JP5758479B2 - Diagnostic marker for neuropsychiatric disorder, diagnostic method, and therapeutic drug evaluation method - Google Patents

Description

  The present invention relates to a method for determining a disease and a diagnostic agent using a gene whose expression level specifically varies in patients with psychiatric disorders such as schizophrenia. The present invention also relates to a method for searching for a prophylactic / therapeutic agent for psychiatric disorders such as schizophrenia using the gene. Furthermore, the present invention relates to a method for searching a candidate gene for treatment target of mental illness.

Schizophrenia is a mental disorder that develops prominently from puberty to early 20s. The prevalence of schizophrenia is about 1% per population, and the influence of genetic factors is estimated to be around 50%. It accounts for about 60% of all hospitalized patients in Japan.
The main symptom of schizophrenia is that thoughts, emotions and behaviors cannot be combined into one purpose. Patients with schizophrenia develop symptoms such as hallucinations and delusions due to poor interpersonal relationships (positive symptoms). When calming measures are taken for this symptom, negative symptoms such as excessive sleepiness, malaise, lethargy, and withdrawal will appear. Thereafter, these symptoms recover through a recovery period, but about 80% of patients relapse due to psychological and social stress, and the disease becomes intractable as this cycle is repeated. Existing treatments for mental illness can transiently suppress symptoms, but there are few treatments that can improve the essence of the disease state. Therefore, there is an urgent need to develop a method that can accurately determine a mental illness such as schizophrenia and to develop a medicine that can prevent or treat the disease.

  The outline of the onset mechanism of psychiatric disorders such as schizophrenia is considered as follows: (1) Specific mutation of genetic information or modification of expression control system; (2) Change of cell biological function chain ; (3) Functional changes in brain information processing system; (4) Changes in physiological characteristics (reflected in EEG, eye search movement, startle response filtering, cognitive function test results), and (5) Psychiatric symptoms Express. However, since the current diagnostic criteria do not include items (1) to (4), there is a problem that the diversity of biological factors is not considered in the treatment strategy. In other words, in the current diagnosis, only the presence or absence of (5) by the doctor's interview is considered, so among the patients diagnosed with mental illness, there are various patients with different biological factors. This has hindered the development of objective and highly reproducible determination methods and essential therapeutics for mental illness.

  Search for genes with different expression levels between patients who have developed the disease of interest and patients who have not developed the disease, and use the resulting genes as targets for diagnosis of the disease and search for therapeutic agents The differential gene analysis used is widely performed. However, as described above, even if a gene having a difference in expression level between a psychiatric patient and a non-psychiatric person based on the diagnosis name alone is analyzed, noise caused by diversity of biological factors It is difficult to obtain genes that are directly linked to the onset of mental illness. Therefore, development of a new diagnosis method for mental illness based on biological characteristics and a differential gene analysis method is desired.

  On the other hand, CaMKIIα hetero-deficient animals are known to be able to be suitable model animals for schizophrenia because they exhibit strong working memory impairment regardless of age or learning experience (Patent Document 1). ).

JP 2006-327951 A

  An object of the present invention is to provide a novel method for determining a psychiatric disorder based on biological characteristics and a method for searching for a candidate gene for treatment, and further, using a gene obtained by using the search method, It is to provide a method for searching for a new substance capable of preventing or treating a disease.

The present inventors first identified a gene that characterizes a CaMKIIα heterodeficient animal, which is a model animal for schizophrenia. Then, the expression level of the gene was measured for a patient diagnosed with a psychiatric disorder such as schizophrenia through a doctor's inquiry, and hierarchical clustering was performed on the expression level. As a result, gene expression approximated to CaMKIIα hetero-deficient animals was obtained. A cluster of psychiatric patients with a pattern could be identified. Furthermore, by comparing the gene expression profiles of psychiatric patients included in this cluster with those of non-psychiatric non-affected individuals outside the cluster, a gene whose expression level fluctuates in psychiatric disorders was found and the present invention was completed. It came.
Based on the above findings, the present invention has been completed.

That is, the present invention relates to the following.
[1] In the subject of determination,
CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY9, CCND1, CPN1, ND Determining whether or not the subject is suffering from mental illness using as an index the expression level of at least one gene selected from the group consisting of PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1 and NPNT A method for determining mental illness, comprising:
[2] Expression levels of CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, and PCDH8 are used as an index. 1] The method of description.
[3] The method according to [1], wherein it is determined that there is a high possibility that the subject of the determination is suffering from a mental illness when the following expression level variation is confirmed for the gene serving as an index:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; and increased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.
[4] The method according to [1], wherein the mental illness is selected from the group consisting of schizophrenia, bipolar mood disorder, schizophrenia disorder, developmental disorder, and depression.
[5] The method according to [4], wherein the mental illness is schizophrenia.
[6] The method according to claim 1, wherein the gene expression level is measured using any substance selected from the following (i) to (iii):
(I) a nucleic acid probe or primer capable of specifically detecting a transcription product of a gene serving as an index;
(Ii) an antibody that specifically recognizes the translation product of the gene as an indicator; and (iii) a ligand or receptor that specifically binds to the translation product of the gene as an indicator.
[7] The method of [1], wherein the expression level in the hippocampus is used as an index.
[8] A diagnostic agent for mental illness comprising any of the following substances (i) to (iii): (i) ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, TDO2, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, NPIL A nucleic acid probe or primer capable of specifically detecting a transcript of at least one gene selected from the group;
(Ii) an antibody that specifically recognizes the translation product of at least one gene selected from the group; and (iii) a ligand or receptor that specifically binds to the translation product of at least one gene selected from the group. body.
[9] The agent according to [8], comprising any group selected from the following (ia) and (iia).
(Ia) Specific detection of each transcript of CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C and PCDH8 A group of nucleic acid probes or primers that may be; and A group of antibodies that specifically recognize each translation product of.
[10] A method for searching for a substance capable of preventing or treating mental illness, comprising the following steps:
(I) a test substance,
ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, TDO2, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, 24 Contacting with a cell capable of measuring the expression level of any gene selected from the group consisting of ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, and NPNT;
(II) measuring the expression level of the gene selected in the above (I) in the cell contacted with the test substance, and comparing the expression level with the expression level of the gene in the control cell not contacted with the test substance And (III) on the basis of the comparison result of (II) above, selecting the test substance that caused the following expression level variation for the gene as a substance capable of preventing or treating mental illness:
Increased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or decreased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.
[11] The method of [10], wherein the cell is a hippocampal cell.
[12] The method according to [10], wherein the expression level of the gene to be measured in the cell contacted with the test substance is in any of the following states:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or increased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.
[13] The method according to [10], wherein the contact between the test substance and the cell is performed in a living body of a non-human mammal.
[14] The method according to [13], wherein the expression level of the gene to be measured in the hippocampus of a non-human mammal to which the test substance is administered is in any of the following states:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or increased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.
[15] The method according to [13], wherein the non-human mammal is a CaMKIIα hetero-deficient animal.
[16] The method according to [10], wherein the gene to be measured is TDO2, and the expression level of TDO2 is measured using a radioisotope-labeled tryptophan.
[17] The gene expression level is determined by immunohistochemical staining, radioimmunoassay, competitive assay, enzyme-linked immunosorbent assay, western blot, flow cytometry analysis, RT-PCR, in situ hybridization, The method according to [10], which is measured by Northern blot hybridization method, dot blot hybridization method, PET method, MRI method, or near infrared spectroscopy (NIRS).
[18] The method according to [10], wherein the mental illness is selected from the group consisting of schizophrenia, bipolar mood disorder, schizophrenia disorder, developmental disorder and depression.
[19] The method of [18], wherein the mental illness is schizophrenia.
[20] A method for searching for a substance capable of preventing or treating mental illness, comprising the following steps:
(I) contacting the test substance with neural progenitor cells;
(II) comparing the degree of maturation of a neural progenitor cell contacted with a test substance into a mature neuron with that of a control neural progenitor cell not contacted with the test substance; and (III) (II) above Based on the comparison result, a test substance that promotes the maturation of neural progenitor cells into mature neurons is selected as a substance that can prevent or treat mental illness.
[21] The method of [20], wherein the neural progenitor cells are derived from the hippocampus.
[22] The method according to [20], wherein the contact between the test substance and the neural progenitor cell is performed in a living body of a non-human mammal.
[23] The method of [22], wherein the non-human mammal is a CaMKIIα hetero-deficient animal.
[24] The method according to [20], wherein the mental illness is selected from the group consisting of schizophrenia, bipolar mood disorder, schizophrenia disorder, developmental disorder and depression.
[25] The method according to [24], wherein the mental illness is schizophrenia.
[26] CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY8, CCND1, CPY8 A nucleic acid comprising a nucleic acid probe or primer capable of specifically detecting a transcription product of two or more genes selected from the group consisting of PDYN, PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1 and NPNT A group of probes or primers.
[27] CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY8, CCND1, ADCY8, CCND1 , PDYN, PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1, and a group of nucleic acid probes including a nucleic acid probe capable of specifically detecting a transcription product of two or more genes selected from the group consisting of NPNT A nucleic acid array in which is immobilized.
[28] CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY8, CCND1, CPY8 A group of antibodies comprising an antibody that specifically recognizes each translation product of two or more genes selected from the group consisting of PDYN, PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1, and NPNT.
[29] CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY8, CCND1, CPY8, CCND1 , PDYN, PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1, and a group of antibodies including antibodies that specifically recognize each translation product of two or more genes selected from the group consisting of NPNT are fixed Antibody array.
[30] A method for searching a candidate gene for treatment target of psychiatric disorder, comprising the following steps:
(I) identifying a gene whose expression level is significantly different from that of a wild-type control animal in a model animal for mental illness;
(II) Hierarchical clustering analysis of human population including humans diagnosed as having or not suffering from mental illness based on psychiatric symptoms based on the expression level of the gene identified in (I) in the patient To identify a cluster of accumulated psychiatric patients; and (III) in the psychiatric patients included in the clusters identified in (II), the expression level is significantly higher than that of non-psychiatric patients Identifying different genes as candidate treatment targets for psychiatric disorders.
[31] The method of [30], wherein the mental illness is schizophrenia.
[32] The method according to [30], wherein the model animal is a CaMKIIα gene hetero-deficient animal.

