JP2005278490A - Method for determining biological marker involving schizophrenia and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining the expression amount of a biological marker (e.g. gene) involving schizophrenia in order to contribute to objective diagnosis of schizophrenia using genetic expression, etc., as index and utilization of the same. <P>SOLUTION: The determination method comprises determining whether the expression amount of a nucleic acid encoding VLDLR in a subject and/or the protein is kept in a prescribed range or not. The determination method comprises a measuring step for obtaining a determination value by measuring the expression amount of the nucleic acid encoding VLDLR in a sample collected from the subject and/or the protein and a determination step for determining whether the determined value obtained by the measuring step is kept in the prescribed range or not. According to the determination method, the expression amount of VLDLR which is a biological marker involving the schizophrenia is kept in the prescribed range or not can objectively be determined. Consequently, the determination method is utilizable for objective diagnosis for schizophrenia. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for determining a biological marker involved in schizophrenia (schizophrenia) and use thereof.

  Schizophrenia (schizophrenia) is one of endogenous psychosis or functional (sexual) psychosis that is considered to involve genetic factors in its fragility (vulnerability). It is a mental illness with an extremely high frequency of 0.8%. Symptoms of this schizophrenia include positive symptoms such as hallucinations and delusions, emotional dullness, negative symptoms such as reduced motivation, social withdrawal, and cognitive dysfunction. Social losses due to disease are immeasurable.

  For this reason, many laboratories around the world have been actively investigating schizophrenia treatments and diagnostic methods. As pharmacological treatments, phenothiazine compounds, butyrophenone compounds, benzamide compounds, imino compounds, Dibenzyl compounds, thiepine compounds, indole compounds and serotonin / dopamine receptor blockers have been developed. On the other hand, the diagnosis method of schizophrenia is the delusional type, dismantled type, tension type, indistinguishable in the latest American diagnostic standard DSM-IV (American Psychiatric Association Diagnostic Manual 4th edition) Since it is defined only by psychosymptomatology such as type, the final diagnosis must be only a symptom diagnosis depending on the subjectivity of the doctor in charge, and the accuracy of the diagnosis is not perfect.

  Under such circumstances, chromosomal mapping and identification of schizophrenia causative genes are currently being actively pursued, and several biological markers that can be used to diagnose schizophrenia have been reported. (For example, see Patent Documents 1 to 3).

  For example, in Patent Document 1, as a method for diagnosing whether or not a subject suffers from schizophrenia (schizophrenia), a sample containing nucleic acid and / or protein is collected from the subject, and DNA topoisomerase I or the like is collected. And a method for diagnosing whether or not the subject is suffering from schizophrenia using a quantitative value of the protein or nucleic acid as an index. ing.

  Patent Document 2 discloses that in a sample collected from a subject, a nucleic acid that defines a gene whose expression level changes due to schizophrenia (for example, a vascular endothelial growth factor precursor) and / or the expression level due to schizophrenia. Disclosed is a method for statistically evaluating the expression level of a nucleic acid that defines a gene whose expression level changes due to schizophrenia, including a step of quantifying the expression level of a protein encoded by the nucleic acid that defines the gene whose level changes Yes.

Patent Document 3 describes a diagnostic agent for schizophrenia comprising an anti-basic fibroblast growth factor antibody as an active ingredient, and measures basic fibroblast growth factor in serum using the diagnostic agent. By this, schizophrenia can be diagnosed.
JP 2001-245661 A (publication date: September 11, 2001) JP 2003-38198 A (publication date: February 12, 2003) JP 2003-212795 A (publication date: July 30, 2001)

  As described above, schizophrenia is difficult to distinguish from depression and manic depression because of the peculiarity of the disease state, and an appropriate treatment cannot be performed unless the diagnosis is accurate. In addition, establishment of a comprehensive treatment system for early diagnosis, treatment, rehabilitation activities, and prevention of recurrence is desired. In particular, in recent years, a new generation of antipsychotic drugs with few side effects has been developed, and it has become possible to treat schizophrenia with appropriate diagnosis and treatment.

