JP2010004848A - Method for evaluating attention deficit hyperactivity disorder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which attention deficit hyperactivity disorder can readily and accurately be inspected by using a new marker gene, and to provide a method for identifying a causative agent and the component of a therapeutic agent, of the attention deficit hyperactivity disorder, by using the expression of the marker gene as an index. <P>SOLUTION: The evaluation method decides if a subject is in a state of the attention deficit hyperactivity disorder or has the risk of onset of the attention deficit hyperactivity disorder when the expression of Pmch gene in a biosample isolated from the subject is measured and the gene expression is abnormal. The method for identifying a factor causing the abnormality of the expression of the Pmch gene as the causative factor of the attention deficit hyperactivity disorder, and the method for identifying a material ameliorating the abnormality of the expression of the Pmch gene as a remedy of the attention deficit hyperactivity disorder are also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for evaluating attention deficit / hyperactivity disorder using the expression level of Pmch gene as an index, and a method for screening a drug component for attention deficit / hyperactivity disorder.

  Attention deficit hyperactive disorder (ADHD) is one of the developmental disorders characterized by hyperactivity, inattention and impulsivity, and is a diagnostic statistical manual for mental disorders (DSM) by the American Psychiatric Association -IV) and the World Health Organization (ICD-10) diagnostic criteria are classified as neurobehavioral disorders. It has features such as difficulty in maintaining attention, time lag, and poor ability to gather information. This disorder is said to have a strong genetic factor, but it has been found that early detection and early treatment (appropriate treatment and environmental improvement) can alleviate behavioral abnormalities. Symptoms such as hyperactivity, carelessness, and impulsivity are usually confirmed by 7 years of age and are generally said to occur in 4-12% of school-age children, but carelessness is not noticeable In the case of dominant type, it is often not diagnosed in childhood. If ADHD is diagnosed early and appropriate measures are not taken, it may interfere with life at home and school, and may cause secondary mental disorders. Therefore, it is important to perform appropriate treatment by early diagnosis especially in childhood.

  Diagnosis of ADHD is carried out based on interviews and behavioral standards like other mental illnesses, and useful biological markers for the diagnosis of ADHD have not yet been developed. In particular, since the behavior of children is complex, it is often difficult to diagnose ADHD, so the development of markers that can be objectively and biologically diagnosed is strongly desired.

  For example, the following methods have been proposed as several methods for diagnosing or evaluating ADHD.

  A technique for measuring an individual's peripheral temperature and measuring a value indicating ADHD (Patent Document 1), and a method for assessing a subject's attention state by scoring the subject's response to auditory stimuli ( Patent Document 2), a method of diagnosing attention deficit / hyperactivity disorder (AD-HD) by assessing dopamine transporter in at least one region of the central nervous system (Patent Document 3), PRKCB1 gene and expression product Diagnosis of specific mutations and autism and related disorders based on these mutations (Patent Document 4). However, an objective and clear method for diagnosing ADHD has not been established, and since it has characteristics that tend to depend on the environment, ambiguous results may occur in many cases. Some genes are also used as biological markers, but they have not been widely accepted.

In addition, as a model animal of hyperactivity disorder, in the maintenance process of the original strain of hepatitis, liver cancer, human Wilson disease model LEC rats promoted at the Center for Experimental Biology, Hokkaido University,
1) Shake your head constantly while walking.
2) Suddenly make a swiveling motion or move the head greatly backwards.
3) When a normal rat holds its tail and lifts its body, it stretches its limbs and then begins to squeeze out of the body after a short while. Individuals with abnormal behavior such as turning the body to the left and right and twisting around the tail, sometimes rotating in a circle in the air, were found. There is known a “hyperactivity disorder model rat (WKAH Wig)” that is isolated and maintained (Patent Document 5).

  On the other hand, the following is known for the Pmch gene.

  Pmch (pro-melanin-concentrating hormone) gene is a neuropeptide-glycine-glutamic acid (NGE), neuropeptide-glutamic acid-isoleucine (NEI), melanin-concentrating hormone (MCH) It is a gene containing a plurality of protein coding sequences, and is known to have a large amount of expression mainly in the hypothalamus. MCH is secreted as a peptide-type neurohormone MCH consisting of 19 amino acids after pre-pro-MCH (PMCH) containing NGE and NEI is processed.

