JP2003235557A - Method for analyzing nucleic acid specifying gene changing expression level with schizophrenia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for statistically evaluating the expression level of a nucleic acid specifying a gene changing the expression level with schizophrenia in order to contribute to objective diagnosis of the schizophrenia using the genetic expression as an index. <P>SOLUTION: The method for analyzing whether or not the expression level of the nucleic acid specifying the gene changing the expression level with the schizophrenia in a subject is statistically within the range of the expression of a normal human is provided. The method comprises a step of determining the expression level of the nucleic acid specifying the gene changing the expression level with the schizophrenia and/or the expression level of a protein encoded with the nucleic acid specifying the gene changing the expression level with the schizophrenia in a sample collected from the subject. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing whether or not the expression level of a nucleic acid that defines a gene whose expression level changes due to schizophrenia is statistically within the range of expression levels in healthy individuals. .

[0002]

2. Description of the Related Art Schizophrenia is a mental illness that develops in adolescents in about 0.8% of the population, and has a long medical history.
The social loss of schizophrenia has been immeasurable. Therefore, many laboratories around the world have been actively researching methods for treating and diagnosing schizophrenia, and for the treatment, since the development of dopamine receptor antagonists such as chlorpromazine , Has made great strides. In contrast, the diagnosis of schizophrenia is a paranoid type even in the latest diagnostic standard in the United States, DSM IV.
Since it is defined only by psychological symptomology such as disassembly type, tension type, and indistinguishable type, the final diagnosis must depend on the subjectivity of the doctor in charge, and the accuracy of the diagnosis is not sufficient. Under these circumstances, chromosomal mapping of the causative gene of schizophrenia and its identification are currently being actively pursued, but there is no definitive report.

[0003]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and it is possible to contribute to the objective diagnosis of schizophrenia using gene expression as an index by measuring nucleic acid. To aim.

[0004]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present invention provides that the expression level of a nucleic acid that regulates a gene whose expression level is changed by schizophrenia in a subject is statistically It provides a method of analyzing whether or not it is within the range. This method comprises, in a sample collected from a subject, a nucleic acid that defines a gene whose expression level changes due to schizophrenia and / or an expression level of a protein encoded by the nucleic acid that defines a gene whose expression level changes due to schizophrenia. Including the step of quantifying.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, in schizophrenia patients, the expression levels of the 14 protein genes shown in Table 1 below, that is, messenger RNA, are statistically significantly changed. It was made based on the discovery of the present inventors. As described in detail in Experimental Examples below, the present inventors have found these genes by comparing the expression levels of about 3000 genes in autopsy brains of schizophrenic patients and normal subjects. In the present specification, the "nucleic acid defining a gene whose expression level changes due to schizophrenia" means a nucleic acid defining the genes shown in Table 1.

[0006]

[Table 1]       Protein encoded by nucleic acid GenBank No (1) Protein tyrosine phosphatase Sigma U35234   (Protein tyrosine phosphatase sigma) (2) Interleukin 10 precursor M57627   (Interleukin-10 precursor (IL-10)) (3) Tyrosine protein kinase receptor TIE-2 L06139   (Tyrosine-protein kinase receptor TIE-2) (4) EPH-related receptor tyrosine kinase ligand 5 L38734   (EPH-related receptor tyrosine kinase ligand 5) (5) Type 1 interferon receptor X77722   (Type I interferon receptor (IFN-R)) (6) Protein kinase ATR U49844   (Protein kinase ATR) (7) STAT2 U18671   (Signal transducer and activator of transcription 2) (8) High affinity nerve growth factor receptor precursor X03541   (High-affinity nerve growth factor receptor precursor) (9) OX40L receptor precursor X75962   (OX40L receptor precursor) (10) STAT4 L78440   (Signal transducer and activator of transcription 4) (11) KIAA0096 D43636 (12) Purine-rich single-stranded DNA-binding protein alpha M96684   (Purine-rich single-stranded DNA-binding protein alpha) (13) OX2 membrane glycoprotein precursor X05323   (OX2 membrane glycoprotein precursor) (14) CDC25B M81934 (15) Guanine nucleotide binding protein G (I) / G (S) / G (O) U31383       Gamma-10 subunit   (Guanine nucleotide-binding protein G (I) / G (S) / G (O)     gamma-10 subunit) (16) PKU-alpha AB004884 (17) Epidermal growth factor receptor M34309   (Epidermal growth factor receptor) (18) Wnt-8B X91940 (19) Glycyl-tRNA synthetase D30658   (Glycyl tRNA synthetase) (20) Melanoma antigen P15 U19796   (Melanoma antigen P15) (21) Transcription initiation factor eIF-2 alpha subunit U26032   (Translation initiation factor eIF-2 alpha subunit) (22) Curin homolog 2 U83410   (Cullin homolog 2 (CUL2)) (23) Transmembrane proteinaceous precursor X87852   (Transmembrane protein sex precursor) (24) GTP-binding nuclear protein RAN M31469   (GTP-binding nuclear protein RAN (TC4)) (25) Adenylate kinase isoenzyme 1 J04809   (Adenylate kinase isoenzyme 1 (AK1)) (26) Cell cycle protein P38-2G4 homolog U59435   (Cell cycle protein P38-2G4 homolog) (27) Interferon gamma receptor J03143   (Interferon-gamma receptor (IFNR-gamma; IFNGR1)) (28) Neurofibrilma tosis 2 L27133   (Neurofibromatosis 2 (NF2)) (29) Cadherin 6 precursor (CDH6) D31784   (Cadherin 6 precursor (CDH6)) (30) Bispecific protein phosphatase 8 U27193   (Dual-specificity protein phosphatase 8) (31) Arginine / serine rich splicing factor 7 L22253   (Arginine / serine-rich splicing factor 7) (32) PIRIN Y07867 (33) RAD51C cleavage protein AF029670   (RAD51C truncated protein) (34) Vascular endothelial growth factor receptor 1 X51602   (Vascular endothelial growth factor receptor 1) (35) DNA topoisomerase 1 J03250   (DNA topoisomerase I (TOP1)) (36) Basic fibroblast growth factor precursor M34186   (Basic fibroblast growth factor receptor precursor) (37) Laminin alpha 4 subunit precursor X70904   (Laminin alpha 4 subunit precursor) (38) Protein kinase DYRK4 Y09305   (Protein kinase DYRK4) (39) Somatostatin receptor type 2 M81830   (Somatostatin receptor type 2 (SS2R)) (40) Transferrin receptor X01060   (Transferrin receptor (TFRC))

