JP3771502B2 - Methods for analyzing nucleic acids that regulate genes whose expression levels change due to schizophrenia - Google Patents

Description

【００２８】
【表４】

【００２９】
例えば、ここではアトラスヒト癌1.2 アレイの位置E-14-fに存在して、以下の解析で有意に精神分裂病に伴って遺伝子発現が変化していた、インターロイキン１０前駆体（Interleukin-10 precursor : GenBank No.M57627）mRNAの定量とその処理、診断可能性について解説する。
【００３０】
（１）複数の精神分裂病患者に共通性をもって量的変化を示す遺伝子を確定するために、精神分裂病患者（サンプル群Ｓ；N=6 ）と非精神患者（サンプル群Ｃ；N=6 ）から得られた発現シグナルのデータを以下の例に従い解析した。この遺伝子シグナルを含む本DNA アレイ上のすべての遺伝子RNA シグナルをBAS5000 イメージアナライザーで測定、定量した。ハイブリダイゼーションによって得た、発現シグナルのデータを図１に示す。結果、インターロイキン１０前駆体（Interleukin-10 precursor : GenBank No.M57627）mRNAの測定シグナル強度は各サンプルで；
精神分裂病患者（サンプル群Ｓ；N=6 ）；S1=9.0, S2=8.3, S3=13.0, S4=14.4,S5=17.6, S6=23.4
非精神病者（サンプル群Ｃ；N=6 ）；C1=0.0, C2=1.7, C3=0.8, C4=5.6, C5=7.0, C6=15.8
となった。
【００３１】
（２）グローバルノーマライゼーションにより、遺伝子発現シグナルの総和がアレイ、サンプルによらず一定（総計３００００）と仮定することで、アレイ間のシグナル強度の標準化を行うと、インターロイキン１０前駆体mRNAの補正シグナル強度は各サンプルで；
精神分裂病患者（サンプル群Ｓ）；S1=27.7, S2=36.9, S3=23.2, S4=37.9, S5=36.0, S6=57.4
非精神病者（サンプル群Ｃ）；C1=0.0, C2=4.2, C3=4.6, C4=18.3, C5=9.0, C6=19.9
となった。
【００３２】
（３）スチューデントのt 検定による有意差検定を用いて、シグナルデータ強度に関する解析を精神分裂病患者（群Ｓ１からＳ６）６例と非精神病患者（群Ｃ１からＣ６）６例に対して行うと平均値と標準偏差は；
精神分裂病患者（サンプル群Ｓ）；36.4±12.0
非精神病患者（サンプル群Ｃ）； 8.8±8.6
でスチューデントｔ検定によるとこれら２群の平均値が異なることが判明する（Ｐ＜0.001 ）。実際、本実施例では、シグナル強度20.0以下のものは全て精神分裂病のもので、20.0以下のものは非精神分裂病となり、この基準で判定してよいことがわかる。マン−ホイットニーの検討によっても、これらの群に順位関係にかたよりがあることが判明した（ｐは0.05以下）。
【００３３】
（４）実際に、未知の検体から上記DNA アレイ解析によって得られたインターロイキン１０前駆体（Interleukin-10 precursor : GenBank No.M57627）mRNAの標準化シグナル強度が36.4-12.0x1.64=16.72以下である場合、正規分布を仮定した精神分裂病患者（サンプル群Ｓ）の分布と対照して９５％以上の統計学的有意性をもって、精神分裂病患者群に属すると見做せず、結果「正常」と判定される。また、標準シグナル強度が8.8+8.6x1.64=22.904 以上である場合、正規分布を仮定した非精神分裂病患者（サンプル群Ｃ）の分布と対照して９５％以上の統計学的有意性をもって正常群に属さない、つまり「精神分裂病」と判定される。22.904未満、16.72 以上のシグナルが得られた場合は、擬陽性と判定される。
【００３４】
同様の基準で選定された表１に記載の遺伝子は、上記のように精神分裂病の診断指標として特に有用であり、ここに得られた複数の遺伝子発現量を検定することで、精神分裂病に対する診断の信頼性は更に向上する。
【００３５】
【発明の効果】
本発明の方法によれば、被験者が精神分裂病に罹患しているか否かを客観的に診断することができる。該方法は、従来の主観的な診断方法に比べて、極めて精度が高い。
【図面の簡単な説明】
【図１】図１は、インターロイキン１０前駆体の発現パターンを、精神分裂病患者と非精神病者において比較を行った結果を示す、BAS5000 により検出したハイブリダイゼーション後のシグナルの写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for analyzing whether the expression level of a nucleic acid defining a gene whose expression level changes due to schizophrenia is statistically within the range of the expression level of a healthy person.