  MCH has the function of agglomerating skin and scale melanin (pigments) in fish to lighten the body color. On the other hand, it is said that mammals are mainly involved in feeding regulation. MCH-deficient mice have decreased food intake and reduced body weight, and have a high metabolic rate (Non-patent Document 1), and transgenic mice that overexpress MCH have increased food intake and insulin resistance and mild obesity are observed. There is also a report (Non-Patent Document 2).

Further, as a patent application related to MCH, a method for promoting fattening of fish (Patent Document 6) characterized by stimulating the production of melanin-concentrating hormone in fish to be bred to increase appetite of fish, MCH or a derivative thereof Or a salt or SLC-1 or a salt thereof, a screening method for a compound or a salt thereof that changes the binding property between MCH or a salt thereof and SLC-1 or a salt thereof (Patent Document 7), melanin aggregation Novel arylamines that are antagonists of hormone receptor 1 (MCH R1), pharmaceutical compositions containing said compounds, methods for their preparation, and their use in drugs for the treatment of obesity, diabetes, depression and / or anxiety (Patent Literature) 8) is known. That is, the inventions of these patent applications relate to antagonists of melanin-concentrating hormone receptor 1 (MCH R1), and are mostly used for the treatment of obesity, metabolic disorders, food disorders, depression and the like.
JP 2003-33355 A Special table 2008-510580 gazette Special table 2003-503325 gazette Special table 2008-504838 JP 2001-186828 A JP 2004-298059 A JP 2001-141728 A Special table 2007-509158 gazette Shimada M. et al, 1998, Nature 396: 670-674 Ludeig D S. et al, 2001, Clin. Invest. 107, 379-386

  An object of the present invention is to provide a method capable of simply and accurately examining attention deficit / hyperactivity disorder using a novel marker gene. Another object of the present invention is to provide a method for identifying a causative factor of attention deficit / hyperactivity disorder and a method for identifying a therapeutic drug component for attention deficit / hyperactivity disorder using the expression of the marker gene as an index. .

The present inventors searched for a marker specific for ADHD using the brain of a hyperactivity disorder model rat, and found that the Pmch (pro-melanin-concentrating hormone) gene in the midbrain of the hyperactivity disorder model rat. newly found that the expression of (GenBank Accession No. NM _{_} 012625) is significantly increased.

  From these facts, the present inventors have been convinced that the Pmch gene is a gene that serves as a disease marker for attention deficit hyperactivity disorder. Furthermore, the present inventors have found that the expression modulation of the Pmch gene can also be detected in plasma.

  Therefore, we are convinced that screening using the increase in expression of Pmch gene and the increase in protein (MCH) encoded by Pmch gene as an index is effective in preventing attention deficit hyperactivity disorder and searching for therapeutic agents. As a result, the present invention has been completed.

  That is, in order to solve the above problems, the present application measures the expression of the Pmch gene in a biological sample isolated from a subject, and the subject is in a state of attention deficit / hyperactivity disorder when the gene expression is modulated. Or an evaluation method characterized by evaluating that the risk of developing attention deficit hyperactivity disorder is high.

  In this evaluation method, the biological sample is preferably plasma.

  The present application is also a method for screening a causative factor of attention deficit / hyperactivity disorder, in which a candidate factor is allowed to act on a cell that normally expresses the Pmch gene, and a factor that causes modulation of the expression of the Pmch gene is used as a target factor. Provided is a causal factor screening method for attention deficit / hyperactivity disorder characterized by specifying.

  The present application further relates to a method for screening a therapeutic drug component for attention deficit / hyperactivity disorder, wherein a candidate substance is allowed to act on cells exhibiting Pmch gene expression modulation, and a substance that improves Pmch gene expression modulation is a target substance The screening method characterized by specifying as follows is provided.

  In the above evaluation method and screening method, “Pmch gene expression modulation” means an increase in Pmch gene expression. For example, the amount of MCH protein in a biological sample is 10% higher than that of a healthy subject. % Or more, specifically 30% or more, more specifically 50% or more, particularly 70% or more, and most specifically 90% or more.