The genes listed in Table 1 are (1) signal intensity, (2) gene expression change rate ("average expression level of patient group /
Whichever is larger, "average expression level of normal group" or "average expression level of normal group / average expression level of patient group", see Experimental Example),
And (3) the gene determined to be particularly useful as a diagnostic index for schizophrenia by taking into account all p values obtained by the test of the difference in the average expression level of the gene between the patient group and the normal group. . The “p value” is the probability that a certain statistic will be measured under the null hypothesis. However, depending on the accuracy of the diagnosis, the indicator gene may be selected using a criterion other than such a strict criterion (for details, see Experimental Examples). Specifically, only the p-value,
Alternatively, the nucleic acid to be used as an index may be selected based on only the gene expression change rate.

When only the p value is used as a reference, the p value is 0.5 or less, preferably 0.4 or less, more preferably 0.3 or less, more preferably 0.25 or less, more preferably 0.2 or less, more preferably 0.15 or less, more preferably A gene having 0.10 or less, more preferably 0.05 or less can be selected as an index. More preferably, the p value is 0.02 or less, 0.01 or less, and 0.
Genes of 005 or less, 0.025 or less, 0.002 or less, or 0.001 or less may be selected as an index. When only the gene expression change rate is used as a reference, the gene expression change rate is 1.1 or more,
Preferably 1.2 or more, more preferably 1.25 or more, more preferably 1.3 or more, more preferably 1.4 or more, more preferably 1.5 or more, more preferably 1.6 or more, more preferably 1.7 or more, more preferably 1.75 or more, more preferably 1.8 or more, more preferably 1.9 or more, more preferably
2.0 or more genes can be selected as an index. Furthermore, preferably, the gene expression change rate is 2.1 or more, 2.2 or more, 2.
25 or more, 2.5 or more, 3 or more, 4 or more, 5 or more, 6 or more,
7+, 7.5+, 8+, 9+, 10+, 15+,
20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70
As described above, 75 or more genes can be selected as an index. For example, depending on the diagnostic accuracy, instead of the genes shown in Table 1, or together with these genes, the expression levels of the genes shown in Table 2 below may also be used as an index.

[0009]

[Table 2]       Protein encoded by nucleic acid GenBank No (1) Protein phosphatase 2A B56 beta (PP2A) L42374   (Protein phosphatase 2A B56-beta (PP2A)) (2) Protein phosphatase 2A B56 alpha L42373   (Protein phosphatase 2A B56-alpha) (3) Janus Kinase 2 (JAK2) AF005216   (Janus kinase 2 (JAK2)) (4) Signal transducing adapter molecule U43899   (Signal transducing adaptor molecule (STAM)) (5) CDC37 homolog U63131   (CDC37 homolog) (6) MAP kinase activation death domain protein U77352   (MAP kinase-activating death domain protein) (7) Serine / threonine protein phosphatase 2B S46622       Catalytic subunit gamma   (Serine / threonin protein phosphatase 2B      catalytic subunit gamma) (8) Protein kinase A anchoring protein AF037439   (Protein kinase A anchoring protein) (9) Ephrin A3 precursor (EFNA3) U14187   (Ephrin A3 precursor (EFNA3)) (10) Sodium-dependent proline transporter S80071   (Sodium-dependent proline transporter) (11) CD45 antigen Y00638   (CD45 antigen) (12) Lysosome alpha glucosidase precursor Y00839   (Lysosomal alpha-glucosidase precursor) (13) Herpesvirus entry mediator U70321   (Herpes virus entry mediator (HVEM)) (14) Serine / threonine protein kinase receptor R4 precursor L11695   (Serine / threonine-protein kinase receptor R4     precursor (SKR4)) (15) ARG2 (Arginase II) U82256   (ARG2 (Arginase II)) (16) Cytotoxic T-lymphocyte proteinase M36118   (Cytotoxic T-lymphocyte proteinase) (17) Activin receptor type I precursor Z22534   (ACTIVIN RECEPTOR TYPE I PRECURSOR (EC 2.7.1.-)    (ACTR-1)) (18) Phenylethanolamine N-methyltransferase J03727   (Phenylethanolamine N-methyltransferase (PNMTase))

