alzheimer's disease, comprising administering a therapeutically effective amount of bortezomib to the subject. (Item 21) A method for treating a human subject suffering from Alzheimer's disease, comprising administering a therapeutically effective amount of bumetanide to the subject. (Item 22) A method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of furosemide. (Item 23) A method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of torasemide. [Brief explanation of the drawing]

[0009] [Figure 1]This figure shows the statistically significant genes when comparing the AD group and the non-ADD group, based on the first study ("Study 1"). From Study 1, it was revealed that there are 2103 statistically significant genes for P values ​​less than 0.1; 1099 statistically significant genes for P values ​​less than 0.05; 285 statistically significant genes for P values ​​less than 0.01; and 6 statistically significant genes for P values ​​less than or equal to 0.001.

[0010] [Figure 2] This figure shows the top six statistically significant genes (P ≤ 0.001) for six AD cases and two non-ADD cases, based on Study 1. Squares represent the AD population, while circles represent the non-ADD population.

[0011] [Figure 3] This figure shows examples of the top 10 statistically significant genes (P ≤ 0.01) based on Study 1. (A) Raw TPM data showing the AD population as squares and the non-ADD population as circles. (B) Mean TPM data showing the AD population as squares and the non-ADD population as circles. Error bars are standard deviations. (C) Percentage change in gene expression (%Ch) when comparing AD to control (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0012] [Figure 4] This figure shows the genes ranked 11th to 20th in statistical significance (P ≤ 0.01) at 1% overlap probability, based on Study 1. (A) Raw TPM data showing the AD population as squares and the non-ADD population as circles. (B) Mean TPM data showing the AD population as squares and the non-ADD population as circles. Error bars are standard deviations. (C) Percentage change in gene expression (%Ch) when comparing AD to control (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0013] [Figure 5]This figure shows genes ranked 21st to 30th based on Study 1, with statistical significance (P ≤ 0.01) at 1% overlap probability. (A) Raw TPM data, with the AD population shown as squares and the non-ADD population as circles. (B) Mean TPM data, with the AD population shown as squares and the non-ADD population as circles. Error bars represent the standard deviation. (C) Percentage change in gene expression (%Ch) when comparing AD with control (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0014] [Figure 6] This figure shows genes ranked 31st to 40th based on Study 1, with statistical significance (P ≤ 0.01) at 1% overlap probability. (A) Raw TPM data, with the AD population shown as squares and the non-ADD population as circles. (B) Mean TPM data, with the AD population shown as squares and the non-ADD population as circles. Error bars represent the standard deviation. (C) Percentage change in gene expression (%Ch) when comparing AD with control (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0015] [Figure 7] This figure shows the percentage change (%Ch) in gene expression for the top 40 genes, based on Study 1.

[0016] [Figure 8] This figure, based on Study 2 ("Study 2"), shows the number of statistically significant differentially expressed genes in the training set (Cylinder 1 - lighter shaded) versus the number of statistically significant differentially expressed genes in the validation set (Cylinder 2 - darker shaded) for various levels of statistical significance P < 0.001, 0.01, 0.05, and 0.10.

[0017] [Figure 9]This figure shows the gene networks for (A)PAN3, (B)PSMB9, (C)TTC26, (D)ZNF444, (E)NHLH1, (F)URB2, and (G)ADAM20, based on Study 2.

[0018] [Figure 10] This figure shows network metrics for cross-validated genes based on Study 2: (A) number of edges; (B) mean node degree; and (C) mean local clustering coefficient.

[0019] [Figure 11] This figure, based on Study 2, compares non-ADD (n=3) with non-dementia controls (NDC; n=5) and shows the number of differentially expressed genes in the non-ADD population compared to the NDC population. [Modes for carrying out the invention]

[0020] Detailed description of the invention definition In this application, certain terms are used and have the meanings set forth below.

[0021] As used herein, “administer” means, with respect to a drug, to deliver that drug to the body of a subject by any known method. Specific modes of administration include, but are not limited to, intravenous, oral, sublingual, transdermal, subcutaneous, intraperitoneal, and intrathecal administration.