  Further, other terms and concepts in the above invention are defined in detail in the description of the embodiments of the invention and the examples. The terms are basically based on the IUPAC-IUB Commission on Biochemical Nomenclature, or based on the meanings of terms commonly used in the field. Various techniques used for carrying out the invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the techniques that clearly indicate the source. For example, genetic engineering and molecular biology techniques are described in J. Sambrook, EF Fritsch & T. Maniatis, "Molecular Cloning: A Laboratory Manual (2nd edition)", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995; edited by the Japanese Biochemical Society, “Sequel Biochemistry Experiment Course 1, Genetic Research Method II”, Tokyo Chemical Doujin (1986); Laboratory Lecture 2, Nucleic Acid III (Recombinant DNA Technology) ", Tokyo Chemical Doujin (1992); R. Wu ed.," Methods in Enzymology ", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. Ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. ed., "Methods in Enzymology", Vol. 15 3 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987), etc. It can be carried out by substantially the same method or modification method.

  According to the present invention, whether or not the subject is in a state of attention deficit / hyperactivity disorder by checking the expression modulation of the Pmch gene for a biological sample (eg, plasma) isolated from the subject, Alternatively, it is possible to easily and accurately evaluate whether or not the risk of developing attention deficit / hyperactivity disorder is high. This makes it possible to select an appropriate and effective treatment method or the like for a subject in attention deficit / hyperactivity disorder or a high-risk person thereof.

  Further, according to the present invention, the causative factor of attention deficit / hyperactivity disorder is identified using the expression modulation of the Pmch gene as an index. The causal factor identified by this makes it possible to effectively carry out treatment for preventing attention deficit / hyperactivity disorder and suppressing progression of symptoms.

  Furthermore, according to the present invention, a therapeutic drug component for attention deficit / hyperactivity disorder is specified, which greatly contributes to the development of an effective therapeutic drug for attention deficit / hyperactivity disorder.

  The attention deficit / hyperactivity disorder evaluation method of the present invention is directed to a biological sample (for example, plasma) isolated from a subject, by confirming the expression modulation of the Pmch gene, thereby allowing the subject to develop attention deficit / hyperactivity disorder. Evaluate whether the patient is in a state or at risk of developing attention deficit hyperactivity disorder. It should be noted that the expression of the Pmch gene in a biological sample (for example, plasma) can be easily carried out by a person who has a normal blood test technique, and the determination of the expression requires the involvement of medical personnel. do not do.

  Specifically, the Pmch gene expression modulation can be determined by measuring the transcription product of the Pmch gene. When measuring mRNA as a transcription product of the Pmch gene, a technique known in the art for detecting and measuring the expression of a specific gene according to known genetic engineering and molecular biology techniques, for example, in situ hybridization , Northern blotting, dot blot, RNase protection assay, RT-PCR, Real-Time PCR (Journal of Molecular Endocrinology, 25, 169-193 (2000) and references cited therein), DNA array analysis method (edited by Mark Shena) , "Microarray Biochip Technology", Eaton Publishing, 2000) etc., and can be carried out by detecting and measuring the expression level of KLF5 mRNA.

For example, a method for detecting the mRNA level of the Pmch gene using an oligonucleotide probe (in the case of Northern blot analysis, at least the following steps:
(a) preparing RNA from a biological sample of a subject;
(b) electrophoretically separating the RNA prepared in step (a);
(c) hybridizing the RNA separated in step (b) with an oligonucleotide probe under stringent conditions;
(d) using the labeled amount of the oligonucleotide probe hybridized to RNA in step (e) as an indicator of the expression level of Pmch mRNA, and comparing the result with a normal biological sample; and
(e) a step of using a higher Pmch mRNA expression level as an indicator of attention deficit hyperactivity disorder compared to a normal biological sample;
It is characterized by including.

  Since the oligonucleotide probe hybridizes with Pmch mRNA under stringent conditions (for example, the conditions described in JP-T-10-508186 and JP-T9-511236), the oligonucleotide probe accurately matches any region of mRNA. A DNA sequence consisting of complementary sequences is used. Such a DNA sequence can also be obtained, for example, by cleaving Pmch cDNA with an appropriate restriction enzyme. Alternatively, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411-418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acid Res. 25: 3440-3444 Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. : 109; Beaucage (1981) Tetra. Lett. 22: 1859; can be synthesized in vitro by well-known chemical synthesis techniques as described in US Pat. No. 4,458,066.