In the method of the present invention, a subject suffers from schizophrenia by using the expression level of a gene or a fragment thereof that meets such criteria and / or a protein or a fragment thereof encoded by these genes as an index. It can also be used for the purpose of objectively diagnosing whether or not there is.
In order to carry out the method of the present invention, first, a sample containing a nucleic acid or a protein is collected from a subject to be diagnosed as to whether or not he / she has schizophrenia. As used herein, the term "schizophrenia" refers to any type of schizophrenia, including delusional schizophrenia, demolition schizophrenia, catatonic schizophrenia, and indistinguishable schizophrenia. included. As used herein, the term “subject” refers to a human, and particularly to a patient who is the subject of diagnosis according to the method of the present invention. In the present specification, the “test animal” is an animal other than human, and particularly means a mouse, rat, guinea pig, dog, rabbit, monkey, chimpanzee, etc. suitable as an experimental animal.

In the method of the present invention, the proteins shown in Tables 1 and 2 above, most preferably the proteins shown in Table 1 above or fragments thereof, and / or nucleic acids encoding these proteins or fragments thereof or the nucleic acids At least one protein and / or nucleic acid selected from the fragments of. "Nucleic acids defining the genes encoding the proteins listed in Table 1 and nucleic acids complementary to the nucleic acids"
Typically means the messenger RNA and cDNA of these proteins. Further, any polynucleotide such as a regulatory sequence or polyadenyl sequence may be contained at the end and / or inside of the translation region of these messenger RNAs or cDNAs. When the proteins listed in Table 1 can be encoded by a plurality of alleles, all the alleles, their transcripts and cDNAs are defined as "a nucleic acid and a nucleic acid that define the genes encoding the proteins listed in Table 1. Included in "complementary nucleic acid". The “fragment” of a nucleic acid means a nucleic acid defining a gene encoding a protein or a polynucleotide containing a part of the nucleic acid, and typically, a messenger RNA of the protein described in Table 1 or a restriction fragment of cDNA. Can be. In order to quantify the expression level of a gene to be used as an index, first, a “sample containing nucleic acid or protein” is collected from a subject. Nucleic acids and proteins are widely contained in the body, and as long as they have the same gene, they are often subject to the same control. Therefore, in addition to the brain, any sample including tissues, cells, and body fluids collected from subjects. Can be a “sample containing nucleic acid or protein”. Preferred samples include biopsy brain, autopsy brain, cerebrospinal fluid, blood.

The preferred sample for use in the method of the invention is a biopsy sample taken from a limbic brain site, preferably a cingulate biopsy sample. However, since the above genes are also expressed in other brain regions and peripheral tissues such as blood, these tissues can also be used as a biopsy sample in the method of the present invention. Methods using these various tissues, as well as biopsy samples, should be understood to be within the scope of the present invention. As used herein, the term “nucleic acid” refers to a polynucleotide consisting of any simple nucleotide and / or modified nucleotide, such as cDNA, messenger RNA, total RNA, hnR.
NA, etc. are included. “Modified nucleotide” includes inosine, acetyl cytidine, methyl cytidine, methyl adenosine, and phosphoric acid ester including methyl guanosine,
It also includes nucleotides that can be acquired posteriorly by the action of ultraviolet rays and chemical substances. When quantifying a nucleic acid, an operation of extracting a nucleic acid from the sample is usually performed after collecting a sample from a subject. As a method for extracting nucleic acids from biological components, for example, phenol extraction, ethanol precipitation, or any other extraction method can be used. When extracting messenger RNA, it may be applied to an oligo dT column. When the amount of the nucleic acid is small, an operation of amplifying the nucleic acid may be performed, if necessary. The amplification operation can be performed by, for example, a polymerase chain reaction (hereinafter abbreviated as PCR) such as reverse transcription polymerase chain reaction (RT-PCR). Further, as described below, the amplification operation may be performed as a quantification operation, or may be combined with the quantification operation.

If necessary, after performing the extraction operation and / or the amplification operation, at least one nucleic acid selected from the group consisting of nucleic acids defining the genes encoding the proteins listed in Table 1 or Table 2 or Quantify the fragment. Any method known in the art can be used to quantify nucleic acids, including quantitative PCR, Southern blotting, Northern blotting, RNA digestion enzyme protection mapping and combinations thereof. For quantitative PCR, typically
A method of internally labeling the amplification product with a radioactive substance, eg, ³² P-labeled nucleotides, may be used. In addition, a method of end-labeling an amplification product with a primer labeled with a radioactive substance can also be used. Labeled amplification products can be separated from free radionucleotides or primers using well known methods including gel filtration, alcohol precipitation, trichloroacetic acid precipitation, physical adsorption to glass filters and the like. Then, the amplification product is quantified by operations such as liquid scintillation, autoradiography, and imaging plate (Bioimaging Analyzer (BAS; Fuji Photo Film) etc.) with or without electrophoresis or hybridization. Instead of the radioactive substance, a fluorescent substance or a luminescent substance may be used as a labeling substance, and the amplified product may be quantified using a spectrofluorometer, a fluorescence microplate reader, or a CCD camera. Also, if you do not incorporate the labeling substance during the PCR operation, ethidium bromide, SYBR Green IT
The amplification product can also be detected using an intercalating fluorescent dye such as M or PicoGreen ™ (Molecular Probes).