[0002]
[Prior art]
Schizophrenia is a mental illness that occurs in about 0.8% of the population in adolescence and has a long history, making social losses due to schizophrenia immeasurable.
For this reason, many laboratories around the world have been actively investigating the treatment and diagnosis of schizophrenia since the development of dopamine receptor antagonists such as chlorpromazine. Has made great strides.
On the other hand, the diagnosis method of schizophrenia is prescribed only in psychological symptomology such as delusion type, dismantling type, tension type, and indistinguishable type in DSMIV which is the latest diagnostic standard in the United States. The final diagnosis must depend on the subjectivity of the attending physician, and the accuracy of the diagnosis is not perfect.
Under such circumstances, chromosomal mapping and identification of schizophrenia causative genes are currently being actively pursued, but there is no definitive report.
[0003]
[Problems to be solved by the invention]
The present invention has been made to solve the above-described problems, and an object thereof is to contribute to an objective diagnosis of schizophrenia using gene expression as an index by measuring a nucleic acid.
[0004]
[Means for Solving the Problems]
In order to solve the above problems, the present invention analyzes whether the expression level of a nucleic acid that defines a gene whose expression level changes in a subject due to schizophrenia is statistically within the range of the expression level of a healthy person. Provide a method. In the method, the expression level of a nucleic acid that regulates a gene whose expression level changes due to schizophrenia and / or a protein encoded by a nucleic acid that regulates a gene whose expression level changes due to schizophrenia is measured in a sample collected from a subject A step of quantifying.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have found that the expression levels of the genes of 14 proteins listed in Table 1 below, that is, messenger RNA, are statistically significantly changed in schizophrenic patients. It was made based on. As described in detail in the experimental examples below, the present inventors found these genes by comparing the expression levels of about 3000 genes in autopsy brains of schizophrenic patients and normal individuals. In the present specification, “a nucleic acid that defines a gene whose expression level changes due to schizophrenia” means a nucleic acid that defines a gene shown in Table 1.
[0006]
[Table 1]
Nucleic acid encoded protein 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) Neurofibrosis 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))
[0007]
The genes listed in Table 1 are: (1) signal intensity, (2) gene expression change rate (“average expression level of patient group / average expression level of normal group” and “average expression level of normal group / average of patient group” The larger of “expression level” (see experimental example)), and (3) schizophrenia by taking into account all p-values obtained by testing the difference in the average expression level of genes in patient and normal groups It is a gene that is judged to be particularly useful as a diagnostic index. The “p value” refers to the probability that a certain statistic is measured under the null hypothesis.
However, depending on the accuracy of diagnosis, the indicator gene may be selected using a standard other than the strict standard (for details, refer to the experimental example).
Specifically, a nucleic acid to be used as an index may be selected based on only the p value or only the gene expression change rate.
[0008]
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 0.10 or less, More preferably, a gene that is 0.05 or less can be selected as an index. More preferably, genes having a p value of 0.02 or less, 0.01 or less, 0.005 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 Can be selected as an index using a gene of 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. Further 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 or more, 7.5 or more, 8 or more, 9 or more, 10 or more, 15 As described above, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 75 or more genes can be selected as an index.
For example, depending on the diagnostic accuracy, the expression levels of the genes in Table 2 below may be used as an index instead of or together with the genes listed in Table 1.
[0009]
[Table 2]
Nucleic acid encoded protein 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) Lysosomal 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))
[0010]
The method of the present invention can determine whether or not a subject suffers from schizophrenia by using as an index the expression level of genes or fragments thereof that meet such criteria and / or proteins or fragments thereof encoded by these genes. It can also be used for the purpose of objective diagnosis.
In order to carry out the method of the present invention, first, a sample containing nucleic acid or protein is collected from a subject to be diagnosed as to whether or not he / she suffers from schizophrenia.
As used herein, the term “schizophrenia” includes any type of schizophrenia, including delusional schizophrenia, dismantle schizophrenia, tension schizophrenia, and indistinguishable schizophrenia. included.