The oligonucleotide probe is labeled by a radioisotope (RI) method or a non-RI method, but it is preferable to use a non-RI method. Examples of the non-RI method include a fluorescence labeling method, a biotin labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method. As the fluorescent substance, those capable of binding to the base moiety of the oligonucleotide can be appropriately selected and used. However, cyanine dyes (eg, Cy Dye ^TM series Cy3, Cy5 etc.), rhodamine 6G reagent, N-acetoxy- N ² -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used. As the labeling method, a method known in the art (for example, random prime method, nick translation method, DNA amplification by PCR, labeling / tailing method, in vitro transcription method, etc.) can be appropriately selected and used. For example, a functional group (for example, primary aliphatic amino group, SH group, etc.) is introduced into the HRF oligonucleotide, and the above-mentioned label is bound to such a functional group to prepare a labeled oligonucleotide probe.

Moreover, the amount of Pmch mRNA can also be measured by using a DNA microarray. This method comprises at least the following steps:
(a) preparing RNA from a biological sample of a subject;
(b) a step of preparing labeled cDNA from the RNA prepared in step (a),
(c) contacting the labeled cDNA prepared in step (b) with a DNA microarray;
(d) using the labeled amount of labeled cDNA hybridized to the capture probe of the DNA microarray in step (c) as an indicator of the amount of Pmch mRNA, and comparing it with the result of a normal biological sample; and
(e) using a higher amount of Pmch mRNA compared to a normal biological sample as an indicator of attention deficit / hyperactivity disorder;
It is characterized by including.

  When measuring the amount of Pmch mRNA using a microarray, for example, cDNA is synthesized using mRNA isolated from a biological sample of a subject as a template and PCR amplified. At that time, labeled dNTP is incorporated into labeled cDNA. The labeled cDNA is brought into contact with the macroarray, and the cDNA hybridized with the capture probe of the microarray is detected. Hybridization can be performed by dispensing a 96- or 384-well plastic plate and spotting a labeled cDNA aqueous solution on the microarray. The amount of spotting can be about 1 to 100 nl. Hybridization is preferably performed in the temperature range of room temperature to 70 ° C. for 6 to 20 hours. After completion of hybridization, washing is performed using a mixed solution of a surfactant and a buffer to remove unreacted labeled cDNA. As the surfactant, sodium dodecyl sulfate (SDS) is preferably used. As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used, and a citrate buffer solution is preferably used.

Furthermore, the amount of Pmch mRNA can also be measured by the RT-PCT method. This method comprises at least the following steps:
(a) preparing RNA from a biological sample of a subject;
(b) using the RNA prepared in step (a) as a template and synthesizing cDNA using a primer set;
(c) using the amount of cDNA synthesized in step (b) as an indicator of the amount of Pmch mRNA and comparing it with the result of a normal biological sample; and
(d) using a high amount of Pmch mRNA compared to a normal biological sample as an indicator of attention deficit hyperactivity disorder;
It is characterized by including.

  For designing the primer set to be used, commercially available software for primer design, such as Oligo ™ [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)], etc. can also be used.

Probes and primers used in the method of evaluating attention deficit hyperactivity disorder to target more than humans can be designed based on the rat PMCH DNA sequence (GenBank / NM _{_} 012625), synthesized and used. In addition, a human homologue of the rat Pmch gene is obtained by screening a human cDNA library using a probe prepared based on a known rat Pmch sequence, or by RT-PCR using human RNA as a template using a primer set. Probes and primers can also be prepared from this human gene sequence.

  Another method for determining the modulation of the expression of the Pmch gene is to quantify the amount of the expression product MCH of the Pmch gene. Such protein quantification is carried out in accordance with known genetic engineering and molecular biology techniques, using techniques known in the art for detecting and measuring specific protein amounts, such as in situ hybridization, Western blotting, various immunizations, and the like. It can be performed by histological methods.