When quantitative PCR is not carried out, most commonly, a sample containing a nucleic acid is subjected to electrophoresis, subjected to Southern blotting or Northern blotting, and then quantified using a probe labeled with a detectable labeling substance. To do. Further, in the case of quantifying many kinds of nucleic acids at the same time, a DNA chip or a DNA microarray may be used together with these methods or in place of these methods. The expression level of the gene may be indirectly estimated by quantifying the protein produced from the messenger RNA (gene) instead of or together with the quantification of the nucleic acid. It will often be useful to quantify the protein encoded by the nucleic acid, rather than the nucleic acid, to diagnose schizophrenia by the methods of the invention.

As a method for extracting a protein from a tissue and a method for quantifying a protein, any method well known in the art can be used. For protein quantification, Western blotting, enzyme immunoassays including solid-phase enzyme immunoassays, immunocytochemistry, immunohistochemistry can be used. It should be noted that in the present specification, since only the outline of the most general operation is illustrated, various modifications of the above method or completely different methods can be used. Currently, by combining electrophoresis equipment, PCR equipment, etc., nucleic acid extraction, amplification,
Since there are commercially available devices that perform separation and quantification fully automatically,
It is also preferable to use such a device. By using such a device, schizophrenia can be diagnosed in the same manner as a normal clinical test. Following the quantification of a predetermined nucleic acid and / or protein, it is determined whether or not the subject has schizophrenia using the quantified value as an index. When diagnosing with a single nucleic acid and / or protein quantitative value as an index, set an appropriate threshold value with reference to the normal value, and if it is above or below the threshold value, schizophrenia is possible It is highly likely. For example, when the amount increases in a schizophrenic patient, it can be determined that the possibility of schizophrenia is high if the amount exceeds the set threshold value.

As described below, the threshold value may be selected according to the desired diagnostic accuracy. Non-schizophrenic healthy group (hereinafter,
When the distribution of gene expression levels in both the normal group) and the schizophrenia patient group (hereinafter simply referred to as the patient group) is known, for example, an individual who has collected the nucleic acid or protein to be quantified is normal. 10% chance to belong to a group
, 5%, or 1%. When only the distribution of gene expression levels in the normal group is known, such a quantitative value can be obtained for the nucleic acid or protein under the assumption that the individual who collected the nucleic acid or protein to be quantified belongs to the normal group. The probability or probability (hereinafter, p-value, which is typically a two-sided probability, but may be a one-sided probability) is 10%, 5%, or 1%. The amount or concentration of nucleic acid or protein is set as a threshold value. You can Even when only the distribution of gene expression levels in a patient group is clear, analysis can be performed by the same statistical method. The calculation of the p-value is done, for example, by
It can be calculated by a test method such as t-test or non-parametric test. To clarify the statistical distribution of gene expression levels in the normal group and / or patient group,
Typically, the expression level of at least 5 individuals, preferably 10 individuals, more preferably 20 individuals, still more preferably 50 individuals, most preferably 100 individuals should be measured.

Further, if necessary, it is possible to more accurately determine whether or not the subject is suffering from schizophrenia by using any of various statistical methods. The diagnostic method used is, of course, also within the scope of the method of the invention. In the specification of the present application, "the step of statistically analyzing whether or not the quantitative value is within the range of the normal group" means a statistical step as specifically described below. When the quantitative value of a single nucleic acid and / or protein is used as an index, as described in detail in the experimental examples below,
The expression level in the patient group was high (signal was 10 or more; see experimental example), and the absolute gene expression change rate of both (see example) was 1.
A nucleic acid and / or protein having a p-value of 5% or more and 5% or less in the average difference test is preferably used as an index. In the case of determining the quantitative values of a plurality of nucleic acids and / or proteins as an index, an appropriate threshold value is set for each nucleic acid and / or protein, as in the case of using a single nucleic acid and / or protein as an index. , Check whether the expression level of each gene is above or below the threshold value. Depending on the desired accuracy of determination, if the quantified value of one nucleic acid and / or protein is above or below the threshold value, it can be determined that there is a possibility of schizophrenia. If the quantified value of two or more nucleic acids and / or proteins is above or below the threshold value, it can be determined that the possibility of schizophrenia is higher. When it is desired to make a definitive determination, schizophrenia is determined when the quantitative value of more nucleic acids and / or proteins is above or below the threshold value. Furthermore, the determination method of the present invention can be used in combination with a conventional subjective diagnosis method.

In addition, by some method, data on quantitative values of nucleic acid and / or protein that can be used as an index in the diagnostic method of the present invention is collected from a patient who is definitely deficient in schizophrenia. If it is possible, it is possible to make a definite decision only by the diagnostic method of the present invention. The subject of the invention lies in providing an objective diagnostic method for schizophrenia and not in the individual extraction, amplification and analysis operations specifically described herein. Therefore, it should be noted that a diagnostic method using an operation other than each of the above operations also belongs to the scope of the present invention. As described above, when the method of the present invention is used, the subject suffers from schizophrenia by using the expression level of the nucleic acid (gene) and the biological product (protein) derived from the nucleic acid (gene) as indicators. Whether or not it can be objectively diagnosed. Therefore, the method of the present invention is applied to a method for determining the usefulness of a model animal for schizophrenia other than humans, and a method for determining the efficacy of a drug in drug screening using such a model animal. obtain. To determine the usefulness of a model animal for schizophrenia, similar to the above-mentioned diagnostic method, based on the expression of a predetermined gene, it is diagnosed whether the test animal develops schizophrenia, If the animal develops schizophrenia, it is judged to be useful as a model animal for schizophrenia.