  According to the present invention, it is possible to accurately and objectively determine whether or not the subject suffers from schizophrenia. Further, according to the present invention, a therapeutic target gene for the disease based on the biological characteristics of the mental disease is newly provided, and an unprecedented approach to a medicine capable of preventing or treating the mental disease by using the gene Can be screened.

A method for searching for a specific gene that characterizes CaMKIIα HKO mice is schematically shown. The expression in the hippocampus of a wild-type mouse (40 weeks old) and a CaMKIIα HKO mouse (12 weeks old) is shown for genes corresponding to 130 probe sets whose expression levels are significantly changed in CaMKIIα HKO mice. Green indicates a decrease in expression level, and red indicates an increase in expression level. The expression of 10 genes whose expression levels are significantly changed in CaMKIIα HKO mice and schizophrenia patients in the hippocampus of wild-type mice (40 weeks old) and CaMKIIα HKO mice (40 weeks old) is shown. The result of the hierarchical clustering analysis based on the expression level of 10 genes is shown. The mark at the right end of each column indicates the patient with the corresponding disease. It can be seen that a cluster in which schizophrenic patients, bipolar mood disorder patients, and schizophrenia patients accumulate is formed. A cluster of depressed patients and suicides is not formed. The search method of the psychiatric disorder change gene using donor selection based on a cluster is shown typically. The expression level in the hippocampus of CD24 found in the present invention is shown. White column / circle: donor not diagnosed with mental illness, black column / circle: schizophrenia, gray column / circle: donor not diagnosed with psychiatric disorder in the schizophrenia cluster. The expression level in the hippocampus of CALB1 (Encinas JM et al., Proc. Natl. Acad. Sci. USA, 103, p. 8233-8238 (2006)), which is the most typical marker of mature granule cells, is shown. White column: specific control in negative cluster, black column: schizophrenia patient in positive cluster, gray column: donor not diagnosed with psychiatric disorder in positive cluster. It shows that BrdU-labeled cells are increased in the hippocampal dentate gyrus of CaMKIIα HKO mice. It shows that PSA-NCAM positive cells are increased in the hippocampal dentate gyrus of CaMKIIα HKO mice. Reduced expression of CALB1 (Encinas JM et al., Proc. Natl. Acad. Sci. USA, 103, p. 8233-8238 (2006)), the most representative marker of mature granule cells, in the hippocampus of CaMKIIα HKO mice Indicates that

1． Method for determining mental illness
CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY9, CCND1, CPN1, ND PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1 and NPNT
A method for determining a mental illness comprising determining whether or not the subject is suffering from a mental illness using as an index the expression level of at least one gene selected from the group consisting of:

  In the present specification, the mental illness refers to a disease caused by a functional or organic disorder of the brain or heart. Psychiatric disorders include schizophrenia with features associated with working memory impairment, bipolar mood disorder, schizoaffective disorder, developmental disorders (such as autism, Asperger syndrome, attention deficit hyperactivity disorder), depression Diseases can be mentioned. The mental illness is preferably schizophrenia.

  The subject of determination by the method of the present invention is usually a human.

  In the present specification, genes used in the present invention are collectively referred to as “mental disease-related genes”. The existence of any psychiatric disorder-related gene is known. Nucleotide sequences and polypeptide sequences of each psychiatric disorder-related gene are also known and registered in public databases such as GenBank and can be used by those skilled in the art. The mental disease-related gene used in the present invention is usually derived from a human. Mental illness related genes are listed in Table 1.

  Table 1 shows GenBank accession numbers of typical polynucleotide (mRNA or cDNA) sequences and polypeptide sequences of each psychiatric disorder-related gene (from human). Each sequence is shown in the sequence listing. In the present specification, the nucleotide sequence is described as a DNA sequence unless otherwise specified. However, when the polynucleotide is RNA, thymine (T) is appropriately read as uracil (U).

  Among the above mental disease-related genes, 10 genes of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and TDO2 are the genes compared to the wild type in CaMKIIα heterodeficient animals. It was selected from 130 genes that show biological characteristics (gene expression in the hippocampus) of the manipulated animals regardless of their age or learning experience. Hierarchical clustering of the expression levels of these genes makes it possible to clearly discriminate between the genetically engineered animal and the wild-type animal, and to distinguish between a psychiatric patient (especially a schizophrenia patient) and a healthy person. CaMKIIα heterozygous animals show strong working memory impairment regardless of age or learning experience, and this trait is in common with the characteristic symptoms of schizophrenia. That is, the CaMKIIα heterodeficient animal is a schizophrenia model having both a strong working memory disorder, which is a symptom characteristic of schizophrenia, and a biological factor of the disorder. By using the above 10 genes, robust working memory disorder among patients diagnosed as exhibiting psychiatric symptoms peculiar to psychiatric disorders (eg schizophrenia) through dialogue with medical staff To eliminate patients who do not have biological characteristics (gene expression changes) related to cancer (outliner), and to select patients who have psychiatric symptoms based on a common biological basis This makes it possible to determine a more definitive mental illness.

  Among the mental disease-related genes, CUTL2, ZNF492, PIP3-E, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C and PCDH , Patients who have manifested psychiatric symptoms peculiar to psychiatric disorders (for example, schizophrenia), and have biological characteristics (gene expression changes) related to the above-mentioned strong working memory impairment of CaMKIIα heterozygous animals Has been identified as a gene exhibiting a significant change in expression level compared to non-psychiatric patients, and so far, there has been a clear association between expression level changes in the hippocampus and working memory disorders or molecular conditions of mental disorders It has never been done. By using any of the genes selected from the 20 genes described above, mental disorders that have both mental symptoms of mental disorders and biological characteristics (gene expression changes) associated with robust working memory impairment (for example, Patients with schizophrenia can be clearly distinguished from non-psychiatric patients.

  Among the psychiatric disease-related genes, TDO2, DSP, IL-1R1, and NPNT show significant changes in expression levels in CaMKIIα heterodeficient animals compared to wild-type animals and are responsible for working memory impairment. It is a gene expressed specifically in the hippocampal dentate gyrus, and has been identified as a corresponding human ortholog. By using any gene selected from the above four genes, a patient with a mental illness (eg, schizophrenia) can be clearly identified.

  The determination method of the present invention is characterized by using the expression level of one or more genes selected from the above-mentioned mental disease-related genes as an index.

  The number of genes whose expression level is used as an index in the determination method of the present invention is not particularly limited, and the expression level of at least one gene selected from the above-mentioned mental disease-related genes may be used as an index. For the purpose of improving the accuracy of the test, selected from the above mental disease-related genes, for example, 2 or more, preferably 5 or more, more preferably 10 or more, still more preferably 15 or more, still more preferably 20 or more, even more preferably May use as an index the expression level of 30 or more, most preferably 32 genes.

In a preferred embodiment, ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, and TDO2 are selected from two or more, preferably 5 or more, more preferably 8 or more, and most preferably 10 The gene expression level is used as an index. In particular, using the expression level of all the 10 genes described above as an index, and subjecting a human population including psychiatric patients to hierarchical clustering analysis, a patient population having both psychiatric symptoms and biological characteristics of psychiatric disorders is obtained. Since it accumulates in one cluster, it is solid from patients diagnosed as having manifested psychiatric symptoms peculiar to psychiatric disorders (for example, schizophrenia) as superficial symptoms through dialogue with medical professionals, etc. This makes it possible to determine a more definitive mental illness that excludes patients (outliners) who do not have biological characteristics (gene expression changes) related to working memory impairment.

  In another preferred embodiment, the group consisting of CUTL2, ZNF492, PIP3-E, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, PCDH8 An expression level of 2 or more, preferably 5 or more, more preferably 10 or more, further preferably 15 or more, and most preferably 20 selected from the above is used as an indicator.

  Specifically, in the determination method of the present invention, i) the expression level of at least one gene selected from the above-mentioned mental disease-related gene in the tissue of the determination subject is measured, and ii) based on the measured expression level Determining whether the subject is suffering from a mental illness.

  The measurement of the expression level in step i) is performed by measuring the expression level of the gene product of each gene. The gene product is a comprehensive meaning of a transcription product and a translation product. Transcript refers to RNA generated from a psychiatric disorder-related gene through a transcription process, preferably mRNA (immature mRNA, mature mRNA, etc.), more preferably transcription / processing process from immature mRNA. It refers to the mature mRNA produced through A translation product refers to a protein (polypeptide) produced from a mental illness-related gene through a transcription / translation process. The translation product may be unmodified or post-translationally modified. Examples of post-translational modifications include modifications with phosphoric acid, sugars or sugar chains, phospholipids, lipids, nucleotides, and the like.