Specifically, it can be carried out using an antibody that specifically recognizes MCH. The antibody is a polyclonal antibody or a monoclonal antibody, and includes all the whole molecules capable of binding to the epitope of MCH, Fab, F (ab ′) ₂ , Fv fragment, and the like. For example, in the case of a polyclonal antibody, such an antibody can be obtained from serum after immunizing an animal using a protein or a partial fragment thereof as an immunogen. Alternatively, it can be prepared by introducing an expression vector for eukaryotic cells incorporating the Pmch gene into an animal muscle or skin by injection or gene gun, and then collecting serum. As the animal, mouse, rat, rabbit, goat, chicken and the like are used.

  Monoclonal antibodies are known monoclonal antibody production methods (“monoclonal antibodies”, Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; “Monoclonal Antibody” James W. Goding, third edition, Academic Press, 1996) Can be manufactured according to

These antibodies are used after being labeled with a labeling substance. As the labeling substance, an enzyme, a radioisotope, or a fluorescent dye can be used. The enzyme is not particularly limited as long as it satisfies the conditions such as a large turnover number, stability even when bound to an antibody, and specific coloring of a substrate, and is used for normal EIA. Enzymes such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase and the like can also be used. Moreover, an enzyme inhibitor, a coenzyme, etc. can also be used. These enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound. As the substrate, a known substance can be used according to the type of enzyme used. For example, when peroxidase is used as an enzyme, 3,3 ′, 5,5′-tetramethylbenzidine can be used, and when alkaline phosphatase is used as an enzyme, paranitrophenol or the like can be used. As the radioisotope, those used in usual RIA such as ¹²⁵ I and ³ H can be used. As the fluorescent dye, those used in usual fluorescent antibody methods such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) can be used.

  Methods using such antibodies can be performed, for example, by immunostaining, eg, tissue or cell staining, immunoelectron microscopy, immunoassay, eg, competitive or noncompetitive immunoassay, radioimmunoassay (RIA), fluorescence Immunoassay (FIA), luminescent immunoassay (LIA), enzyme immunoassay (EIA), ELISA, etc. can be used, and the measurement may be performed with or without BF separation. it can. RIA, EIA, FIA, and LIA are preferable, and a sandwich type assay is also included. The sandwich type assay may be a simultaneous sandwich type assay, a forward sandwich type assay or a reverse sandwich type assay.

  One embodiment of the diagnostic method using such an antibody is a method for detecting the binding between the antibody and MCH in a liquid phase system. For example, the labeled antibody is brought into contact with a biological sample (eg, plasma) to bind the labeled antibody and MCH, and the conjugate is separated. Separation can be performed by known separation means (chromatography, solid phase method, etc.). A method according to a known Western blot method can also be employed. When an enzyme is used as the label, the label signal is measured by adding a substrate that develops color by enzymatic action and optically measuring the amount of degradation of the substrate to determine the enzyme activity. The amount of antibody is calculated by conversion and comparison with the standard value. When using a radioactive isotope, the radiation dose emitted by the radioactive isotope is measured with a scintillation counter or the like. In addition, when a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.

  Another method of diagnosis in a liquid phase system is to contact a primary antibody and a biological sample (eg, plasma) to bind the primary antibody and MCH, and to bind the labeled secondary antibody to the conjugate. The label signal in the human conjugate is detected. Alternatively, in order to further enhance the signal, an unlabeled secondary antibody may be first bound to the primary antibody + MCH conjugate, and a labeling substance may be bound to the secondary antibody. Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylating the secondary antibody and avidinizing the labeling substance. Alternatively, an antibody (tertiary antibody) that recognizes a partial region (for example, Fc region) of the secondary antibody may be labeled and the tertiary antibody may be bound to the secondary antibody. The primary antibody and the secondary antibody may both be monoclonal antibodies, or one of the primary antibody and the secondary antibody may be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed as described above.