In the present specification, the “subject” means a human, particularly a patient who is a diagnostic target of the method of the present invention.
In the present specification, the “test animal” refers to animals other than humans, and particularly means mice, rats, guinea pigs, dogs, rabbits, monkeys, chimpanzees and the like suitable as experimental animals.
[0011]
In the method of the present invention, the protein described in Table 1 and Table 2 above, most preferably the protein described in Table 1 above or a fragment thereof, and / or nucleic acid encoding these protein or fragment thereof or a fragment of the nucleic acid. Quantifying at least one selected protein and / or nucleic acid.
“Nucleic acids defining genes encoding the proteins listed in Table 1 and nucleic acids complementary to the nucleic acids” typically mean messenger RNAs and cDNAs of these proteins. Moreover, arbitrary polynucleotides, such as a regulatory sequence and a polyadenyl sequence, may be contained in the terminal and / or inside of the translation region of these messenger RNAs or cDNAs. When the protein described in Table 1 can be encoded by a plurality of alleles, all the alleles, their transcripts and cDNAs are “the nucleic acids defining the genes encoding the proteins listed in Table 1 and the nucleic acids Included in “complementary nucleic acids”.
A “fragment” of a nucleic acid means a nucleic acid that defines a gene encoding a protein or a polynucleotide containing a part of the nucleic acid, and is typically a restriction fragment of the messenger RNA or cDNA of the protein described in Table 1. It can be.
In order to quantify the expression level of a gene to be used as an indicator, 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 are the same gene, they are often subject to the same control, so in addition to the brain, any sample containing tissues, cells, or body fluids collected from the subject Can be a “sample containing nucleic acid or protein”. Preferred samples include biopsy brain, autopsy brain, cerebrospinal fluid, blood.
[0012]
A preferred sample for use in the method of the present invention is a biopsy sample taken from a limbic brain region, preferably a zonal 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 biopsy samples in the method of the present invention. It should be understood that not only biopsy samples but also methods using these various tissues are within the scope of the present invention.
In the present specification, the term “nucleic acid” includes polynucleotides composed of any simple nucleotide and / or modified nucleotide, such as cDNA, messenger RNA, total RNA, hnRNA, and the like. “Modified nucleotides” include inosin, acetylcytidine, methylcytidine, methyladenosine, and methylguanosine, as well as nucleotides that can be generated by the action of ultraviolet light or chemical substances.
When quantifying a nucleic acid, after a sample is collected from a subject, an operation for extracting the nucleic acid from the sample is usually performed. As a method for extracting a nucleic acid from a biological component, for example, any extraction method other than phenol extraction and ethanol precipitation can be used. When extracting messenger RNA, it may be applied to an oligo dT column.
When the amount of nucleic acid is small, an operation of amplifying the nucleic acid may be performed as necessary. The amplification operation can be performed, for example, by 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 quantitative operation or also as a quantitative operation.
[0013]
After performing an extraction operation and / or an amplification operation, if necessary, at least one nucleic acid selected from the group consisting of nucleic acids defining genes encoding the proteins listed in Table 1 or Table 2 or a fragment thereof Quantify.
Any method known in the art can be used to quantify nucleic acids, including quantitative PCR, Southern blotting, Northern blotting, RNA digestive enzyme protection mapping methods, and combinations thereof.
Quantitative PCR typically involves radioactive materials, such as ³² A method of internally labeling the amplification product with nucleotides labeled with P can be used. In addition, a method in which an amplification product is end-labeled using a primer labeled with a radioactive substance can be used. Labeled amplification products can be separated from free radioactive nucleotides or primers using well known methods including gel filtration, alcohol precipitation, trichloroacetic acid precipitation, glass filter, and the like. Subsequently, the amplification product is quantified by operations such as liquid scintillation, autoradiography, imaging plate (bioimaging analyzer (BAS; Fuji Photo Film), etc.) with or without electrophoresis and hybridization. Instead of radioactive substances, fluorescent substances or luminescent substances may be used as labeling substances, and amplification products may be quantified using a spectrofluorometer, a fluorescent microplate reader, or a CCD camera. When the labeling substance is not incorporated during the PCR operation, the amplification product can be detected using an intercalating fluorescent dye such as ethidium bromide, SYBR Green ITM, PicoGreenTM (Molecular Probes).