  The measurement of the expression level in step i) is performed by an in vitro method for measuring the expression level of the mental disease-related gene in a biological sample collected from the determination subject, or the expression of the mental disease-related gene in the determination subject's living body. This can be done by in vivo methods that measure levels directly.

  When measuring the expression level of a gene transcript by an in vitro method, first, a gene transcript is prepared from a biological sample. Preparation of the transcription product can be performed by a method well known in the art, or may be performed using a commercially available kit.

  Examples of the biological sample derived from the subject include, but are not limited to, tissues, cells, liquid components, and the like. The biological sample is separated from the subject by biopsy or the like so as not to cause ethical problems.

  The tissue is not particularly limited as long as it can express a gene. For example, the brain (eg, cerebrum, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, Adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeletal muscle, Examples include lymph nodes. The tissue is preferably the brain (eg, cerebrum, cerebellum), peripheral blood, etc., and more preferably the hippocampus (eg, hippocampal dentate gyrus) in the cerebrum.

  The cell is not particularly limited as long as it can express a gene. Hepatocyte, spleen cell, nerve cell, glial cell, pancreatic β cell, bone marrow cell, mesangial cell, Langerhans cell, epidermal cell, epithelial cell, goblet Cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, blood cells, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or between Or a precursor cell, stem cell or cancer cell of these cells. Preferably, the cell is a nerve cell, a glial cell, a blood cell, or a precursor cell or stem cell thereof, more preferably a nerve cell, a glial cell, or a precursor cell or stem cell thereof.

  In the method of the present invention, among the cells described above, cells present in the hippocampus (eg, hippocampal dentate gyrus) are preferably used. In addition, among cells existing in the hippocampus, cells that possess nuclear DNA and whose mRNA expression level changes in response to stimulation of endogenous factors (referred to as “mRNA-expressing cells” in this specification) Are preferably used.

  The liquid component is not particularly limited as long as it can confirm gene expression, and examples thereof include cerebrospinal fluid, blood, serum, plasma, semen, saliva, urine, and sweat. In particular, when the translation product of a psychiatric disorder-related gene is a liquid component such as a cytokine (eg, CXCL12), the amount of the translation product in the liquid component separated from the determination subject is used as an index.

  In a specific embodiment, the amount of CXCL12 protein in blood separated from the determination subject is used as an indicator.

  The expression level of the transcription product can be measured by a method well known in the art, for example, Northern blot hybridization method, dot blot hybridization method, RT-PCR method, nucleic acid array and the like. In the Northern blot hybridization method and the dot blot hybridization method, a probe capable of specifically detecting a transcription product of a mental illness-related gene as an index described in the section of “2. In the RT-PCR method, a primer capable of specifically detecting a transcription product of a psychiatric disorder-related gene as an index described in the section of “2. Diagnostic agent for mental illness” described later is used as a nucleic acid array. Each of the nucleic acid arrays described in the section “2.

  When measuring the expression level of the transcript, the measured value can be corrected by a known method. The correction makes it possible to compare the expression levels of transcripts in a plurality of independent biological samples more accurately. The correction of the measured value is performed by correcting the measured value of the expression level of the gene based on the measured value of the expression level of a gene (for example, a housekeeping gene) whose expression level does not vary greatly in the biological sample. . Examples of genes whose expression levels do not vary greatly include GAPDH and β-actin.

  When measuring the expression level of a translation product of a gene by an in vitro method, first, a sample capable of measuring the expression level of the translation product is prepared from a biological sample. When measurement of the expression level of a translation product is performed by ELISA, Western blotting, spot method, aggregation method, immunological method such as antibody array described later, surface plasmon resonance, etc., a liquid sample is usually prepared from a biological sample. For example, when the biological sample is a tissue or a cell, a solubilizate is prepared by mechanically crushing them or treating them with a solubilizer. When measuring the expression level of translation products by an immunological method such as flow cytometry or immunological tissue staining, biological samples such as tissues and cells are appropriately fixed, and stained with antibodies. Is done. Thus, when measuring the expression level of a translation product of a mental disease-related gene by an immunological method, the translation of the mental disease-related gene described in the section of “2. An antibody that specifically recognizes the product can be used. Moreover, the translation product of a mental disease-related gene can also be detected by detecting specific binding between a ligand or a receptor that specifically binds to the translation product of a later-described mental disease-related gene and the translation product by surface plasmon resonance or the like. Can be measured.

When the measurement of the expression level in step i) is performed by an in vivo method, the expression level of the translation product of the gene in the tissue is usually measured. In this case, the expression level of the translation product can be measured by a method well known in the art. For example, positron emission tomography (PET) can be used. When measuring the expression level of a translation product of a psychiatric disorder-related gene by PET, a substance having affinity for the translation product and labeled with a positron-emitting nuclide is administered to the subject, and the substance By detecting the gamma rays emitted when the positrons generated from annihilation disappear, the distribution of the substance in the living body, that is, the change in the expression level of the translation product can be measured non-invasively with a PET image. Examples of the substance include a nucleic acid that specifically binds to a transcription product of a mental illness-related gene described in the section of “2. Diagnostic agent for mental illness”, an antibody that specifically recognizes the translation product, and the translation product. A ligand or a receptor that specifically binds to can be used. Examples of positron emitting nuclides include ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ O, ⁴⁵ Ti, ⁶² Cu, ⁶⁸ Ga, and ⁸² Rb. When the translation product of a psychiatric disorder related gene is a membrane protein (eg, receptor, channel, etc.), PET is preferably used. Examples of such mental disease-related genes include IL-1R1, HTR2C, ATP1A3 and the like.

  In a specific embodiment, the determination method of the present invention comprises administering a positron-emitting nuclide-labeled ligand that specifically binds to IL-1R1 to a subject to be determined, and changing the expression level of IL-1R1 in the subject to PET. And measuring with an image, and using the measured change in the expression level of IL-1R1 as an indicator. Alternatively, a metal nanoparticle labeled ligand that specifically binds to IL-1R1 is administered to a subject to be determined, a change in the expression level of IL-1R1 in the subject is measured by an MRI image, and measured It includes using a change in the expression level of IL-1R1 as an indicator.

  In addition, the expression level of a translation product of a psychiatric disorder-related gene can be determined by measuring the activity of the translation product in a biological sample.

  “Translation product activity” refers to the biological activity of the translation product. Common methods for measuring the activity of translation products include, for example, cyclase activity (Zor et al., Proc. Natl. Acad. Sei. USA, 63, p. 918 (1969)), kinase activity (Park SY et al., J. Biol. Chem. 275, p. 19768-19777 (2000)), protease activity (Meng, L, et al., Proc. Natl. Acad. Sci. USA, 96, p. 10403- 10408 (1999)), cell growth regulatory activity (Macleod, RJ et al., Proc. Natl. Acad. Sei. USA, 88, p. 552-556 (1991)), protein-protein interaction (edited by Masato Okada et al., “Proceedings of protein experiments”, Yodosha, p. 172 (2001)), nuclease activity, chemokines, chemokine receptors (Zhou N et al., J. Biol. Chem., 276, p. 42826-42833, (2001)), cytokines, cytokine receptors (Piek E et al., J. Biol. Chem., 276, p. 19945-19953 (2001)), transcription factors (Zhao F et al., J. Biol. Chem. , 276, p. 40755-40760 (2001)), cell adhesion factor (Fujiwara H et al., J. Biol. Chem., 276, p. 17550-1 7558 (2001)), extracellular matrix protein (Miyazaki K et al., Proc. Natl. Acad. Sci. USA, 90, p. 11767 (1993)), phosphatase (Aoyama K et al., J. Biol. Chem) , 276, p. 27575-27583 (2001)), ion channels (Hamill, OP et al., Pfluegers Arch., 391, p. 85-100 (1981)) and the like. The activity of the translation product of TDO2 can be measured using radioisotope-labeled tryptophan by the method described in, for example, Salter M et al., Biochem. Pharmacol. 49, p. 1435-1442 (1995).

  In a preferred embodiment, in step i), the expression level of a psychiatric disorder-related gene in the hippocampus is measured in both in vitro and in vivo methods.

  In step ii), it is determined whether or not the subject is suffering from a mental illness using the measured expression level as an index. As shown in the examples below, in patients with mental illness (eg schizophrenia), TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL -1R1 and NPNT expression levels are reduced compared to healthy individuals, ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121 The expression levels of DKFZp434G1615 and KIAA1651 are increased. The above determination is made based on such a correlation between the expression level of a psychiatric disorder-related gene and the morbidity of the psychiatric disorder.

  For example, the expression level of a psychiatric disorder-related gene in a subject is compared to a human who is not afflicted with a mental illness (eg, a healthy person) (negative control) and a patient who is afflicted with a mental illness (positive control). Alternatively, a correlation diagram between the expression level of a mental disease-related gene and the morbidity of the mental disease may be prepared in advance, and the expression level of the mental disease-related gene in the target patient may be compared with the correlation diagram. Comparison of expression levels is preferably performed based on the presence or absence of statistical significance.

Then, for the psychiatric disorder-related gene as an index, if the following expression level variation is confirmed, it can be determined that the subject of the determination is relatively likely to have a mental illness:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT;
Increased expression level: ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.

