JP2022054158A - Methods for testing onset risk of schizophrenia, kits therefor, and mammalian animal models of schizophrenia - Google Patents

Abstract

To provide methods for evaluating likelihood of schizophrenia onset using mutation in EP400 gene as an indicative, and methods for preparing mammalian animal models of schizophrenia.SOLUTION: Disclosed is a method for testing the liability of a subject to develop schizophrenia by detecting the presence or absence of a mutation in EP400 gene in the DNA in a sample taken from the subject, based on a criterion that when a subject has a mutation in EP400 gene the subject is likely to develop schizophrenia. Also disclosed is a schizophrenia mammalian model having a mutation introduced in an ortholog of human EP400 gene and exhibiting human schizophrenia-like behavior abnormality.SELECTED DRAWING: None

Description

There is an application for application of Article 30, Paragraph 2 of the Patent Law. ▲ 1 ▼ August 21, 2nd year of Reiwa https: // www. c-linkage. co. jp / jpn116 / pdf / blog_all. Announced through pdf ▲ 2 ▼ September 14, 2nd year of Reiwa https: // www. jspn. or. jp / models / basics / indexes. php? file = activity / R2 / 116_day2. Announced through pdf

The present invention relates to a method for testing the likelihood of developing schizophrenia based on the presence or absence of mutation in the EP400 (E1A binding protein p400) gene in DNA derived from a sample collected from a subject. Furthermore, the present invention relates to a kit for evaluating the susceptibility to developing schizophrenia, which can be used in the method. Furthermore, the present invention relates to a model mammal for schizophrenia, a method for producing the model animal, and a use of the model animal.

Schizophrenia is a mental illness in which the ability to organize thoughts / behaviors and emotions / emotions for one purpose continues to decline for a long period of time, and the condition is thought to be derived from impaired brain function. There is. The frequency of developing schizophrenia is about 0.8 to 1.2% of the population, and it is not a rare disease.

Generally, positive symptoms, negative symptoms, and cognitive dysfunction are known as symptoms of schizophrenia. Typical symptoms of positive symptoms include delusions, hallucinations, and thought disorders, which mainly appear in the acute phase of schizophrenia. On the other hand, typical symptoms of negative symptoms include emotional flattening, poor thinking, lack of motivation, and autism, and even if the positive symptoms are alleviated by treatment in the acute phase, the negative symptoms often remain. .. Symptoms of cognitive dysfunction include poor memory, attention, poor concentration, and impaired judgment. The definition of "schizophrenia" herein follows those commonly used in the art. Commonly used diagnostic criteria for schizophrenia include WHO (World Health Organization) international classification of diseases "ICD-10" and American Psychiatric Association "DSM-5", which are used clinically. Has been done.

The cause and mechanism of schizophrenia are unknown, but there are theories that it is due to genetic factors, that it is related to the imbalance of neurotransmitters in the brain, and that it is due to environmental factors. In addition to these, there are theories that viral infections during pregnancy are associated, epigenetic abnormalities are associated, and chronic inflammation of the nervous system is associated. However, there is still no consensus on the causes and mechanisms of schizophrenia.

The importance of the genetic background in the onset of schizophrenia has been pointed out in the past, and twin studies and family studies have been conducted. In recent years, whole exome sequencing (WES) using a large number of samples has reported a genome showing a significant association with the development of schizophrenia. However, those regions (Common risk variants) identified by WES have a low contribution to disease risk (odds ratio), and many unclear points remain regarding the effects on the pathophysiology of schizophrenia (non-patent). Document 1).

On the other hand, although there are reports on gene mutations (Rare risk variants) that have a high odds ratio and are expected to have a significant effect on the onset of schizophrenia (Non-Patent Document 2, etc.), the number is extremely small. Therefore, there is still insufficient knowledge about the genetic factors involved in the development of schizophrenia.

In addition, in order to develop a therapeutic drug for schizophrenia, a model animal for schizophrenia is required to evaluate its efficacy by animal experiments. So far, as model mice for schizophrenia, a pharmacological animal model to which methamphetamine or amphetamine has been administered (Non-Patent Document 3), a neurodevelopmental animal model imitating perinatal infection (Non-Patent Document 4), etc. have been proposed. However, its scientific and clinical validity is controversial.

Many of the schizophrenia model animals reported so far are produced by drug administration. At present, no consensus genetic background for schizophrenia has been found, and although Non-Patent Document 5 is an example of a report on a schizophrenia model mouse prepared from genetic modification, such a report is also available. poor. Therefore, there is still a demand for schizophrenia model animals produced by a genetic approach.

Prata DP et al. Unravelling the genetic basis of schizophrenia and bipolar disorder with GWAS: A systematic review. J Psychiatr Res. 2019 Jul; 114: 178-207. Takata A et al. Loss-of-function variants in schizophrenia risk and SETD1A as a candidate susceptibility gene. Neuron. 2014 May 21; 82 (4): 773-80. Shimatsuna, Toru Nishikawa. Monoamine disorder / amphetamine model. Molecular psychiatry 2005 5: 58-63. Masayoshi Kawai, Envoy Takei. Theory of viral infection of schizophrenia. Molecular Psychiatry 2004 4: 56-64. Tsuboi D et al. Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity. Nat Neurosci. 2015 May; 18 (5): 698-707

Therefore, an object of the present invention is a method for identifying a gene associated with the susceptibility to the onset of schizophrenia, and further, based on the finding, a method for diagnosing schizophrenia and a method for evaluating the susceptibility to the onset of schizophrenia. Is to provide. Further, it is also an object of the present invention to provide a model animal for schizophrenia useful for the development of a drug for schizophrenia.

In order to achieve the above object, the present inventors focused on a family in which a large number of patients with schizophrenia existed for three generations, and investigated genes related to the susceptibility to developing schizophrenia. .. The family line will be described in detail in the following examples. Specifically, several subjects (subjects with schizophrenia and subjects without schizophrenia) were selected from the above-mentioned family, and the samples (blood) collected from the subjects were used to treat the disease. Whole Exome Sequencing (WES) (Funayama M et al., Lancet, 2015, 14: 274-282, Kim HJ et al.,, A powerful tool for detecting a single gene responsible for Nature, 2013, 495: 467-473) was performed. As a result, we found that the mutation of proline at position 2,805 of the EP400 gene to leucine is associated with the development of schizophrenia.

Furthermore, the present inventors created mice having a mutation in EP400 by using a gene editing method (CRISPR / CAS9), imitating this mutation (EP400: p.Pro2715Leu). Behavioral analysis of the mice showed schizophrenia-like behavioral abnormalities in humans. Furthermore, when the immune tissue staining of the brain axons of the mice was performed, morphological changes in nerve cells in the central nervous system were confirmed. Therefore, it was found that mice with such mutations can be used as model animals for human schizophrenia.