  Still another embodiment in the case of using an antibody is a method for testing the binding between the antibody and MCH in a solid phase system. This method in the solid phase system is a preferable method for detecting a very small amount of MCH and simplifying the operation. That is, in this solid-phase method, the antibody is immobilized on a resin plate or membrane, the subject's plasma is brought into contact with the immobilized antibody, unbound proteins are washed away, and then the antibody + MCH conjugate remaining on the plate In this method, a labeled anti-antibody is bound to and the signal of the labeled antibody is detected. This method is a so-called “sandwich method”. When an enzyme is used as a marker, this method is widely used as an “ELISA (enzyme linked immunosorbent assay)”. Both of the two types of antibodies can be monoclonal antibodies, or one of them can be a polyclonal antibody.

  In addition, in the evaluation method of this invention, it can also be set as a more accurate diagnostic method by combining and performing 2 or more of each method illustrated above.

  Next, the method for screening the causative factor of attention deficit / hyperactivity disorder of the present invention is the method of causing a candidate factor to act on a cell that normally expresses the Pmch gene and causing the expression modulation of the Pmch gene to be expressed. Identified as a causal factor.

  In this method, “cells that normally express the Pmch gene” are, for example, cells existing in the body of normal animals (laboratory animals such as rats, mice, rabbits, cats, dogs, and pigs), or these animals. Cells that have been isolated from and maintained in culture conditions. Preferably, cells of animal brain tissue (cerebral cortex, linear body, midbrain, etc.) are targeted.

  “Candidate factors” include drugs and compounds that are assumed as causative factors for attention deficit / hyperactivity disorder, food and drink materials, various stresses, and the like. In addition, when eating and drinking materials and stress are used as candidate factors, attention-deficit / hyperactivity disorder can be reduced by confirming the modulation of Pmch gene expression in plasma isolated from cells or animals. Causative factors can be identified. Modulation of Pmch gene expression can be the same as that employed in the evaluation method.

  The drug component screening method for attention deficit / hyperactivity disorder of the present invention specifies a substance that improves the Pmch gene expression modulation as a target substance by causing a candidate substance to act on cells exhibiting Pmch gene expression modulation.

  In this screening method, “cells exhibiting modulation of Pmch gene expression” means, for example, cells of the brain (particularly the midbrain) of the hyperactivity disorder model rat (Patent Document 16) as shown in the Examples, or In other words, animal cells treated with the causative factor specified by the causal factor screening method of attention deficit hyperactivity disorder. These cells may be allowed to act on the candidate substance in a state existing in the animal individual, or may be allowed to act on the candidate substance in a state isolated from the animal individual. When a candidate substance is allowed to act on cells in an animal individual, the animal is injected or ingested with the candidate substance. When a candidate substance is allowed to act on cells under culture conditions, a method such as adding the candidate substance to the cell culture medium can be employed.

  Candidate substances used in this screening method include, for example, organic or inorganic compounds (particularly low molecular weight compounds), proteins, peptides and the like. These substances have known functions and structures and may be unknown. Alternatively, the “combinatorial chemical library” is an effective means as a candidate substance group for efficiently specifying a target substance. A combinatorial chemical library is a collection of various chemical compositions generated by combining many chemical “building blocks”, such as reagents, by chemical or biological synthesis. For example, a linear combinatorial chemical library, such as a peptide library, is formed by combining a set of building blocks (amino acids) in all possible ways for a given compound length (ie, peptide size). The Numerous chemical compositions can be synthesized through such combinatorial mixing of chemical building blocks. For example, systematic combinatorial mixing for 100 commutative chemical building blocks results in 100 million tetramer compounds or 10 billion pentamer compounds (eg, Gallop et al., (1994) 37 (9): 1233-1250). The preparation and screening of combinatorial chemical libraries is well known in the art (see, eg, US Pat. Nos. 6,004,617; 5,985,365). Various commercially available libraries can also be used.

  Hereinafter, as examples, experimental data confirming the relationship between attention deficit / hyperactivity disorder and expression of the Pmch gene will be shown to explain the feasibility of the present invention, but the present invention is limited to the following examples. It is not a thing.

Comparison of Pmch gene expression levels in the brains of hyperactivity disorder model rats and normal rats
Cerebral cortex, striatum, and midbrain samples (obtained from the Center for Experimental Biology, Hokkaido University) isolated from each of 3 4-week-old hyperactivity disorder model rats, 4-week-old WKAH rats that do not exhibit hyperactivity disorder pathology Total RNA was extracted from cerebral cortex, striatum, and midbrain samples extracted from each of the three individuals, and gene expression analysis was performed by DNA microarray analysis.

