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WO2014129437A1 - Procédé d'inspection de troubles mentaux et trousse d'inspection - Google Patents

Procédé d'inspection de troubles mentaux et trousse d'inspection Download PDF

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Publication number
WO2014129437A1
WO2014129437A1 PCT/JP2014/053710 JP2014053710W WO2014129437A1 WO 2014129437 A1 WO2014129437 A1 WO 2014129437A1 JP 2014053710 W JP2014053710 W JP 2014053710W WO 2014129437 A1 WO2014129437 A1 WO 2014129437A1
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WIPO (PCT)
Prior art keywords
shati
mental disorders
mental
subject
cpg island
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Ceased
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PCT/JP2014/053710
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English (en)
Japanese (ja)
Inventor
淳美 新田
恭介 宇野
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University of Toyama NUC
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University of Toyama NUC
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Priority to JP2015501446A priority Critical patent/JPWO2014129437A1/ja
Publication of WO2014129437A1 publication Critical patent/WO2014129437A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a test method and test kit for mental disorders based on the methylation status of CpG islands related to the mental disorder-related protein Shati gene.
  • Non-patent Document 1 Diagnosis of mental disorders by detecting Shati's nucleic acid has been proposed (Patent Document 2).
  • Non-patent Document 2 Epigenetics is a field for pursuing symptoms that appear for the first time due to both genetic and environmental factors, and has been attracting attention as related to mental disorders such as depression and schizophrenia (Non-patent Document 2).
  • the present invention provides a test method for in vitro diagnosis of mental disorders such as drug dependence, schizophrenia, and depression, and a test kit used for the test, and a method / tool for easily and early finding mental disorders The issue is to provide.
  • the present inventors have clarified the expression control mechanism of Shati from the viewpoint of epigenetics, established a test method for mental disorders using the methylation state of CpG island in Shati as an index, and completed the present invention. It came. Hereinafter, the present invention will be described in detail.
  • 1st invention of this invention is a test
  • the mental disorder is caused by any of drug dependence, schizophrenia, and depression, and the sample is a biological sample collected from a mammal.
  • the second invention of the present invention is a test kit for a psychiatric disorder characterized by including a specific primer set for detecting a methylation state in a CpG island of a Shati gene of a sample collected from a subject.
  • a third invention of the present invention is a method for evaluating a psychiatric disorder therapeutic agent and / or preventive agent, comprising the following detection step (3) and evaluation step (4).
  • Methylation status and test of samples collected from subjects in the absence of the test compound using as an index the methylation status of the Shati gene in the CpG island of samples collected from individuals with mental disorders and non-mental disorders
  • An evaluation step of evaluating whether or not the test compound is a therapeutic and / or prophylactic agent for mental disorders by comparing the methylation state of a sample collected from the subject in the presence of the compound.
  • the mental disorder is caused by any of drug dependence, schizophrenia, and depression, and the sample is a biological sample collected from a mammal.
  • the state of mental disorder can be objectively determined. Moreover, it can be evaluated whether a test compound becomes a psychiatric disorder therapeutic agent and / or a preventive agent by setting it as the parameter
  • the individual CpG methylation status in the CpG island of the Shati gene decreases the CpG methylation rate when the subject is mentally ill or when schizophrenia progresses. Can be used.
  • the subject is not particularly limited as long as it is a mammal, but examples include rodents such as mice and rats; rabbits; cats; dogs; Preferred subjects are rodents or humans.
  • the sample may be tissue or blood collected from a living body, and blood is particularly preferable.
  • the blood may be a specific blood component such as whole blood or platelets.
  • the CpG island of the Shati gene is not particularly limited as long as the ratio of the methylated DNA of the CpG island in the promoter region of the Shati gene can be seen.
  • SEQ ID NO: 1 which is the mouse Shati CpG island, or the Shati CpG island of human It is preferable that the base sequence of SEQ ID NO: 4 is included.
  • the method used to detect the methylation state of Shati DNA is not particularly limited, such as a method of concentrating methylated DNA, a method of using base substitution by bisulfite treatment, or a method of using a methylation-sensitive restriction enzyme.
  • a method utilizing base substitution by treatment is preferred.
  • the bisulfite treatment for example, a commercially available kit product such as EZ DNA Methylation Kit (Zymo Research) may be used.
  • the target DNA region may be PCR amplified, cloned, and sequence analysis may be performed.
  • “Methyl Primer Express” or the like may be used for designing a primer for methylation detection and unmethylation detection that uses a target DNA region for PCR amplification.
  • Specific examples of the primer include the following.
  • SEQ ID NO: 1 When SEQ ID NO: 1 is the target DNA region, the following primer set is used after bisulfite treatment ⁇ Shati BSP up> Sequence: 5′-gggattataggagattattagtg-3 ′ (SEQ ID NO: 2) ⁇ Shati BSP down> Sequence: 5′-aaaaaaccccacaatacataccccc-3 ′ (SEQ ID NO: 3)
  • SEQ ID NO: 4 When the human Shati CpG island of SEQ ID NO: 4 is the target DNA region, the following primer set may be used with the DNA of SEQ ID NO: 5 after bisulfite treatment as the analysis region.
  • PCR amplification using the above primers may be performed by a known method, for example, “Epi Taq HS (Takara Bio)” and “Custom Primer (Invitrogen)” using “PCR Thermal Cycler Dice Gradient (Takara Bio). It is sufficient to perform PCR by “. Further, the PCR product may be subjected to gel electrophoresis, and DNA may be extracted from the target band using “QIAquick Gel Extraction Kit (Qiagen)” or the like.
  • DNA may be incorporated into a vector and amplified by a known method.
  • DNA is ligated to a vector using “pGEM-T Easy Vector System (Promega)” and “E. coli DH5 ⁇ Competent Cells (Takara) is used. Bio) and may be cultured in an LB / Amp liquid medium.
  • transformation E. coli according to the boiling method transformation E. coli according to the boiling method. Plasmid DNA is extracted from E. coli, and the target DNA may be purified by phenol chloroform extraction and ethanol precipitation.