The "test animal" includes mice, rats, and monkeys, but any animal other than human can be the "test animal". The diagnosis of schizophrenia in non-human animals has been more difficult, making the method extremely useful. Furthermore, in such model animals,
After administration of a candidate substance for an anti-schizophrenia drug, a predetermined nucleic acid and / or protein is quantified as described above, and if the schizophrenia is cured or ameliorated, the candidate substance has an anti-schizophrenic action. You can judge that you are doing. That is, by applying the diagnostic method of the present invention, a candidate substance for an anti-schizophrenia drug can be easily and accurately screened. The "anti-schizophrenia drug candidate substance" can be any substance desired by the tester.

Further, the diagnostic method of the present invention can be applied to a psychological test performed for the purpose of examining the existence of legally responsible ability or for other purposes. Hereinafter, the present invention will be described in more detail with reference to Experimental Examples and Examples, but the scope of the present invention is not limited in any sense.

[0021]

[Examples] The genes identified by the present inventors that can be diagnostic indicators will be described. In this study, post-mortem autopsy brains of the cingulate gyrus of patients with schizophrenia (sample group S) and autopsy brains of the cingulate gyrus of people without mental illness (sample group C).
RNA was extracted from the tissue using and the quality was first assayed.

Six RNA samples judged to be of high quality were selected from each group, labeled with activated phosphorus by reverse transcription reaction from individual RNAs (12 cases in total), and used as a probe. By reacting with three types of DNA microarrays, the expression levels of multiple genes were collectively measured and patterned (molecular expression profiling). Used 3
The types of DNA microarrays are Atlas Human 1.2 Array, Atlas Human 1.2 Array II, and Atlas Human Cancer 1.
There are 2 arrays (each array contains 1176 genes). By using these three kinds of arrays, a total of about 3000 gene expression changes are evaluated and assayed.

High temperature at 65 degrees Celsius and low temperature concentration (0.3xSSC), 1
It was removed from the DNA array using a non-specific hybridization signal by washing for more than an hour. Then, the radioactivity signal corresponding to each gene spot is
It was measured and quantified with a BAS5000 image analyzer manufactured by Fuji Photo Film Co., Ltd. In order to correct the variation in signal intensity due to the experimental procedure that occurs between the DNA microarray sheets corresponding to each sample, the sum of all gene expression signals on the array is calculated, and the sum is constant regardless of the array and sample RNA. The signal was standardized by assuming (total of 30000) (commonly called global normalization).

The signal corresponding to each gene spot is
It was measured and quantified with a BAS5000 image analyzer manufactured by Fuji Photo Film Co., Ltd. In order to correct the variation in signal intensity between the DNA microarray sheets corresponding to each RNA sample, the sum of all gene expression signals on the sheet is calculated, and the sum is assumed to be constant regardless of the sheet and sample RNA. Therefore, the signal intensity was standardized.

In order to determine a gene showing a quantitative change in common with a plurality of schizophrenic patients, schizophrenic patients (sample group S; N = 6) and non-psychotic patients (sample group C;
The expression signal data obtained from N = 6) were analyzed.
In addition, the statistical analysis of the signal intensity data was performed using the significant difference test by Student's t-test.

In this experimental example, the rate of gene expression change (“average expression level of S group / average expression level of C group” and “C group”) was determined for genes whose mean signal intensity in the schizophrenia group exceeds 10. The average expression level of S / average expression level of S group), whichever is greater), is 1.5 times or more, and the p value is 0.05 or less. Based on the above, it was determined that the expression level of the relevant gene was significantly changed in schizophrenia when the p value was 0.01 or less. Table 1 above is a list listing the former genes, and Table 2 above is a list listing the latter genes. The genes listed in Table 1 selected on this basis are:
It is particularly useful as a diagnostic index for schizophrenia, and the genes listed in Table 2 are also useful as a diagnostic index for schizophrenia. Table 3 shows detailed statistical data such as the rate of change in gene expression and p-value for the genes in Table 1.
Table 4 shows detailed statistical data such as the gene expression change rate and p value of the genes in Table 2.

[0027]

[Table 3]

[0028]

[Table 4]

For example, here, the interleukin-10 precursor (which is present at position E-14-f in the Atlas human cancer 1.2 array and whose gene expression was significantly changed in association with schizophrenia in the following analysis ( Interleukin-10 precursor: GenBank
No.M57627) Quantification of mRNA, its processing, and diagnostic possibility are explained.