  The cerebral cortex for 3 individuals was combined and powdered in liquid nitrogen, and about 100 mg was divided for total RNA extraction. The striatum and midbrain were similarly performed.

  Total RNA was prepared using RNeasy Mini kit (Qiagen) according to the attached protocol. The extracted total RNA was confirmed to be of quality used for DNA microarray analysis by gel electrophoresis and absorbance measurement. For DNA microarray analysis, 150 ng of total RNA extracted from cerebral cortex, striatum, and midbrain samples were mixed and used for analysis, and hyperactivity disorder model rats were compared with normal rats.

  DNA microarray analysis is divided into three tasks: labeling, hybridization, and scanning. First, cDNA was synthesized from the above-mentioned total RNA 450 ng (150 ng each of total RNA extracted from cerebral cortex, striatum, and midbrain sample) using Low RNA Input Linear Amp Kit (Agilent Technologiest). Furthermore, Cy3-dCTP (manufactured by PerkinElmer) was added from the cDNA to prepare labeled cRNA. Next, the labeled cRNA was fragmented, and the fragmented labeled cRNA was hybridized with a probe on a chip (Whole Rat Genome Oligo Microarray Kit G4131F, manufactured by Agilent Technologiest). Further, the chip was washed, and gene expression analysis was performed using a microarray scanner (G2565BA, manufactured by Agilent Technologiest) and GeneSpring ver 7.3. Even when Cy5-dCTP was used as a fluorescent label, cRNA was labeled in the same manner.

  Fig. 1 shows the expression level (Signal value) of the Pmch gene in the brains (cerebral cortex, striatum, midbrain) of hyperactivity disorder model rats and normal rats obtained from DNA microarray analysis. In contrast to normal rats labeled with Cy3-dCTP and hyperactivity disorder model rats labeled with Cy5-dCTP, normal rats are labeled with Cy5-dCTP, and hyperactivity disorder model rats are labeled with Cy3-dCTP. Two results are shown when labeled with dCTP. It was confirmed that the expression level of the Pmch gene increased by about 2 to 5 times the expression level in the normal rat, regardless of which fluorescent label was used.

  As can be seen from the results in FIG. 1, the expression of the Pmch gene is higher in the brain of the hyperactivity disorder model rat than in the normal rat brain, so that it can be applied as a marker gene for ADHD. it was thought.

Comparison of Pmch gene expression levels in cerebral cortex, striatum, and midbrain of hyperactivity disorder model rats and normal rats by RT-PCR Total RNA of cerebral cortex, striatum, and midbrain extracted in Example 1 Was used for RT-PCR analysis. First, cDNAs of cerebral cortex, striatum, and midbrain were synthesized from 1 ng of total RNA using AffinityScript QPCR cDNA Synthesis Kit (Stratagene).

Primers specific for the Pmch gene were designed. The primer design is as shown below. Primer synthesis was commissioned by Hokkaido System Science.
Pmch-1 (Rat)
Forward Primer: 5'-gccttttctttgttttctcacg-3 '(SEQ ID NO: 1)
Reverse Primer: 5'-aagactgctggtcctttcagag-3 '(SEQ ID NO: 2)
Using the above primers, PCR was performed according to the attached protocol using Ex Taq Hot Start Version (manufactured by TaKaRa) using cerebral cortex, striatum, and midbrain cDNA as templates. The PCR reaction was repeated 27 cycles.

  After the PCR reaction, 1.8% agarose gel electrophoresis was performed, and the target gene was detected by ethidium bromide staining. For detection, a UV transilluminator (ATTO) was used to measure the intensity of the gene band.

  The results obtained by RT-PCR analysis are shown in FIG. As can be seen from the intensity values of each band, the expression of Pmch gene was increased in the midbrain of hyperactivity disorder model rats.

  As can be seen from the results in Fig. 2, the Pmch gene is an applicable gene as a marker gene for ADHD because its expression in the midbrain of hyperactive disorder model rats is higher than that in normal rats. It was thought that there was.