[0022] Furthermore, in the present invention, the various agents can be formulated using one or more conventionally used pharmaceutically acceptable carriers. Such carriers are well known to those skilled in the art. For example, oral delivery systems include tablets and capsules. These may contain additives such as binders (e.g., hydroxypropyl methylcellulose, polyvinylpyrrolidone, other cellulosic substances, and starch), excipients (e.g., lactose and other sugars, starch, dicalcium phosphate, and cellulosic substances), disintegrants (e.g., starch polymers and cellulosic substances), and lubricants (e.g., stearate and talc). Injectable drug delivery systems include, for example, liquids, suspensions, gels, microspheres, and polymer injections, and may contain additives such as solubility-altering agents (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprylactone and PLGA). Implantation systems include rods and discs, and may contain additives such as PLGA and polycaprylactone.

[0023] As used herein, “Alzheimer’s disease” means the simultaneous presence of the following three conditions: (i) dementia; (ii) amyloid plaques; and (iii) neurofibrillary tangles. Dementia may be diagnosed at any time during a person’s lifetime. Cerebral amyloid plaques and neurofibrillary tangles may be diagnosed, for example, during autopsy. This definition of Alzheimer’s disease is provided by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) and is publicly known as the “optimal criteria.” All subjects from whom samples have been taken and studied, and whose data are presented herein, are autopsy-confirmed patients with and without Alzheimer’s disease.

[0024] As used herein, a gene's expression level is considered to match that of a corresponding synchronized cell derived from an AD patient if its expression level is the same as, or close to, that of the same gene in the same synchronized cell derived from an AD patient. For example, suppose the TPM scale of gene X in synchronized cells derived from an AD patient is 10, and the TPM scale of that gene X in the same type of cell derived from a non-ADD patient synchronized in the same manner is 100. The gene X expression level in the subject should match that of the AD expression level of gene X if, for example, the AD expression level was less than 50, less than 40, less than 30, less than 20, or ideally, 10 or less.

[0025] As used herein, “culturing” lymphocytes is achieved, for example, by culturing them in a growth factor environment that allows for a certain temperature and cell growth. In another embodiment, “culturing” lymphocytes is carried out under conditions that preserve lymphocyte viability (e.g., conditions described herein with respect to proliferation). In one embodiment, the temperature, salinity, and protein environment that allows for cell growth is RPMI 1640 medium containing 10% fetal bovine serum ("FBS") and 1% penicillin ("PS") at 37°C. In one embodiment of the present invention, the step of culturing the lymphocytes is carried out over a period of more than 3 hours. Preferably, the step of culturing the lymphocytes is carried out over a period of more than 6 hours (e.g., overnight). B lymphocytes may be cultured until they reach overconfluent, i.e., high density / μl. This high density is determined as a plateau, typically greater than 90% on the growth curve. The lymphocytes are then starved overnight.

[0026] Methods for obtaining lymphocytes from the blood of a subject are known, including, for example, flow cytometry, Ficoll (a hydrophilic polysaccharide for separating layers of blood), and gradient centrifugation. Furthermore, in the methods of this subject, the lymphocytes (e.g., B lymphocytes) may be used in an immortalized or primary (i.e., unimmortalized) form. Methods for immortalizing lymphocytes (e.g., B lymphocytes) are known, including, for example, treating the lymphocytes with Epstein-Barr virus ("EBV").

[0027] As used herein, “culturing” dermal fibroblasts is achieved, for example, by culturing them in a growth factor environment that allows for a certain temperature and cell growth. In another embodiment, “culturing” dermal fibroblasts is carried out under conditions that preserve dermal fibroblast viability (e.g., conditions described herein with respect to proliferation). In one embodiment, the temperature, humidity and protein environment that allows for cell growth is 37°C and DMEM medium containing 10% fetal bovine serum ("FBS") and 1% penicillin ("PS"). In one embodiment of the present invention, the step of culturing the dermal fibroblasts is carried out for more than 3 hours. Preferably, the step of culturing the dermal fibroblasts is carried out for more than 6 hours (e.g., overnight).

[0028] Methods for obtaining cutaneous fibroblasts from the blood of a subject are known, including, for example, skin punch biopsy and growing cells from explants. When cell confluence reaches 100%, the cells are passaged. Typically, after two passages, the fibroblasts are purified to a rate of over 95%.

[0029] As used herein, cells “derived” from a subject are cells that arise through culture and / or other physical manipulations performed on cells directly removed from that subject. For example, cultured dermal fibroblasts derived from a subject are those dermal fibroblasts that arise through culture of a sample of skin cells (e.g., those contained in a punch biopsy) directly removed from that subject.