  For this reason, the schizophrenia diagnostic methods disclosed in Patent Documents 1 to 3 described above are not sufficient, and a biological marker that can diagnose schizophrenia more easily and accurately is developed. At the same time, in order to contribute to an objective diagnosis of schizophrenia using gene expression and the like as an index, a method for statistically evaluating biological markers involved in schizophrenia has been strongly demanded.

  The present invention has been made in view of the above problems, and its purpose is to contribute to objective diagnosis of schizophrenia using gene expression or the like as an index, and a biological marker involved in schizophrenia It is to provide a method for determining the expression level (for example, a gene or the like) and use thereof.

  As a result of intensive studies in view of the above problems, the present inventors collected peripheral blood from each of schizophrenic patients and healthy individuals, and performed quantitative RT-PCR. As a result, blood VLDLR of schizophrenic patients was analyzed. The present inventors have found that the expression level of (Very Low Density Lipoprotein Receptor) is significantly lower than that of healthy subjects, and based on this phenomenon, the present invention has been completed.

  That is, the present invention includes the following 1) to 11) as industrially useful methods or substances.

1) A method for determining whether or not the expression level of a biological marker involved in schizophrenia in a test subject organism is within a predetermined range, wherein schizophrenia in a sample collected from the test subject organism A measurement step for determining the quantitative value by measuring the expression level of the biological marker involved in the determination, and a determination step for determining whether the quantitative value obtained by the measurement step is within the predetermined range; And the biological marker includes at least one substance selected from the following (a) to (e):
(A) a nucleic acid encoding an ultra-low density lipoprotein receptor (b) a nucleic acid having a base sequence complementary to the nucleic acid of (a) (c) a fragment of (a) or (b) (d) an ultra-low density lipo Fragment of protein receptor (e) (d) 2) The determination method wherein the predetermined range in 1) above is the range of the expression level of the biological marker involved in schizophrenia in a healthy organism.

  3) The determination method in which the sample in 1) or 2) is peripheral blood.

  4) In the determination method of any one of 1) to 3) above, the biological marker is mRNA of a very low density lipoprotein receptor, and the measurement step includes a reverse transcription polymerase chain reaction step Method.

  5) In the determination method of any one of 1) to 4) above, the reverse transcription polymerase chain reaction step is complementary to at least a part of the base sequence of the nucleic acid of any one of (a) to (c). A step of adding to the PCR reaction system an oligonucleotide, the oligonucleotide having a 5 ′ end modified with a fluorescent substance and a 3 ′ end modified with a substance that quenches the fluorescence of the fluorescent substance .

  6) A determination kit for carrying out the determination method according to any one of 1) to 5) above.

7) The determination kit according to 6), comprising at least one substance selected from the following (i) to (iii):
(I) an oligonucleotide complementary to at least a part of the base sequence of any one of the nucleic acids (a) to (c) above, wherein the 5 ′ end is modified with a fluorescent substance, and the 3 ′ end is Oligonucleotides modified with a substance that quenches the fluorescence of the fluorescent substance (ii) Primer for amplifying the base sequence of any one of the nucleic acids (a) to (c) (iii) Reagents for nucleic acid amplification 8) Using the determination method or determination kit according to any one of 1) to 7) above, whether the test organism is schizophrenia or the test organism may be affected by schizophrenia. A diagnostic method for diagnosing whether or not there is.

  9) The diagnostic method according to 8), wherein the test organism is a human.

  10) A model animal determination method for determining whether a test animal is useful as a model animal for schizophrenia, wherein the determination method or determination kit according to any one of 1) to 7) above is used. A diagnostic process for diagnosing whether or not a test animal suffers from schizophrenia, and the test animal is a model of schizophrenia, using as an indicator that the test animal suffers from schizophrenia in the diagnostic process. And a determination process for determining that the animal is useful as an animal.