(1) In order to determine a gene showing a quantitative change in common with a plurality of schizophrenic patients, schizophrenic patients (sample group S; N = 6) and non-psychotic patients (sample group C; The expression signal data obtained from N = 6) was analyzed according to the following example. This DNA containing this gene signal
All gene RNA signals on the array were measured and quantified by BAS5000 image analyzer. The expression signal data obtained by hybridization is shown in FIG. As a result, Interleukin-10 precursor (Interleukin-
10 precursor: GenBank No.M57627) mRNA measurement signal intensity in each sample; Schizophrenia patients (sample group S; N = 6); S1 = 9.0, S2 =
8.3, S3 = 13.0, S4 = 14.4, S5 = 17.6, S6 = 23.4 Non-psychotic (Sample group C; N = 6); C1 = 0.0, C2 = 1.7,
C3 = 0.8, C4 = 5.6, C5 = 7.0, C6 = 15.8.

(2) As a result of global normalization, assuming that the sum of gene expression signals is constant regardless of the array or sample (total of 30,000), the signal intensity between the arrays is standardized, and interleukin 10 precursor mRNA Corrected signal intensity of each sample; Schizophrenia patients (sample group S); S1 = 27.7, S2 = 36.9,
S3 = 23.2, S4 = 37.9, S5 = 36.0, S6 = 57.4 Non-psychotic (Sample group C); C1 = 0.0, C2 = 4.2, C3 = 4.
6, C4 = 18.3, C5 = 9.0, C6 = 19.9.

(3) Using the Student's t-test for significant difference, analysis of signal data intensity was performed for 6 schizophrenic patients (groups S1 to S6) and 6 nonpsychotic patients (groups C1 to C6). The mean and standard deviation are: schizophrenic patients (sample group S); 36.4 ± 12.0 non-psychotic patients (sample group C); 8.8 ± 8.6 according to Student's t-test, the mean values of these two groups are different Is found (P <0.001). In fact, in this example, all signals with a signal intensity of 20.0 or less are those of schizophrenia, and those with a signal intensity of 20.0 or less are non-schizophrenia, and it is understood that this criterion may be used for the determination. The Mann-Whitney study also revealed that these groups had a bias in the rank relationship (p is 0.05 or less).

(4) Actually, Interleukin-10 precursor (GenBank No. M57627) mR obtained from the unknown sample by the above DNA array analysis.
When the standardized signal intensity of NA is 36.4-12.0x1.64 = 16.72 or less, with a statistical significance of 95% or more in comparison with the distribution of schizophrenic patients (sample group S) assuming a normal distribution, Not considered to belong to the schizophrenia patient group, and the result is judged to be "normal". Also, the standard signal intensity is 8.8+
If it is 8.6x1.64 = 22.904 or more, 9 against the distribution of non-schizophrenic patients (sample group C) assuming a normal distribution
Does not belong to the normal group with a statistical significance of 5% or more,
In other words, it is judged as "schizophrenia". Less than 22.904, 16.7
A false positive is obtained if two or more signals are obtained.

The genes listed in Table 1 selected according to the same criteria are particularly useful as a diagnostic index for schizophrenia as described above. By assaying the expression levels of a plurality of genes obtained here, The reliability of the diagnosis for schizophrenia is further improved.

[0035]

According to the method of the present invention, it is possible to objectively diagnose whether or not a subject has schizophrenia. The method is much more accurate than conventional subjective diagnostic methods.

[Brief description of drawings]

FIG. 1 is a photograph of signals after hybridization detected by BAS5000 showing the results of comparison of the expression pattern of interleukin-10 precursor in schizophrenic patients and non-psychotic patients.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hitoshi Takahashi             7-10 Kannan-cho, Niigata City, Niigata Prefecture (72) Inventor Shuji Iriya             25 of 11 of 2 Kasuya, Setagaya-ku, Tokyo (72) Inventor Nako Kawamura             2-3 Kodaidai, Niigata City, Niigata Prefecture F term (reference) 2G045 AA29 AA35 CB01 DA13 FB02                       JA00                 4B024 AA11 CA04 CA11 HA08 HA12                       HA20                 4B063 QA19 QQ02 QQ08 QQ21 QQ53                       QQ79 QR08 QR14 QR20 QR32                       QR42 QR56 QR62 QR72 QR77                       QS25 QS32 QS34 QS36 QX02

Claims (5)