[0030] As used herein, “diagnose Alzheimer’s disease” means determining that a symptomatic human subject has a greater than 50% probability of having Alzheimer’s disease. Preferably, “diagnose Alzheimer’s disease” means determining that a subject has a greater than 60%, greater than 70%, greater than 80%, or greater than 90% probability of having Alzheimer’s disease. As used herein, the phrase “determine whether a subject has Alzheimer’s disease” is synonymous with the phrase “diagnose Alzheimer’s disease.”

[0031] As used herein, “diagnose non-ADD” means determining that, with respect to a symptomatic human subject, there is a greater than 50% probability that the subject has non-ADD. Preferably, “diagnose non-ADD” means determining that there is a greater than 60%, greater than 70%, greater than 80%, or greater than 90% probability that the subject has non-ADD. As used herein, the phrase “determine whether the subject has non-ADD” is synonymous with the phrase “diagnose non-ADD.”

[0032] As used herein, a gene is differentially expressed between corresponding synchronized cells from AD patients and corresponding synchronized cells from non-ADD patients if, for example, the TPM scale of the gene in synchronized cells from AD patients differs from that in cells of the same type from non-ADD patients synchronized in the same manner. For example, gene X should be differentially expressed between corresponding synchronized cells from AD patients and corresponding synchronized cells from non-ADD patients if its TPM scale is 10 in synchronized cells from AD patients and 100 in cells of the same type from non-ADD patients synchronized in the same manner.

[0033] As used herein, a drug "favorably" affects the expression level of a gene, and that expression level correlates with AD if the drug either decreases or increases the expression toward a level that correlates with non-AD status. For example, if the expression level of gene X is lower in AD patients than in non-AD patients, a drug that favorably affects the expression level of that gene should increase its expression level. Similarly, if the expression level of gene X is higher in AD patients than in non-AD patients, a drug that favorably affects the expression level of that gene should decrease its expression level.

[0034] As used herein, “measuring” a gene expression level means quantitatively determining its expression level by any means to do so (e.g., total RNA sequencing (20 million reads, 2 x 75 bp PE)). Preferably, the measurement of a gene expression level is achieved by measuring the number of RNA transcripts of that gene per million total RNA transcripts (i.e., “TPM” via FastQ data, and FPKM predictions per sample) present in the RNA population derived from the cells being studied. For example, measuring the expression level of gene X in a synchronized cell population may yield a result of 50 TPM.

[0035] As used herein, subjects suffering from "non-Alzheimer's disease" means subjects exhibiting dementia, such as Parkinson's disease, Huntington's disease, and frontotemporal dementia.

[0036] As used herein, a “population” of cells includes any number of cells that enable the operations and studies required to evaluate gene expression. In one embodiment, the population of cells includes at least 1,000,000 cells. In another embodiment, the population of cells includes between 100,000 and 1,000,000 cells, between 10,000 and 100,000 cells, between 1,000 and 10,000 cells, between 100 and 1,000 cells, between 10 and 100 cells, and fewer than 10 cells (e.g., one cell).

[0037] As used herein, the term “subject” includes, but is not limited to, mammals (e.g., humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, rats, and mice). If the subject is human, the subject may be of any age. For example, the subject may be 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, or 90 years or older. The methods of the present invention are intended for all subjects, preferably humans (and preferably symptomatic).

[0038] As used herein, a human subject "suspected of having AD or non-ADD" is a subject exhibiting at least one symptom consistent with both AD and non-ADD (e.g., dementia).

[0039] As used herein, “synchronizing” a population of cells means placing at least the majority of the cells in that population into the same cell cycle stage (i.e., G1, S, G2, or M phase, and preferably G1, S, or G2 phase). In one embodiment, the step of synchronizing a population of cells means placing at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or preferably at least 99%, of the cells in that population into the same cell cycle stage. In another embodiment, synchronizing a population of cells means placing the cells in that population into the same cell cycle stage in which they would be if they were cultured to overconfluence and then starved. Confluenced cells and then serum starvation typically results in the cells being arrested in the G0 / G1 phase [1-3].

[0040] Dosage, i.e., “therapeutically effective amount,” is used in connection with the present invention and includes, for example, a single dose and two or more doses (i.e., divided doses). In one embodiment, the therapeutically effective amount of a drug approved for a non-Alzheimer’s indication is the approved dose and administration regimen for that non-Alzheimer’s indication.