  Alternatively, a cut-off value of the expression level of the mental disease-related gene is set in advance, and the expression level of the mental disease-related gene in the subject is compared with this cut-off value. For example, if the mental disease related gene as an index is TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, or NPNT, When the expression level of a psychiatric disorder-related gene is equal to or lower than the cut-off value, it can be determined that the possibility of suffering from a psychiatric disorder is relatively high. On the other hand, when the psychiatric disorder-related gene as an index is ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615 or KIAA1651 When the expression level of the psychiatric disorder-related gene in the subject is equal to or higher than the cut-off value, it can be determined that the possibility of suffering from psychiatric disorder is relatively high. The “cut-off value” is a value that can satisfy both of high diagnostic sensitivity (prevalence of correct diagnosis) and high diagnostic specificity (prevalence of nondiagnostic diagnosis) when a disease is determined based on the value. For example, the expression level of a psychiatric disorder-related gene that shows a high positive rate in subjects suffering from mental illness and a high negative rate in subjects not suffering from mental illness, as a cut-off value It can be set.

  When the expression level of two or more mental disease-related genes is used as an index, it is preferable to perform a hierarchical clustering analysis on the expression level of a population including a person to be determined, a mental disease patient, and a non-psychiatric patient. The expression level of the psychiatric disorder-related gene in humans other than the subject of determination may be a value measured at the same time as the subject or may be a value determined in advance. As a result of the hierarchical clustering analysis, when the determination target person belongs to a cluster in which mental disease patients are accumulated, it can be determined that the target person is highly likely to suffer from mental disease. In particular, when using the expression level of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, and TDO2 as an index, hierarchical clustering analysis of the expression level for the population, A population of patients with both psychiatric symptoms and biological characteristics accumulates in one cluster. Therefore, biological features (genes) related to strong working memory impairment among patients diagnosed as having manifested psychiatric symptoms peculiar to psychiatric disorders (for example, schizophrenia) through dialogue with medical professionals. It becomes possible to determine a more definitive mental illness by excluding patients (outliner) who do not have (expression change).

2． The diagnostic agent for mental illnesses The present invention also provides a diagnostic agent for mental illnesses that can perform the determination method of the present invention.
The diagnostic agent of the present invention includes any substance selected from the following (i) to (iii):
(I) a nucleic acid probe or primer capable of specifically detecting a transcript of at least one gene selected from the group consisting of the above-mentioned mental disease-related genes;
(Ii) an antibody specifically recognizing a translation product of at least one gene selected from the mental disease-related gene; and (iii) specific to a translation product of at least one gene selected from the mental disease-related gene. Ligand or receptor that binds to

  The probe refers to a nucleic acid molecule that is used for detection of a target nucleic acid molecule by specific hybridization to a target nucleic acid molecule (for example, mRNA, cDNA, chromosomal DNA, etc. of a psychiatric disorder-related gene). A primer refers to a nucleic acid molecule that serves as a starting point from which a polynucleotide chain extends during a nucleic acid synthesis reaction.

  Examples of the nucleic acid probe or primer of (i) include a nucleic acid molecule having a nucleotide sequence of at least one gene selected from the group consisting of the above mental disease-related genes, a partial sequence thereof, or a complementary sequence thereof.

The “gene nucleotide sequence” is a full-length nucleotide sequence of chromosomal DNA, mRNA, or cDNA encoding the gene, and is not particularly limited, but is preferably a full-length nucleotide sequence of mRNA or cDNA.
Each gene is composed of a structural gene encoding a protein and a regulatory gene that regulates its expression, and is not particularly limited, but preferably each gene is a structural gene.
The mRNA is immature mRNA or mature mRNA, and is not particularly limited, but is preferably mature mRNA.

  When the nucleic acid molecule has a partial sequence of the nucleotide sequence of a psychiatric disorder-related gene, the length of the partial sequence is not particularly limited and can be appropriately selected depending on the purpose of use of the nucleic acid molecule, but is usually 15 bp. Set as above.

  When nucleic acid molecules are used as probes, the length of the partial sequence of each nucleic acid molecule is sufficient to detect the target nucleic acid molecule or to achieve specific hybridization to the target nucleic acid molecule. Although not particularly limited, for example, it may be at least 15 bp, preferably at least 20 bp, more preferably at least 25 bp, even more preferably at least 30 bp or more.

  When the nucleic acid molecule is used as a primer in a PCR method or the like, the length of the partial sequence contained in the nucleic acid molecule is not particularly limited as long as it is sufficient for the extension reaction of the target nucleic acid molecule, but usually 15 to 100 bp, Preferably it is 18-50 bp, More preferably, it is 18-35 bp. The primer (set) is not particularly limited, but is preferably selected so that the size of the specific gene amplification product is 50 to 1000 bp, more preferably 100 to 600 bp, from the viewpoint of gene amplification reaction efficiency. The primer (set) can be easily designed by those skilled in the art based on the nucleotide sequence of the psychiatric disorder-related gene to be amplified.

  The nucleic acid molecule may be DNA or RNA.

  The configuration of the nucleic acid molecule is not particularly limited as long as it has the nucleotide sequence described above or a partial sequence thereof or a complementary sequence thereof. For example, the nucleic acid molecule includes i) a nucleic acid molecule consisting of the above-described nucleotide sequence or a partial sequence thereof or a complementary sequence thereof; ii) a nucleotide sequence in addition to the above-described nucleotide sequence or a partial sequence thereof or a complementary sequence thereof; It can be a nucleic acid molecule comprising a sequence.

  As one embodiment of the nucleic acid molecule of ii), for example, the nucleic acid molecule introduced into an arbitrary vector (for example, a subcloning vector, an expression vector, etc.) as an insert can be mentioned. For example, when an expression vector is used as the above arbitrary vector, the nucleic acid molecule as an insert may be linked to an expression regulatory element so as to be functional. Examples of expression control elements include any promoter. For example, when expression in E. coli is intended, promoters such as lac and taq are preferably used, and expression in mammalian cells is intended. In this case, promoters such as CAG promoter, SRα promoter, EF1α promoter, CMV promoter, PGK promoter, U6 promoter, SV40 promoter, and early or late promoter of adenovirus are preferably used.

  Nucleic acid molecules can be prepared by methods well known in the art. For example, a nucleic acid molecule having a size of about 100 bp or less can be prepared by an organic chemical method. In addition, when the size of the nucleic acid molecule is large, it can also be prepared by a biological method (including a cell system and a cell-free system). Specifically, as a biological method, a method using a nucleic acid synthase (for example, PCR, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) (for example, refer to WO00 / 56877 pamphlet), LAMP (Loop-mediated isothermal amplification) (for example, see WO00 / 28082 pamphlet), any vector (eg, plasmid, BAC, YAC, phage DNA, etc.) containing each nucleic acid molecule in the group of nucleic acid molecules as an insert And a method for amplifying the vector.

The nucleic acid molecule may be modified. The modification is not particularly limited, and examples thereof include fluorescence (FITC, rhodamine, Texas red, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy. -4,5-dichloro-6-carboxyfluorescein (JOE), hexochloro-6-carboxyfluorescein (HEX), 6-carboxy-tetramethyl-rhodamine (TAMRA), etc.) label, biotin label, digoxigenin label, Examples include alkaline phosphatase labeling and radioisotope ( ³² P, ³ H) labeling.

  In one embodiment, the nucleic acid probe or primer of (i) includes a plurality of nucleic acid probes or primers that can specifically detect each transcription product of two or more genes selected from the group consisting of the above mental disease-related genes, It can be provided as a group of nucleic acid probes or primers.

  A group of nucleic acid probes or primers means a collection of isolated nucleic acid probes or primers that are ready for use at one time, for example, each nucleic acid on a single or multiple solid phases. The probe or primer may be fixed, or each nucleic acid probe or primer may be dissolved in a single or a plurality of liquid phases. For example, the group of nucleic acid probes or primers may be a group in which each nucleic acid probe or primer is individually dispensed on a plate such as a microtiter plate.

  The number of nucleic acid probes or primers included in the group of nucleic acid probes or primers (that is, the number of types of nucleic acid probes or primers) is not particularly limited, but the group is selected from the above-mentioned mental disease-related genes, for example 2 or more, preferably 5 or more, more preferably 10 or more, still more preferably 15 or more, still more preferably 20 or more, even more preferably 30 or more, and most preferably 34, each transcript of a gene is specifically detected. Nucleic acid probes or primers that may be included.

  In one preferred embodiment, the nucleic acid probe or primer of (i) is 2 or more, preferably 5 selected from the group consisting of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and TDO2. As described above, it can be provided as a group of nucleic acid probes or primers including a plurality of nucleic acid probes or primers capable of specifically detecting each transcript of 8 or more, most preferably 10 genes.

  In another preferred embodiment, the group consisting of CUTL2, ZNF492, PIP3-E, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, PCDH8 A plurality of nucleic acid probes or primers capable of specifically detecting each transcription product of 2 or more, preferably 5 or more, more preferably 10 or more, more preferably 15 or more, and most preferably 20 or more genes selected from Can be provided as a group of nucleic acid probes or primers.

  In one embodiment, the group of nucleic acid probes is used as a nucleic acid array. That is, the present invention provides a nucleic acid array on which the group of nucleic acid probes is immobilized. Each nucleic acid probe in the nucleic acid array may be double-stranded, but is preferably single-stranded. In addition, the support for the nucleic acid array is not particularly limited as long as it is a support that is usually used in the art, and examples thereof include membranes (for example, nylon membrane), glass, plastic, and metal.

  In a preferred embodiment, the proportion of nucleic acid probes that can specifically detect the transcription product of the mental disease-related gene among the types of nucleic acid probes included in the nucleic acid array of the present invention is, for example, 10% or more, preferably 20 % Or more, more preferably 40% or more, still more preferably 80% or more.