That is, the present invention is as follows.
[1]
The presence or absence of a mutation in the human EP400 gene in the DNA in the sample derived from the subject is detected, and the subject's susceptibility to schizophrenia is tested based on the criteria that if the mutation is present, schizophrenia is likely to occur. Method.
[1']
A method of detecting the presence or absence of a mutation in the human EP400 gene in DNA in a sample derived from a subject and diagnosing that schizophrenia is likely to occur if the mutation is present, or a method of assisting the diagnosis.
[2]
The method according to [1], wherein the presence or absence of a mutation in exon 48 of the human EP400 gene in DNA in a sample derived from a subject is detected.
[2']
The method according to [1'], wherein the presence or absence of a mutation in exon 48 of the human EP400 gene in DNA in a sample derived from a subject is detected.
[3]
The method according to [1] or [2], wherein the presence or absence of a mutation in cytosine at position 8414 of the human EP400 gene in DNA in a sample derived from a subject is detected.
[3']
The method according to [1'] or [2'], wherein the presence or absence of a mutation of cytosine at position 8414 to thymine in the DNA of a sample derived from a subject is detected.
[4]
Detects the presence or absence of a mutation in human EP400 protein to leucine at position 2805, [
1] The method according to any one of [3].
[4']
Detects the presence or absence of a mutation in human EP400 protein to leucine at position 2805, [
The method according to any one of 1'] to [3'].
[5]
The method according to any one of [1] to [4], wherein the subject is a human suspected of developing schizophrenia.
[6]
A diagnostic kit for schizophrenia containing a set of PCR primers capable of amplifying nucleotides in the entire coding region of the human EP400 gene.
[7]
The diagnostic kit for schizophrenia according to [6], which comprises a PCR primer set capable of amplifying the cytosine-containing region at position 8414 of the human EP400 gene.
[8]
A nucleotide sequence derived from the human EP400 gene, a probe capable of specifically hybridizing to a nucleotide sequence in which the 8414th nucleotide of the gene is mutated to chimin, and a nucleotide sequence derived from the human EP400 gene, the EP400 gene. The kit according to [6] or [7], further comprising a probe capable of specifically hybridizing to a nucleotide sequence in which the 8414th nucleotide of the above remains cytosine.
[9]
A non-human mammalian model of schizophrenia that has a mutation in the Ep400 gene and exhibits behavioral abnormalities that are indicators of human schizophrenia.
[10]
The model according to [9], wherein the Ep400 gene has a mutation in the region corresponding to exon 48 of the human EP400 gene.
[11]
The model mammal for schizophrenia according to [9] or [10], wherein the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene of the model mammal is mutated to thymine. [12]
The model mammal is a mammal selected from the group consisting of mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, goats, mini pigs, pigs, sheep, cows, monkeys and chimpanzees [9]-[. 11] The model mammal according to any one of.
[13]
The model mammal according to any one of [9] to [12], wherein the model mammal is a mouse, and cytosine at position 8144 of the mouse Ep400 gene is mutated to thymine.
[14]
The model mammal according to any one of [9] to [12], wherein the model mammal is a mouse, and proline at position 2715 of the mouse Ep400 protein is mutated to leucine.
[15]
The model mammal according to any one of [9] to [14], which exhibits anxious behavior as the behavioral abnormality.
[16]
A method for producing a model mammal for schizophrenia showing behavioral abnormalities that are indicators of human schizophrenia by introducing a mutation into the Ep400 gene.
[17]
The method according to [16], wherein a mutation is introduced into a region of the Ep400 gene corresponding to exon 48 of the human EP400 gene.
[18]
The method according to [16] or [17], wherein the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene of the model mammal is mutated to thymine by introducing the mutation.
[19]
The method according to [16] or [17], wherein the amino acid corresponding to proline at position 2805 of the human EP400 protein of the model mammal is mutated to leucine by introducing the mutation.
[20]
The method according to any one of [16] to [19], wherein the mutation is introduced by a gene editing method using Crispr / CAS-9.
[21]
The model mammal is a mammal selected from the group consisting of mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, goats, mini pigs, pigs, sheep, cows, monkeys and chimpanzees [16]-[. 20] The method according to any one of.
[22]
The method according to any one of [16] to [21], wherein the model mammal is a mouse.
[23]
The method according to any one of [16] to [22], which exhibits anxious behavior as the behavioral abnormality.
[24]
[9] A method for screening a therapeutic or preventive drug for schizophrenia using the model mammal according to any one of [9] to [15], wherein the following steps;
When the behavioral abnormality is improved as compared with (1) the step of administering the test substance to the model mammal and (2) the control group to which the test substance was not administered, the test substance is used for schizophrenia. The process of selecting as a candidate substance for a therapeutic or preventive drug,
How to include.

According to the present invention, the presence or absence of a mutation in the human EP400 gene in DNA in a sample derived from a subject, preferably the presence or absence of a mutation in the human EP400 gene exon 48, particularly preferably the 8414th (80th exon 48) thymine of the human EP400 gene. By detecting the presence or absence of a mutation in thymine, the susceptibility of the subject to schizophrenia can be evaluated. In addition, by detecting the presence or absence of a mutation in the human EP400 gene of the subject, preferably the presence or absence of a mutation in the human EP400 gene Exxon 48, particularly preferably the presence or absence of a mutation in the 8414th cytosine of the human EP400 gene to schizophrenia, other In addition to the presence or absence of clinical symptoms peculiar to schizophrenia (positive symptoms, negative symptoms, cognitive dysfunction, etc. described above), the subject can determine the susceptibility to developing schizophrenia. The mutation of cytosine at position 8414 of the human EP400 gene to thymine results in the mutation of proline at position 2805 of the human EP400 protein to leucine. In addition, a kit capable of detecting the mutation in the human EP400 gene is useful for evaluating the susceptibility to developing schizophrenia and assisting in diagnosis.

Further, according to the present invention, there is provided a model mammal for schizophrenia in which the EP400 gene is mutated and exhibits behavioral abnormalities that are indicators of human schizophrenia. In the model mammal of schizophrenia, the nucleotide of the region corresponding to exon 48 of the human EP400 gene of the mammal is preferably mutated, and particularly preferably 8414 of the human EP400 gene of the mammal.
The nucleotide corresponding to the th (80th exon 48) cytosine is mutated to thymine. In the following examples, a mouse model mouse for schizophrenia was prepared by mutating cytosine at position 8144 (80th position of Exxon 47) of the mouse EP400 gene to thymine, and the mouse EP400 protein was produced in the model mouse for schizophrenia. The 2715th proline of the mouse is mutated to leucine. In addition, such schizophrenia model mammals can also be used to screen for therapeutic or prophylactic agents of schizophrenia.

FIG. 1a is a family tree of three generations of a family tree in which schizophrenia frequently occurs. In FIG. 1a, the square symbol is male, the circle symbol is female, and the black symbol is the subject diagnosed with schizophrenia (I-3, I-4, I-9, II- in FIG. 1a). 2, II-3, II-5, II-10, II-12, II-13, II-17, III-2, III-3). Figure 1b: Missense mutation in EP400 isolated with schizophrenia (chr12: 132064747; hg38 C> T, ENST00000389561.7 p.P2805L). Figure 1c: Location of familial missense mutations in exon 48 of the EP400 gene within a highly conserved disorder binding region among mammals. Figure 1d: PONDR's CaN-XT algorithm predicted that p.P2805L would cause elongation of the disorder region of the EP400 protein. Genotype and hindlimb clenching of mutant-introduced mice (Ep400-p.P2715L # 1 and Ep400-p.P2715L # 2). A silent mutation that disrupts the Ep400 missense mutation (p.P2715L, red arrow) and the protospacer flanking motif (PAM; green arrow) was confirmed by Sanger sequencing. All mutant-introduced mice showed hindlimb clenching (yellow arrow) from 3 months of age. Figure 3a: Open field test of Ep400-p.P2715L # 1 mice (n = 6) and wild-type (WT) littermates (n = 6). At 3 months of age, there was no significant difference in center time compared to wild-type mice, but at 6 months of age, the center time was significantly reduced. Figure 3b: Social interaction test of Ep400-p.P2715L # 1 mice (n = 6) and wild-type (WT) littermates (n = 6). At 3 months of age, there was no significant difference in the number of Stretched Approach behaviors to confirm stimulated mice that were later caged with their necks extended compared to wild-type mice, but at 6 months of age, there was no significant difference in the Stretched Approach. The number of times was significantly increased. The results of histological analysis of the brain of a mutant-introduced mouse (Ep400-p.P2715L # 1) are shown. FIG. 4a: HE staining of brain tissue. Mutation-introduced mouse brains showed no significant changes compared to wild-type (WT) littermates. CS = coronal section. SS = sagittal section. DG = Hippocampal dentate gyrus. White box: There was no morphological abnormality of the nerve cell body. Green scale bar = 1000 μm. White scale bar = 50 μm. FIG. 4b: Immunofluorescent staining of the hippocampal dentate gyrus. White box: Expression of Ep400 was widely observed in the nucleus of neurons, and the expression pattern was less than that of wild-type mice. Red: Ep400. Green: Map2 (nerve cell marker). Blue: DAPI. White scale bar = 50 μm. The results of histological analysis of the spinal cord of the mutant-introduced mouse (Ep400-p.P2715L # 1) are shown. Figure 5a: 3 month old Ep400-p.P2715L # 1 mice showed no clear difference in HE staining compared to wild-type (WT) littermates, but KB and TB staining showed a decrease in axonal diameter. Indicated. HE = hematoxylin-eosin staining. KB = Kluber-Vallera stain. TB = toluidine blue stain. Yellow scale bar = 200 μm. White scale bar = 50 μm. Orange scale bar = 10 μm. Figure 5b: TEM analysis. Ep400-p.P2715L # 1 mice (n = 2) showed no morphological abnormalities in the myelin sheath and had no significant difference in G ratio compared to wild-type (WT) littermates, but significant axonal diameter. The decrease was also confirmed by TEM analysis. Orange scale bar = 10 μm. Red scale bar = 1 μm. The results of histological analysis of the spinal cord of the mutant-introduced mouse (Ep400-p.P2715L # 2) are shown. FIG. 6a: 3-month-old Ep400-p.P2715L # 2 mice showed reduced axonal diameter by KB and TB staining compared to wild-type (WT) littermates. KB = Kluber-Vallera stain. TB = toluidine blue stain. Yellow scale bar = 200 μm. White scale bar = 50 μm. Orange scale bar = 10 μm. Figure 6b: TEM analysis. Ep400-p.P2715L # 2 mice (n = 2) showed no morphological abnormalities in the myelin sheath and had no significant difference in G ratio compared to wild-type (WT) littermates, but significant axonal diameter. The decrease was also confirmed by TEM analysis. Orange scale bar = 10 μm. Red scale bar = 1 μm. Harmful variants of EP400 detected in Japanese (schizophrenia patients and controls). Rare variants are represented by lollipops and show the number of alleles with schizophrenia patients (upper panel) and controls (lower panel) variants. Rare variant = variant with 0.001 <MAF <0.005 in any database. Ultra-rare variant = variant with MAF <0.001 in all databases. New variant = variant that is not registered in all databases.