[0041] As used herein, “treating” a subject suffering from a disorder includes, but is not limited to, (i) reversing the progression of the disorder, (ii) slowing, halting, or reversing the progression of the symptoms of the disorder, (iii) reducing the likelihood of the disorder recurring, and / or (iv) reducing the likelihood of the symptoms of the disorder recurring in the future. In a preferred embodiment, treating a subject suffering from a disorder means (i) reversing the progression of the disorder, ideally to the point of eliminating the disorder, and / or (ii) reversing the progression of the symptoms of the disorder, ideally to the point of eliminating the symptoms.

[0042] Treatment for AD can be measured according to a number of clinical endpoints, including, but not limited to, (a) reducing, stabilizing, or delaying the progression of dementia, (ii) synaptic loss, (iii) amyloid plaques, and / or (iv) neurofibrillary tangles, and / or (b) conveniently affecting the expression levels of genes whose expression levels correlate with AD.

[0043] Embodiments of the present invention The present invention provides an accurate gene-based method for determining whether a human subject has AD or non-ADD when the subject is suspected of having AD or non-ADD. The method of the present invention is at least in part based on the remarkable discovery that synchronizing a suitable cell population of a patient and then measuring the expression levels of genes differentially expressed between AD cells and non-ADD cells makes it possible to accurately diagnose the patient as having either AD or non-ADD. The present invention also provides a method for treating AD using agents that modify the expression of certain genes.

[0044] Specifically, the present invention relates to a method for determining whether a human subject has AD or not has ADD when the subject is suspected of having AD or not having ADD, (a) A process of synchronizing a suitable population of cells derived from the subject; and (b) A step of measuring the expression levels of genes known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients in the obtained synchronized cell population, The present invention provides a method that includes (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD.

[0045] In one embodiment of the method of this subject, suitable cells derived from the subject are cultured skin cell fibroblasts. In another embodiment, suitable cells derived from the subject are cultured B lymphocytes (preferably immortalized B lymphocytes).

[0046] Methods for synchronizing cell populations are known in the art. In one embodiment of the method of the subject, synchronizing a suitable cell population involves culturing the cells until they become overconfluent, and then starving the resulting overconfluent cells.

[0047] Ideally, in the method of this subject, it is known that the gene is differentially expressed with a significant margin. In one embodiment, it is known that the gene is differentially expressed by at least 50% between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. Preferably, it is known that the gene is differentially expressed by at least 100% between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. Another way to express the degree of differential expression is "% change" or "%Ch", where [AD] 発現 -Non-ADD 発現 / Non-ADD 発現 It is equal to ].

[0048] In another preferred embodiment of the method of the subject, the gene is selected from the group consisting of CFAP97, LINC01393, ZNF623, HAUS2, PAN3, PSMB9, ZFP28, TTC26, RFESDP1, ZNF444, WASF2, NHLH1, NPPA-AS1_3, NORAD, URB2, ADAM20, ZCWPW2, AC004057.1, AC092651.1, ACP6, ACP2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, ASXL2, and IL18R1.

[0049] In one embodiment, the gene expression levels shown in Table 9, individually or collectively (e.g., one, two or more, three or more, four or more, etc.), indicate AD. In a preferred embodiment, the gene expression levels shown in Table 10, individually or collectively (e.g., one, two or more, three or more, four or more, etc.), indicate AD. For example, as shown in Table 10, PSMB9 expression levels greater than 18 TPM indicate AD. In another embodiment, the expression levels indicating AD for each other gene disclosed herein are readily determined based on the data provided.

[0050] In a further preferred embodiment of the method of the subject, step (b) includes measuring the expression levels of a plurality of genes (each gene is known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients). The plurality of genes (e.g., at least two genes, at least five genes, at least 20 genes, at least 100 genes, and at least 1,000 genes) can be of any suitable size. Preferably, each of the plurality of genes is known to be differentially expressed by at least 50% (and more preferably at least 100%) between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients. In yet another preferred embodiment of the method of the subject, the plurality of genes comprises two or more genes selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70.

[0051] In the method of this subject, in which the expression levels of multiple genes are measured, the expression levels measured in step (b) are "matching" the expression levels in corresponding synchronized cells derived from AD patients, for example, if, with respect to at least the majority of the gene expression levels measured, each such level independently matches the expression level of that gene in corresponding synchronized cells derived from AD patients.