  As a form of the nucleic acid array, a form well known in the art can be used. For example, an array in which nucleic acids are directly synthesized on a support (so-called affiliometric method), an array in which nucleic acids are immobilized on the support ( So-called Stanford method), fiber type array, electrochemical array (ECA) and the like.

  Affymetrix array refers to a nucleic acid chip in which a nucleic acid probe of a certain length is mounted on a silicon support by photolithography technology and solid phase nucleic acid synthesis technology. This array can be prepared by, for example, synthesizing nucleic acid molecules one base at a time on a support by repeating a process of covering the support with a light shielding plate called a mask and exposing the support. This array can fix nucleic acid probes having the nucleotide sequence of interest at high density, so that genes can be comprehensively detected. Nucleic acid probes can be fixed perpendicular to the support, so that hybridization efficiency is high. Quantitative and reproducibility. It has the advantage of being excellent in.

  The Stanford-type array refers to a nucleic acid array in which nucleic acid probes are attached to a support by covalent bonds or non-covalent bonds. This array is produced, for example, by spotting a nucleic acid probe such as cDNA or synthetic oligo DNA prepared in advance on a support. Compared to the Affymetrix array, this array has more cross-hybridization when a large amount of unpurified cDNA is contained in one spot. However, these disadvantages include the use of synthetic oligo DNA purified as a nucleic acid probe or the covalent attachment of a nucleic acid probe to a support. It can be improved by doing. On the other hand, the present array has advantages such as that an arbitrary nucleic acid probe can be mounted and the running cost is low, as compared with an affiliometric array. The Stanford DNA chip also has the advantage of being easy to make.

  The fiber type array refers to an array in which fibers containing nucleic acid probes are assembled. This array can be produced, for example, by impregnating a hollow fiber with a nucleic acid probe, bundling hollow fibers impregnated with different nucleic acid probes, and slicing the aggregate of the hollow fibers. This array has advantages such as high reproducibility because it can be produced in large quantities with the same quality, and high sensitivity because DNA is fixed in a flexible structure and hybridization efficiency is high.

  Electrochemical array (ECA) refers to a nucleic acid array that electrochemically detects hybridization. Unlike other nucleic acid arrays, the present array detects nucleic acids by using, for example, an intercalator without labeling the nucleic acids. Examples of the detection method include a method of measuring a difference in current generated when an intercalating agent is inserted, and a method of detecting luminescence generated from an intercalating agent that emits light when a voltage difference occurs. This array has advantages such as direct measurement in mRNA, excellent sensitivity and quantification, no need for PCR, and high efficiency of hybridization due to no sample labeling.

  The measurement of the transcription product of a psychiatric disorder-related gene using a nucleic acid array can be performed, for example, by labeling the sample mRNA and then hybridizing it to the nucleic acid array, although it depends on the form of the nucleic acid array used. The label may directly emit a detectable signal or may be a substance that specifically binds to a substance that emits a detectable signal. Examples of labels that emit a signal that can be directly detected include fluorescent substances, alkaline phosphatase, and radioisotopes. Examples of a substance that specifically binds to a substance that emits a detectable signal include biotin, avidin, digoxigenin, and an antibody. Examples of the labeling method include a method of incorporating a labeled nucleotide while replicating a sample nucleic acid using a nucleic acid synthase, and a method of directly binding to a sample nucleic acid by a chemical reaction. In addition, in the case of electrochemical detection, labeling of a nucleic acid is not required. For example, an intercalation that emits light by measuring a current difference using an intercalating agent or when a voltage difference occurs. By detecting the luminescence generated from the agent, the transcript of the psychiatric disorder-related gene can be measured.

  A person skilled in the art can appropriately select an appropriate nucleic acid array among the above-described nucleic acid arrays according to the purpose of the assay, and can also select these nucleic acid arrays by methods well known in the art, for example, as described above. Can be produced.

  The type of antibody (ii) is not particularly limited. For example, the antibody may be a polyclonal antibody or a monoclonal antibody. Also, each antibody in the group of antibodies of the present invention can be a chimeric antibody, a humanized antibody, a human antibody, or an epitope-binding fragment thereof. When post-translational modification of a translation product plays an important role in its function, the antibody is preferably one that can distinguish between an unmodified translation product and a modified translation product.

  When a part of the antibody against the translation product of the psychiatric disease-related gene is commercially available as a polyclonal antibody or a monoclonal antibody, these commercially available products can be used. Polyclonal antibodies or monoclonal antibodies can also be prepared by methods well known in the art. Specifically, the polyclonal antibody is, for example, a translation product of a psychiatric disorder-related gene or a partial peptide thereof (length is usually 6 amino acids or more, such as 8 amino acids or more, preferably 10 amino acids or more, more preferably 12 amino acids or more). ) As an antigen, and optionally together with Freund's Adjuvant, mammals (eg, mice, rats, hamsters, guinea pigs, rabbits), and serum from this immunized mammal (immune immunized product) It can produce by collect | recovering. Monoclonal antibodies are prepared by preparing hybridomas (fusion cells) from antibody-producing cells obtained from the immunized animal and myeloma cells (myeloma cells), cloning the hybridomas, and using them as antigens for immunization of mammals. It can be produced by selecting clones that produce monoclonal antibodies that exhibit specific affinity for them.

The epitope-binding fragment refers to a portion including the antigen-binding region of the antibody described above or a portion derived from the region, and specifically F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody). , ScFv (single chain Fv), dsFv (disulphide stabilized Fv) and the like.

The antibody may be modified. The modification is not particularly limited, and examples thereof include fluorescence (FITC, rhodamine, Texas red, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy. -4,5-dichloro-6-carboxyfluorescein (JOE), hexochloro-6-carboxyfluorescein (HEX), 6-carboxy-tetramethyl-rhodamine (TAMRA), etc.) label, biotin label, digoxigenin label, Alkaline phosphatase label, radioisotope ( ¹²⁵ I, ³² P, ³ H, positron emitting nuclide ( ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ O, ⁴⁵ Ti, ⁶² Cu, ⁶⁸ Ga, ⁸² Rb)) label, iron, etc. And labeling with metal nanoparticles.

  In one embodiment, the antibody of (ii) can be provided as a group of antibodies including a plurality of antibodies that specifically recognize translation products of two or more genes selected from the group consisting of the above-mentioned mental disease-related genes.

  The group of antibodies means a collection of isolated antibodies, which can be used for a single use, for example, each antibody immobilized on a single or multiple solid phases The antibody may be dissolved in a single or a plurality of liquid phases. For example, the antibody group may be a group in which each antibody is individually dispensed on a plate such as a microtiter plate.

  The number of antibodies included in the antibody group (that is, the number of antibody types) is not particularly limited, but the group is selected from the above-mentioned mental disease-related genes, for example, 2 or more, preferably 5 or more, Preferably, it may contain an antibody that specifically recognizes each translation product of 10 genes, more preferably 15 or more, even more preferably 20 or more, even more preferably 30 or more, and most preferably 32 genes.

  In one preferred embodiment, the antibody of (ii) is ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, and TDO2 or more, preferably 5 or more, more Preferably, it can be provided as a group of antibodies comprising a plurality of antibodies that specifically recognize each translation product of 8 or more, most preferably 10 genes.

  In another preferred embodiment, the group consisting of CUTL2, ZNF492, PIP3-E, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, PCDH8 As a group of antibodies, comprising a plurality of antibodies specifically recognizing each translation product of 2 or more, preferably 5 or more, more preferably 10 or more, more preferably 15 or more, most preferably 20 selected from Can be provided.

  In one embodiment, the group of antibodies is used as an antibody array. That is, the present invention provides an antibody array in which the group of antibodies is immobilized. The support for the antibody array is not particularly limited as long as it is a support that is usually used in the art. For example, a membrane (eg, nitrocellulose membrane, nylon membrane), glass, plastic, metal (eg, gold thin film) ) And the like.

  In a preferred embodiment, of the types of antibodies included in the antibody array of the present invention, the proportion of the antibody specifically recognizing the translation product of the mental disease-related gene is, for example, 10% or more, preferably 20% or more, More preferably, it is 40% or more, and further preferably 80% or more.

  The measurement of gene translation products (for example, post-translationally modified protein, unmodified protein) by an antibody array varies depending on the form of the antibody array to be used.For example, a translation product extracted from a sample is labeled, This can then be done by reacting with each antibody on the antibody array. The label may directly emit a detectable signal or may be a substance that specifically binds to a substance that emits a detectable signal. Examples of labels that emit a signal that can be directly detected include fluorescent substances, alkaline phosphatase, and radioisotopes. Examples of a substance that specifically binds to a substance that emits a detectable signal include biotin, avidin, digoxigenin, and an antibody. Examples of the labeling method include a method of directly binding to a translation product by a chemical reaction. When the antibody array is produced on a gold thin film, the presence or absence of binding of the translation product to the antibody can be detected by a known method such as surface plasmon resonance without labeling the translation product.

  An antibody array can be prepared by immobilizing an antibody on a support by a method well known in the art. Examples of the method for immobilizing the antibody on the support include a method of immobilizing by covalent bond and a method of immobilizing by electrostatic bond, but considering the reproducibility of the experiment, a method of immobilizing by covalent bond is preferable. . When the support is glass, plastic, gold thin film, etc., the antibody is immobilized on the support by reacting and binding the functional group of the amino acid contained in the antibody to the functional group on the support surface. You can also.