[0014]
When quantitative PCR is not performed, most commonly, a sample containing a nucleic acid is subjected to electrophoresis, subjected to Southern blot or Northern blot, and then quantified using a probe labeled with a detectable labeling substance.
In addition, when 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 instead of these methods.
The expression level of the gene may be estimated indirectly by quantifying the protein produced from the messenger RNA (gene) instead of or together with the nucleic acid quantification. To diagnose schizophrenia by the method of the present invention, it will often be useful to quantify the protein encoded by the nucleic acid rather than the nucleic acid.
[0015]
As a method for extracting protein from tissue and a method for quantifying protein, any method known in the art can be used. For protein quantification, Western blotting, enzyme immunoassay including solid phase enzyme immunoassay, immunocytochemistry, and immunohistochemistry can be used.
In the present specification, only the outline of the most general operation is illustrated, and various modifications of the above method and completely different methods can be used.
At present, there are commercially available devices for performing full-automatic extraction, amplification, separation, and quantification of nucleic acids in combination with an electrophoresis device, a PCR device, etc., and it is also preferable to use such a device. If such an apparatus is used, it becomes possible to diagnose schizophrenia as in the case of a normal clinical test.
Following a predetermined nucleic acid quantification and / or protein quantification, whether or not the subject suffers from schizophrenia is determined using the quantified value as an index.
When diagnosing with the quantitative value of a single nucleic acid and / or protein as an index, an appropriate threshold is set with reference to the normal value, and if it is above or below this threshold, it may be schizophrenia High nature. 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 a set threshold.
[0016]
As described below, the threshold value may be selected according to the desired diagnostic accuracy.
When the distribution of gene expression levels in the group of healthy non-schizophrenic patients (hereinafter referred to as normal group) and the group of patients with schizophrenia (hereinafter simply referred to as patient group) is clear, for example, quantification The threshold is set so that the probability that the individual from whom the nucleic acid or protein to be collected belongs to the normal group is 10%, 5%, or 1%.
When only the distribution of gene expression levels in the normal group is known, such quantitative values are obtained for the nucleic acid or protein under the assumption that the individual from whom the nucleic acid or protein to be quantified is collected belongs to the normal group. The threshold value is the amount or concentration of nucleic acid or protein that has a probability of being 10%, 5%, or 1%. Can do.
Even when only the distribution of gene expression levels in the patient group is clear, the same statistical method can be used for analysis.
The p value can be calculated by a test method such as t test or nonparametric test.
In order to clarify the statistical distribution of the expression level of the gene in the normal group and / or patient group, typically, at least 5 individuals, preferably 10 individuals, more preferably 20, more preferably 50 individuals, Most preferably, the expression level of 100 individuals should be measured.
[0017]
In addition, if necessary, it is possible to more accurately determine whether or not a subject suffers from schizophrenia using a variety of statistical methods. Diagnosis using such methods The method naturally also belongs to the scope of the method of the present invention. In the present specification, the “step of statistically analyzing whether 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, the expression level of the patient group is large as shown in detail in the following experimental example (signal is 10 or more; see the experimental example) It is preferable to use as an index a nucleic acid and / or protein having an absolute gene expression change rate of both (see Examples) of 1.5 times or more and a p value of 5% or less in an average difference test.
When determining the quantitative value of a plurality of nucleic acids and / or proteins as an index, an appropriate threshold 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. Then, it is examined whether the expression level of each gene is above or below the threshold value.
Depending on the desired determination accuracy, if the quantitative value of one nucleic acid and / or protein is above or below a threshold value, it can be determined that there is a possibility of schizophrenia. If the quantitative values of two or more nucleic acids and / or proteins are above or below a threshold, it can be determined that the possibility of schizophrenia is even higher. When it is desired to make a definitive determination, it is determined that the subject has schizophrenia when more nucleic acid and / or protein quantification values are above or below a threshold value.
Furthermore, the determination method of the present invention can be used in combination with a conventional subjective diagnosis method.
[0018]
Moreover, if it is possible to collect data on quantitative values of nucleic acids and / or proteins that can be used as an index in the diagnostic method of the present invention from a patient who is clearly determined to have schizophrenia by any method, It is also possible to make a definitive determination only by the diagnostic method of the present invention.