  11) A screening method for screening a candidate substance for an anti-schizophrenia drug, the process of providing a test substance to a model animal for schizophrenia, and the determination method or determination kit according to any one of 1) to 7) above The test substance is anti-schizophrenia using the process of diagnosing whether the schizophrenia of the model animal has been cured or improved as an index and whether the schizophrenia of the model animal is cured or improved. And a process for determining that the substance is a candidate substance for a disease drug.

  In addition, as shown in the Examples described later, the biological marker (VLDLR) used in the present invention is considered to be a pathogenic susceptibility gene for major depression as well as schizophrenia. It can be said that the determination method, determination kit, diagnosis method, model animal determination method, and screening method can be similarly used for major depression.

  As described above, according to the determination method according to the present invention, the expression level of the biological marker involved in schizophrenia in the test target organism (for example, human or laboratory animal) is within the range of that of the healthy organism. Whether it is included or not can be determined accurately and simply. For this reason, by using the determination method according to the present invention, it is possible to diagnose whether or not the subject suffers from schizophrenia or is likely to suffer.

  The present invention provides a method for easily and accurately determining whether the expression level of a biological marker involved in schizophrenia in a test subject organism is within that range of a healthy organism, a method for using the method, and the like To do. For this reason, in the following description, first, a method for determining a biological marker according to the present invention (hereinafter, sometimes simply referred to as “determination method” for convenience of description) will be described, followed by determination as its usage. The kit, diagnostic method, model animal determination method, and screening method will be described in this order.

[1] Biological Marker Determination Method The biological marker determination method according to the present invention is based on whether or not the expression level of a biological marker involved in schizophrenia in a test target organism is within a predetermined range. As long as it includes at least a measurement step and a determination step, other specific steps, materials, conditions, and the like are not particularly limited. Hereinafter, each process will be described in detail.

[1-1] Measurement Step The “measurement step” may be a step in which a quantitative value is obtained by measuring the expression level of a biological marker related to schizophrenia in a sample collected from a test subject organism. In this measurement step, any one of the above (a) to (e) is quantified. In order to quantify the expression level of the biological marker to be used as an index, first, a sample is collected from the test target organism. There is a need. For this reason, for example, a collection step of collecting a sample from the test target organism may be included in the previous stage of the main measurement step.

  Here, the “test target organism” refers to all organisms showing symptoms of schizophrenia, and is a concept including a subject and a test animal. In the present specification, the “subject” means a human, particularly a patient who is a subject of the method of the present invention. In the present specification, the “test animal” refers to animals other than humans, and particularly means mice, rats, guinea pigs, dogs, rabbits, monkeys, chimpanzees and the like suitable as experimental animals.

  Further, the “sample” may be a sample collected from the test subject organism, and for example, any sample containing tissues, cells, and body fluids extracted from the test subject organism can be the “sample” in the present invention. . Examples of preferable samples include blood, particularly peripheral blood, but since the biological marker referred to in the present invention is expressed in other than blood, other samples such as biopsy brain, autopsy brain, cerebrospinal fluid, etc. Methods using various tissues should also be understood to be within the scope of the present invention.

  The “biological marker involved in schizophrenia” is a biological marker whose expression level is changed (increased or decreased) by suffering from schizophrenia, the above-mentioned (a) to (a) to (b) It is sufficient that at least one substance selected from (e) is included.

  (A) may be any nucleic acid that encodes a very low density lipoprotein receptor (hereinafter sometimes referred to as VLDLR). Moreover, (b) should just be a nucleic acid which has a base sequence complementary to the nucleic acid of (a). That is, typical examples of (a) or (b) include VLDLR mRNA and cDNA. Moreover, arbitrary polynucleotides, such as a regulatory sequence and a polyadenyl sequence, may be contained in the terminal and / or inside of the translation region of these mRNA or cDNA. Also, if the VLDLR can be encoded by multiple alleles, all alleles, their transcripts, and cDNA are included in such nucleic acids. In the present specification, the term “nucleic acid” includes a polynucleotide comprising any simple nucleotide and / or modified nucleotide, such as cDNA, mRNA, total RNA, hnRNA, and the like. “Modified nucleotides” include inosin, acetylcytidine, methylcytidine, methyladenosine, and methylguanosine, as well as nucleotides that can be generated by the action of ultraviolet light or chemical substances.