  Examples of the ligand or receptor (iii) include those listed in Table 1. These ligands and receptors are both known, and when they are proteins, those skilled in the art obtain amino acid sequences from public databases and produce them as recombinant proteins using well-known genetic engineering techniques. I can do it. Since the receptor is usually a membrane protein, it is preferably obtained as a soluble receptor containing a region essential for specific ligand binding for use as a diagnostic agent of the present invention. A soluble receptor can be obtained by producing the extracellular portion of the receptor using genetic engineering techniques.

The ligand and receptor may be modified. The modification is not particularly limited, and examples thereof include fluorescence (FITC, rhodamine, Texas red, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy. -4,5-dichloro-6-carboxyfluorescein (JOE), hexochloro-6-carboxyfluorescein (HEX), 6-carboxy-tetramethyl-rhodamine (TAMRA), etc.) label, biotin label, digoxigenin label, Alkaline phosphatase label, radioisotope ( ¹²⁵ I, ³² P, ³ H, positron emitting nuclide ( ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ O, ⁴⁵ Ti, ⁶² Cu, ⁶⁸ Ga, ⁸² Rb)) label, iron, etc. And labeling with metal nanoparticles.

  By using the substances (i) to (iii) described above and measuring the expression level of a mental disease-related gene in a subject to be determined, the determination method of the present invention can be easily carried out.

In addition to the substances (i) to (iii) described above, the diagnostic agent of the present invention can be combined with additional elements necessary for measurement and storage to make a diagnostic kit for mental illness. For example, if the diagnostic agent of the present invention contains the substance (i), additional elements include blotting buffer, labeling reagent, blotting membrane, chromogenic substrate, 10 × PCR reaction buffer, 10 × MgCl ₂ It may further include an aqueous solution, a 10 × dNTPs aqueous solution, Taq DNA polymerase (5 U / μL), reverse transcriptase, positive control, negative control, instructions describing the diagnostic protocol, psychiatric disease criteria data, and the like. If the diagnostic agent of the present invention contains the substance (ii), as additional elements, labeled secondary antibody, chromogenic substrate, blocking solution, washing buffer, ELISA plate, blotting membrane, positive control, negative control In addition, it can further include instructions describing the diagnostic protocol, data for criteria for determining mental illness, and the like. These elements can be mixed in advance if necessary. Moreover, a preservative and preservative can also be added to each element as needed.

3． Screening method I
The present invention provides a method (search method I of the present invention) for searching for a substance capable of preventing or treating a mental illness, which comprises measuring the expression level of the mental illness-related gene. The search method I of the present invention binds to a transcript (mRNA) of a psychiatric disorder-related gene (cleaves or degrades as necessary), or binds to an expression regulatory site of the gene, or controls the expression of the gene. To regulate the post-translational modification of a translation product of a psychiatric disorder-related gene or a substance that can increase or decrease the expression level of the transcription product or translation product of the gene by regulating the expression or function of other genes This makes it possible to identify substances that increase or decrease the activity expression of the translation product.

  The number of genes whose expression level is measured in the search method I of the present invention is not particularly limited, and the expression level of at least one gene selected from the above-mentioned mental disease-related gene group may be measured. For the purpose of more reliably identifying a substance having a higher therapeutic effect on mental illness, selected from the above-mentioned group of mental illness related genes, for example, 2 or more, preferably 5 or more, more preferably 10 or more, more preferably 15 or more Even more preferably, the expression level of 20 or more genes, even more preferably 30 or more, and most preferably 32 genes may be measured.

The search method I of the present invention specifically includes the following steps:
(I) contacting a test substance with a cell capable of measuring the expression of any gene selected from the mental disease-related genes;
(II) measuring the expression level of the gene selected in the above (I) in the cell contacted with the test substance, and comparing the expression level with the expression level of the gene in the control cell not contacted with the test substance And (III) on the basis of the comparison result of (II) above, selecting the test substance that caused the following expression level variation for the gene as a substance capable of preventing or treating mental illness:
Increased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or decreased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.

  The test substance to be used in the search method I of the present invention may be any known compound or novel compound, for example, using nucleic acid, carbohydrate, lipid, protein, peptide, low-molecular-weight organic compound, combinatorial chemistry technique. Compound libraries, random peptide libraries, natural components derived from microorganisms, animals and plants, marine organisms, and the like.

  Cells that can be used in the screening method I of the present invention are mammalian cells. In this specification, examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, domestic animals such as pigs, cows, goats, horses, and sheep, and pets such as dogs and cats. , Primates such as humans, monkeys, orangutans and chimpanzees.

  The “cell capable of measuring expression” refers to a cell capable of directly or indirectly evaluating the expression level of the transcription product or translation product of the measurement target gene. Examples of the cells that can directly evaluate the expression level of the transcription product or translation product of the measurement target gene include cells that can naturally express the measurement target gene, while the expression of the transcription product or translation product of the measurement target gene. Examples of the cell whose level can be indirectly evaluated include a cell that enables a reporter assay for the transcriptional regulatory region of the gene to be measured.

  The cell that can naturally express the transcription product or translation product of the psychiatric disorder-related gene to be measured is not particularly limited as long as it can potentially express the transcription product or translation product. Examples of the cells include those listed in the section “1. Determination method of mental illness”. The cell is preferably a nerve cell, a glial cell, a blood cell, or a progenitor cell or stem cell thereof, more preferably a nerve cell, a glial cell, or a progenitor cell or stem cell thereof. In the search method of the present invention, hippocampal (eg, hippocampal dentate gyrus) cells are preferably used among the cells described above.

In one preferred embodiment, the cell whose expression can be measured has an expression level of a psychiatric disorder-related gene to be measured in any of the following states:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or increased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.

  A substance capable of preventing or treating a mental illness can be obtained by selecting a test substance having an action of eliminating a state in which the expression level of a psychiatric related gene is reduced or increased using the above cells.

  CaMKIIα-deficient cells can be used as the cells in which the expression level of the psychiatric disorder-related gene is reduced or increased as described above. `` CaMKIIα deficiency '' refers to a state where the CaMKIIα gene is functionally insufficient, that is, a state where the normal function of the CaMKIIα gene cannot be fully exerted, the CaMKIIα gene is not expressed at all, or the CaMKIIα gene is not The expression level of the CaMKIIα gene product is reduced to such an extent that the normal function normally cannot be exhibited, or the function of the CaMKIIα gene product is completely lost, or the CaMKIIα gene cannot exhibit its normal function. The state where the function of is lowered is mentioned.

  Examples of CaMKIIα-deficient cells include CaMKIIα homo-deficient cells or CaMKIIα hetero-deficient cells, preferably CaMKIIα hetero-deficient cells. CaMKIIα-deficient cells can be obtained, for example, by collecting cells from CaMKIIα-deficient animals (CaMKIIα-deficient animals and the like). Alternatively, CaMKIIα-deficient cells can also be obtained by introducing a targeting vector for the CaMKIIα gene into cells and deleting the CaMKIIα gene by homologous recombination.

  For example, hippocampal cells isolated from CaMKIIα gene heterodeficient animals expressed TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1 and NPNT Levels are decreasing and expression levels of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615 and KIAA1651 are increased .

  A cell that enables reporter assay for the transcriptional regulatory region of the gene to be measured is operatively linked to the transcriptional regulatory region of a psychiatric disorder-related gene (for example, DNA consisting of a base sequence of about 2 kbp upstream from the transcription start point). A cell containing a generated reporter gene (eg, GFP gene). Since it expresses physiological transcriptional regulatory factors for psychiatric disorder-related genes and is considered more appropriate by evaluating the regulation of expression of psychiatric disorder-related genes, the cells to be measured are neurons, glial cells, or precursors thereof. Cells or stem cells are preferred. In the search method of the present invention, cells of the hippocampus (eg, hippocampal dentate gyrus) are preferably used.

  Contact between the test substance and the cell can be performed in vitro or in vivo.

  Contact between the test substance and the cell in vitro is performed in a culture medium. The culture medium is appropriately selected depending on the cells capable of measuring the expression of sarcopodin. For example, a minimal essential medium (MEM) containing about 5 to about 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), etc. It is. The culture conditions are appropriately determined in the same manner. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is about 12 to about 72 hours.

  Contact between a test substance and cells in vivo can be performed by administering the test substance to a non-human mammal orally / parenterally.

In one preferred embodiment, the expression level of a psychiatric disorder-related gene to be measured in the hippocampus of a non-human mammal to which a test substance is administered is in any of the following states:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or increased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.

  A substance capable of preventing or treating mental illness can be obtained by selecting a test substance having the action of eliminating the state in which the expression level of a psychiatric disorder-related gene in the hippocampus is reduced or increased using the mammal. .

  Examples of such mammals include CaMKIIα-deficient animals (for example, homo-deficient animals or hetero-deficient animals, preferably hetero-deficient animals).

  In the hippocampus of CaMKIIα heterodeficient animals, the expression levels of TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1 and NPNT are reduced. The expression levels of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615 and KIAA1651 are increased.

  The measurement of the expression level of a psychiatric disorder-related gene in step (II) can be performed according to the method described in the section “1. Determination method of psychiatric disorder”. Specifically, immunohistological staining method, radioimmunoassay method, competitive assay method, enzyme-linked immunosorbent assay method, Western blot method, flow cytometry analysis, RT-PCR method, in situ hybridization method, Northern blot hybridization Or a dot blot hybridization method, a method for measuring the activity of a translation product, or the like can be used. When cells containing a reporter gene are used, the expression level is measured based on the signal intensity of the reporter gene.