The subject of the invention resides in providing an objective diagnostic method for schizophrenia, not in the individual extraction, amplification and analysis operations specifically described herein. Therefore, it should be noted that diagnostic methods using operations other than the above operations also belong to the scope of the present invention.
As described above, according to the method of the present invention, a subject suffers from schizophrenia by using the expression level of a nucleic acid (gene) and a biological product (protein) derived from the nucleic acid (gene) as an index. It can be objectively diagnosed whether or not it exists.
Therefore, the method of the present invention is applied to a method for determining the usefulness of model animals for schizophrenia other than humans, and a method for determining the effectiveness of drugs in drug screening using such model animals. obtain.
In order to determine the usefulness of a model animal for schizophrenia, as in the above-described diagnosis method, based on the expression of a predetermined gene, it is diagnosed whether the test animal has developed schizophrenia, and the animal If it develops schizophrenia, it determines with it being useful as a model animal of schizophrenia.
[0019]
“Test animals” include mice, rats, and monkeys, but any animal other than humans can be a “test animal”.
Diagnosis of schizophrenia has been more difficult in animals other than humans, so the method is very useful.
Furthermore, after administering a candidate substance for an anti-schizophrenic drug to such a model animal, the predetermined nucleic acid and / or protein is quantified as described above, and if the schizophrenia is cured or improved, the candidate It can be judged that the substance has an anti-schizophrenic action. That is, if the diagnostic method of the present invention is applied, a candidate substance for an anti-schizophrenia drug can be easily and accurately screened.
The “candidate substance for anti-schizophrenic drug” may be any substance desired by the examiner.
[0020]
In addition, the diagnostic method of the present invention can be applied to a psychiatric test performed for the purpose of examining the presence or absence of legal responsibility or for other purposes.
EXAMPLES Hereinafter, although an experiment example and an Example demonstrate this invention further in detail, it does not limit the scope of the present invention in any meaning.
[0021]
【Example】
A gene that can be a diagnostic index identified by the present inventors will be described.
In this study, RNA was extracted from tissues using postmortem autopsy brains (sample group S) after death of schizophrenic patients and autopsy brains (sample group C) of people with no mental illness, First, the quality was tested.
[0022]
Select 6 samples of RNA judged to be of high quality for each group, label activated radioactive phosphorus from individual RNAs (total 12 cases) by reverse transcription reaction, and use this as a probe, DNA microarray from Clontech, By reacting with the three types, we performed batch measurement and patterning (molecular expression profiling) of the expression levels of multiple genes. The three types of DNA microarrays used are Atlas Human 1.2 Array, Atlas Human 1.2 Array II, and Atlas Human Cancer 1.2 Array (each array contains 1176 genes). By using these three types of arrays, a total of about 3000 changes in gene expression are evaluated and tested.
[0023]
It was removed from the DNA array using a non-specific hybridization signal by washing for 1 hour or more under a low temperature (0.3xSSC) at a high temperature of 65 ° C. Thereafter, the radioactivity signal corresponding to each gene spot was measured and quantified with a BAS5000 image analyzer of Fuji Photo Film. To correct for signal intensity variability due to experimental techniques occurring 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 or sample RNA. The signal was standardized by assuming (total 30000) (commonly called global normalization).
[0024]
The signal corresponding to each gene spot was measured and quantified with a BAS5000 image analyzer from Fuji Photo Film. To correct for signal intensity variability between the DNA microarray sheets corresponding to each RNA sample, calculate the sum of all gene expression signals on the sheet and assume that the sum is constant regardless of the sheet or sample RNA. Thus, the signal intensity was standardized.
[0025]
Obtained from schizophrenic patients (sample group S; N = 6) and non-psychotic patients (sample group C; N = 6) to establish genes that show quantitative changes in common to multiple schizophrenic patients The obtained expression signal data was analyzed. In addition, statistical analysis of signal intensity data was performed using a significant difference test by Student's t test.