Analysis of Pmch gene expression in plasma Blood was collected from normal rats, centrifuged into plasma and serum, and each was stored at -80 ° C. Dissolve the frozen plasma, transfer about 500 μl to a new tube, add 500 μl of Solution D (5.5 M Guanidium thiocyanate, 25 mM Sodium citrate, 0.5% Lauryl sarcosine, 0.1 M 2-Mercaptoethanol), mix, and then add a phenol solution (Phenol: 500 μl of Chloroform: Isoamyl alcohol (25: 24: 1)) was added and mixed well. Collect the supernatant by centrifugation, add 20 μl of 3M Sodium Acetate (pH 5.5) to this supernatant, add 500 μl of TE saturated phenol and 100 μl of chloroform, mix well, and then mix at 0 ° C for 15 minutes. I left it alone. The supernatant was collected by centrifugation, and an equal amount of 2-propanol was added to the supernatant, mixed well, and then allowed to stand at 0 ° C. for 15 minutes, and a precipitate was obtained by centrifugation. 100 μl of sterilized water was added to the precipitate and dissolved, and then total RNA was purified using an RNeasy Mini kit (Qiagen) according to the attached instructions.

  The extracted total RNA was confirmed to be of quality used for RT-PCR analysis by gel electrophoresis and absorbance measurement.

  Next, plasma cDNA was synthesized from 1 ng of total RNA using AffinityScript QPCR cDNA Synthesis Kit (Stratagene).

Primers specific for the Pmch gene were designed. The primer design is as shown below. Primer synthesis was commissioned by Hokkaido System Science.
Pmch-1 (Rat)
Forward Primer: 5'-gccttttctttgttttctcacg-3 '(SEQ ID NO: 1)
Reverse Primer: 5'-aagactgctggtcctttcagag-3 '(SEQ ID NO: 2)
Pmch-2 (Rat)
Forward Primer: 5'-tacggagcagcaaacaggatggcgaagatg-3 '(SEQ ID NO: 3)
Reverse Primer: 5'-ttcttctgaaaggatgttgtggaccagcag-3 '(SEQ ID NO: 4)
PCR reaction was performed using Ex Taq Hot Start Version (manufactured by TaKaRa) using the above primers and plasma cDNA as a template according to the attached protocol. The PCR reaction was repeated for 38 cycles.

  After the PCR reaction, 1.8% agarose gel electrophoresis was performed, and the target gene was detected by ethidium bromide staining. For detection, a UV transilluminator (ATTO) was used to measure the intensity of the gene band.

  The results obtained by RT-PCR analysis are shown in FIG. Pmch-1 primer was used for brain cDNA, but the target DNA band could not be detected by RT-PCR analysis using plasma cDNA as a template. However, when the primer design was changed and examined, it was found that the Pmch gene could be detected in plasma with the primer of Pmch-2 primer.

  As can be seen from the results in FIG. 3, the Pmch gene was found to be detectable even in easily collected plasma, so it was considered to be a gene applicable as a marker gene for mental illness.

It shows the expression level (Signal value) of Pmch gene in the brain (cerebral cortex, striatum, midbrain) of hyperactivity disorder model rat and normal rat obtained from DNA microarray analysis. RT-PCR analysis shows Pmch gene expression in cerebral cortex, striatum, and midbrain of hyperactivity disorder model rats and normal rats. It shows Pmch gene expression in plasma by RT-PCR analysis.

Claims (4)

Measure the expression of the Pmch gene in a biological sample isolated from the subject, and if this gene expression is modulated, the subject is in the state of attention deficit hyperactivity disorder or the risk of developing attention deficit hyperactivity disorder An evaluation method characterized by evaluating that the property is high. The evaluation method according to claim 1, wherein the biological sample is plasma. A screening method for causative factors of attention deficit / hyperactivity disorder, characterized by allowing a candidate factor to act on a cell that normally expresses the Pmch gene and identifying the factor that causes Pmch gene expression modulation as a target factor And a causal factor screening method for attention deficit hyperactivity disorder. A method for screening a drug component for attention-deficit / hyperactivity disorder, wherein a candidate substance is allowed to act on cells exhibiting Pmch gene expression modulation, and a substance that improves Pmch gene expression modulation is identified as a target substance. A screening method characterized.