[Claims] 1. A method for analyzing whether or not the expression level of a nucleic acid that defines a gene whose expression level changes due to schizophrenia in a subject is statistically within the range of the expression level of healthy subjects, In a sample collected from a subject, a nucleic acid (including a fragment thereof and a nucleic acid complementary to the nucleic acid) defining a gene whose expression level changes due to schizophrenia and / or a gene whose expression level changes due to the schizophrenia A step of measuring the expression level of a protein (including a fragment thereof) encoded by the defined nucleic acid to obtain a quantitative value, and a step of statistically analyzing whether the quantitative value is within the range of a normal group, Nucleic acids that define genes whose expression level changes due to schizophrenia are the following protein groups: (1) Protein tyrosine phosphatase sigma (GenBank
No.U35234) (2) Interleukin 10 precursor (GenBank No.M5762
7) (3) Tyrosine protein kinase receptor TIE-2 (GenB
ank No.L06139) (4) EPH-related receptor tyrosine kinase ligand 5
(GenBank No.L38734) (5) Type 1 interferon receptor (GenBank No.X
77722) (6) Protein kinase ATR (GenBank No.U49844) (7) STAT2 (GenBank No.U18671) (8) High affinity nerve growth factor receptor precursor (GenBan
k No.X03541) (9) OX40L receptor precursor (GenBank No.X75962) (10) STAT4 (GenBank No.L78440) (11) KIAA0096 (GenBank No.D43636) (12) Purine-rich single-stranded DNA-binding protein alpha (Ge
nBank No.M96684) (13) OX2 membrane glycoprotein precursor (GenBank No.X05323) (14) CDC25B (GenBank No.M81934) (15) Guanine nucleotide binding protein G (I) / G (S) / G
(O) Gamma-10 subunit (GenBank No.U31383) (16) PKU-alpha (GenBank No.AB004884) (17) Epidermal growth factor receptor (GenBank No.M34309) (18) Wnt-8B (GenBank No.X91940) ) (19) Glycyl-tRNA synthetase (GenBank No.D30658) (20) Melanoma antigen P15 (GenBank No.U19796) (21) Transcription initiation factor eIF-2 alpha subunit (GenB)
ank No.U26032) (22) Curine homolog 2 (GenBank No.U83410) (23) Transmembrane proteinaceous precursor (GenBank No.X87852) (24) GTP-binding nuclear protein RAN (GenBank No.M31469) (25) Adenylate kinase isoenzyme 1 (GenBank No.J
04809) (26) Cell cycle protein P38-2G4 homolog (GenBank No.U
59435) (27) Interferon gamma receptor (GenBank N
o.J03143) (28) Neurofibrillosis 2 (GenBank No.L27
133) (29) Cadherin 6 precursor (CDH6) (GenBank No. D3178
4) (30) Bispecific protein phosphatase 8 (GenBank N
o.U27193) (31) Arginine / serine rich splicing factor 7
(GenBank No.L22253) (32) PIRIN (GenBank No.Y07867) (33) RAD51C cleavage protein (GenBank No.AF029670) (34) Vascular endothelial growth factor receptor 1 (GenBank No.X51
602) (35) DNA topoisomerase 1 (GenBank No.J03250) (36) Basic fibroblast growth factor precursor (GenBank No.M
34186) (37) Laminin alpha 4 subunit precursor (GenBan
k No.X70904) (38) Protein kinase DYRK4 (GenBank No.Y09305) (39) Somatostatin receptor type 2 (GenBank No.M81
830) (40) Transferrin receptor (GenBank No.X0106
0) which is a nucleic acid contained in the nucleic acid group having the GenBank accession number in the above parentheses. 2. A diagnostic method for diagnosing whether a subject is suffering from schizophrenia, comprising a step of collecting a sample containing a nucleic acid and / or a protein from the subject; At least one nucleic acid that is a nucleic acid (including a fragment thereof and a nucleic acid complementary to the nucleic acid) that defines a gene whose expression level changes due to schizophrenia, and / or a protein (including a fragment thereof) encoded by the nucleic acid A step of quantifying the content of at least one protein, and a nucleic acid that defines a gene whose expression level changes due to schizophrenia, and / or a nucleic acid that defines a gene whose expression level changes due to schizophrenia Diagnosing whether the subject is suffering from schizophrenia using the quantitative value of the protein as an index. Nucleic acids that define genes whose expression level changes more are the following protein groups: (1) Protein tyrosine phosphatase sigma (GenBank
No.U35234) (2) Interleukin 10 precursor (GenBank No.M5762
7) (3) Tyrosine protein kinase receptor TIE-2 (GenB
ank No.L06139) (4) EPH-related receptor tyrosine kinase ligand 5
(GenBank No.L38734) (5) Type 1 interferon receptor (GenBank No.X
77722) (6) Protein kinase ATR (GenBank No.U49844) (7) STAT2 (GenBank No.U18671) (8) High affinity nerve growth factor receptor precursor (GenBan
k No.X03541) (9) OX40L receptor precursor (GenBank No.X75962) (10) STAT4 (GenBank No.L78440) (11) KIAA0096 (GenBank No.D43636) (12) Purine-rich single-stranded DNA-binding protein alpha (Ge
nBank No.M96684) (13) OX2 membrane glycoprotein precursor (GenBank No.X05323) (14) CDC25B (GenBank No.M81934) (15) Guanine nucleotide binding protein G (I) / G (S) / G
(O) Gamma-10 subunit (GenBank No.U31383) (16) PKU-alpha (GenBank No.AB004884) (17) Epidermal growth factor receptor (GenBank No.M34309) (18) Wnt-8B (GenBank No.X91940) ) (19) Glycyl-tRNA synthetase (GenBank No.D30658) (20) Melanoma antigen P15 (GenBank No.U19796) (21) Transcription initiation factor eIF-2 alpha subunit (GenB)