[0026]
In this experimental example, the gene expression change rate (“average expression level of group S / average expression level of group C” and “average expression level of group C” and “average expression level of group C” for genes whose average signal intensity of schizophrenia group exceeds 10 For the medium signal with a p value of 0.05 or less and a moderate signal of 10 or less, the gene expression change rate is 2.0 times or more. When the p value was 0.01 or less, it was determined that the expression level of the relevant gene was significantly changed in schizophrenia. Table 1 above is a list describing the former genes, and Table 2 above is a list describing the latter genes. The genes listed in Table 1 selected according to this criterion are particularly useful as diagnostic indicators for schizophrenia, and the genes listed in Table 2 are also useful as diagnostic indicators for schizophrenia. Table 3 shows detailed statistical data such as gene expression change rate and p-value for the genes in Table 1. Table 4 shows detailed statistical data such as gene expression change rate and p-value for the genes in Table 2.
[0027]
[Table 3]

[0028]
[Table 4]

[0029]
For example, the interleukin-10 precursor (Interleukin-10), present here at position E-14-f of the Atlas human cancer 1.2 array and significantly altered gene expression with schizophrenia in the following analysis. precursor: GenBank No.M57627) Explains the quantification of mRNA, its processing, and the possibility of diagnosis.
[0030]
(1) In order to establish a gene that shows 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; N = 6). The expression signal data obtained from (1) was analyzed according to the following example. All gene RNA signals on this DNA array containing this gene signal were measured and quantified with a BAS5000 image analyzer. The expression signal data obtained by hybridization is shown in FIG. As a result, the measurement signal intensity of interleukin-10 precursor (GenBank No. M57627) mRNA is different for each sample;
Schizophrenic 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
It became.
[0031]
(2) By assuming that the sum of gene expression signals is constant regardless of the array and sample (total 30000) by global normalization, and standardizing the signal intensity between arrays, the correction signal of interleukin 10 precursor mRNA Intensity for each sample;
Schizophrenic 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
It became.
[0032]
(3) Using a significant difference test by Student's t-test, signal data intensity analysis is performed on 6 schizophrenic patients (groups S1 to S6) and 6 nonpsychotic patients (groups C1 to C6). Mean value and standard deviation are;
Schizophrenic patients (sample group S); 36.4 ± 12.0
Non-psychotic patients (sample group C); 8.8 ± 8.6
The Student t test reveals that the mean values of these two groups are different (P <0.001). In fact, in this example, it is understood that all signals with a signal intensity of 20.0 or less are schizophrenia, and those with a signal intensity of 20.0 or less are non-schizophrenia, which may be determined based on this criterion. The Mann-Whitney study also revealed that these groups have a ranking relationship (p is 0.05 or less).
[0033]
(4) Actually, the standardized signal intensity of interleukin-10 precursor (GenBank No. M57627) mRNA obtained from the unknown specimen by the above DNA array analysis is 36.4-12.0x1.64 = 16.72 or less. In some cases, the distribution of patients with schizophrenia (sample group S) assuming a normal distribution is not considered to belong to the schizophrenic patient group with a statistical significance of 95% or more. Is determined. In addition, when the standard signal intensity is 8.8 + 8.6x1.64 = 22.904 or more, it has a statistical significance of 95% or more in contrast to the distribution of non-schizophrenic patients (sample group C) assuming a normal distribution. It is determined not to belong to the normal group, that is, “schizophrenia”. If a signal less than 22.904 or greater than 16.72 is obtained, it is determined as a false positive.
[0034]
The genes listed in Table 1 selected according to the same criteria are particularly useful as a diagnostic index of schizophrenia as described above. By testing the expression levels of a plurality of genes obtained here, schizophrenia The reliability of the diagnosis is further improved.
[0035]
【The invention's effect】
According to the method of the present invention, it can be objectively diagnosed whether or not a subject suffers from schizophrenia. The method is extremely accurate compared to conventional subjective diagnostic methods.
[Brief description of the drawings]
FIG. 1 is a photograph of a signal after hybridization detected by BAS5000, showing the result of comparison of the expression pattern of interleukin-10 precursor in schizophrenic patients and non-psychotic patients.