1. 1. Test method and diagnostic method of the present invention The present invention is a method for testing the susceptibility of a subject to schizophrenia by detecting the presence or absence of a mutation in the human EP400 gene of the subject (hereinafter, "the test method of the present invention"). Also called.). Preferably, the test method of the present invention is the presence or absence of a mutation in exon 48 of the human EP400 gene in DNA in a sample derived from a subject, particularly preferably at position 8414 of the human EP400 gene.
The presence or absence of a mutation in cytosine of exon 48 (80th) to thymine can be detected, and if there is a mutation, it can be performed according to the criteria that schizophrenia is likely to occur. As used herein, the "Xth of the EP400 gene" means the Xth nucleotide counting from the first nucleotide of the coding sequence (CDS). Further, in the present specification, "the Y-th amino acid of the EP400 protein" means that it is the Y-th amino acid counting from the starting methionine of the protein.

Further, the present invention is a method of diagnosing the subject's susceptibility to schizophrenia by detecting the presence or absence of a mutation in the human EP400 gene of the subject, or a method of assisting the diagnosis (collectively, "the present invention". The method of diagnosing the invention ”is provided. Preferably, the diagnostic method of the present invention relates to the presence or absence of a mutation in exon 48 of the human EP400 gene in DNA in a sample derived from a subject, particularly preferably to thymine of cytosine at position 8414 (eighth of exon 48) of the human EP400 gene. This can be done by detecting the presence or absence of a mutation and, if it has a mutation, diagnosing that it is likely to develop schizophrenia.

EP400 (E1A binding protein p400) is a component of the NuA4 histone acetyltransferase complex, which is involved in transcriptional activation of selected genes by acetylation of histones H4 and H2A in nucleosomes. This modification can alter the nucleosome-DNA interaction and facilitate the interaction of the modified histone with other proteins that positively regulate transcription. EP400 may be required for transcriptional activation of E2F1 and MYC target genes during cell proliferation. NuA4 complex ATPase and helicase activity
At least in part, the meeting of RUVBL1 and RUVBL2 with EP400 is believed to have contributed. EP400 can also control ZNF42 transcriptional activity. EP400 is also a component of the SWR-1-like complex that specifically mediates the removal of histones H2A, Z / H2AFZ from nucleosomes from nucleosomes. However, to date, there are no reports suggesting a link between EP400 and schizophrenia.

The human EP400 protein consists of 3123 amino acid residues and is encoded by a nucleic acid consisting of 9372 nucleotides. For the nucleotide sequence of the cDNA of human EP400, GenBank Accession No. NM_015409 (reference sequence hg38) can be referred to, and the nucleotide sequence is shown as SEQ ID NO: 1. The amino acid sequence of EP400 encoded by the CDS is shown as SEQ ID NO: 2. The cDNA sequence of EP400 in non-human mammals is also known. For example, the cDNA sequence of mouse EP400 can refer to GenBank Accession No. NM_029337 (reference sequence mm10), and the sequence is shown as SEQ ID NO: 3. The amino acid sequence of mouse EP400 encoded by the CDS is shown as SEQ ID NO: 4. Further, in the present specification, unless otherwise specified (such as described as "mouse EP400"), the position of the nucleotide is described based on the nucleotide sequence of the human EP400 cDNA, but the corresponding nucleotide in the non-human mammalian ortholog is described. Sequences, nucleotide positions, etc. are also included in the description.

As shown in the examples below, when a subject suffering from schizophrenia in a family with frequent onset of schizophrenia was analyzed, the subject's EP400 gene at position 8414 (80th of exon 48) was analyzed. ) Cytosine was shown to be mutated to thymine. Therefore, the most important feature of the method of the present invention is to detect the presence or absence of a mutation in the EP400 gene of a subject, preferably the presence or absence of a mutation in exon 48, and particularly preferably the presence or absence of a mutation in cytosine at position 8414 of the EP400 gene to thymine. This contributes to the risk assessment of schizophrenia. The gene mutation results in a mutation in which proline at position 2805 is replaced with leucine in the encoded human EP400 protein.

The "subject" to be tested in the present invention may be a human suspected of having schizophrenia or a human who does not show any symptoms of schizophrenia. Since even a person who does not show symptoms of schizophrenia at the time of examination can predict the likelihood of developing schizophrenia in the future, the present invention is used as a means of genetic diagnosis for evaluating the risk of schizophrenia. be able to.

As used herein, the term "prone to develop schizophrenia" is used in the future for subjects who do not show any symptoms of schizophrenia at the time of examination or who are suspected of developing schizophrenia but whose diagnosis has not been confirmed. It means that the likelihood of developing schizophrenia is higher than that of the general population of subjects.

The diagnostic method of the present invention can also be used for the purpose of assisting the definitive diagnosis of schizophrenia in a subject suspected of developing schizophrenia. As already mentioned, the diagnosis of schizophrenia is mainly based on the presence or absence of positive symptoms, negative symptoms, and mental symptoms such as cognitive dysfunction. Such diagnoses are made with caution by skilled psychiatrists, but such diagnostic methods based on psychiatric symptoms lack objectivity. Since the diagnostic method of the present invention is carried out by detecting the presence or absence of a mutation in the human EP400 gene, it contributes to the objective and simple diagnosis of schizophrenia. The accuracy of the diagnostic result can be improved by combining the diagnostic method of the present invention with the presence or absence of the mental symptom.

For subjects with a confirmed diagnosis of schizophrenia, it is preferable to treat schizophrenia. The main treatment for schizophrenia is drug treatment. Drugs used to treat schizophrenia include first-generation psychotropic drugs such as chlorpromazine, haloperidol, levomepromazine, sulpiride, thimiperon, propericiadin, and zotepine, as well as risperidone, olanzapine, quetiapine, peropirin, aripiprazole, bronanceline. , Clozapine, parisperidone, acenapine, and second-generation psychotropic drugs such as brexpiprazole. Accordingly, the present invention also provides a method for diagnosing and treating schizophrenia, which comprises performing drug treatment in a subject diagnosed with schizophrenia.

Furthermore, the present invention relates to the presence or absence of a mutation in the human EP400 gene in DNA derived from a sample collected from a subject, preferably the presence or absence of a mutation in the exon 48 of the human EP400 gene, particularly preferably to thymine at position 8414 of the human EP400 gene. Also provided is a method of collecting data for assessing the likelihood of developing schizophrenia, including the step of detecting the presence or absence of mutations. Data can be collected by a method known per se.

The sample used in the present invention is not particularly limited as long as it is a sample collected from a subject, and examples thereof include blood, plasma, serum, saliva, urine, skin, nails, hair, bone marrow fluid, oral mucosa, and organs. .. Preferably, it is blood, plasma, serum or saliva. These samples can be obtained by a method known per se, for example, serum or plasma can be prepared by collecting blood from a subject according to a conventional method and separating the humoral component. The DNA in the sample can be obtained by extracting genomic DNA from the sample, and for example, genomic DNA can be isolated from blood collected from a subject by ordinary blood sampling by a phenol extraction method or the like. At that time, for example, a commercially available genomic DNA extraction kit or device such as the QI Amp Circulating Nucleic Acid Kit may be used.

Detection of mutations in the human EP400 gene specifically amplifies, for example, the entire coding region of the human EP400 gene, preferably the region of exon 48 of the human EP400 gene, particularly preferably the region containing cytosine at position 8414 of the human EP400 gene. Mutations can be detected by amplifying the region by PCR using the obtained primer set and determining the base sequence of the obtained amplified product by a method known per se. Regarding the primer set, refer to the following 2. Will be explained in detail in. Examples of the method for determining such a base sequence include a Sanger method and a method using a next-generation sequencer (NGS). For the method using the next-generation sequence, for example, the separate volume of experimental medicine "Next-generation sequence analysis standard", 2014 (Yodosha), etc. can be referred to. NGS used for this purpose include Illumina equipment (eg MiSeq, HiSeq2500), Life Technologies equipment (eg Ion Proton, Ion PGM), and Roche Diagnostic. Devices manufactured by Roche Diagnostic (eg, GS FLX +, GS Junior) can be mentioned, but are not limited to these devices.

The method for determining the base sequence using NGS differs depending on the type of NGS, and can be carried out, for example, according to the manual of each company (eg, Nextera ^R XT DNA Library Prep Reference Guide). In addition, paired-end analysis is preferably used for sequencing the obtained samples.

Alternatively, the presence or absence of mutation in the human EP400 gene can be determined by any polymorphism analysis method known in the art, and examples thereof include a method using a PCR method. As a method using the PCR method, the nucleic acid to be analyzed is amplified by the PCR method and the mutation of the amplification product is detected by fluorescence or luminescence, PCR-RFLP (restriction fragment length polymorphism). Method, PCR-SSCP (single strand conformation polymorphism) method (Orita, M. et al., Proc. Natl. Acad. Sci., USA, 86, 2766-2770 (1989), etc.)
, PCR-SSO (specific sequence oligonucleotide) method, ASO (allele specific oligonucleotide) hybridization method (Saiki, Nature, which is a combination of PCR-SSO method and dot hybridization method)
324, 163-166 (1986), etc.), TaqMan®-PCR method (Livak, KJ, Genet Anal, 14,143 (1999), Morris, T. et al., J. Clin. Microbiol ., 34,2933 (1996)), real-time PCR method, digital PCR method (eg, droplet digital PCR (ddPCR) method, micropore distribution type digital PCR method, etc.) and the like can be mentioned.

Other methods include a cycling probe method, an Invader (registered trademark, Third Wave Technologies) method (Lyamichev V et al., Nat Biotechnol, 17,292 (1999)), and a method using FRET (Fluorescence Resonance Energy Transfer) (Heller, Academic Press Inc, pp. 245-256 (1985), Cardullo et al., Proc. Natl. Acad. Sci. USA, 85, 8790-8794 (1988), International Publication No. 99/28500, JP-A-2004- 121232, etc.), methods using DNA chips or microarrays (Wang DG et al., Science 280, 1077 (1998), etc.), Southern blot hybridization method, dot hybridization method (Southern, E., J. Mol) .Biol. 98,
503-517 (1975)) and the like.

In order to detect a small amount of mutant DNA, a method using the PCR method is preferable from the viewpoint of simplicity and sensitivity. In addition, unlike conventional real-time PCR, digital PCR (eg, ddPCR), which is a next-generation PCR that does not have to worry about amplification efficiency, enables absolute quantification, has high accuracy and high sensitivity, and has excellent processing performance, is available. Especially preferable.

2. 2. Kit for Diagnostic of Schizophrenia The present invention provides a kit for evaluating the risk of developing schizophrenia or a kit for diagnosing schizophrenia (collectively referred to as "kit of the present invention"). .. The kit is based on the above 1. A kit suitable for carrying out the test method of the present invention or the diagnostic method of the present invention, the nucleotide of the entire coding region of the human EP400 gene, preferably the cytosine at position 8414 (exon 48, position 80) of the human EP400 gene. Contains a set of PCR primers capable of amplifying the region containing. Such a PCR primer set is a set of forward and reverse primers of about 15 to about 30 bases capable of specifically amplifying the region containing the 8414th nucleotide of the EP400 gene. Such a primer can be designed by a method known per se. For example, it can be designed using a PCR primer design tool (eg Primer3, etc.) based on the sequence information of the EP400 gene, and as a specific example, a forward primer consisting of 5'-TCATCAAAATGCAGAAGCAGA-3'(SEQ ID NO: 5). And, but not limited to, a primer set consisting of a reverse primer consisting of 5'-TGCTGCTTTGAAAACAAA-3'(SEQ ID NO: 6).

Each primer in the above PCR primer set consists of nucleic acid, and the nucleic acid is preferably a single-stranded nucleic acid. Examples of the nucleic acid include DNA, RNA, and a polymer in which RNA and DNA are mixed. When RNA is contained, the base sequence thereof is "T (thymine)" in the DNA sequence.
Should be read as "U (uracil)". The above primer can be prepared by any chemical synthesis method well known to those skilled in the art, for example, can be prepared by using an enzyme such as a nuclease, or a commercially available DNA / RNA automatic synthesizer (Applied Biosystems). It can also be manufactured using a company, Beckman, etc.). These primers may be obtained by further modifying the constituent nucleic acids as desired. For example, the above-mentioned primer is a labeling substance at the 5'end or 3'end for facilitating detection and amplification of the primer (for example, a fluorescent molecule, a dye molecule, a radioactive isotope, an organic compound such as digoxigenin or biotin). And / or an additional sequence (such as a loop primer moiety used in the LAMP method) may be included. The primer may be phosphorylated or amineated at the 5'end. Further, the primer may contain only a natural base or may contain a modified base. Examples of the modified base include, but are not limited to, deoxyinosine, deoxyuracil, and S-modified base. moreover,
The primer may contain any derivative oligonucleotide including a phosphorothioate bond, a phosphoroamidate bond, or the like, or may contain a peptide-nucleic acid (PNA) containing a peptide nucleic acid bond.

These primers can be provided as a solid in a dry state or in an alcohol-precipitated state, or can be provided in a dissolved state in water or a suitable buffer solution (eg, TE buffer solution, etc.).

The kit of the present invention may further include a probe set for detecting mutations in the EP400 gene. Such probe sets are: (a) a probe capable of specifically hybridizing to a cytosine at position 8414 of the EP400 gene mutated to thymine, and (b) a nucleotide at that position unmutated (ie, remains cytosine). Includes probes that can specifically hybridize to (is). As such a probe set, an oligonucleotide consisting of a continuous nucleotide sequence of about 5 to about 30 bases, preferably about 7 to about 20 bases, which hybridizes with a partial nucleotide sequence containing the 8414th nucleotide of the EP400 gene is used. Be done.

The sequence of the probe can be appropriately designed according to the target sequence. For example, EP400 mentioned above
The sequence consisting of 5 or 7 nucleotides for detecting the mutation of cytosine at position 8414 of the gene includes the sequence enclosed in the box in Table 1 and the sequence complementary to the sequence. In the table, the bold T indicates a mutant nucleotide, and the mutation changes the codon (CCG) encoding proline to leucine (CTG). Examples of the probe that can specifically hybridize the nucleotide at the corresponding position to the normal one, which is a pair of the probe, include the sequence surrounded by the box in Table 2 and the sequence complementary to the sequence. Be done. Thus, in one embodiment, the probe set comprises (or consists of) any of the sequences listed in Table 1 or a sequence complementary to that sequence, and any of the sequences listed in Table 2 or A probe set consisting of a probe containing (or consisting of) a sequence complementary to the sequence can be mentioned, but the probe set is only an example, and the probe is appropriately considered in consideration of the target sequence and its length and the like. Can be designed.

Labeling material can be attached to each probe. Examples of the labeling substance include fluorescent dyes and the like. Various fluorescent dyes are commercially available, for example, 6-FAM (fluorescein), HEX, TE, Quasar 670, Quasar 570, Quasar 705, Pulsar 650, TET, HEX, VIC, JOE, CAL Fluor Orenge. , CAL Fluor Gold, CAL Fluor Red, Texas Red, Cy, Cy5 and so on. It is preferable that the fluorescent dye bound to the normal probe and the fluorescent dye bound to the mutant probe are different. By adopting this configuration, the normal probe hybridizes to the EP400 gene fragment containing normal (non-mutated) nucleotides, and the mutant probe hybridizes to the EP400 gene fragment containing mutated nucleotides. Two types of fluorescence derived from the fluorescent dye of the probe are detected.

It is preferable that each probe is further bound to a quencher capable of quenching the fluorescence from the fluorescent substance. The quencher is not particularly limited as long as it can quench the fluorescence from the fluorescent dye, and may be a fluorescent dye or a non-fluorescent dye, but a non-fluorescent dye is preferable from the viewpoint of detection accuracy. There can be. Specific quenchers include, for example, 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), Eclipse Dark Quencher, Iowa black FQ (IBFQ), minor groove binder (MGB), non- Fluorescent quencher (NFQ) and the like can be mentioned.

Examples of the building blocks of the probe include ribonucleotides and deoxyribonucleotides. The nucleotide residue contains sugars, bases and phosphoric acid as components. Ribonucleotides have a ribose residue as a sugar and bases adenine (A), guanine (G), cytosine (C) and uracil (U) (which can also be replaced with thymine (T)). Deoxyribonucleotide residues have deoxyribose residues as sugars and bases such as adenine (dA), guanine (dG), cytosine (dC) and thymine (dT) (which can also be replaced with uracil (dU)). Have.

The kit of the present invention is used for the PCR reaction in the above-mentioned detection method of the present invention in addition to the above-mentioned primer set and, if necessary, the above-mentioned normal probe and the above-mentioned mutant probe set. Reagents such as DNA extraction reagents, DNA polymerases, dNTPs, reaction buffers, nucleic acids containing target regions that are positive controls for PCR, and containers.
Can include appliances, instructions, etc. In addition, the above-mentioned primer set or probe set may be included in the kit as a probe set and / or a primer set in which each primer or each probe is stored in a coexisting state so as not to adversely affect the reaction. can.

3. 3. The model mammal of schizophrenia of the present invention and the method for producing the same The present invention is a model mammal of schizophrenia having a mutation in the EP400 gene and showing behavioral abnormalities that are indicators of human schizophrenia. (Hereinafter, also referred to as "a model mammal for schizophrenia of the present invention"). The model mammal for schizophrenia of the present invention preferably has a nucleotide mutation corresponding to exon 48 of the human EP400 gene of the model mouse, and particularly preferably to cytosine at position 8414 of the human EP400 gene of the model mouse. The corresponding nucleotide is mutated to thymine.

As used herein, "behavioral disorders that are indicators of human schizophrenia" include, but are not limited to, anxious behavior. The anxiety behavior exhibited by the model mammal of schizophrenia of the present invention is considered to reflect the anxiety tendency that is always seen in the symptoms of human schizophrenia. The model mammal for schizophrenia of the present invention is particularly excellent in that it exhibits behavioral abnormalities corresponding to the characteristics of human schizophrenia.

As shown in the examples below, the present inventors created a model mouse in which the 8144th cytosine of the mouse EP400 gene (corresponding to the 8414th cytosine of the human EP400 gene) was mutated to thymine. The mutation is that the 2175th proline of the EP400 protein of the model mouse is mutated to leucine.

The schizophrenia model mice produced in the examples showed the Hind-Limbclasping sign, which is a sign of abnormalities in the central nervous system. The model mammals for schizophrenia of the present invention do not show the Hind-Limbclasping sign immediately after birth, and the findings are seen after 3 months of age. Considering that mice become pregnant in about two months after birth, that time is almost the same as the age at which humans are prone to develop schizophrenia. Furthermore, when the behavioral analysis of the model mouse was performed, it was found that anxiety was enhanced (shortening of the central stay time in the open field test, No of Stretched Approach in the social interaction test (stretching the neck to the stimulated mouse and confirming its existence). An increase in the number of actions performed) was obtained. In addition, a histological analysis of the central nervous system of the model mouse revealed morphological changes in nerve cells (shortening of the nerve axis cord diameter) in the central nervous system. Observed.

Therefore, the schizophrenia model mammal produced by the present invention exhibits human schizophrenia-like behavioral abnormalities. As already mentioned, anxiety tendencies are almost always seen in human schizophrenia. Furthermore, the shortening of the nerve axon diameter of nerve cells in the central nervous system reflects microstructural abnormalities in the axons and myelin sheath that have been repeatedly suggested in human schizophrenia. The model mammal for schizophrenia of the present invention shows a long-term but mild abnormality of the central nervous system and does not die from the abnormality of the central nervous system. Even in human schizophrenia, patients rarely die due to abnormalities in the central nervous system itself, and it is said that lifestyle-related diseases due to lack of self-care associated with schizophrenia affect lifespan. In this respect as well, it is suggested that the schizophrenia model mammal of the present invention may imitate human schizophrenia.

In addition, since the schizophrenia model mammal of the present invention exhibits behavioral abnormalities similar to human schizophrenia, it can be used for research on the onset mechanism of schizophrenia and the progress of symptoms of the disease.

The animal species that is the source of the model mammal for schizophrenia of the present invention is a mammal other than human, and it is preferable to introduce a mutation into the EP400 gene in the mammal, preferably to Exxon 48 of the human EP400 gene. Introducing the mutation into the corresponding region, particularly preferably in the mammal, is not particularly limited as long as it is a mammal capable of introducing the mutation into the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene, but is not particularly limited, for example. Examples include rodents such as mice, rats, hamsters and guinea pigs, primate animals such as red-tailed monkeys, crab monkeys, Japanese monkeys and chimpanzees, cows, horses, dogs and cats. From the viewpoint of ease of handling, rodents are preferable, and mice are more preferable.

Examples of the method for producing the model mammal of the present invention include the following methods. For example, RNA encoding CRISPR-Cas9 and a guide RNA targeting the EP400 gene are injected into a fertilized egg, and the EP400 gene of the mammal is subjected to deletion, insertion or substitution, preferably substitution, which occurs during non-homologous end binding. In particular, it is possible to introduce a mutation into the nucleotide of the region corresponding to exon 48 of the human EP400 gene of the mammal, and particularly preferably to the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene of the mammal. can. Further, in addition to the above RNA, a donor DNA into which a mutation has been introduced in advance can be introduced into a fertilized egg, and a mutation can be introduced into the EP400 gene by homologous recombination between the donor DNA and the genomic DNA. By transplanting the fertilized egg thus obtained into the uterus of a pseudo-pregnant female animal, a mutation was introduced into the EP400 gene of the animal, preferably corresponding to Exxon 48 of the human EP400 gene of the real thing. It is possible to obtain a model mammal for schizophrenia in which a mutation has been introduced into a region thereof, particularly preferably, a mutation has been introduced into the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene. Alternatively, the donor DNA is introduced into ES cells, and homologous recombination between the donor DNA and genomic DNA is performed to prepare ES cells in which the mutation is introduced into the EP400 gene.
By injecting the ES fine into the scutellum of a mouse to produce a chimeric animal and mating the chimeric animal with a wild-type animal (and, if necessary, further mating offspring), the human EP400 gene of the animal. The mutation was introduced into the region corresponding to Exxon 48 of the human EP400 gene of the animal, particularly preferably to the nucleotide corresponding to the 8414th cytosine of the human EP400 gene of the animal. A model mammal for schizophrenia introduced with is produced.

The schizophrenia model mammal of the present invention prepared as described above can be used, for example, for the treatment of schizophrenia and / or the screening of preventive agents. The schizophrenia model mammal of the present invention is very useful in that it can be used for the treatment of schizophrenia and / or the screening of preventive agents. That is, the present invention is a method for screening a therapeutic or preventive drug for schizophrenia.
(1) The step of administering the test substance to the model mammal of the schizophrenia of the present invention, and (2) the behavioral abnormality that is an index of human schizophrenia as compared with the control group to which the test substance was not administered. Provided is a method including a step of selecting the test substance as a candidate substance for a therapeutic or preventive drug for schizophrenia when the substance is improved.

The administration of the test substance to the model mammal of schizophrenia in the step (1) is oral or parenteral (for example, subcutaneous injection, intramuscular injection, local injection (eg, intraventricular administration), intraperitoneal administration, etc. ) Can be done. The test substance may be produced as a parenteral preparation such as an injection, a suspension, or an infusion by mixing with a pharmaceutically acceptable carrier according to conventional means. Pharmaceutically acceptable carriers that may be included in the parenteral preparation include, for example, isotonic solutions containing saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Examples include an aqueous solution for injection.

Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which include antioxidants, buffers, bacteriostatic agents, isotonic agents and the like. May be. Examples thereof include aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The pharmaceutical product can be encapsulated in a container at a unit dose or a plurality of doses like an ampoule or a vial. In addition, the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried and stored in a state where it can be dissolved or suspended in a suitable sterile vehicle immediately before use.

The test substance to be orally administered may be produced as a solid preparation such as a powder, a tablet, a granule, a coated tablet, a powder or a capsule, or a liquid preparation such as a liquid, a suspension, an emulsion or a syrup. The test substance is an excipient, a binder, a lubricant, a surfactant, a diluent, a preservative, a stabilizer, a flavoring agent, a moisturizer, a preservative, an antioxidant, etc., according to a conventional method. Can be prepared.

In step (2), "not administered the test substance" means, for example, when the test substance is administered in the form of a preparation, a preparation containing the same components other than the test substance is administered. This includes the administration of a drug containing a substance known to have no therapeutic or preventive effect on schizophrenia, which is different from the substance, or the administration of none of these agents. Further, as the model animal to which the test substance used as a control in step (2) has not been administered, a separate model animal prepared by the same method may be used, or the same model before administration of the test substance may be used. Animals may be used. As a result of the comparison in the step (2), when the behavioral abnormality that is an index of human schizophrenia is improved, the test substance can be selected as a candidate substance for a therapeutic or preventive drug for schizophrenia. Behavioral abnormalities that are indicators of human schizophrenia include, but are not limited to, anxiety behavior.

Evaluation of delay in progression of such symptoms or improvement of symptoms is performed based on experiments using animals such as behavioral experiments (eg, open field test, social interaction test, Y-shaped maze test, novel object search test, etc.). Alternatively, it may be based on histological analysis of the central nervous system.

Examples of the test substance used for the above screening include cell extracts, cell culture supernatants, microbial fermentation products, marine organism-derived extracts, plant extracts, purified proteins or crude proteins, peptides, non-peptide compounds, and synthetic small molecules. Examples include compounds and natural compounds.

The test substances are also (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) It can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches are applicable to small molecule library of peptides, non-peptide oligomers, or compounds (Lam (1997). ) Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angelw. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angelw. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem . 37: 1233-51). Compound libraries can be found in solutions (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555- 6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698, No. 5,403,484, and No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; US Patent Application No. 2002103360).

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1: Analysis of gene mutations in a family with frequent schizophrenia

Example 1: Analysis of gene mutations in multiple families of schizophrenic patients 1. Family of schizophrenia analyzed by the present invention The present invention originated from the discovery of one of the largest schizophrenia-prone families in Japan. There are many schizophrenia patients in the family tree of the patient, and the family tree is shown in FIG. 1a. In FIG. 1a, the square symbol is male, the circle symbol is female, and the black symbol is the subject diagnosed with schizophrenia (I-3, I-4, I-9, II-2 in FIG. 1a). , II-3, II-5, II-10, II-12, II-13, II-17, III-2, III-3).

The above subjects (I-3, I-4, I-9, II-2, II-3, II-5, II-10, II-12, II-13, II-17, III-2, FIG. 1a) III-3) shows positive symptoms such as illusion, delusion, and thought disorder, which are the main symptoms of schizophrenia, and negative symptoms such as inaction, autism, and loss of motivation, and is an international diagnostic standard for psychiatric disorders (ICD). He was diagnosed with schizophrenia by -10 and DSM-5).

2. 2. Samples for DNA Extraction 7 patients from this family (I-9, II-3, II-5, II-12, II-17, for whole exome sequencing (WES) and Sanger sequencing (I-9, II-3, II-5, II-12, II-17, Analysis was performed on III-2, III-3), and 3 non-affected persons (I-13, II-11, II-20). A total of 10 mL of peripheral blood was collected from each subject, and genomic DNA was extracted from blood lymphocytes using the QIAamp DNA Midi Kit (Qiagen).

3. 3. Whole Exome Sequencing 10 subjects from this family (I-9, II-3, II-5, II-12, II-17, III-2, III-3, I-13, II-11, II For -20), a library was prepared using the SureSelect Exome Target Enrichment System v5 kit (Agilent Technologies), and WES was performed using HiSeq 2500 (Illumina).

4. Selection of causative mutations
In order to detect the pathogenic substitutions in this family from the WES data, we selected the harmful variants that meet the following criteria as candidate genes for the etiology.
(1) Variants that result in stop gain mutations, stop loss mutations, silent mutations, or splice site mutations in GENCODE basic version 19 downloaded from the UCSC Genome Browser (2) Alternative allergens at variant loci in the following databases. Frequency is 0.1% or less-Data of 1000 genome project on total population announced in October 2014-Exome data of 6515 of NIH National Cardiopulmonary Blood Research Institute (evs.gs.washington.edu / EVS)
Exome data from the Exome Aggregation Consortium (ExAC) Human genomic variation database (HGVD) Exome data from 1208 individuals collected in Japan-Recovered from whole-genome sequencing of 2049 healthy subjects SNV allele frequency (2KJPN) (ijgvd.megabank.tohoku.ac.jp)
(3) Mutations not included in the UCSC segment overlapping region (4) Rare SNVs defined as harmful by the functional annotation program (Polyhen_2, SIFT, PROVEN or Mutation Taster)
After selection by this criterion, one locus remained as a candidate.

5. Sanger Sequencing In 10 subjects (I-9, II-3, II-5, II-12, II-17, III-2, III-3, I-13, II-11, II-20) Sanger sequencing of one candidate locus was performed. All primers were designed using Primer3. Primers consisting of the nucleotide sequence set forth in SEQ ID NO: 5: forward, 5'-TCATCAAATGCAGAAGCAGA-3 and the nucleotide sequence set forth in SEQ ID NO: 6: reverse, 5'-TGCTGCTTTGAAAACAAA-3' were designed here. It is one of the primers. Genomic DNA (5 ng) was amplified by PCR in a 20 μL reaction solution using KOD FX Neopolymerase (Toyobo). Using the BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems), the sequence reaction was performed according to the manufacturer's instructions. After cleaning with CleanSEQ (Agen Court), the product is ABI Genetic Analyzer.
Separation was performed on 3130 (Applied Biosystems) and the electrophoretic flow diagram was visually evaluated using 4Peaks software (nucleobytes.com/4peaks/) (Fig. 1b).

6. Identification of the causative mutation As a result of Sanger sequencing, the same mutation (EP400, chr12: 132064747; C> T) was found in all 7 subjects presenting with the disease. On the other hand, none of the three subjects who did not present the disease showed this mutation. Therefore EP400 in subjects with schizophrenia of this family
It was thought that the dysfunction of the disease was likely to be behind the etiology. This base sequence mutation is EP400
It has been strongly suggested that it is associated with the etiology of schizophrenia because it causes mutations in amino acid residues (Pro2805Leu).

7. Evaluation of in silico function of EP400 mutation (Pro2805Leu) The amino acid sequences of the identified human EP400 protein mutation sites and their vicinity were compared with the corresponding amino acid sequences of other mammals, birds and fish. As a result, the mutation site was located in a highly conserved disorder region among mammals (Fig. 1c).
Therefore, in order to investigate the possibility of functional changes due to mutations in the EP400 protein, the importance of mutations was scored using various prediction programs (SIFT, PROVEN, Mutation Taster, M-CAP). As a result, in all programs, the mutation is "damaging" (SIFT score = 0.007;
deleterious, PROVEN score =? 2.67; deleterious, MutationTaster score = 0.994; disease-causing, M-CAP score = 0.254; damaging). In addition, when the disorder region was predicted using the Predictor of Natural Disordered Regions (PONDR) program, the CaN-XT algorithm in PONDR predicted that the disorder region would be extended by the mutation (Fig. 1d).

Example 2: Preparation of Ep400 knockout mouse and its characteristic analysis 1. Preparation of Knockout Mouse Using CRISPR-Cas9 Gene Editing System In the mouse ortholog (Ep400) of the human EP400 gene, a guide RNA was designed in the region in exon 47 corresponding to the family mutation site identified in Example 1, and the mouse was prepared. A mutation in the Ep400 gene was introduced. Two mutations that do not cause protein substitution (c.8130C) because CRISPR-Cas9 required gene modification of the PAM sequence required for genome recognition in order to stably introduce the required gene mutation. > G, c.8142C> T) Designed and line 1 (5'-CCACCACAGCCCCCCACCACCGCAGGCGCAGCCAGGTCCCCTACAGCAACCAGCGCAAGTGCAAGTACAGACTCCACAGCC-3': SEQ ID NO: 9), Line 2 (5'-GATGCAGCTGCCACCACACAGCCACCACCGCACCAGCCAAG Nucleotides were synthesized.

A single guide RNA was designed using the ATUM gRNA design tool (atum.bio/eCommerce/cas9/input) (5'-CACTTGCGCTGGTTGCTGTG-3': SEQ ID NO: 11). Single-stranded oligonucleotides, single-guide RNA, crRNA, and tracrRNA (FASMAC) were injected into wild-type fertilized eggs using a microinjection system. After injecting RNA, fertilized eggs were cultured in vitro up to the blastulatory stage and transplanted into the female uterus of a pseudopregnant recipient. PCR amplification was performed using mouse-derived KOD FX neopolymerase (Toyobo) to confirm the genotype of the mouse, and the product was cloned into a T vector (Takara Bio Inc.) for sequence analysis. The PCR primers were forward 5'-TGGAGGGTGGCTTATGGTTA-3'(SEQ ID NO: 12) and reverse 5'-GTCACTGTGGTGCCTGTGAG-3' (SEQ ID NO: 13).

2. 2. Confirmation of mutation introduction in mutant-introduced mice The electrical flow chart in the upper part of Fig. 2 shows the prepared mutant-introduced mice Line 1 (Ep400-p.P2715L # 1) and Line 2 (Ep400-p.P2715L # 2). , Ep400 gene exon 47 indicates that mutations that do not cause amino acid substitutions of c.8130C> G and c.8142C> T, which are required for gene modification, have been introduced (green arrow). The upper part of FIG. 2 shows that the introduction of the c.8144C> T mutation synthesizes a protein having an amino acid substitution of Pro2715Leu (red arrow). The mutation-introduced mice produced by the above procedure have a homozygous Pro2715Leu mutation (Ep400 ^{p.P2715L / p.P2715L} ).

In addition, as shown in the photograph of the mouse in the lower part of FIG. 2, the Line 1 and Line 2 mice into which the Pro2715 Leu mutation was introduced showed the Hind-limb clasping sign (yellow arrow) from the time of 3 months of age. Hind-limb clasping sign is a behavior in which rodents clasp their hind limbs. This Hind-limb
Clasping signs are often observed in mice with disorders of the cerebellum, basal ganglia, and neocortex and indicate signs of central nervous system abnormalities. Considering that mice become pregnant in about 2 months after birth, it is almost the same as the onset time of schizophrenia in humans.

3. 3. Behavioral test 6 mutant-introduced mice (Ep400 ^{p.P2715L / p.P2715L} ) Line 1 (Ep400-p.P2715L # 1) and 6
Behavioral tests (open field test, social interaction test) were performed using wild-type mice (Ep400 ^WT ). Mice were first tested at 3 months of age and retested at 6 months of age.

Figure 3 shows the results of an open field test to evaluate anxiety-like behavior. The mouse was placed in a 50 cm x 50 cm x 40 cm clean box. The range of 60% of the center is defined as the center range. After a 15 minute rest period, the mice were gently placed outwardly in the perimeter area. The mice were observed for 10 minutes with video tracking. Video data was analyzed using Bara Baby X, LNSOFT and ImageJ Software (National Institutes of Health) to calculate center time. Mouse position was tracked every 2 seconds in 2 frames. Center dwell time was calculated as the ratio of movement in the central range to total travel time.

As can be seen in FIG. 3a, at 3 months of age, there was no significant difference in the central residence time between the mutant-introduced mice (Ep400-p.P2715L # 1) and the wild-type mice (Ep400 ^p.WT ). In the 6-month-old mutant-introduced mice, the central residence time was shorter than that in the wild-type mice. This result indicates that the mutant-introduced mice continue to search within the gauge and have a tendency to maintain anxiety. Thus, the unclear findings at 3 months of age show a significant difference at 6 months, suggesting that the mutant-introduced mice can reproduce the progressive characteristics of human schizophrenia.

A social interaction test was conducted to evaluate sociality. As a preparation, the test mice were placed in cages for 7 days. The stimulated mice were then placed in the same cage as the test mice, monitored by video. After a 15 minute stimulation time, the stimulated mice were removed and video monitoring was discontinued. These videos were analyzed using BORIS software 4.1.4.

The results are shown in FIG. 3b. The number of Stretched Approaches indicates the number of behaviors in which the stimulated mouse is later placed in a cage and stretches its neck to confirm. At 3 months of age, there was no significant difference in the number of Stretched Approaches between mutant-introduced and wild-type mice, but at 6-month-old, the number of Stretched Approaches in mutant-introduced mice was higher than that of wild-type mice. It increased significantly, indicating an increase in anxiety. The increased anxiety tendency observed in mutant-introduced mice is thought to mimic the anxiety seen in the positive symptoms of human schizophrenia. In addition, the fact that the findings that were not clear at 3 months after birth showed a significant difference at 6 months suggests that the mutant-introduced mice can reproduce the progressive characteristics of human schizophrenia.

4. Histological analysis of mouse central nervous system tissue The brain and spinal cord of fresh mice (3 months old) were fixed with 10% formalin for 72 hours, embedded in paraffin and sectioned at 5 μm intervals. After deparaffinization, tissue slides were stained with hematoxylin-eosin (HE) to visualize the structure. In addition, immunohistochemical staining was performed on brain tissue sections to examine the expression of Ep400 and the neuron marker Map2. Tissue slides were deparaffinized and rehydrated and washed with Tris buffered saline (TBS). 20 tissue sections for antigen recovery
Microwave treated for minutes. After blocking for 1 hour at room temperature, the cells were incubated with the primary and secondary antibodies for 1 hour at room temperature. As primary antibodies, polyclonal anti-EP400 (HPA016704, Atlas Antibodies) and polyclonal anti-MAP2 (ab5392, Abcam), and as secondary antibodies, goat anti-chicken IgY H & L Alexa Fluor 488 (ab150169, Abcam) and goat anti-rabbit IgG H & L Alexa Fluor. 555 (ab150078, Abcam) were used respectively. Nuclei were DAPI stained. BZ-9000
Fluorescence-stained images were taken using an all-in-one fluorescence microscope (KEYENCE).

The results are shown in FIGS. 4 and 5a. As a result of HE staining, the mutant-introduced mice (Fig. 5a, upper left) in the brain tissue (Fig. 4a) of the coronal section (CS), sagittal section (SS) and hippocampal dentate gyrus (DG), and the spinal cord tissue (Fig. 5a upper left). No morphological difference was observed between Ep400-p.P2715L # 1) and wild-type mice (WT). As a result of immunofluorescent staining, the expression of Ep400 was shown especially in the nerve cell nucleus in the 3-month-old mouse brain (Fig. 4b). The expression pattern of the Ep400 protein was not different between the mutant-introduced mice and the wild-type mice (Fig. 4b).

Next, spinal cord sections were stained with Kluber Valera (KB) and toluidine blue (TB) to detect the structure of the myelin sheath. Sections were observed and photographed using a BZ-9000 all-in-one fluorescence microscope (Keyence).

The results of two strains of mutant-introduced mice (Ep400-p.P2715L # 1 and Ep400-p.P2715L # 2) are shown in FIGS. 5a and 6a, respectively. KB staining (upper right and middle of each figure) and TB staining (lower of each figure) showed no morphological abnormality in the myelin sheath in any of the mutant-introduced mice, but the axon diameter was higher than that of the wild-type mouse. Was observed to decrease.

5. Transmission electron microscopy (TEM)
Samples were immobilized at room temperature for 72 hours with 2% glutaraldehyde (Nacalai Tesque) in 0.1 M sodium cacodylic acid buffer (cacodylic acid buffer pH 7.4) containing 1 mM CaCl ₂ and 1 mM MgCl ₂ . .. Samples were washed with cacodylic acid buffer and then post-fixed in 1% OsO ₄ (Nacalai Tesque) in cacodylic acid buffer for 60 minutes at 4 ° C. They were then washed with cacodylic acid buffer, dehydrated with a graded series of ethanol and acetone, and embedded in Quetol 651 epoxy resin (Nisshin EM). The sample embedded in the resin was cut into sections using a diamond knife on an ultramicrotom (Richard-Jung). Ultrathin sections were collected on a grid and stained with uranyl acetate and lead citrate. Samples were tested at 80 kV under a transmission electron microscope (JEM-1230, JEOL Ltd.). Axon diameters and G ratios (bare axon diameter / myelinated axon diameter) of randomly selected axons in the white matter of the spinal cord were measured using ImageJ (axon diameter; n = 100, G ratio; n = 30). An F-test was applied to confirm the normal distribution data. Student's t-test and Welch's t-test were applied for comparison. The significance threshold was set to 0.05.

The results of two strains of mutant-introduced mice (Ep400-p.P2715L # 1 and Ep400-p.P2715L # 2) are shown in FIGS. 5b and 6b, respectively. As a result of TEM analysis, none of the mutant-introduced mice showed morphological abnormalities in the myelin sheath, and the G ratio was normal, but a significant decrease in axon diameter was confirmed. Such changes reflect axonal and myelin sheath microstructural abnormalities that have been repeatedly suggested in human schizophrenia. It is also believed that the mutagenesis mice of the present invention mimic some of the clinical and biological features of human schizophrenic patients.

Example 3: Correlation Analysis of Rare Variants of the Human EP400 Gene with Schizophrenia To identify unique harmful variants of EP400 that correlate with schizophrenia, the accumulation of 23 harmful variants identified by the target sequence was tested. .. Fisher's direct test was used to estimate the P-value. Bonferroni correction was performed and the significance threshold was 0.002174.

Of the 23 variants, 6 are new variants that are not registered in any database (HGVD2.30, 4.7KJPN, gnomADexome), and 10 variants are registered in at least one database with MAF <0.001. It was a very rare variant, and 7 species were rare variants registered in at least one database with 0.001 <MAF <0.005. The positions of these variants on the EP400 gene are schematically shown in FIG. There were 13 EP400 variants detected only in patients with schizophrenia, although there was no significant difference in the range tested.

To test the difference in loading of the human EP400 gene, three different analyzes (CMC, Madsen-Browning, and SKAT-O) were performed using the Efficient and Parallelizable Association Container Toolbox (EPACTS) algorithm. Public Database (1000 Genomes Project, all population data released in October 2014; NIH NHLBI 6515 Exome Data (http://evs.gs.washington.edu/EVS/); Exome Aggregation Consortium 65000 Ex Somme data; HGVD exome data of 1208 people in Japan; Minor allele frequency in SNV allele frequency (https://ijgvd.megabank.tohoku.ac.jp) in the whole genome sequence data of 2049 healthy Japanese people. Common variants with (MAF) greater than 0.005 and in-house control samples with greater than 0.005, with a call rate of 90% or less or a genotyping quality of 99 or less were excluded. The significance threshold was set to 0.05.

Participants with schizophrenia had a higher frequency of adverse mutations (7.37% (21 of 285)) compared to controls (3.15% (14 of 445)). As a result of the correlation test of rare variants using the EPACTS algorithm, the accumulation of rare variants was confirmed by the SKAT-O algorithm.

We have found that mutations in the EP400 gene are associated with the development of schizophrenia. Using this finding, by detecting a mutation in the EP400 gene in a subject, it became possible to evaluate the likelihood of developing schizophrenia in the subject. The method of the present invention makes it possible to easily predict the likelihood of developing schizophrenia. The present invention also provides a model mammal for schizophrenia in which the EP400 ortholog gene is mutated. Such schizophrenia model mammals can be used to screen for therapeutic or prophylactic agents of schizophrenia.

Claims (24)

The presence or absence of a mutation in the human EP400 gene in the DNA in the sample derived from the subject is detected, and the subject's susceptibility to schizophrenia is tested based on the criteria that if the mutation is present, schizophrenia is likely to occur. Method. The method according to claim 1, wherein the presence or absence of a mutation in exon 48 of the human EP400 gene in DNA in a sample derived from a subject is detected. The method according to claim 1 or 2, wherein the presence or absence of a mutation in cytosine at position 8414 of the human EP400 gene in DNA in a sample derived from a subject is detected. The method according to any one of claims 1 to 3, wherein the presence or absence of a mutation of proline No. 2805 of the human EP400 protein to leucine is detected. The method according to any one of claims 1 to 4, wherein the subject is a human suspected of developing schizophrenia. A diagnostic kit for schizophrenia containing a set of PCR primers capable of amplifying nucleotides in the entire coding region of the human EP400 gene. The diagnostic kit for schizophrenia according to claim 6, comprising a PCR primer set capable of amplifying the cytosine-containing region at position 8414 of the human EP400 gene. A nucleotide sequence derived from the human EP400 gene, a probe capable of specifically hybridizing to a nucleotide sequence in which the 8414th nucleotide of the gene is mutated to chimin, and a nucleotide sequence derived from the human EP400 gene, the EP400 gene. The kit according to claim 6 or 7, further comprising a probe capable of specifically hybridizing to a nucleotide sequence in which the 8414th nucleotide remains cytosine. A model mammal for schizophrenia that has a mutation in the EP400 gene and exhibits behavioral abnormalities that are indicators of human schizophrenia. The model mammal for schizophrenia according to claim 9, which has a mutation in the region of the Ep400 gene corresponding to exon 48 of the human EP400 gene. The model mammal for schizophrenia according to claim 9 or 10, wherein the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene of the model mammal is mutated to thymine. Claims 9-11, wherein the model mammal is a mammal selected from the group consisting of mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, goats, mini pigs, pigs, sheep, cows, monkeys and chimpanzees. The model mammal according to any one of the above items. The model mammal according to any one of claims 9 to 12, wherein the model mammal is a mouse, and cytosine at position 8144 of the mouse Ep400 gene is mutated to thymine. The model mammal according to any one of claims 9 to 12, wherein the model mammal is a mouse, and proline at position 2715 of the mouse Ep400 protein is mutated to leucine. The model mammal according to any one of claims 9 to 14, which exhibits anxious behavior as the behavioral abnormality. A method for producing a model mammal for schizophrenia showing behavioral abnormalities that are indicators of human schizophrenia by introducing a mutation into the Ep400 gene. 16. The method of claim 16, wherein a mutation is introduced into the region of the Ep400 gene corresponding to exon 48 of the human EP400 gene. The method of claim 16 or 17, wherein by introducing the mutation, the nucleotide corresponding to cytosine at position 8414 of the human EP400 gene of the model mammal is mutated to thymine. The method according to claim 16 or 17, wherein the amino acid corresponding to proline at position 2805 of the human EP400 protein of the model mammal is mutated to leucine by introducing the mutation. The method according to any one of claims 16 to 19, wherein the mutation is introduced by a gene editing method using Crispr / CAS-9. 16-20, wherein the model mammal is a mammal selected from the group consisting of mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, goats, mini pigs, pigs, sheep, cows, monkeys and chimpanzees. The method according to any one of the above. The method according to any one of claims 16 to 21, wherein the model mammal is a mouse. The method according to any one of claims 16 to 22, which exhibits anxious behavior as the behavioral abnormality. A method for screening a therapeutic or prophylactic agent for schizophrenia using the model mammal according to any one of claims 9 to 15, wherein the following steps;
When the behavioral abnormality is improved as compared with (1) the step of administering the test substance to the model mammal and (2) the control group to which the test substance was not administered, the test substance is used for schizophrenia. The process of selecting as a candidate substance for a therapeutic or preventive drug,
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