  Moreover, (c) should just be a fragment of the nucleic acid of (a) or (b). The “nucleic acid fragment” means a nucleic acid that defines a gene encoding VLDLR or a polynucleotide containing a part of the nucleic acid, and typically includes a VLDLR mRNA or cDNA fragment.

  Specific examples of such a nucleic acid include a gene encoding human VLDLR (accession #: Genomic NT — 008413, mRNA NM — 003383) and a fragment thereof. The NCBI site is http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=7436. The gene encoding VLDLR that can be used in the present invention is not limited to the gene encoding human VLDLR, and genes encoding VLDLR in various experimental animals can be preferably used. It goes without saying that which animal VLDLR gene is used can be appropriately selected according to the test subject organism.

  (D) is a very low density lipoprotein receptor, that is, a protein of VLDLR. Moreover, (e) should just be a fragment | piece of the protein of (d), and includes partial protein, polypeptide, and oligopeptide. Specific examples of such proteins include human VLDLR protein (accession #: NP_003374) and fragments thereof.

  Schizophrenia is a mental illness formerly called schizophrenia, and includes delusional schizophrenia, disorganized schizophrenia, tension schizophrenia, and indistinguishable schizophrenia. Any type of schizophrenia (schizophrenia) is included.

  Further, in this measurement step, when the nucleic acids (a) to (c) are quantified, after the sample is collected from the test target organism, usually, an operation of extracting the nucleic acid from the sample is performed. As a method for extracting nucleic acid from this sample (biological component), for example, any extraction method other than phenol extraction and ethanol precipitation can be used. Moreover, when extracting mRNA, you may apply to an oligo dT column. When the amount of nucleic acid is small, an operation of amplifying the nucleic acid may be performed as necessary. The amplification operation can be performed by, for example, a polymerase chain reaction (hereinafter simply referred to as PCR) such as reverse transcription polymerase chain reaction (RT-PCR). Further, as described below, the amplification operation may be performed as a quantitative operation or also as a quantitative operation.

  After performing the extraction operation and / or the amplification operation as necessary, at least one nucleic acid selected from the group consisting of the nucleic acids (a) to (c) is quantified. To quantify nucleic acids, use any technique known in the art, including quantitative PCR, quantitative RT-PCR, Southern blotting, Northern blotting, RNA digestive enzyme protection mapping, and combinations thereof be able to.

As quantitative PCR, typically, a method of internally labeling an amplification product using a nucleotide labeled with a radioactive substance (for example, ³² P) or a fluorescent substance can be used. In addition, a method in which an amplification product is end-labeled using a primer labeled with a radioactive substance or a fluorescent substance can be used. Labeled amplification products are separated from free radioactive (or fluorescently added) nucleotides or primers using well-known methods including gel filtration, alcohol precipitation, trichloroacetic acid precipitation, physical adsorption on glass filters, etc. can do.

  Subsequently, when radioactive materials are used, operations such as liquid scintillation, autoradiography, and imaging plates (bioimaging analyzer (BAS; Fuji Photo Film), etc.) with or without electrophoresis and hybridization are performed. Quantify the amplification product. When a fluorescent substance or a luminescent substance is used as a labeling substance instead of a radioactive substance, the amplification product may be quantified using a spectrofluorometer, a fluorescent microplate reader, a CCD camera, or the like.

  When the biological marker is VLDLR mRNA, this measurement step may include a quantitative RT-PCR step. In addition, so-called real-time PCR can be performed as quantitative PCR and quantitative RT-PCR. As the real-time PCR, conventionally known methods such as an incalator method, TaqMan probe method, cycling probe method and the like can be used.