  • the sequence may be determined by a known method, for example, using “BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems)”, PCR is performed using T7 or SP6 primer, and Sequence ScannerA) A sequence analysis may be performed.
  • the methylation state of the sample collected from the subject is compared with the methylation state in the CpG island of the Shati gene of the sample collected from the individual with mental disorder and non-mental disorder.
  • the data on the methylation status of a plurality of patients with normal psychiatric disorders and healthy subjects are collected in advance, and the subject is compared with the average data to thereby determine whether the subject has psychiatric disorders. It can be inspected.
  • it may be compared with the average data of patients for each of a plurality of types of mental disorders to check which mental disorder is applicable.
  • the subject (subject) is examined by comparing with average data of healthy subjects, drug dependent patients, schizophrenia patients, and depressed patients. Or even if it is not average data, it can also be examined by using multivariate analysis.
  • the evaluation step (4) of the present invention relates to a method for evaluating a psychiatric disorder therapeutic agent and / or preventive agent. Specifically, using the methylation status of the CatG island of the Shati gene of samples collected from individuals with mental illness and non-psychotic illness as an index, the methylation status of the sample in the absence of the test compound and the presence of the test compound This is a step of comparing the methylation state of samples and evaluating whether or not the test compound is a therapeutic agent and / or preventive agent for mental disorders.
  • test compound means any substance that can be an object of evaluation (including screening for a psychiatric treatment and / or prevention agent) as to whether or not it becomes a psychiatric treatment and / or prevention agent.
  • a low molecular compound, a nucleic acid, or a polypeptide can be used.
  • the low molecular weight compound, nucleic acid, polypeptide or the like may be extracted and purified from a natural product, or may be artificially synthesized.
  • purified thing but an unpurified thing can also be used as a test compound.
  • a novel substance but a well-known substance or its improvement may be sufficient. For example, it is possible to evaluate an existing therapeutic agent or prophylactic agent or a derivative thereof.
  • a detection step (3) is first performed. In the absence and presence of the test compound, the methylation state in the CpG island of the Shati gene of the sample collected from the subject is detected. Sample collection, methylation state detection means, and the like are the same as those in the detection step (1).
  • the methylation state of the CpG island collected from a mentally or non-mental mouse or human individual is used as an index, and the methyl of the sample in the absence or presence of the test compound thus detected is used as an index. Compare the conversion status. From the result, it is evaluated whether or not the test compound is a psychiatric disorder therapeutic agent and / or preventive agent. Specifically, for example, data on methylation status of a plurality of mice and humans with mental disorders and healthy mice and humans is collected in advance, and the average data and the like is used as an index for samples before and after administration of the test compound. Compare methylation status. Thereby, it can be evaluated whether a test compound becomes a therapeutic agent and / or preventive agent of a mental disorder.
  • the therapeutic agent and / or preventive agent for mental disorders is a therapeutic agent or preventive agent for drug dependence, schizophrenia and depression. In addition, even if it is not average data, it is also possible to evaluate by multivariate analysis.
  • the primer set for detecting the CpG island of the Shati gene can be a psychiatric disorder test kit.
  • the kit may include various reagents, enzymes, buffers, reactor materials, and / or instructions that are suitable for each methylation state detection means.
  • CpG island is as follows. -% GC> 50 ⁇ Length: 300-2000bp Observed / Expected CpG> 0.6 As a result of the analysis of this condition, a CpG island was obtained from ⁇ 648, which is a promoter, to +1342, which is intron I (FIG. 1).
  • ⁇ Amplification of target sequence> Phenol / chloroform extraction To 200 ⁇ L of mouse whole blood, 200 ⁇ L of a tris / phenol / chloroform mixed solution was added and mixed by inversion, followed by centrifugation at 4 ° C. and 20000 g for 5 minutes. The supernatant was collected in a new tube, an equal amount of chloroform was added, mixed with stirring, and centrifuged again at 4 ° C. and 20000 g for 5 minutes, and the supernatant was collected in a new tube.
  • DNA was divided with DraI (Takara Bio), and the DNA was purified by phenol chloroform extraction and ethanol precipitation.
  • the purified DNA is subjected to bisulfite treatment using “EZ DNA Methylation Kit (Zymo Research)” and “PCR Thermal Cycler Gradient (Takara Bio) using“ Epi Taq HS (Takara Bio) ”and“ Custom Primer (invitrogen) ”. Bio) ”.
  • the reaction conditions are as follows.
  • PCR was performed using T7 or SP6 primers using “BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems)”. The reaction conditions are as follows. • 1 minute at 96 ° C. • 25 cycles of (96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes). After PCR precipitation, the PCR product was dissolved in HiDi-formamide and dissolved in 3130 Genetic Analyzer ( The sequence reaction was performed by Applied Biosystems). The sequence was analyzed by Sequence Scanner (Applied Biosystems), and methylated cytosine and unmethylated cytosine (thymine) were discriminated (FIG. 4). Differences in methylation patterns were observed between the physiological saline administration group (bottom of FIG. 4) and the methamphetamine administration group (top of FIG. 4).
  • the target region is determined in human and the primer is designed, and the base sequence consisting of SEQ ID NO: 4 of human DNA is subjected to bisulfite treatment and the base sequence consisting of SEQ ID NO: 5 is analyzed. The area. As a result, CpG islands from ⁇ 1726, which is the promoter, to +2010, which is intron 2 (FIG. 7).
  • Whether or not the subject suffers from a psychological disorder such as drug dependence, schizophrenia, depression, etc. by examining the methylation status of the Shati gene in the blood of animals such as humans or mice as subjects. You can know the progress of the medical condition. Therefore, it is useful for the treatment and development of therapeutic drugs for mental disorders such as drug dependence, schizophrenia, and depression.