ank No.U26032) (22) Curine homolog 2 (GenBank No.U83410) (23) Transmembrane proteinaceous precursor (GenBank No.X87852) (24) GTP-binding nuclear protein RAN (GenBank No.M31469) (25) Adenylate kinase isoenzyme 1 (GenBank No.J
04809) (26) Cell cycle protein P38-2G4 homolog (GenBank No.U
59435) (27) Interferon gamma receptor (GenBank N
o.J03143) (28) Neurofibrillosis 2 (GenBank No.L27
133) (29) Cadherin 6 precursor (CDH6) (GenBank No. D3178
4) (30) Bispecific protein phosphatase 8 (GenBank N
o.U27193) (31) Arginine / serine rich splicing factor 7
(GenBank No.L22253) (32) PIRIN (GenBank No.Y07867) (33) RAD51C cleavage protein (GenBank No.AF029670) (34) Vascular endothelial growth factor receptor 1 (GenBank No.X51
602) (35) DNA topoisomerase 1 (GenBank No.J03250) (36) Basic fibroblast growth factor precursor (GenBank No.M
34186) (37) Laminin alpha 4 subunit precursor (GenBan
k No.X70904) (38) Protein kinase DYRK4 (GenBank No.Y09305) (39) Somatostatin receptor type 2 (GenBank No.M81
830) (40) Transferrin receptor (GenBank No.X0106
0) which is a nucleic acid contained in the nucleic acid group having the GenBank accession number in the above parentheses. 3. A diagnostic method for diagnosing whether or not a test animal suffers from schizophrenia, comprising a step of collecting a sample containing a nucleic acid and / or a protein from the test animal; At least one nucleic acid that is a nucleic acid (including a fragment thereof and a nucleic acid complementary to the nucleic acid) that defines a gene whose expression level is changed by schizophrenia, and / or
Or a step of quantifying the content of at least one protein that is a protein (including a fragment thereof) encoded by the nucleic acid, a nucleic acid that defines a gene whose expression level changes due to the schizophrenia, and / or the schizophrenia And a step of diagnosing whether or not the test animal is suffering from schizophrenia, using a quantitative value of a protein encoded by a nucleic acid that defines a gene whose expression level changes according to Nucleic acid that regulates the gene whose expression level changes depending on the following protein groups: (1) Protein tyrosine phosphatase sigma (GenBank
No.U35234) (2) Interleukin 10 precursor (GenBank No.M5762
7) (3) Tyrosine protein kinase receptor TIE-2 (GenB
ank No.L06139) (4) EPH-related receptor tyrosine kinase ligand 5
(GenBank No.L38734) (5) Type 1 interferon receptor (GenBank No.X
77722) (6) Protein kinase ATR (GenBank No.U49844) (7) STAT2 (GenBank No.U18671) (8) High affinity nerve growth factor receptor precursor (GenBan
k No.X03541) (9) OX40L receptor precursor (GenBank No.X75962) (10) STAT4 (GenBank No.L78440) (11) KIAA0096 (GenBank No.D43636) (12) Purine-rich single-stranded DNA-binding protein alpha (Ge
nBank No.M96684) (13) OX2 membrane glycoprotein precursor (GenBank No.X05323) (14) CDC25B (GenBank No.M81934) (15) Guanine nucleotide binding protein G (I) / G (S) / G
(O) Gamma-10 subunit (GenBank No.U31383) (16) PKU-alpha (GenBank No.AB004884) (17) Epidermal growth factor receptor (GenBank No.M34309) (18) Wnt-8B (GenBank No.X91940) ) (19) Glycyl-tRNA synthetase (GenBank No.D30658) (20) Melanoma antigen P15 (GenBank No.U19796) (21) Transcription initiation factor eIF-2 alpha subunit (GenB)
ank No.U26032) (22) Curine homolog 2 (GenBank No.U83410) (23) Transmembrane proteinaceous precursor (GenBank No.X87852) (24) GTP-binding nuclear protein RAN (GenBank No.M31469) (25) Adenylate kinase isoenzyme 1 (GenBank No.J
04809) (26) Cell cycle protein P38-2G4 homolog (GenBank No.U
59435) (27) Interferon gamma receptor (GenBank N
o.J03143) (28) Neurofibrillosis 2 (GenBank No.L27
133) (29) Cadherin 6 precursor (CDH6) (GenBank No. D3178
4) (30) Bispecific protein phosphatase 8 (GenBank N
o.U27193) (31) Arginine / serine rich splicing factor 7
(GenBank No.L22253) (32) PIRIN (GenBank No.Y07867) (33) RAD51C cleavage protein (GenBank No.AF029670) (34) Vascular endothelial growth factor receptor 1 (GenBank No.X51
602) (35) DNA topoisomerase 1 (GenBank No.J03250) (36) Basic fibroblast growth factor precursor (GenBank No.M
34186) (37) Laminin alpha 4 subunit precursor (GenBan
k No.X70904) (38) Protein kinase DYRK4 (GenBank No.Y09305) (39) Somatostatin receptor type 2 (GenBank No.M81
830) (40) Transferrin receptor (GenBank No.X0106
0) which is a nucleic acid contained in the nucleic acid group having the GenBank accession number in the above parentheses. 4. A method for judging whether or not a test animal is useful as a model animal for schizophrenia, wherein the test animal is suffering from schizophrenia by the method according to claim 3. A method comprising the steps of diagnosing whether or not the subject animal has schizophrenia and determining that the subject animal is useful as a model animal for schizophrenia. 5. A method for screening a candidate substance for an anti-schizophrenia drug, comprising the step of providing the test substance to a model animal for schizophrenia, and the method according to claim 3, Process of diagnosing whether schizophrenia is cured or ameliorated and if schizophrenia of the model animal is cured or ameliorated, it is determined that the test substance is a candidate substance for anti-schizophrenia drug A method comprising the steps of: