WO2003056006A1 - Method of detecting mutation in human serotonin receptor subtype 2b gene - Google Patents

Abstract

It is intended to provide DNA of a human serotonin receptor subtype 2B gene having a novel mutation. It is also intended to provide a method of detecting a mutation in the human serotonin receptor subtype 2B gene using the novel mutation as described above. Namely, (1) a DNA having a mutation of the base at the 1162-position in the code domain of human serotonin receptor subtype 2B gene into thymine (T); or (2) a DNA being derived from the above DNA by partial modification in a site other than the above base and having such a base length as allowing the detection of the mutation at the above-described base position. A mutation in the human serotonin receptor subtype 2B gene is detected by a method involving the step (a) of analyzing the base at the 1162-position in the code domain of human serotonin receptor subtype 2B gene.

Description

 Description Method for detecting mutations in human sedentary tonin receptor subtype 2B gene

 The present invention relates to a polymorphism of the human seotonin receptor sa: 2B gene involved in psychiatric disorders such as schizophrenia. More specifically, the present invention relates to DNA having a specific polymorphism in the human serotonin receptor subtype 2B gene and its use. In addition, the present invention relates to a method for detecting a mutation in the human seotonin receptor subtype 2B gene using a genetic engineering technique, a nucleic acid used in the method, and a mutation detection kit. INDUSTRIAL APPLICABILITY The present invention can be used for diagnosing whether or not a disease caused by mutation of the human mouth tonin receptor subtype 2B gene has been developed, or for diagnosing the risk of developing the disease. BACKGROUND ART-Schizophrenia and stimulant-induced psychotic disorders are both frequent and cause major disability, resulting in significant losses both for the individual patient and for society as a whole. However, at present, the pathophysiology of both is unknown, so there is no useful test method for diagnosis, and there is no treatment that is appropriate for the pathophysiology. What is certain about the pathology of both diseases is that it is a multifactorial disease that develops due to the complex association of various factors, and pathophysiological searches have been conducted using various approaches. However, the diagnosis category is not based on pathophysiology, it is simply a so-called syndrome. As a result, it is considered that one diagnosis includes various pathologies, and this point is a major obstacle for confirmatory pathophysiological search.

In order to control to some extent the heterogeneity of the pathological conditions included in this one diagnostic category, some stratification was performed using traditional genetic methodologies. Research has been promoted based on the hypothesis that it may be possible to identify genetic factors (Nozakio Ozaki: Molecular genetics research on mental illness: Near completion of the Human Genome Project Psychological Neurology Journal 103 532-537, 2001). Among them, individual differences between cases where stimulant use induces mental illness symptoms and cases where frequent use does not induce prominent psychotic symptoms may be due to some biological basis. It is likely to be common to the biological basis of positive symptoms such as hallucinations and delusions in schizophrenia (Lieberman JA, Kinon BJ, Loebel AD: Dopam inergic mechanisms in idiopathic and drug -induced psychoses. Schizophr B ull 16 (1): 97-110, 1990). Numerous facilities, including ours, provide insights from pharmacological effects of stimulants and other psychotic symptoms-inducing substances and pharmacological effects of antipsychotics that suggest a link between psychotic symptoms and serotonin neurotransmission. Thus, serotonin-related genetic polymorphisms may lead to differences in the manifestation of psychotic symptoms (Nozaki Ozaki: Mental illness and serotonin. Modern Medicine 46 329-332, 1999).

The present inventors have already identified a new polymorphism associated with amino acid substitution present in the 5A subunit of the serotonin receptor gene and found that this mutation is strongly correlated with schizophrenia (Iwata N, N. Ozaki, T. Inada, Goldman D: Association of a 5-HT5A receptor polymorphism, Prol 5Ser, to schizophrenia. Molecular Psychiatry 6 (2): 217-219, 2001). In mice deficient in the 5A gene, differences in response to administration of the psychotic symptom-inducing substance, LSD, have been found from the cellular level to the behavioral level. Lappalainen et al. Also reported that polymorphic markers at serotonin receptor subtype 1B were associated in a population with alcohol use disorder and antisocial personality disorder (Lappalainen J, Long JC, Eggert M, Ozaki N, Robin RW, Brown GL, Naukkar inen H, Yirkkunen M, Linnoi la M, Go ldman D: Linkage of antisocial alcohol ism to the serotonin 5-HT1B recept or gene in 2 populations. Archives of General Psychiatry 55 ( 11): 989-99 4, 1998). However, the serotonin receptor gene has only been reported at this time, and there are 13 subtypes in humans, and it is necessary to consider other subtypes.

 Of these various serotonin receptor subtypes, 2B is mainly expressed in peripheral organs, and recent genetically modified animal studies have shown that it is deeply involved in heart development. At the same time, this gene is also expressed in the brain, but its function in the central nervous system remains unclear because no ligand has been developed that specifically binds to this receptor. Rather, substances that have been thought to be ligands for serotonin receptor subtype 2C actually have the same affinity as 2B, so until now it was thought to be an action of 2C, feeding, anxiety, and reproductive behavior. It can be understood that 2B may have some effect at the same time. In addition, 2C has been thought to be related to the pathophysiology of schizophrenia and substance use disorders, and many researchers, including us, have examined it, but no definitive results have been obtained.

Recently, some ligands specific to 2B have been developed to some extent, and the results of behavioral experiments using these substances show that this receptor is associated with anxiety. (Duxon MS, Kennett GA, Lightowler S, Blackburn TP, Fone KC: Activation of 5-HT2B receptors in the medial amygdala cau ses anxiolys is in the social interaction test in the rat.Neuropharmacol ogy 36 (4- 5): 601-8, 1997). However, no studies have been conducted on behaviors such as impulsive behavior or alcohol consumption.Model animal experiments on psychosis-like symptoms and analysis of behavior changes due to administration of antipsychotics or psychotic substances or stimulants have been described earlier. The central role of this gene has not yet been determined, including for mutant mice. On the other hand, as mentioned above, although 2C has been previously examined based on the hypothesis that schizophrenia and stimulant-induced psychotic disorder or antisocial , In addition to the fact that it is currently negative, considering the homology of 2B and 2C Thus, 2B may be considered as a candidate gene for these conditions.

 Although existing reports on the mutation search of the 2B gene in humans have been made in patients with childhood-onset obsessive-compulsive disorder, it has not been possible to find a function-related mutation (Kim SJ, Veenstra- VanderWeele J, Hanna GL, Gonen D, Leventhal BL, Cook EH Jr: Mutation screening of human 5-HT (2B) receptor gene in early-onset obsessive-compulsive disorder.Mol Cell Probes 14 (1): 7-52,

2000). Disclosure of the invention

 The present invention has been made in view of the above background, and has been made in consideration of the DNA of a human osteotonin receptor subtype 2B gene with a novel mutation, the protein encoded by the DNA, and the human osteotonin receptor subtype 2B gene. It is an object of the present invention to provide a method for detecting a mutation, a nucleic acid used in the method, and a mutation detection kit. Furthermore, it is another object of the present invention to provide a method for diagnosing a disease caused by a mutation in the human sedentary tonin receptor subtype 2B gene using the detection of the mutation, and a diagnostic kit.

The present inventors conducted a mutation search for schizophrenic patients with respect to the human serotonin receptor subtype 2B gene in order to achieve the above object. As a result, five single nucleotide polymorphisms (SNPs) were identified in these samples, four of which involved amino acid substitutions. Two relatively common mutations with amino acid substitutions (polymorphisms) were examined for their association with schizophrenia and stimulant-induced psychiatric disorders. Mutation of the coding region 1162). From this, detection of the mutation in the human serotonin receptor subtype 2B gene is a useful tool for diagnosis of schizophrenia, etc., selection of treatment, and pre-symptomatic diagnosis (understanding risk of onset). It was thought to be. The present invention has been completed based on such knowledge, and has the following structure Become.

 [1] (1) DNA obtained by mutating the base at position 1162 of the coding region to thymine (T) in the human mouth tonine receptor subtype 2B gene, or

 (2) DNA that is partially modified at a site other than the base in the DNA, and has a base length capable of detecting mutation at the base site.

 [2] A protein encoded by the DNA of (1) or (2) of [1].

 [3] (a): a step of analyzing the nucleotide at position 1162 of the coding region of the human mouth tonin receptor subtype 2B gene,

 A method for detecting a mutation in the human sebocyte tonin receptor subtype 2B gene, comprising:

 [4] The method according to [3], wherein the step (a) includes the following step (al):

 (al): a step of amplifying a DNA region containing the base region at position 1162 of the code region in the human sedentary tonin receptor subtype 2B gene.

 [5] (a): a step of analyzing the nucleotide at position 1162 of the coding region of the human mouth tonin receptor subtype 2B gene,

 A method for diagnosing a disease caused by a mutation in the human sedentary tonin receptor subtype 2B gene, comprising:

 [6] The diagnostic method according to [5], wherein the step (a) includes the following step (al): (al): a base at position 1162 of a coding region in a human sedentary tonin receptor subtype 2B gene Amplifying the DNA region containing the site.

 [7] The diagnostic method according to [5] or [6], wherein the disease is schizophrenia or a substance use disorder.

 [8] Nucleic acid for analyzing the nucleotide at position 1162 of the coding region of the human mouth tonine receptor subtype 2B gene, which is a nucleic acid for analyzing the coding region of position 1162 in the human mouth tonin receptor subtype 2B gene. A nucleic acid having a sequence complementary to a DNA region containing a base site.

[9] coding region of human serotonin receptor subtype 2B gene nucleotide at position 1162 A nucleic acid for analyzing

 Specific for a DNA fragment derived from a region containing the base at position 1162 of the human setotonin receptor subtype 2B gene and capable of being amplified by PCR using the primers of SEQ ID NOs: 14 and 15 Nucleic acids that hybridize to

 [10] A kit for detecting a mutation in the human serotonin receptor subtype 2B gene, the kit comprising the nucleic acid of [8] or [9].

 [11] The kit according to [10], further comprising a reagent for amplifying a DNA region containing the nucleotide at position 1162 of the human sedentary tonin receptor subtype 2B gene.

 It should be noted that the DNA in the present invention is not limited to double-stranded DNA, and is used to include single-stranded DNA (sense strand and antisense strand) constituting the DNA. Further, the DNA of the present invention includes DNA having an arbitrary nucleotide sequence in consideration of codon degeneracy. Further, the form is not limited, and includes cDNA, genomic DNA, and synthetic MA.

 In the present specification, the human serotonin receptor is abbreviated as "5-HT", the human serotonin receptor subtype 2B is abbreviated as "5-ΗΠΒ", and the human serotonin receptor subtype 2Β gene is abbreviated. Therefore, it is also referred to as “5- {2} gene”.

 Further, the nucleotide at position 1162 of the coding region of the human sedentary tonin receptor subtype 2Β gene is abbreviated as “base 1162”. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows the sequence of the primer designed to search the entire amino acid coding region of the human sedentary tonin receptor subtype 2 gene, the position of the type I DNA corresponding to each primer, and the amplification by each primer set. 3 is a table showing the lengths of DNA fragments.

Figure 2 shows information on the human serotonin receptor subtype 2Β gene polymorphisms (SNPs), a set of primers used to detect each polymorphism by the SSCP method, and PCR of each polymorphism. -The following table summarizes the primer set used for typing in the RFLP method, the restriction enzymes used in the RFLP method, the fragment size, and the frequency of polymorphism.

FIG. 3 is a table summarizing the results of association analysis of relatively common polymorphisms F173L and R338W with schizophrenia and stimulant-induced psychotic disorders. * 1: Fisher's exact test, df = l, P = 0.000006 (2-tail),% ² = 17.1, OR = 5.18 (Sheehe), 95% CI [2.20-12.24], * 2: Fisher's exact test, df = l, P 0.00 0.000001 (2-tail),% ² = 26.6, OR = 56.1 (Sheehe), 95% CI [7.70-408] The best mode for carrying out the invention

 A first aspect of the present invention relates to a DNA having the following constitution. (1) It is a DNA obtained by mutating the base at position 1162 of the coding region to thymine (T) in the human cervical tonin receptor subtype 2B gene. Or (2) a partially modified DNA at a site other than the base in the DNA, and having a base length capable of detecting a mutation at the base site.

 Human serotonin receptor subtype 2B (5-HT2B) is one of the human serotonin receptors (5-HT). The 5-HT group has 5-HT1, 5-HT2, and other types, the former including 5-HT1A, 1B, and ID, and the latter including subtypes 2A, 2B, and 2C.

 5-HT2B had many unknowns, partly because it was identified last in the 5-II group. Recently, a specific agonist of this receptor, the angyu gonist, has been developed, and it has been clarified that it controls anxiety and alcohol intake in rats and mice (Du Tsutsu MS, Kennett GA , Lightowler S, Blackburn TP, Feme KC: Activation of 5-HT2B receptors in the medial amygdala causes anxiolys is in the social interaction test in the rat. Neuropharmacology 36 (4-5): 6 01-8, 1997) „

In GenBank, etc., normal (not including the mutation identified in the present invention) human Sequence information of the toserotonin receptor subtype 2B gene (GenBank ACCESS ION No. X 77307) and sequence information of the coding region (GenBank ACCESS ION No. 475197) are provided.

 The DNA in (1) contains a mutation (position 1162 from the 5 'end of the coding region) of the newly identified coding region (mutation from cytosine (C) to thymine (T)). It is the MA of the human cleft tonin receptor subtype 2B gene (hereinafter also referred to as “mutated 5-HT2B gene”). This DNA can be used as a test DNA in a method for detecting the mutation of the human sedentary tonin receptor subtype 2B gene described below. That is, it is useful for diagnosis of schizophrenia and the like, and is useful in giving an index such as a risk of suffering from the disease. On the other hand, it is also useful in that the DNA of (1) can be used in a drug screening system for schizophrenia and the like described below. As a specific example of the MA of (1), MA consisting of the nucleotide sequence of SEQ ID NO: 1 can be mentioned. This DNA is a full-length DNA of the human serotonin receptor subtype 2B gene in which the base at position 1162 (base 1162) of the coding region is mutated to thymine (T).

 Here, as long as it contains the base at position 162, mutation of the base can be detected and used for diagnosis of schizophrenia and the like. From this, it can be said that even a DNA or a DNA fragment partially modified at a site other than the base at position 1162 in the DNA of (1) can be similarly used as a test target and useful. Therefore, the present invention further provides such a modified DNA or DNA fragment (that is, the DNA of the above (2); hereinafter, these are also collectively referred to as “modified DNA”). Here, "a part of the DNA is modified" means that a part of the bases constituting the DNA is deleted, substituted, inserted or added. The number of bases required for the modification is, for example, 1 to 100, preferably 1 to 20, and more preferably 1 to 10.

Specific examples of the modified DNA include a DNA having the nucleotide sequence of SEQ ID NO: 2. In this DNA, the base at position 162 of the coding region is mutated to thymine (T). It is a DNA consisting of the coding region of the human seotonin receptor subtype 2B gene. As described in Examples below, five single nucleotide substitution polymorphisms were identified in the coding region of the 5- 5- gene. From this, a DNA with at least one of these base substitutions can be mentioned as an example of the modified DNA. Specifically, in the sequence of SEQ ID NO: 1, the base at position 388 of the coding region is replaced with T (thymine), and in the sequence of SEQ ID NO: 2, the base at position 388 of the coding region is replaced with T (thymine). DNA that has been replaced, DNA in which the base at position 505 in the coding region in the sequence of SEQ ID NO: 1 has been replaced by A (adenine), DNA in which the base at position 517 in the coding region is replaced with C (cytosine) in the sequence of SEQ ID NO: 1, and the base in position 517 in the coding region in the sequence of SEQ ID NO: 2 is C (cytosine). DNA in which the base at position 742 in the code region of the sequence of SEQ ID NO: 1 has been replaced with C (cytosine), and the base in position 742 of the coding region has C (cytosine) in the sequence of SEQ ID NO: 2 ) Can be exemplified. When the modified DNA is used as a test DNA or a standard (control) when detecting a mutation at position 1-162 of the human sedentary tonin receptor subtype 2B gene (5-HT2B gene), the relevant mutation is detected. It must have a possible base length. Examples of the base length here include a length of 10 bp or more, preferably 20 bp or more, and more preferably 30 bp or more. More specific base lengths include 100 bp to 1000 bp, preferably 100 to 500 bp, and more preferably 200 to 300 bp.

 The modified DNA can be used as a DNA primer for specifically amplifying a part (or all) of the mutant 5-HT2B gene in the sample, or a DNA probe for detecting the mutant 5-HT gene. It is also useful in that respect. Such a DNA primer and the like can be used as a nucleic acid for polymorphism analysis described below.

The above DNA or modified DNA is obtained from an appropriate genomic DNA library or cDNA library. The probe can be prepared from the ligation gene using a probe, a primer, and the like that can specifically hybridize to the mutant 5- 5- gene. Also, dNTPs (dATP, dGTP, dCTP, dTTP) can be synthesized by PCR using at least a part of the mutant 5-ΗΠΒ gene as a starting material.

 The genomic DNA library or the cDNA library used for preparing the DNA of the present invention may be any library containing the DNA of the present invention or the modified DNA.For example, a commercially available DNA library may be used. can do. Alternatively, a cDNA library prepared by extracting a mRNA from a suitable cell and using it as a type III by a well-known method can be used.

 Another aspect of the present invention relates to a vector holding the DNA of the present invention (including the modified DNA). Any vector can be used as long as it can introduce the DNA of the present invention. However, appropriate vectors may be used depending on the purpose of use (cloning, protein expression) and the type of host cell. Preferably, the vector is selected. The DNA of the present invention can be introduced into a vector by a well-known method using restriction enzymes and DNA ligase (Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York). it can.

Yet another aspect of the present invention relates to a transformant carrying the DNA of the present invention (including the modified DNA). That is, the present invention relates to a transformant obtained by transforming a host cell with the DNA of the present invention. For example, the DNA of the present invention can be prepared by the calcium phosphate method, Elect-Mouth Boret-Sillon (Potter, H. et al., Proatl. Acad. Sci. USA 81, 7161-7165 (1998)), Lipofexion (Feigner, PL Natl. Acad. Sci. USA 84, 7413-7417 (1984)), microinjection (Graes smann, M. & Graes smann, A., Proc. Natl. Acad. Sci. USA 73, 366). -370 (1976)) and the like. Further, the transformant of the present invention can be obtained by transforming a host cell with the vector of the present invention. Wear. Various host cells can be used depending on the purpose. For example, CH0 cells, COS cells, HeLa cells, insect cells, yeast, and the like can be used. Further, not only eukaryotic cells but also prokaryotic cells such as Escherichia coli can be used.

 By culturing the transformant of the present invention under appropriate conditions, it is possible to produce a large amount of an expression product (protein) of the DNA of the present invention. It can be used for a drug screening system for etc.

 A second aspect of the present invention relates to a protein encoded by the DNA (including the modified DNA) of the present invention. Specific examples include a protein consisting of the amino acid sequence of SEQ ID NO: 3 (corresponding to the DNA of SEQ ID NO: 1). This protein is an expression product of the 5-HT2B gene having a mutation in the coding region at position 1162. The compound acting on the protein of the present invention is a candidate for an agonist or antagonist for human serotonin receptor subtype 2B (5-HT2B) having a mutation at position 1162 of the coding region. Therefore, using these proteins, it is possible to construct a drug screening system for diseases caused by the mutation of the 5-ΗΠΒ gene as described later.

 The present invention also includes proteins functionally equivalent to the above-mentioned proteins (for example, a protein having the amino acid sequence of SEQ ID NO: 3). The term “function” as used herein refers to a function involved in binding (sensitivity) to serotonin. Therefore, a protein that retains the three-dimensional structure involved in binding to serotonin and has the same degree of binding of serotonin to it is included.

As long as the protein is functionally equivalent, for example, a protein having an amino acid sequence of SEQ ID NO: 3 in which one or more amino acids are deleted, substituted, added, and / or inserted, is also included in the present invention. It is contained in. The position of the mutation in the amino acid sequence is not particularly limited as long as it is functionally equivalent, and mutation may occur at a plurality of positions. The term “plurality” here means, for example, a number corresponding to within 10% of all amino acids. Preferably, it is a number corresponding to within 5% of all amino acids. More preferably, the number is within 1% of all amino acids.

 The protein of the present invention includes both proteins with and without sugar chains.

 Among the proteins of the present invention, those that exist in nature can be prepared as natural proteins by performing operations such as extraction and purification. For example, it can be prepared from human cells and organs. The cell used herein is not particularly limited as long as it produces or expresses the protein of the present invention. For example, a nerve cell or a cell forming a placenta can be used. Further, among the proteins of the present invention, those produced or expressed in organisms other than human can be prepared from the organism.

 Further, the protein of the present invention can also be prepared as a recombinant protein using genetic engineering techniques. That is, it can be prepared by transforming MA encoding the protein of the present invention into a suitable host cell and collecting the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. When preparing as a recombinant protein, various modifications are possible. For example, a DNA encoding the protein of the present invention and another appropriate DNA are simultaneously introduced into a vector, and the protein of the present invention and the peptide or protein encoded by the other DNA are ligated. The obtained recombinant protein can be obtained. Here, other DNAs can be inserted into the vector in advance. Such modifications can facilitate the extraction and purification of the recombinant protein or add a biological function.

 The protein of the present invention can also be prepared by chemical synthesis. For example, it can be synthesized by a well-known method for synthesizing peptides, such as a solid phase method.

A third aspect of the present invention provides: (a) a step of analyzing the base at position 1162 of the coding region of the human oral tonin receptor subtype 2B gene; The present invention relates to a method for detecting a mutation of a type 2B gene.

 In the present invention, the base at position 1162 (base 1162) in the coding region (GenBank ACCESSION No. 475197) of the human serotonin receptor subtype 2B gene (5-ΗΠΒ gene, GenBank ACCESSION No. 475197) was analyzed. You. At position 1162, there are at least two types of polymorphisms, cytosine (C) and thymine (T). Therefore, "analyzing the nucleotide at position 1162 of the coding region of the human serotonin receptor subtype 2B gene" is synonymous with "analyzing the polymorphism at the relevant position". It particularly means to determine whether it is cytosine or thymine.

 Mutation between cytosine and thymine at position 1162 is accompanied by amino acid mutation. Specifically, the 388th amino acid of the 5-HT2B expression product becomes arginine (Arg, R) or tributophan (Trp, W) depending on the polymorphism of the base. In this specification, the polymorphism at the base position 1162 of the 5-HT2B gene is also referred to as “R388W”.

 Analysis of the base at position 1162 can be performed on the quotient allele. That is, the genotype of the test subject is either the allele, which is a type of cytosine at position 116'2, the type of both alleles is a type of thymine at position 1162, or a type having an allele of position 1162 of cytosine and thymine. Can be detected. Based on this polymorphism information, a diagnosis of a disease caused by a mutation in the 5-ΗΠΒ gene can be made.

The method for analyzing the base at position 1162 is not particularly limited. For example, a PCR-RFLP (restriction fragment length polymorphism) method using a PCR (polymer erase chain reaction) method, a PCR method, -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): ASO (allele specific oligonucleotide: allele specific oligonucleotide) combining PCR-SS0 method and dot hybridization method Hybridization method (Saiki, Nature, 324, 163-166 (19 86)), or TaqMan-PCR method (Livak, KJ, Genet Anal, 14, 143 (1999), Morris, T. et al., J. Clin. Microbiol., 34, 2933 (1996)), Invader method (Lyamichev V et al., Nat Biotechnol, 17, 292 (1999)), MALDI-TOF / MS (matrix) method using primer extension method (Haff LA, Smirnov IP, Genome Res 7,378 (1997)), RCA (rolling cicle am pliiication) method (Lizardi PM et al., Nat Genet 19, 225 (1998)), method using DNA chip or microarray (Wang DG et a, Science 280, 1077 (1998), etc.) Known methods such as a primer extension method, a Southern blot hybridization method, and a dot hybridization method (Southern, E., J. Mol. Biol. 98, 503-517 (1975)) can be used. Further, analysis may be performed by directly sequencing the 1162 salt S moiety. Incidentally, these methods can be used in any combination.

 When the amount of the test DNA is small, it is preferable to analyze by a PCR-RFLP method using a PCR method from the viewpoint of detection sensitivity or accuracy. Alternatively, any of the above analysis methods can be applied after the test DNA has been amplified in advance by the PCR method or a gene amplification method based on the PCR method. That is, the following step (al) can be contained in the above step (a).

 (al): A step for amplifying a DNA region containing the base region at position 1162 of the code region in the human mouth tonine receptor subtype 2B gene.

 On the other hand, when analyzing a large number of test DNAs, in particular, the TaqMan-PCR method, the Invader method, the MALDI-TOF / MS (matrix) method using the primer extension method, the RCA (rolling particle amplification) method, or It is preferable to use a method using a DNA chip or a microarray.

The test subject, 5-HT2B gene, can be prepared from the blood, skin cells, mucous membrane cells, hair, etc. of the subject using known extraction methods and purification methods. In addition, as long as it contains a 1162 nucleotide portion, it can be used as a test subject regardless of whether it is full-length DNA or partial DNA. Can be. In other words, the step (step (a)) of analyzing the base at the position 1162 of the coding region of the 5-HT2B gene using a DNA fragment of any length as long as the DNA fragment contains the position 1162 of the coding region of the 5-HT2B gene is performed. Can be performed.

 Using the mRNA that is a transcription product of the 5-HT2B gene, the base at position 126 of the code region of the 5-HT2B gene can also be analyzed. For example, after extracting and purifying the mRNA of the 5-ΗΠΒ gene from the blood, urine, etc. of the subject, Northern blotting (Molecular Cloning, Third Edition, 7.42, Cold Spring Harbor Laboratory Press, New York), DNA block method (Molecular Cloning, Third Edition, 7.46, Cold Spring Harbor Laboratory Press, New York), RT-PCR method (Molecular Cloning, Third Edition, 8.4, Cold Spring Harbor Laboratory Press, New York), DNA chip By performing a method using (DNA array), analysis of 1162 bases using mRNA as a starting material, that is, polymorphism analysis at the position can be performed.

 Furthermore, since the mutation at position 1162 of the 5-HT2B gene coding region involves an amino acid change, polymorphism analysis can also be performed using the expression product of the coding region. „In this case, even a partial peptide can be used as a test sample as long as it contains an amino acid corresponding to the base at position 1162. Specifically, the mutation at position 1162 in the 5-HT2B gene coding region is expressed as To change the amino acid at position 388 of the product (5-HT2B) (to produce arginine (Arg) or tributophan (Trp)), a part of the expression product containing at least the amino acid at position 388 (including protein, A partial protein or a partial peptide) can be used as a test sample.

Examples of the method of analyzing using the expression product of the 5-HT2B gene include a method of analyzing amino acids of a polymorphic portion, and a method of performing an immunological analysis by utilizing a change in a three-dimensional structure. As the former, a well-known amino acid sequence analysis method (a method using the Edman method) can be used. The latter includes a monoclonal antibody or a polyclonal antibody having a specific binding activity to any polymorphic 5-ΗΠΒ gene expression product. ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, immunoprecipitation, immunodiffusion, etc., using the body can be used.

 The polymorphism information obtained by executing the above-described detection method of the present invention can be used for diagnosis of a disease caused by a mutation in the serotonin receptor subtype 2B gene. That is, the present invention provides (a): a step of analyzing the base at position 1162 of the coding region of the human serotonin receptor subtype 2B gene, comprising the steps of: Methods for diagnosing are also provided.

 Here, the term "disease caused by mutation in the human serotonin receptor subtype 2B gene" refers to a disease that occurs when the expression level of the normal human serotonin receptor subtype 2B gene is reduced or not expressed. This includes schizophrenia and substance-dependent disorders (such as stimulant-induced psychotic disorders). The “diagnosis of a disease caused by a mutation in the human serotonin receptor subtype 2B gene” includes not only examining whether or not the above-mentioned disease has been developed, but also based on an objective index of the gene mutation. This includes assessing the underlying medical condition, as well as assessing the likelihood of developing the disease in the future (probability of developing the disease) and the immediate onset risk (onset vulnerability). In other words, by the diagnostic method of the present invention, it is possible to determine that the subject is suffering from a disease caused by a mutation in the human oral stomatin receptor subtype 2B gene, grasp the symptoms, or evaluate the risk of developing the disease. it can. Above all, the ability to evaluate the risk of onset is extremely clinically significant. Knowing the risk of disease in advance will enable appropriate precautionary measures to be taken. The information obtained from the above diagnosis can be used to select an appropriate treatment, improve the prognosis, improve the patient's quality of life (Q0L), or reduce the risk of onset.

By periodically performing the diagnostic method of the present invention, it is possible to monitor a change in the risk of developing a disease caused by a difference in the human seotonin receptor subtype 2B gene. As a result of such monitoring, certain external factors (environmental factors, If there is a correlation between the risk factor and the increase in the risk of onset, it is considered possible to identify the external factor as a risk factor and reduce the risk of onset based on this information.

 Using the polymorphism information at the position 1162 of the coding region of the 5-HT2B gene, it is possible to treat (including prophylactic treatment) a disease caused by mutation of the 5-ΗΠΒ gene. For example, if a mutation at the polymorphic site of the 5-ΗΠΒ gene is found, introducing a gene without mutation into the living body and expressing it will allow the normal 5-ΗΠΒ gene to exhibit its inherent function. This can be expected to reduce the symptoms of the disease caused by the mutation of the 5-HT2B gene, suppress the onset of the disease, and reduce the risk of developing the disease. A similar therapeutic effect can be expected by introducing an antisense strand against the mRNA of the 5-HT2B gene having a mutation and suppressing the expression of the mRNA.

 The gene or antisense strand can be introduced by, for example, a method using a plasmid for gene introduction or a plasmid vector, an electrification method (Potter, H. et al., Roc. Natl. Acad. Sci. USA 81, 716 7165 (1984)), Lipofexion (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1984)). & Graessmann, A., Proc. Nat and Acad. Sci. USA 73, 366-370 (1976)). Using these methods, a desired gene or the like can be directly introduced into a living body (in vivo method) or indirectly (ex vivo method).

 A fourth aspect of the present invention provides a nucleic acid for analyzing the base at position 1162 of the coding region of 5-HT2B gene (herein, referred to as “nucleic acid for polymorphism analysis”).

In the analysis method to which the polymorphism analysis nucleic acid (PCR-RFLP method, PCR-SSCP, TaqMan-PCR method, Invader method, etc. described above) is applied, the polymorphism portion to be analyzed (ie, 5-H It is designed to specifically amplify (primer) or specifically detectable (probe) DNA containing DNA or its corresponding mRNA (base 1162 of T2B gene coding region). Examples of the nucleic acid for polymorphism analysis of the present invention include a DNA sequence having a sequence complementary to the DNA sequence of a certain region including the nucleotide at position 162 of the coding region of 5- 5- gene, and containing the polymorphic portion. Nucleic acids (primers) designed to specifically amplify fragments can be mentioned. More specifically, the following primer set used in Examples described later can be exemplified as the nucleic acid for polymorphism of the present invention.

Forward primer

 5'-TTCCAACGAACAGAGAGCCT-3 '(SEQ ID NO: 14)

Reverse primer

 5'-CCCGGTAATTGCAGGTGATA-3 '(SEQ ID NO: 15)

 In addition, as another example of the nucleic acid (probe or primer) for polymorphism analysis of the present invention, the primer set (SEQ ID NO: 14 and SEQ ID NO: 15) containing the base at position 1162 of 5-HT2B gene is used. Nucleic acid specifically hybridizing to a DNA fragment derived from a region that can be amplified by the PCR method.

 In the case of a nucleic acid for performing the PCR-RFLP method, for example, when the PCR amplification product has a specific polymorphism so that the genotype is identified when the PCR amplification product is treated with a restriction enzyme, the polymorphic portion is used. Is designed so that a specific restriction enzyme site is formed. Examples of such a nucleic acid for polymorphism analysis include the following primer set used in Examples described later.

Forward primer

5'-TTCCAACGAACAGAGAGCCT-3 '(SEQ ID NO: 14)

Reverse primer

 5'-GATATATCGGCCAAATGCATCTC-3 '(SEQ ID NO: 22)

For the probe and the primer, a DNA fragment or an RNA fragment is appropriately used depending on the analysis method. The base length of the probe and the primer may be a length at which each function is exhibited, and an example of the base length of the primer is about 15 to 30 bp, preferably It is about 20 to 25 bp.

 In the case of primers, there may be some mismatches with the sequence of type III as long as they can specifically hybridize to the amplification target and amplify the target DNA fragment. The degree of mismatch is one to several, preferably one to five, and more preferably one to three.

The nucleic acid (primer, probe) for polymorphism analysis of the present invention can be synthesized by a known method such as the phosphodiester method. In addition, as a labeling substance and a labeling method when used as a probe, known ones can be used. Examples of the labeling substance include a radioactive isotope such as ³² P and a fluorescent substance such as fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.The labeling method is alkaline phosphatase. 5 'end labeling using T4 polynucleotide kinase, 3' end labeling using T4 DNA polymerase Klenow fragment, nick translation method, random primer method (Molecular Cloning, Third Edition, Chapter 9 , Cold Spring Harbor Laboratory Press, New York).

A fifth aspect of the present invention is a human serotonin receptor subtype 2B, comprising a nucleic acid (nucleic acid for polymorphism analysis) for analyzing the nucleotide at position 1162 of the coding region of the human serotonin receptor subtype 2B gene. Provide a kit for detecting gene mutation. The kit can be used for diagnosis of a disease caused by a mutation in the 5-HT2B gene, a treatment method for the disease (selection of a drug, selection of an administration method, etc.), and a preventive diagnosis (judgment of a disease risk). The kit may be composed of one or more reagents (buffers, reaction reagents, detection reagents, etc.) according to the kit usage. For example, a kit can be constructed by combining reagents for amplifying DNA containing the base at position 1162 of the coding region of the 5-HT2B gene. In addition, the method for detecting a mutation in the human serotonin receptor subtype 2B gene of the present invention is suitable for easy implementation. It is preferable to use a mutation detection kit.

 Using the polymorphism information on 5-HT2B, it is possible to construct a screening system for drugs used in the treatment of diseases caused by mutations in the 5-ΗΠΒ gene. For example, a screening system can be constructed by bringing a candidate compound into contact with a 5-HT2B gene expression product having a polymorphism that increases the risk of schizophrenia, etc., and selecting one that specifically binds. . By using such a screening system, it is possible to search and obtain agonists and antagonists for 5-ΗΠΒ. That is, such a screening system can be used for the development of a drug to be used for treating a disease caused by a mutation of the 5-ΗΠΒ gene. Specifically, the following screening methods can be exemplified.

 (A) contacting the candidate compound with the expression product of the serotonin receptor subtype 2B gene, wherein the base at position 162 of the code region is cytosine,

 (B) a step of recovering the compound bound to the expression product.

 By performing these steps, it is possible to select and obtain a compound that specifically binds to the expression product of the 5-HΠΒ gene having a mutation at position 1162 of the code region. There is a high possibility that the obtained compound can be used as a drug (agonist or angonist) acting on 5-HT2B having a mutation. Here, the expression product of the serotonin receptor subtype 2B gene only needs to include at least a portion corresponding to the nucleotide at position 162 in the coding region, and in addition to the expression product of the 5-ΗΠΒ gene (evening protein) itself, It may be a partial protein or partial peptide containing a portion.

 On the other hand, a screening system that performs the following steps ((C) and (D)) in addition to the above steps ((A) and (B)) can be constructed.

 (C) a step of bringing the candidate compound into contact with an expression product of the serotonin receptor subtype 2B gene in which the base at position 162 of the coding region is thymine,

(D) recovering the compound bound to the expression product, The compound obtained as a result of step (D) is a compound that binds to the expression product of a (normal) gene having no mutation at position 1162 of the coding region. Therefore, if the compound obtained as a result of step (D) is excluded from the compounds obtained as a result of the above steps (A) and (B), the compound having a mutation at position 1162 of the coding region 5 -A compound that specifically binds only to the expression product of the HT2B gene, that is, a 5-HT2B receptor having a mutation can be selected. It is very likely that this compound can be used as an agonist and an agonist for the 5-HT2B receptor having a mutation, in other words, it can be used for the treatment of diseases caused by mutations in the 5-ΗΠΒ gene. Very high. Thus, combining steps (A) and (B) with steps (C) and (D) allows efficient screening of drug candidates for diseases caused by 5-HT2B gene mutation. Becomes possible.

 In addition, any of steps (A) and (B) and steps (C) and (D) may be performed first. That is, the order of steps (A), (B), (C), and (D) may be the order of steps (C), (D), (A), and (B). It does not need to be performed continuously, and other steps (for example, a purification step, a heat treatment step, etc.) can be interposed in the middle.

 The contact between the expression product of the specific DNA and the candidate compound in the above step (A) or (C) can be performed, for example, as follows. First, the specific DNA is prepared and inserted into an appropriate vector. Using this vector, a host cell is transformed to obtain an expression product. Then, the candidate compound is brought into contact with the expression product. The manner in which the expression product can be obtained differs depending on the host vector used.For example, when the expression product is secreted, the expression product was purified from the culture solution to obtain the expression product, which was labeled, immobilized, etc. Later, it can be used.

Here, the candidate compounds to be subjected to the above-mentioned screening system include natural proteins extracted from plants, animals, bacteria, etc., natural peptides, natural polymer compounds, etc. Examples include synthetic proteins, synthetic peptides, synthetic high molecular compounds, synthetic low molecular compounds, and the like.

 A system using Escherichia coli (Molecular Cloning, Third Edition, chapter 15, Cold Spring Harbor Laboratory Press, New York) and a baculovirus were used to express the 5-ΗΠΒ gene (or a part thereof). System (Ausebel, FM et al .: Curr. Protocol Mol. Biol., Unit 16.11, Wiley Interscience, 0 'Rely. DR et al .: Baculovirus Expression Vectors (A Laboratory Manual, Oxford University Press, 1994) ) Can be used.

 When the E. coli system is used, pET vectors such as pET-3c and pET-8c (Novagen), pBAD plasmid (Invitrogen), and pGEX plasmid (Amersham Pharmacia biotech) are used as expression vectors. If a baculovirus system is used, PVL1392 (Pharmingen), Mbac (Stratagene), BlueBacHisA (Invitrogen) can be used.

 The 5-ΗΠΒ gene (or a part of it) is converted into His-Tag consisting of several histidines, β-D-galactosidase, GST (daltathione S-transferase), thioredoxin, maltose binding protein, Myc, Xpress, Expression of the product as a fusion protein (peptide) with a tag molecule such as FLAG facilitates purification of the expression product.

 Hereinafter, the present invention will be described in more detail with reference to Examples.