[0052] In the method of this subject, the step of measuring the gene expression level can be achieved by any suitable method known in the art. In a preferred embodiment, the step of measuring the gene expression level includes measuring the number of RNA transcripts of that gene per total number of transcripts.

[0053] In a preferred embodiment, the present invention relates to a method for determining whether a human subject has AD or not, when the subject is suspected of having AD or not having ADD, (a) a step of synchronizing a population of cultured skin fibroblasts derived from the subject, wherein the synchronization step includes culturing the fibroblasts until they become overconfluent, and then starving the resulting overconfluent fibroblasts; and (b) A step of measuring the expression levels of each of the following genes in the obtained synchronized fibroblast population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein the step of measuring the expression level of each gene includes measuring the number of RNA transcripts per total number of transcripts. The present invention provides a method that includes (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD.

[0054] In another preferred embodiment, the present invention relates to a method for determining whether a human subject has AD or not has ADD when the subject is suspected of having AD or not having ADD, (a) a step of synchronizing a population of cultured immortalized B lymphocytes derived from the subject, wherein the synchronizing step includes culturing the lymphocytes until they become overconfluent, and then starving the resulting overconfluent lymphocytes; and (b) A step of measuring the expression levels of each of the following genes in the obtained synchronized lymphocyte population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein the step of measuring the expression level of each gene includes measuring the number of RNA transcripts per total number of transcripts. The present invention provides a method that includes (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD.

[0055] The present invention further relates to a method for determining whether a human subject is suffering from AD, non-ADD, or neither disorder ("NDS") when the subject is suspected to be suffering from AD or non-ADD, (a) A process of synchronizing a suitable population of cells derived from the subject; and (b) A step of measuring the expression levels of genes known to be differentially expressed among corresponding synchronized cells derived from AD patients, corresponding synchronized cells derived from non-ADD patients, and corresponding synchronized cells derived from NDS subjects in the obtained synchronized cell population. The method provides a way to determine whether a human subject has AD or not, by including the following: (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD; (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject has not ADD; and (iii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD subject, the subject has neither AD nor non-ADD. Various embodiments of the above diagnostic method for determining whether a human subject has AD or not can be applied to this method with necessary modifications.

[0056] The present invention further provides a method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of a drug known to conveniently affect the expression level of one or more genes whose expression levels correlate with Alzheimer's disease. Preferably, the gene is selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. In one embodiment, the gene is selected from the group consisting of IL18R1, PSMB9, TTC26, WASF2, ACP6, CARNS1, NPPA-AS1_3, SCG2, and SDHD. In another embodiment, the gene is IL18R1, PSMB9, TTC26, WASF2, ACP6, CARNS1, NPPA-AS1_3, SCG2, or SDHD.

[0057] The present invention further provides a method for treating a human subject suffering from Alzheimer's disease, the method comprising administering to the subject a therapeutically effective amount of a drug selected from the group consisting of: carfilzomib (Kyprolis) (登録商標) , Onyx Pharmaceuticals), Bortezomib (Velcade (登録商標) , Takeda Oncology), bumetanide (Bumex (登録商標) , Hoffman-La Roche), Furosemide (Lasix (登録商標) ), torasemide (Demadex (登録商標)), flavin mononucleotide, phosphoric acid, riboflavin, γ-aminobutyric acid, adenosine monophosphate, histidine, L-arginine, cisplatin, clozapine, cyclosporin A, dexamethasone, etanercept, ethanol, filgrastim, glucose, haloperidol, heparin, infliximab, leflunomide, nitric oxide, oxygen, polyethylene glycol, prednisolone, progesterone, tacrolimus, thalidomide, zinc, calcitriol, calcium, serine, acetylcholine, capsaicin, dopamine, histamine, lithium, norepinephrine, succinic acid, formic acid, tromethamine, citric acid, 10Z-hymenialdisine (Tocris), JIB 04 (Tocris), CRT 0066101 (Tocris), celastrol, dihydroeponemycin, noradrenaline bitartrate (Tocris), or any other drug listed in Table 7. In a preferred embodiment, the drug is carfilzomib, which in one embodiment is administered in the manner set forth in the FDA-approved label for one of its approved indications (e.g., in the approved manner for treating multiple myeloma, wherein the formulation is an injection and is administered at a dose of 30 mg or 60 mg). In another preferred embodiment, the drug is bortezomib, which in one embodiment is administered in the manner set forth in the FDA-approved label for one of its approved indications (e.g., in the approved manner for treating multiple myeloma, wherein the formulation is an injection and has a dose of 3.5 mg or 1.3 mg / m 2(to be administered in doses of ). In another preferred embodiment, the agent is bumetanide, which in one embodiment is administered in the manner described in the FDA-approved labeling for one of its approved indications (for example, in the manner approved for the treatment of edema, where the formulation is an oral preparation and is administered in doses of 0.5 mg, 1 mg, or 2 mg daily, every other day, or daily for 3-4 days, followed by a 1-2 day rest period). In another preferred embodiment, the agent is furosemide, which in one embodiment is administered in the manner described in the FDA-approved labeling for one of its approved indications (for example, in the manner approved for the treatment of edema or hypertension, where the formulation is an oral preparation and is administered in doses of 20 mg, 40 mg, 60 mg, or 80 mg per day (for example, 40 mg twice daily)). In another preferred embodiment, the agent is torasemide, which in one embodiment is administered in the manner described in the FDA-approved indication for one of its approved indications (for example, in the manner approved for treating edema or hypertension, where the formulation is an oral agent and is administered in doses of 5 mg, 10 mg, 15 mg, or 20 mg per day).