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  • Chemical & Material Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

L'invention concerne un procédé d'inspection pour l'examen externe de troubles mentaux tels que la pharmacodépendance, la schizophrénie et la dépression; et une trousse d'inspection s'utilisant dans lesdites inspections. Le procédé d'inspection et la trousse d'inspection de troubles mentaux permettent de déterminer si un sujet humain ou une souris présente une maladie mentale telle que la pharmacodépendance, la schizophrénie et la dépression, ainsi que l'évolution de la pathologie par l'examen de l'état de méthylation des gènes Shati du sujet. La présente invention est par conséquent utile dans le traitement de troubles mentaux tels que la pharmacodépendance, la schizophrénie et la dépression, et pour la mise au point de médicaments destinés à traiter ceux-ci.
PCT/JP2014/053710 2013-02-19 2014-02-18 Procédé d'inspection de troubles mentaux et trousse d'inspection Ceased WO2014129437A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2015501446A JPWO2014129437A1 (ja) 2013-02-19 2014-02-18 精神障害の検査方法および検査キット

Applications Claiming Priority (2)

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JP2013-029643 2013-02-19
JP2013029643 2013-02-19

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WO2014129437A1 true WO2014129437A1 (fr) 2014-08-28

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Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4942044B2 (ja) * 2005-03-03 2012-05-30 国立大学法人富山大学 精神障害関連遺伝子及びその利用
JP5876673B2 (ja) * 2010-06-09 2016-03-02 国立大学法人富山大学 精神障害の診断方法および診断薬キット

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KYOSUKE UNO: "DNA Methyl-ka ni yoru Nanjisei Togo Shicchosho no Gen'in Kaimei Oyobi Shindan eno Oyo", KENKYU KADAI BANGO: 23890067, 2011 NENDO KENKYU JISSEKI HOKOKUSHO, KAKEN: DATABASE OF GRANTS-IN-AID FOR SCIENTIFIC RESEARCH, 2012, Retrieved from the Internet <URL:http://kaken.nii.ac.jp/d/p/23890067/2011/3/ja.ja.html> [retrieved on 20140501] *
MIYAZAKI, T. ET AL.: "The reduction of methylation on the upstream reagion of shati/ nat 81 transcription start site is induced by repeated administration of methamphetamine", JOURNAL OF PHARMACOLOGICAL SCIENCES, vol. 124, no. SUPPLE, 15 February 2014 (2014-02-15), pages 183P *

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