 [Example 1] Mutation search of the co-region of human serotonin receptor subtype 2B gene (5-ΗΠΒ gene)

 (1-1) Preparation of test DNA

For schizophrenia patients and stimulant-induced psychotic disorders DNA is from outpatients and inpatients at Fujita Health University Hospital psychiatry and joint research facilities, who have signed a written consent and signed a consent form. Collect 7 ml of peripheral venous blood and use Puregene DNA was purified using a kit (Gentra). For normal controls, DNA was extracted in the same way from those of Fujita Health and Sanitation University workers and students with consent. The diagnosis of the patients was made in accordance with DSM-IV (American Manual for Diagnosis and Statistics of Mental Illness).

 (1-2) Primer synthesis

 In order to search the entire amino acid coding region of the human serotonin receptor subtype 2B gene (5- 遺 伝 子 gene), a total of the following eight sets of primers (primer set 1 to 8) were designed. Primer design is Primer3 software (http: // ww

-genome, wi.mit.edu/cgi-bin/primer/primer3_ww.cgi), and synthesis was performed using an ABI394 nucleic acid synthesizer (ABI).

 <Primer set 1>

 5-ΗΤ2ΒΠ (forward primer 1)

 5'-GAACGGGATTGAATCACAGAA-3 '(SEQ ID NO: 4)

 5-HT2Blb (Reverse primer 1)

One 5'-TCTCGAGTGAAACAGCCAGA-3 '(SEQ ID NO: 5)

 <Primer set 2>

 5-HT2B2f (forward primer 2)

 5'-GGAAATAAACTGCACTGGGC-3 '(SEQ ID NO: 6)

 5-HT2B2b (reverse primer 1)

 5'-AAACAAAGTGAAATACTTACCAAACA-3 '(SEQ ID NO: 7)

 <Primer set 3> '

 5-HT2B3f (forward primer 3)

 5'-TTCAGAGGCTATGTGGCCC-3, (SEQ ID NO: 8)

 5-HT2B3b (reverse primer 3)

5'-CACTCTGGCATTCTCTACATACC-3 '(SEQ ID NO: 9) <Primer set 4>

5-HT2B4f (forward primer 4)

5'-TTGCCATTCCAGTCCCTATT-3 '(SEQ ID NO: 10) 5-HT2B4b (Reverse Primer 1)

5'-ACGAGCAAGGTGTTTCATCC-3 '(SEQ ID NO: 11) <Primer set 5>

5-HT2B5f (forward primer 5)

 5'-AAAAACAAGCCACCTCAACG-3 '(SEQ ID NO: 12)

5-HT2B5b (Reverse primer 1-5)

5'-GAAGGGACACCACATAAGCAA-3 '(SEQ ID NO: 13) primer set 6>

 5-HT2B6f (forward primer 6)

 5'-TTCCAACGAACAGAGAGCCT-3 '(SEQ ID NO: 14)

5-HT2B6b (reverse primer 6)

5'-CCCGGTAATTGCAGGTGATA-3 '(SEQ ID NO: 15)

<Primer set 7>

 5-HT2B7f (forward primer 7)

 5'-AGACATTTCGGGATGCATTTG-3 '(SEQ ID NO: 16) 5-HT2B7b (Reverse Primer 1)

5'-TTGAACTTCGGAGCCTCATTG-3 '(SEQ ID NO: 17)

<Primer set 8>

5-HT2B8f (forward primer one 8)

 5'-TCTGGGCACATTTACATCTCA-3 '(SEQ ID NO: 18) 5-HT2B8b (Reverse Primer 1)

5'-GAGAAGCATCTACACAAGTC-3 '(SEQ ID NO: 19) The position of type III DNA corresponding to each primer and the length of the DNA fragment amplified by each primer set are shown in the table of FIG.

 (1 — 3) Amplification of DNA fragment by PCR

 A PCR reaction was performed using each of the above primer sets. GeneAmp ™ PCR System 9600 and 9700 (both manufactured by ABI) were used for the PCR reaction. The composition of the reaction solution was 5 fx 1 in total, and 0.125 U of Taq DNA polymerase was added to 20 to 50 ng of sample DNA, 50 nM of primer, 50 M of dNTT, and lx Perkin Elmer buffer I, respectively. All reagents were from ABI. The reaction conditions for PCR were heat denaturation at 95 ° C for 3 minutes, followed by a total of 35 cycles of 94: 15 seconds, 60 ° C for 20 seconds, and 72 ° C for 30 seconds. The extension reaction was performed at 72 ° C for 7 minutes.

 (1 —4) Mutation search by SSCP method

 Using DNA from a schizophrenic patient, mutations in the amino acid coding region of the 5ΗΠΒ gene were searched. For mutation search, SSCP (single strand conformation polymorphism) method (Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polylymorphisms of human DNA by gel electrophoresis as s ingle-strand conformation polymorphisms.Proc Natl Acad Sci US A. 86 (8): 2766-70, 1989) was used. The PCR reaction by the SSCP method was performed by adding [a-32P] dCn> (3,000 Ci / mmol) (Amersham) to the above conditions. After completion of the reaction, stop solution (95% for mamide, 10 mM NaOH, 0.05% xylene cyanoK 0.05% bromphenol blue; was caloried to a total volume of 50, and after thermal denaturation at 80 ° C for 5 minutes, 1 ^ 1 was electrophoresed. 0.5X MDE ™ Gel (FMC BioProducts) was used for electrophoresis The electrophoresis apparatus used was a Sequi-Gen GT system (manufactured by Biorad), and 8 W for 8 to 15 hours at room temperature. After the electrophoresis, the gel was dried using a 583 gel drier (manufactured by Bio-Rad) and exposed to Kodak XAR film (manufactured by Kodak).

Bands with different mobilities were cut directly from the gel, and water was added in 1 for 5 minutes at 80 ° C. After heating for 1 minute, a direct sequence was performed using 1 μl as a template. The nucleotide sequence was determined using a dye terminator cycle sequencing kit (ABI) for the reagent and an ABI373 or ABI377 DNA sequencing system (ABI) for the instrument.

 From the results of the above PCR-SSCP, a total of 5 polymorphisms (L113W, 505C> A, F173L, A248P, R388W) were identified in the amino acid coding region of the 5-HT2B gene as shown in the table of FIG. Four of them (L113W, F173L, A248P, R388W) were mutations involving amino acid substitutions. L113W is a polymorphism in which amino acid 113 is substituted from L (leucine) to W (tryptophan), 505OA is a polymorphism in which base 505 in the coding region is substituted from C (cytosine) to A (adenine), F173L is a polymorphism in which the 173rd amino acid is substituted from F (Fenrylalanine) to L (Leucine), and A248P is a polymorphism in which the 248th amino acid is substituted from A (Aralanine) to P (Proline). R388W represents a polymorphism in which amino acid 388 is substituted from R (arginine) to (tributofan).