  In one embodiment, the gene to be measured is TDO2, and the activity of the translation product is measured using radioisotope-labeled tryptophan as the expression level of TDO2. Specifically, the method is performed by quantifying labeled kynurenine obtained as a tissue homogenate, cell, tissue slice, or in vivo TDO active product using L- [ring-2-14C] -tryptophan as a substrate. . Details of the method are described in Salter M et al., Biochem. Pharmacol. 49, p. 1435-1442 (1995).

  Comparison of expression levels can preferably be performed based on the presence or absence of statistical significance. The expression level of the psychiatric disorder-related gene in the control cells not contacted with the test substance is the expression level measured in advance with respect to the measurement of the expression level of the psychiatric disorder-related gene in the cell contacted with the test substance. Although the expression level measured simultaneously may be sufficient, it is preferable that it is the expression level measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

As a result of the comparison, the test substance that caused the following fluctuation in the expression level of the psychiatric disorder-related gene to be measured is selected as a substance that can prevent or treat mental illness.
Increased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or decreased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.

  The compound obtained by the screening method I of the present invention is useful as a candidate substance for the development of a new preventive or therapeutic drug for mental disorders.

Four. Screening method II
As shown in the examples below, in the hippocampus of CaMKIIα heterodeficient animals that are model animals for schizophrenia, while neurogenesis is increased and the number of neural progenitor cells is increased, an index of mature neurons The expression level of CALB1 is decreased. This suggests that the neuroscientific basis of psychiatric symptoms (eg, working memory impairment) in schizophrenia is an impaired maturation of neural progenitor cells in adults, and its repair can improve the psychiatric symptoms.

Therefore, the present invention provides a method (search method II of the present invention) for searching for a substance capable of preventing or treating mental illness, comprising the following steps:
(I) contacting the test substance with neural progenitor cells;
(II) comparing the degree of maturation of a neural progenitor cell contacted with a test substance into a mature neuron with that of a control neural progenitor cell not contacted with the test substance; and (III) (II) above Based on the comparison result, a test substance that promotes the maturation of neural progenitor cells into mature neurons is selected as a substance that can prevent or treat mental illness.

  Examples of the test substance used in the search method II of the present invention include the same test substances used in the search method I of the present invention.

  Neural progenitor cells that can be used in the screening method II of the present invention are mammalian cells.

  Neural progenitor cells refer to undifferentiated cells that can differentiate only into nerves. Neural progenitor cells can be identified and isolated based on the expression of known marker molecules. Representative marker molecules include PSA and NCAM, and neural progenitor cells are positive for PSA and NCAM. The expression of the marker molecule can be measured by an immunological technique (immunohistological staining, flow cytometry, ELISA, Western blot, etc.) using an antibody that specifically recognizes the marker molecule. Neural progenitor cells can be isolated using, for example, a cell sorter, antibody-bound magnetic beads, or the like.

  The neural progenitor cell may be a natural cell that is not established or may be a established cell. The neural progenitor cells may be derived from any tissue such as brain and spine, but in the search method II of the present invention, cells derived from the hippocampus (eg, hippocampal dentate gyrus) are preferably used.

  In one preferred embodiment, the neural progenitor cells used in the screening method II of the present invention are in a state in which the maturation function is impaired. A substance capable of preventing or treating a mental illness can be obtained by using the cells and selecting a test substance having an action of restoring the impaired maturation function.

  CaMKIIα-deficient cells can be used as neural progenitor cells in which the maturation function is impaired as described above. Examples of CaMKIIα-deficient cells include the same cells as those used in the screening method I of the present invention. For example, the maturation function of hippocampal neural progenitor cells isolated from CaMKIIα gene hetero-deficient animals is reduced compared to wild-type control animals.

  Contact between the test substance and neural progenitor cells can be performed in vitro or in vivo.

  Contact between the test substance and the cell in vitro is performed in a culture medium. The culture medium is appropriately selected depending on the cells capable of measuring the expression of sarcopodin. For example, a minimal essential medium (MEM) containing about 5 to about 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), etc. It is. The culture conditions are appropriately determined in the same manner. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is about 12 to about 120 hours.

  In vivo contact between a test substance and neural progenitor cells can be carried out by administering the test substance to a non-human mammal orally / parenterally.

  In one preferred embodiment, neural progenitor cells in the hippocampus of a non-human mammal to which a test substance is administered are in a state where the maturation function is impaired.

  A substance capable of preventing or treating a mental illness can be obtained by selecting a test substance having an action of restoring the damaged maturation function of neural progenitor cells in the hippocampus using the mammal.

  Examples of such mammals include CaMKIIα-deficient animals (for example, homo-deficient animals or hetero-deficient animals, preferably hetero-deficient animals).

  Evaluation of the degree of maturation of neural progenitor cells into mature neurons in step (II) should be performed according to the method described in the section of “1. Can do. Specifically, immunohistological staining method, radioimmunoassay method, competitive assay method, enzyme-linked immunosorbent assay method, Western blot method, flow cytometry analysis, RT-PCR method, in situ hybridization method, Northern blot hybridization Or a dot blot hybridization method, a method of measuring the activity of a marker molecule, or the like can be used. In addition to DSP, IL1R1, NPNT and the like found in the present invention, as a marker molecule for mature neurons, CALB1 known as a representative marker (Encinas JM et al., Proc. Natl. Acad. Sci. USA) , 103, p. 8233-8238 (2006)).

  Comparison of the degree of maturation into mature neurons can preferably be made based on the presence or absence of statistical significance. The degree of maturation of mature neurons in control cells that were not contacted with the test substance was measured in advance with respect to the measurement of the degree of maturation of mature neurons in the cells that were contacted with the test substance. However, it is preferable that they are measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  As a result of the comparison, the test substance that promotes the maturation of neural progenitor cells into mature neurons is selected as a substance that can prevent or treat mental illness.

  The compound obtained by the screening method II of the present invention is useful as a candidate substance for the development of a new preventive or therapeutic drug for mental disorders.

Five. Method for Searching for a Psychiatric Disease Treatment Target Candidate Gene The present invention provides a method for searching for a mental disease treatment target candidate gene (search method III of the present invention), which comprises the following steps:
(I) identifying a gene whose expression level is significantly different from that of a wild-type control animal in a model animal for mental illness;
(II) Hierarchical clustering analysis of human population including humans diagnosed as having or not suffering from mental illness based on psychiatric symptoms based on the expression level of the gene identified in (I) in the patient To identify a cluster of accumulated psychiatric patients; and (III) in the psychiatric patients included in the clusters identified in (II), the expression level is significantly higher than that of non-psychiatric patients Identifying different genes as candidate treatment targets for psychiatric disorders.

  The model animal for mental illness used in the step (I) is usually a mammal. Examples of mammals include those described in the section “3. Screening Method I”. As the model animal, it is preferable to use a genetically homogeneous animal as much as possible. By using homogeneous animals, individual differences in gene expression profiles can be eliminated, and genes with significantly different expression levels compared to wild-type control animals can be accurately identified in psychiatric disease model animals. From this point of view, the model animal is preferably a gene-deficient animal (for example, a gene-deficient animal) in which a gene having a malfunction is identified in advance. Examples of the model animal include the above-mentioned CaMKIIα-deficient animals (preferably CaMKIIα hetero-deficient animals). As described above, CaMKIIα heterodeficient animals are excellent schizophrenia models.

  The measurement and comparison of the expression level in the step (I) can be performed in the same manner as the method described in the above-mentioned section of “1. Method for determining mental illness” or “2. Diagnostic agent for mental illness”. For the purpose of searching for therapeutic target genes for psychiatric disorders, the expression levels are preferably compared in the brain, more preferably in the hippocampus. Preferably, the level of gene expression in humans is assessed in vitro as a non-medical practice using a biological sample isolated from humans.

  In step (II), for a human population including humans diagnosed as having or not having a mental illness based on the expressed psychiatric symptoms, the gene identified in step (I) in the patient Based on the expression level, a hierarchical clustering analysis is performed to identify a cluster of psychiatric patients. “Aggregation” means that at least 50% of the psychiatric patients to be analyzed belong to the same cluster. From this cluster, patients who were diagnosed as having manifested psychiatric symptoms peculiar to mental illness but had biological characteristics (gene expression changes) similar to those of psychiatric animal models (outliner) ) Is excluded, the psychiatric patients belonging to this cluster can be a relatively homogeneous population having the same or similar biological characteristics (gene expression changes) as the psychiatric model animal.

  In step (III), the expression levels of various genes in patients with psychiatric disorders included in the cluster identified in step (II) are compared with those without psychiatric disorders, and genes with significantly different expression levels are treated for psychiatric disorders Identified as a target candidate gene. The measurement and comparison of the gene expression level can be performed by the method described in the section “1. Determination method of mental illness”.

  For example, when a schizophrenia patient is used as a psychiatric patient and a CaMKIIα heterodeficient animal is used as a model animal for schizophrenia, the treatment target candidate genes are CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121. DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, and the like can be obtained. Among these genes, the expression levels of CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C and PCDH8 are significantly reduced in schizophrenic patients, CUTL2, ZNF492, SCD4, RIMS3, RECK , TCF4, DKFZp586E121, DKFZp434G1615 and KIAA1651 expression levels are significantly increased in schizophrenic patients.