[0058] In another preferred embodiment, the agent is one of cisplatin, clozapine, cyclosporine A, dexamethasone, etanercept, filgrastim, haloperidol, heparin, infliximab, leflunomide, prednisolone, progesterone, tacrolimus, thalidomide, or calcitriol, which in one embodiment is administered in the manner described in the FDA-approved indication for one of its approved indications.

[0059] For each of 10Z-himenialdisine, JIB 04, CRT 0066101, cerastrol, dihydroeponemycin, norepinephrine bitartrate, and other non-FDA approved drugs, the preferred route of administration is orally, and the preferred doses are 0.1 mg / kg to 100 mg / kg, 1 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, or 15 mg / kg to 20 mg / kg.

[0060] The present invention will be better understood by referring to the following embodiments, but those skilled in the art will readily recognize that the detailed examples described are merely illustrative of the invention, so that they may be better described in the subsequent claims. [Examples]

[0061] Example 1 - Study 1 [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] Example 2 - Study 2; Synchronized cell cycle gene expression study for Alzheimer's disease; Cross-validation of differential gene expression Alzheimer's disease patients (AD; n=6) and non-Alzheimer's type dementia patients (non-ADD; The initial findings of differential gene expression in synchronized dermal fibroblasts between n=2 and the second batch of samples (AD; n=2; non-AD n=3) cross-correlated with the second batch of samples (AD; n=2; non-AD n=3). For the purpose of separating the two batches of samples, the inventors referred to the first set of samples as the "training set" and the second set of samples as the "validation set".

[0065] method The genes were ranked in order of decreasing statistical significance, i.e., with the highest statistical significance at the beginning (examples in Tables 4 and 5). This ranking was based on t-tests (two-tailed, unequal variances) for two groups of samples, AD and non-ADD. The two lists of genes were compared as described below.

[0066] result The number of statistically significant genes was similar in the training and validation sets (Figure 8), with smaller differences associated with lower statistical significance (P<0.10) and larger differences associated with higher statistical significance (P<0.001). The larger differences associated with higher statistical significance (P<0.001) may be due not only to the different sample sizes in the validation set (5) compared to the training set (8), but also to the different types of non-ADD samples in the two sets. This difference suggests a high degree of diversity in the dysregulation pathways.

[0067] The majority of the genes (n=53) shown in Tables 4 and 5 are under the highest statistical significance (P<0.001), and all are under high statistical significance (P<0.01). The presence of the top 40 genes from the training set (Table 4) was checked against a list of 2,077 genes from the validation set (P<0.10; Figure 8). Similarly, the presence of the top 40 genes from the validation set (Table 5) was checked against a list of 2,103 genes from the training set (P<0.10; Figure 8). Since the top 40 genes in Tables 4 and 5 are under the highest statistical significance, they are very likely to have the greatest impact on the detection, treatment, and pathway dysregulation of Alzheimer's disease. The cross-correlation of the top 40 genes in each set adapts to the diversity in non-ADD samples and compensates for the different sample sizes in the validation set (5) and training set (8) by using larger numbers from the opposite set. The analysis was performed using a gene pool (P<0.10). However, ultimately, only genes with similar statistical significance are considered to represent the core of AD dysregulation.