 [Example 2] SNPs typing by PCR-RFLP method

 The five identified polymorphisms (SNPs) were typed using the PCR-RFLP (restriction enzyme fragment polymorphism) method. The combinations of primers used in the PCR-RFLP method are shown in the tables of FIG. 1 and FIG. For L113W and 505C> A, the products amplified with the same primer as in the above PCR were directly treated with restriction enzymes to determine the gene type. For F173L, A248P, and R388W, PCR was performed using primers to create a new restriction enzyme recognition sequence by artificially introducing a base different from the genomic sequence two bases away from the mutation position. Was done.

<Primer set 9 (for F173L)>

5-HT2B3f (forward primer)

5'-TTCAGAGGCTATGTGGCCC-3 '(SEQ ID NO: 8)

5-HT2B / F173 (reverse primer)

5'-AACCACACCACTGTAATCTTGATTA-3 '(SEQ ID NO: 20) <Primer set 10 (for A248P)>

 5-HT2B4f (forward primer)

 5'-TTGCCATTCCAGTCCCTATT-3 '(SEQ ID NO: 10)

 5-HT2B / A248 (reverse primer)

 5'-GGTGGCTTGTTTTTGACTAAGTAGG-3 '(SEQ ID NO: 21)

 <Primer set 1 1 (for R388W)>

 5-HT2B6f (forward primer)

 5'-TTCCAACGAACAGAGAGCCT-3 '(SEQ ID NO: 14)

 5-HT2B / R388 (reverse primer)

 5'-GATATATCGGCCAAATGCATCTC-3 '(SEQ ID NO: 22)

 Subsequently, treatment with a restriction enzyme was performed to determine the genotype. PCR reaction conditions are the same as above, with a total volume of 51. I let it. The loading buffer was added to the reaction solution, electrophoresed on a 5% acrylamide gel, and the genotype was determined. Electrophoresis was performed using Mini-Protein 2 (Piorad) and Ready-gel JDNA 5% as a gel. . The following related analysis was performed using the obtained tying results.

 [Example 3] Related analysis

F173L and R338W, which are relatively common in the obtained mutations (polymorphisms), were analyzed for their association with schizophrenia and stimulant-induced psychotic disorders. The results are shown in the table in Fig. 3. Evaluation was performed using the χ ² (chi-square) test and Fisher's exact test. For analysis, LINKAGE UTILITY program software (ftp: \ linkage.rockefeller.edu/software/utilities, http://linkage.rockefeller.edu/ott/IinkutiI.htm) was used. As shown in the table of FIG. 3, no significant association was observed for F173L. On the other hand, with respect to R388W, schizophrenia, strong related to both stimulant-induced psychotic disorder was observed (rf respect schizophrenia / = l, P = 0.000006 ( 2-tail), χ 2 = 17.1, OR = 5.18 (Sheehe), 95% CI [2.20-12.24], df = U 0.000001 (2-tail) for stimulant-induced psychotic disorder, χ ² = 26 OR = 56.1 (Sheehe), 95% CI [7.70-408] ). W338 alleles are present in 22% of normal controls (allele frequency 12%), whereas in Japanese schizophrenia only 5% (allele frequency 2%) and in stimulant-induced psychotic disorders, The result was that they were not seen at all. This suggests that the W338 allele acts as a protective factor against psychosis-like symptoms. Mutations with such a strong association have not yet been identified for these diseases. Thus, the mutations provide very useful and specific information about these diseases. By searching for such mutations, it is possible to examine in advance the vulnerability to schizophrenia and stimulant-induced psychiatric disorders, and to provide useful auxiliary information for the diagnosis of these mental illnesses.

 The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. Industrial applicability

According to the present invention, the presence or absence of a mutation (polymorphism) at a specific position of the serotonin receptor subtype 2B gene is determined. This polymorphism is highly associated with the risk (vulnerability) of developing diseases caused by mutations in the serotonin receptor subtype 2B gene, such as schizophrenia and stimulant-induced psychotic disorders. Therefore, the present invention is an effective means for knowing the risk of developing the disease in advance. Further, according to the present invention, useful information for diagnosing these diseases can be obtained, enabling more appropriate treatment and improving the prognosis. And so on. Furthermore, the present invention can be used as a means for elucidating the role of the serotonin receptor subtype 2B gene polymorphism in the pathophysiology of schizophrenia and the like. Drug discovery).

Claims

The scope of the claims

1. (1) DNA obtained by mutating the nucleotide at position 1162 to thymine (T) in the human sedentary tonin receptor subtype 2B gene, or

 5 (2) DNA that is partially modified at a site other than the base in the DNA, and has a base length capable of detecting mutation at the base site.

2. A protein encoded by the DNA according to claim 1 (1) or (2).

10 3. (a): analyzing the nucleotide at position 1162 in the coding region of the human serotonin receptor subtype 2B gene,

 A method for detecting a mutation in the human oral tonin receptor subtype 2B gene.

4. The method of claim 3, wherein said step (a) comprises the following step (al): 15. (al): T in the human serotonin receptor subtype 2B gene, T, coding region. Step to amplify the DNA region containing the base position at the position.

5. (a): analyzing the nucleotide at position 1162 in the coding region of the human oral tonin receptor subtype 2B gene;

 20. A method for diagnosing a disease caused by a mutation in the human oral tonin receptor subtype 2B gene.

6. The diagnostic method according to claim 5, wherein the step (a) includes the following step (al).

25 (al): coding region 1162 in the human serotonin receptor subtype 2B gene Step to amplify the DNA region containing the base position at the position.

7. The diagnostic method according to claim 5, wherein the disease is schizophrenia or a substance use disorder.

8. A nucleic acid for analyzing the nucleotide at position 1162 of the coding region of the human ostial tonin receptor subtype 2B gene. A nucleic acid having a sequence complementary to a DNA region containing the nucleic acid.

9. A nucleic acid for analyzing the base at position 1162 in the coding region of human serotonin receptor subtype 2B gene, comprising:

 Specific for a DNA fragment derived from a region containing the nucleotide at position 1162 of the human sedentary tonin receptor subtype 2B gene and capable of being amplified by PCR using the primers of SEQ ID NO: 14 and SEQ ID NO: 15 Nucleic acids that hybridize to

10. A kit for detecting a mutation in the human bacterium tonin receptor subtype 2B gene, the kit comprising the nucleic acid according to claim 8.

11. The kit according to claim 10, further comprising a reagent for amplifying a DNA region containing the base at position 1162 of the human sedentary tonin receptor subtype 2B gene.