  By applying the method described in the section “3. Screening Method” to the therapeutic target candidate gene obtained by the screening method III of the present invention, a substance capable of preventing or treating a mental illness can be obtained. For example, in a group of psychiatric patients included in the cluster identified in step (II), for a gene whose expression level was significantly decreased compared to non-psychiatric patients, the effect of increasing the expression level of the gene A substance having the above can be a candidate substance capable of preventing or treating a mental illness. In addition, for a gene whose expression level was significantly increased in the patient population as compared with non-affected individuals, a substance having an action of reducing the expression level of the gene can prevent or treat mental illness Can be a candidate substance.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

(Example 1)
Calmodulin-dependent kinase IIα (CaMKIIα) hetero knockout (HKO) mice (The Jackson Laboratory) showed behavioral characteristics such as strong working memory impairment, long-term changes in overactivity in home cages, and anxiety attenuation. These behavioral characteristics were independent of the age of the mice and the presence or absence of learning experience.
For the purpose of characterizing behavioral characteristics at the molecular level, we used 12-week-old mice with no behavioral test experience and 40-week-old mice with experience of behavioral test to determine gene expression in the hippocampus between wild-type mice and CaMKIIα HKO. Comparison was made by GeneChip system (Affimetrix, Mouse Genome 4302.0 Array). As a result, 130 probe sets whose expression levels were significantly changed in CaMKIIα HKO were identified irrespective of the age of the week and experience of behavioral tests (FIGS. 1 and 2).
Compared to reference memory, the characteristic that a disorder specific to working memory is observed is common to CaMKIIα HKO and schizophrenic patients, so it is included in the human sample gene expression database (BioExpress system, Gene Logic) Of 166 hippocampal GeneChip data (based on the GeneChip system (Affimetrix, human U130 Array)), the best 10 probes to characterize 21 schizophrenia patients, and the human GeneChip analysis that can handle the 130 probe set described above Extraction was performed from 57 probes using analysis of variance, and cluster analysis of expression maps was performed. As a result, when using the probe set consisting of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and TDO2, 16 of 18 patients with schizophrenia and 2 patients with bipolar mood disorder A cluster consisting of 2 of them and 1 of 1 patient with schizoaffective disorder was formed (Figs. 3 and 4). The expression of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, and SPATA13 was significantly increased in schizophrenia patients and CaMKIIα HKO mice. PNCK and TDO2 were significantly reduced in expression in schizophrenic patients and CaMKIIα HKO mice.
We compared the difference in gene expression patterns between 19 patients with psychiatric disorders included in this cluster and 30 non-affected psychiatric patients not included in this cluster, and were not affected by age and gender. Twenty-six probes were identified (Figures 5-7, Table 2) that gave significant changes (P <0.001, more than 2-fold change).

  In the table, UP indicates that expression is significantly increased in psychiatric patients, and DOWN indicates that expression is significantly decreased in psychiatric patients.

As a result of analyzing the brain function of the gene corresponding to these 26 probes from the known reports, the change in gene expression level corresponding to 8 probes (CD24 antigen, Cut-like 2, Transcription factor 7-like 2 as gene products) Suggests the promotion of neurogenesis, 4 probes (Reversion-inducing-cysteine-rich protein with kazal motifs (RECK), Chemokine (CXC motif) ligand 12 (CXCL12), Calbindin 28K (CALB1), Protocadherin 8 ( Changes in gene expression levels corresponding to PCDH8 and Arcadlin) were suggested to inhibit neuronal migration or maturation.
From these results, it was suggested that CaMKIIα HKO mice have a common feature with neurological diseases including schizophrenia, and simultaneously suppress adult maturation in the hippocampus and suppress maturation processes.

(Example 2)
Cell neogenesis in the adult hippocampus of CaMKIIα HKO mice used in Example 1 was analyzed by bromodeoxyuridine (BrdU) incorporation method. As a result, it was found that the subglanular zone (SGZ), known as the neurogenesis field, was accelerated about 1.7 times compared to the wild type (FIG. 8). At the same time, the number of PSA-NCAM positive cells, known as a marker for immature granule cells, increased (Figure 9), while the number of mature granule cell markers, Calbindin 28K positive cells, decreased significantly (Figure 10, Encinas JM et al , Proc. Natl. Acad. Sci. USA, 103, p. 8233-8238 (2006)). From the above, it was confirmed at the protein level and histologically that the neurogenesis was promoted in the hippocampus, which was predicted as a transcriptome change in Example 1, and at the same time the suppression of neuronal maturation.
Furthermore, among the probes that showed a marked decrease in expression as a change in mouse GeneChip analysis, tryptophan 2,3-dioxygenase-2, desmoplakin, interleukin 1 receptor type I, 4921511C04Rik, nephronectin and 9930021J17Rik were allen brain atlas ( Allen Institute for Brain Science, Seattle) showed specific expression of hippocampal dentate gyrus, suggesting that these molecules are novel markers related to suppression of granule cell maturation in hippocampal dentate gyrus .

  According to the present invention, it is possible to accurately and objectively determine whether or not the subject suffers from schizophrenia. Further, according to the present invention, a therapeutic target gene for the disease based on the biological characteristics of the mental disease is newly provided, and an unprecedented approach to a medicine capable of preventing or treating the mental disease by using the gene Can be screened.

Claims (12)

In a biological sample collected from a test subject,
CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, ADCY9, CCND1, CPN1, ND Measuring the expression level of at least one gene selected from the group consisting of PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1 and NPNT, and measuring the expression level of the measured gene,
Schizophrenia, bipolar mood disorder, and schizophrenia, including comparison to correlation criteria that test subjects are likely to have mental illness if the following expression level variability is confirmed A test method for the morbidity of any mental illness selected from the group consisting of disorders,
At least, ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, the level of expression of PNCK, the SPATA13 and TDO 2 gene was measured and used as an index, the method:
Decreased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; and increased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651.   2. The method of claim 1, wherein the mental illness is schizophrenia. The method according to claim 1, wherein the expression level of the gene is measured using any substance selected from the following (i) to (iii):
(I) a nucleic acid probe or primer capable of specifically detecting the transcription product of the gene to be measured;
(Ii) an antibody that specifically recognizes the translation product of the measurement target gene; and (iii) a ligand or receptor that specifically binds to the translation product of the measurement target gene.   2. The method according to claim 1, wherein the expression level in cells in the hippocampus is measured. A diagnostic agent for any mental illness selected from the group consisting of schizophrenia, bipolar mood disorder and schizophrenic disorder, comprising any substance selected from the following (i) to (iii):
(I) ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, TDO2, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, E A nucleic acid probe or primer capable of specifically detecting a transcription product of at least one gene selected from the group consisting of THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1 and NPNT;
(Ii) an antibody that specifically recognizes the translation product of at least one gene selected from the group; and (iii) a ligand or receptor that specifically binds to the translation product of at least one gene selected from the group. Body,
The nucleic acid probes or primers (i), at least, ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and nucleic acids respectively capable of specifically detecting each transcription product of a gene of TDO 2 A probe or primer,
Antibodies of (ii), at least, contain ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and each antibody specifically recognizing the translation product of the gene of TDO 2, and
The ligand or receptor (iii), at least, ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and ligand respectively specifically bind to the translation product of the gene of TDO 2 or A diagnostic agent comprising a receptor. 6. The agent according to claim 5 , comprising any group selected from the following (ia) and (iia).
(Ia) ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, and a group of nucleic acid probes or primers capable of specifically detecting each transcription product; and (iia) ADCY8, CCND1 , CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, and a group of antibodies that specifically recognize TDO2 translation products. A method for searching for a substance capable of preventing or treating any mental illness selected from the group consisting of schizophrenia, bipolar mood disorder and schizophrenic disorder, comprising the following steps;
(I) contacting a test substance with cells of a hippocampus of a CaMKIIα-deficient non-human mammal;
(II) CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, DHTR, PC, DHTL2C, PC , ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13, TDO2, DSP, IL-1R1 and NPNT Comparing the level with the expression level of the gene in a control cell not contacted with the test substance; and (III) the test substance that caused the following expression level variation for the gene based on the comparison result of (II) above As a substance capable of preventing or treating mental illness;
Here, the genes whose expression levels are measured and used as indicators include at least the genes of ADCY8, CCND1, CPNE9, LOC284018, NTNG1, PDYN, PIP3-E, PNCK, SPATA13 and TDO 2 :
Increased expression level: TDO2, PNCK, CXCL12, GLRX, EGR4, CD24, THOP1, ATP1A3, DHPS, FLJ10052, HTR2C, PCDH8, DSP, IL-1R1, NPNT; or decreased expression level: ADCY8, CCND1, CPNE9, LOC284018 , NTNG1, PDYN, PIP3-E, SPATA13, CUTL2, ZNF492, SCD4, RIMS3, RECK, TCF4, DKFZp586E121, DKFZp434G1615, KIAA1651. 8. The method according to claim 7 , wherein the test substance is contacted with the cells of the hippocampus of the CaMKIIα-deficient non-human mammal in vivo. 8. The method according to claim 7 , wherein the CaMKIIα-deficient non-human mammal is a CaMKIIα hetero-deficient non-human mammal. Expression levels of T DO2 is measured using a radioactive isotope-labeled tryptophan The method of claim 7 wherein. Gene expression level is immunohistological staining, radioimmunoassay, competitive assay, enzyme-linked immunosorbent assay, western blot, flow cytometry analysis, RT-PCR, in situ hybridization, northern blot high 8. The method according to claim 7 , which is measured by a hybridization method, a dot blot hybridization method, a PET method, an MRI method, or near-infrared spectrometry (NIRS). 8. The method of claim 7 , wherein the mental illness is schizophrenia.