  The “incalator method” is a method in which a reagent (intercalator: SYBRR Green I, EtBr, etc.) that emits fluorescence by binding to double-stranded DNA is added to the PCR reaction system. Since the intercalator binds to the double-stranded DNA synthesized by the PCR reaction and emits fluorescence when irradiated with excitation light, the amount of amplification product produced can be monitored by detecting this fluorescence intensity.

  The “TaqMan probe method” is a method in which an oligonucleotide (TaqMan probe) whose 5 ′ end is modified with a fluorescent substance (such as FAM) and whose 3 ′ end is modified with a quencher substance (such as TAMRA) is added to the PCR reaction system. is there. The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but since the quencher is present on the probe, the generation of fluorescence is suppressed even when irradiated with excitation light. When the TaqMan probe hybridized to the template is decomposed by the 5 ′ → 3 ′ exonuclease activity of TaqDNA polymerase during the extension reaction step, the fluorescent dye is released from the probe and the quencher is released from suppression. As a result, fluorescence is emitted.

  The “cycling probe method” is a highly sensitive detection method using a combination of an RNA (or RNA and DNA chimera) probe and RNase H, and can efficiently detect a specific sequence of a gene fragment during or after amplification. it can. The probe is labeled with a fluorescent substance (reporter) at the 5 'end and a substance (quencher substance) that quenches fluorescence at the 3' end. In this intact state, this probe does not emit strong fluorescence due to quenching. However, when the RNA portion is cleaved by RNaseH after hybridization with a complementary amplification product, the probe becomes intensely fluorescent. . By measuring the fluorescence intensity, the amount of amplification product can be monitored.

  Among these, as shown in Examples described later, it is preferable to use the “TaqMan probe method”. That is, this measurement step includes an RT-PCR step, and the RT-PCR step includes an oligo complementary to at least a part of the base sequence of any one of the nucleic acids (a) to (c). It is preferable to include a step of adding to the PCR reaction system an oligonucleotide which is a nucleotide and whose 5 ′ end is modified with a fluorescent substance and whose 3 ′ end is modified with a quencher substance. According to the above configuration, the present invention can be carried out accurately and simply by preparing a TaqMan probe specific for VLDLR. The TaqMan probe and the primer for PCR can be arbitrarily prepared by selecting a suitable region by a conventionally known method, and are not particularly limited.

  When quantitative PCR is not performed, most commonly, samples containing nucleic acids are subjected to electrophoresis, Southern blot or Northern blot, and then quantified using a probe labeled with a detectable labeling substance. It is also possible to do. In addition, when quantifying many kinds of nucleic acids at the same time, a DNA chip or a DNA microarray may be used together with these methods or instead of these methods. The expression level of the gene may be indirectly estimated by quantifying the protein produced from the mRNA (gene) instead of quantifying the nucleic acid or together with the quantification of the nucleic acid. For the diagnosis of schizophrenia by the method of the present invention, it may be useful to quantify the protein encoded by the nucleic acid rather than the nucleic acid.

  On the other hand, in the present measurement step, when the protein (b) or (e) or a fragment thereof (hereinafter simply referred to as a protein or the like) is quantified, the sample is usually collected from the test organism, An operation to extract proteins and the like is performed. As a method for extracting the protein and the like from a sample (for example, a tissue) and a method for quantifying the protein, any method known in the art can be used. For example, when protein is quantified, Western blotting, enzyme immunoassay including solid phase enzyme immunoassay, immunocytochemistry, and immunohistochemistry can be used. In the present specification, only the outline of the most general operation is illustrated, and various modifications of the above method and completely different methods can be used. At present, there are commercially available devices that perform full-automatic extraction, amplification, separation, and quantification of nucleic acids by combining electrophoresis devices, PCR devices, and the like, and it is also preferable to use such devices. If such an apparatus is used, it becomes possible to make a diagnosis of schizophrenia in the same manner as a normal clinical test.