Claims (6)

A method of analyzing whether 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 a healthy person,
In a sample collected from a subject, a nucleic acid (including a fragment thereof and a nucleic acid complementary to the nucleic acid ) that defines the gene for protein kinase DYRK4 ( GenBank No. Y09305 ) and / or a protein encoded by the nucleic acid that defines the gene ( The method as described above , comprising the step of measuring the expression level (including fragments thereof) to obtain a quantitative value, and the step of statistically analyzing whether the quantitative value is within the range of the normal group. The method according to claim 1, further comprising 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 the following schizophrenia contained in the sample: Measuring the expression level of a protein (including a fragment thereof) encoded by a nucleic acid that defines a gene whose expression level changes due to the schizophrenia, and determining the quantitative value, and the quantitative value is within the range of the normal group Including statistically analyzing whether or not
  Nucleic acids defining genes whose expression levels are changed by schizophrenia are the following protein groups:
(1) Protein tyrosine phosphatase sigma ( GenBank No. U35234 )
(2) Tyrosine protein kinase receptor TIE-2 ( GenBank No.L06139 )
(3) EPH- Related receptor tyrosine kinase ligand 5 ( GenBank No.L38734 )
(4) Type 1 interferon receptor ( GenBank No.X77722 )
(5) Protein kinase ATR ( GenBank No. U49844 )
(6) STAT2 ( GenBank No. U18671 )
(7) High affinity nerve growth factor receptor precursor ( GenBank No.X03541 )
(8) OX40L Receptor precursor ( GenBank No.X75962 )
(9) STAT4 ( GenBank No.L78440 )
( Ten ) KIAA0096 ( GenBank No.D43636 )
( 11 ) Purin rich single chain DNA Binding protein alpha ( GenBank No.M96684 )
( 12 ) OX2 Membrane glycoprotein precursor ( GenBank No.X05323 )
( 13 ) CDC25B ( GenBank No.M81934 )
( 14 ) Guanine nucleotide binding protein  G (I) / G (S) / G (O) gamma -Ten Sub unit ( GenBank No. U31383 )
( 15 ) PKU-alpha ( GenBank No. AB004884 )
( 16 ) Wnt-8B ( GenBank No.X91940 )
( 17 ) Glycyl tRNA Synthetic enzyme ( GenBank No.D30658 )
( 18 ) Melanoma antigen P15 ( GenBank No. U19796 )
( 19 ) Transcription initiation factor eIF-2 Alpha subunit ( GenBank No. U26032 )
( 20 ) Curin homolog 2 ( GenBank No. U83410 )
( twenty one ) Transmembrane proteinaceous precursor ( GenBank No.X87852 )
( twenty two ) GTP Binding nucleoprotein RAN ( GenBank No.M31469 )
( twenty three ) Adenylate kinase isoenzyme 1 ( GenBank No.J04809 )
( twenty four ) Cell cycle proteins  P38-2G4 Homolog ( GenBank No. U59435 )
( twenty five ) Interferon gamma receptor ( GenBank No.J03143 )
( 26 ) Neurofibrosis 2 ( GenBank No.L27133 )
( 27 ) Cadherin 6 precursor ( CDH6 ) ( GenBank No.D31784 )
( 28 ) Bispecific protein phosphatase 8 ( GenBank No. U27193 )
( 29 ) Arginine / Serine-rich splicing factor 7 ( GenBank No.L22253 )
( 30 ) PIRIN ( GenBank No.Y07867 )
( 31 ) RAD51C Cleavage protein ( GenBank No.AF029670 )
( 32 ) Vascular endothelial growth factor receptor 1 ( GenBank No.X51602 )
( 33 ) DNA Topoisomerase 1 ( GenBank No.J03250 )
( 34 ) Laminin alpha 4 subunit precursor ( GenBank No.X70904 )
( 35 ) Somatostatin receptor type 2 ( GenBank No.M81830 )
( 36 ) Transferrin receptor ( GenBank No.X01060 )
In the brackets above GenBank The method as described above, which is a nucleic acid contained in the nucleic acid group indicated by the accession number. A diagnostic method for diagnosing whether or not a test animal suffers from schizophrenia,
Collecting a sample containing nucleic acid and / or protein from the subject animal;
A nucleic acid (including a fragment thereof and a nucleic acid complementary to the nucleic acid) defining a gene of protein kinase DYRK4 ( GenBank No. Y09305 ) and / or a protein encoded by the nucleic acid (including a fragment thereof) in the sample Quantifying the content of