[0068] These initial findings for the 40 most statistically significant genes suggest that approximately 81% of the genes dysregulated in the training set will also be dysregulated in the validation set. However, only about 7.5% of these genes show the same statistical significance in both the training and validation sets (Table 6).

[0069] Those genes that show the same statistical significance in the training and validation sets are very likely to be at the core of the dysregulated pathway, at the core of the genetic biomarkers for AD, and at the core of the therapeutic targets for AD.

[0070] [Table 4-1] [Table 4-2]

[0071] [Table 5-1] [Table 5-2]

[0072] [Table 6-1] [Table 6-2] [Table 6-3]

[0073] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

[0074] [Table 8-1] [Table 8-2]

[0075] Example 3 - Study 2; TPM value Reference range The mean and standard deviation were calculated for each of the two groups—for each gene—with respect to the total transcription per million (TPM) values ​​for Alzheimer's disease (AD) and non-ADD. The reference range was then calculated as mean ± 2 standard deviations, according to Horn and Pesce (Reference Intervals: A User's Guide. Paul S. Horn and Amadeo J. Pesce. Washington, DC: AACC Press, 2005, ISBN 1-59425-035-9). The reference range thus calculated ensures that 95% of all possible values ​​in each population (AD or non-ADD) are considered.

[0076] The gap between AD and non-ADD If there is no overlap between the reference ranges for AD and non-ADD, a gap exists between the two bell-shaped curves, which indicates a clear diagnosis.

[0077] When there is overlap in the reference ranges for AD and non-ADD (light gray in Table 9), the diagnosis may be false positive or false negative. Genes showing overlap within that reference range, i.e., no gap (light gray), were excluded from the final vector diagnosis. Genes showing a mean of 0 in either of the groups (either the AD group or the non-ADD group) were also excluded.

[0078] Cutoff for each gene The cutoff for each of the remaining 26 genes (Table 10) was determined as the midpoint of the gap in the reference range.

[0079] Genetic vector AD diagnosis The AD diagnosis is based on the 26 components / genes of that vector. For each of these components, the last column indicates whether the value is greater than (>) or less than (<) its cutoff value for each gene.

[0080] [Table 9-1] [Table 9-2] [Table 9-3]

[0081] [Table 10-1] [Table 10-2]

[0082] References 1. Chen M et al. “Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming”, PLoS ONE 7(4) (2012). 2. Baghdadchi N. “The Effects of Serum Starvation on Cell Cycle Synchronization”, OSR Journal of Student Research (2013). 3. Hayes O et al. “Cell confluency is as efficient as serum starvation for inducing arrest in the G0 / G1 phase of the cell cycle in granulosa and fibroblast cells of cattle”, Anim. Reprod. Sci.87(3-4):181-92 (2005). 4. Spellman PT et al, ”Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization” Mol. Biol. Cell. 9(12):3273-97(1998).

Claims

1. AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD in synchronized cell populations derived from human subjects A method for determining whether a human subject has Alzheimer's disease ("AD") or non-ADD (non-ADD) when the subject is suspected of having AD, using the expression levels of five or more genes selected from the group consisting of SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70 as an indicator of whether the subject has AD or non-ADD, The five or more genes mentioned above include at least one gene selected from AC004057.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, LINC01393, MAP1LC3B2, NHLH1, NPPA-AS1_3, PAN3, PHBP8, PSMB9, RAB3IP, RPL5, SCG2, SHISA5, SLC45A3, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, and the method is (a) A step of synchronizing a suitable population of cells derived from the subject; and (b) In the synchronized cell population obtained, AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF A step of measuring the expression level of five or more genes selected from a group consisting of 70, wherein the five or more genes include at least one gene selected from AC004057.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, LINC01393, MAP1LC3B2, NHLH1, NPPA-AS1_3, PAN3, PHBP8, PSMB9, RAB3IP, RPL5, SCG2, SHISA5, SLC45A3, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, This indicates that (i) if the expression level measured in step (b) matches the expression level of the gene measured in the corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene measured in the corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD. The above method is AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN 3. A method that does not include the step of measuring the expression level of each of the following genes: PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. method.