The protein kinase DYRK4 nucleic defining a gene (GenBank No.Y09305), and / or the quantitative value of the protein kinase DYRK4 protein encoded by a nucleic acid defining gene of (GenBank No.Y09305) as an index, the subject animal you and a step of diagnosing whether suffering from schizophrenia, the method described above. The method according to claim 3, further comprising at least one 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 in the sample. Quantifying the content of at least one protein that is a nucleic acid and / or a protein (including fragments thereof) encoded by the nucleic acid;
  Using the nucleic acid that defines a gene whose expression level changes due to the schizophrenia and / or the quantitative value of the protein encoded by the nucleic acid that regulates the gene whose expression level changes as a result of the schizophrenia as an indicator, Comprising diagnosing whether or not schizophrenia is affected,
  Nucleic acids defining genes whose expression levels are changed by schizophrenia are the following protein groups:
(1) Protein tyrosine phosphatase sigma ( GenBank No. U35234 )
(2) Tyrosine protein kinase receptor TIE-2 ( GenBank No.L06139 )
(3) EPH- Related receptor tyrosine kinase ligand 5 ( GenBank No.L38734 )
(4) Type 1 interferon receptor ( GenBank No.X77722 )
(5) Protein kinase ATR ( GenBank No. U49844 )
(6) STAT2 ( GenBank No. U18671 )
(7) High affinity nerve growth factor receptor precursor ( GenBank No.X03541 )
(8) OX40L Receptor precursor ( GenBank No.X75962 )
(9) STAT4 ( GenBank No.L78440 )
( Ten ) KIAA0096 ( GenBank No.D43636 )
( 11 ) Purin rich single chain DNA Binding protein alpha ( GenBank No.M96684 )
( 12 ) OX2 Membrane glycoprotein precursor ( GenBank No.X05323 )
( 13 ) CDC25B ( GenBank No.M81934 )
( 14 ) Guanine nucleotide binding protein  G (I) / G (S) / G (O) gamma -Ten Sub unit ( GenBank No. U31383 )
( 15 ) PKU-alpha ( GenBank No. AB004884 )
( 16 ) Wnt-8B ( GenBank No.X91940 )
( 17 ) Glycyl tRNA Synthetic enzyme ( GenBank No.D30658 )
( 18 ) Melanoma antigen P15 ( GenBank No. U19796 )
( 19 ) Transcription initiation factor eIF-2 Alpha subunit ( GenBank No. U26032 )
( 20 ) Curin homolog 2 ( GenBank No. U83410 )
( twenty one ) Transmembrane proteinaceous precursor ( GenBank No.X87852 )
( twenty two ) GTP Binding nucleoprotein RAN ( GenBank No.M31469 )
( twenty three ) Adenylate kinase isoenzyme 1 ( GenBank No.J04809 )
( twenty four ) Cell cycle proteins  P38-2G4 Homolog ( GenBank No. U59435 )
( twenty five ) Interferon gamma receptor ( GenBank No.J03143 )
( 26 ) Neurofibrosis 2 ( GenBank No.L27133 )
( 27 ) Cadherin 6 precursor ( CDH6 ) ( GenBank No.D31784 )
( 28 ) Bispecific protein phosphatase 8 ( GenBank No. U27193 )
( 29 ) Arginine / Serine-rich splicing factor 7 ( GenBank No.L22253 )
( 30 ) PIRIN ( GenBank No.Y07867 )
( 31 ) RAD51C Cleavage protein ( GenBank No.AF029670 )
( 32 ) Vascular endothelial growth factor receptor 1 ( GenBank No.X51602 )
( 33 ) DNA Topoisomerase 1 ( GenBank No.J03250 )
( 34 ) Laminin alpha 4 subunit precursor ( GenBank No.X70904 )
( 35 ) Somatostatin receptor type 2 ( GenBank No.M81830 )
( 36 ) Transferrin receptor ( GenBank No.X01060 )
In the brackets above GenBank A method characterized in that it is a nucleic acid contained in the nucleic acid group indicated by the receipt number. A method for determining whether a test animal is useful as a model animal for schizophrenia, comprising:
The process of diagnosing whether or not the subject animal suffers from schizophrenia by the method according to claim 3 or claim 4 ,
A method comprising determining that the subject animal is useful as a model animal for schizophrenia if the subject animal suffers from schizophrenia. A method for screening candidate substances for anti-schizophrenic drugs,
Providing the test substance to a model animal of schizophrenia,
The method according to claim 3 or 4 , wherein the process of diagnosing whether the schizophrenia of the model animal has been cured or improved, and if the schizophrenia of the model animal has been cured or improved, A method comprising determining that a test substance is a candidate substance for an anti-schizophrenia drug.

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