2. The method according to claim 1, wherein the appropriate cells derived from the subject are cultured skin cell fibroblasts.

3. The method according to claim 1, wherein the appropriate cells derived from the subject are cultured B lymphocytes.

4. The method according to claim 3, wherein the B lymphocytes are immortalized.

5. The method according to claim 1, wherein the step of synchronizing the appropriate population of cells includes culturing the cells until they become overconfluent, and then starving the resulting overconfluent cells.

6. The method according to claim 1, wherein each gene whose expression level is measured is known to be expressed at least 50% differentially between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients.

7. The method according to claim 6, wherein each gene whose expression level is measured is known to be expressed at least 100% differentially between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients.

8. The method according to claim 1, wherein the step of measuring the expression level of a gene includes measuring the number of RNA transcripts of the gene per total number of transcripts.

9. Process (b) is AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9 The method according to claim 1, comprising measuring the expression levels of 20 or more genes selected from the group consisting of RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70.

10. Synchronized fibroblast populations derived from human subjects: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, P A method for determining whether a human subject has Alzheimer's disease ("AD") or non-ADD ("non-ADD"), where the expression levels of five or more genes selected from the group consisting of AN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70 are used as an indicator of whether a human subject has Alzheimer's disease ("AD") or non-ADD ("non-ADD"), wherein the five or more genes are AC004057.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM1 The method comprises at least one gene selected from 49B1, LINC01393, MAP1LC3B2, NHLH1, NPPA-AS1_3, PAN3, PHBP8, PSMB9, RAB3IP, RPL5, SCG2, SHISA5, SLC45A3, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, and the method comprises (a) A step of synchronizing a population of cultured skin fibroblasts derived from the subject, wherein the synchronizing step includes culturing the fibroblasts until they become overconfluent, and then starving the resulting overconfluent fibroblasts; and (b) In the synchronized fibroblast population obtained, AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_ 3. Five types selected from the group consisting of OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, or more A step of measuring the expression levels of many genes, wherein the step of measuring the expression level of each gene includes measuring the number of RNA transcripts of that gene per total number of transcripts, wherein the five or more genes include at least one gene selected from AC004057.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, LINC01393, MAP1LC3B2, NHLH1, NPPA-AS1_3, PAN3, PHBP8, PSMB9, RAB3IP, RPL5, SCG2, SHISA5, SLC45A3, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. It includes, This indicates that (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD. The above method is AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN 3. A method that does not include the step of measuring the expression level of each of the following genes: PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. method.

11. Process (b) is AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, R The method according to claim 10, comprising measuring the expression levels of 20 or more genes selected from the group consisting of AB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70.

12. AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SD in a synchronized B lymphocyte population derived from human subjects. A method for determining whether a human subject has Alzheimer's disease ("AD") or non-ADD (non-ADD) when the subject is suspected of having AD, using the expression levels of five or more genes selected from the group consisting of HD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70 as an indicator of whether the subject has AD or non-ADD, The five or more genes mentioned above include at least one gene selected from AC004057.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, LINC01393, MAP1LC3B2, NHLH1, NPPA-AS1_3, PAN3, PHBP8, PSMB9, RAB3IP, RPL5, SCG2, SHISA5, SLC45A3, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, and the method is (a) A step of synchronizing a population of cultured immortalized B lymphocytes derived from the subject, wherein the synchronizing step includes culturing the B lymphocytes until they become overconfluent, and then starving the resulting overconfluent B lymphocytes; and (b) In the synchronized B lymphocyte population obtained, AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_ 3. Five types selected from the group consisting of OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, or more A step of measuring the expression levels of many genes, wherein the step of measuring the expression level of each gene includes measuring the number of RNA transcripts of that gene per total number of transcripts, wherein the five or more genes include at least one gene selected from AC004057.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, LINC01393, MAP1LC3B2, NHLH1, NPPA-AS1_3, PAN3, PHBP8, PSMB9, RAB3IP, RPL5, SCG2, SHISA5, SLC45A3, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. It includes, This indicates that (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject has AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject does not have ADD. The above method is AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN 3. A method that does not include the step of measuring the expression level of each of the following genes: PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. method.

13. Process (b) is AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, R The method according to claim 12, comprising measuring the expression levels of 20 or more genes selected from the group consisting of AB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70.

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