[1-2] Determination Step The determination step referred to in the present invention may be a step for determining whether or not the quantitative value obtained by the measurement step is within the predetermined range. That is, after a predetermined biological marker (quantification of nucleic acid and / or protein) is quantified in the measurement step, it is determined whether or not the quantification value is within a predetermined range. Here, the “predetermined range” is a range of the expression level of the biological marker related to schizophrenia in a healthy organism (an organism not suffering from schizophrenia, a non-schizophrenia organism), that is, normal A range of values. As the “predetermined range”, a threshold range set based on the normal value may be used. That is, in this determination step, an appropriate threshold range is set with reference to the normal value or normal value of the expression level of the biological marker, and it is determined whether the normal value or the threshold value is exceeded or below the normal value. It can be said that it will do.

  Moreover, what is necessary is just to select the said threshold value according to desired determination precision as follows. For example, when the distribution of gene expression levels in a healthy group of non-schizophrenia (hereinafter referred to as a normal group) and a schizophrenic organism group (hereinafter simply referred to as a patient group) is clarified, for example, The threshold is set so that the probability that the individual from which the nucleic acid or protein to be quantified has belonged to the normal group is 10%, 5%, or 1%. When only the distribution of gene expression levels in the normal group is known, such quantitative values are obtained for the nucleic acid or protein under the assumption that the individual from whom the nucleic acid or protein to be quantified is collected belongs to the normal group. The threshold or the amount or concentration of a nucleic acid or protein whose probability of being (hereinafter referred to as p-value, typically a two-sided probability, but may be one-sided probability) is 10%, 5%, or 1% Can do. Moreover, even when only the distribution of gene expression levels in the patient group is clear, it can be analyzed by the same statistical method.

  The p value can be calculated by a conventionally known test method such as t test or nonparametric test. In order to clarify the statistical distribution of gene expression levels in the normal group and / or patient group, typically, at least 5, preferably 10, more preferably 20, more preferably 50, Most preferably, the expression level of 100 individuals should be measured. In addition, if necessary, it is possible to more accurately determine whether or not the biological marker in the test target organism is within a predetermined range by using any of various statistical techniques. Of course, a determination method using such a technique also belongs to the scope of the method of the present invention.

  Further, in the present specification, “the determination step for determining whether or not the quantitative value obtained by the measurement step is within a predetermined range” is, for example, “the quantitative value obtained by the measurement step is a normal group In other words, it is an "analysis process for statistically analyzing whether the range is within the range of". The “analysis process” means a statistical process as specifically described below.

  First, when determining a quantitative value of a single nucleic acid and / or protein or the like as an index, the expression level of the patient group is large (for example, the signal is 10 or more), and the absolute gene expression change rate of both (“patient group” In the test of the difference between the mean expression level of the normal expression / average expression level of the normal group and the average expression level of the normal group / average expression level of the patient group, whichever is greater) It is preferable to use a nucleic acid and / or protein having a p value of 5% or less as an index. In addition, when determining a quantitative value of a plurality of nucleic acids and / or proteins as an index, an appropriate threshold is set for each nucleic acid and / or protein, and a single nucleic acid and / or protein or the like is used as an index. In the same manner as described above, it is checked whether the expression level of each gene is above or below the threshold value. Depending on the desired determination accuracy, it can be determined whether a quantitative value of one nucleic acid and / or protein is above or below a threshold value. As a result, it can be determined that the patient is likely to have schizophrenia. Moreover, it can be determined whether the quantitative values of two or more nucleic acids and / or proteins are above or below a threshold value, in which case it can be determined that the possibility of schizophrenia is even higher. If you want to make a definitive determination, you can determine whether you have schizophrenia by determining whether the quantitative values of more nucleic acids and / or proteins are above or below the threshold. . Furthermore, the determination method of the present invention can be used in combination with a conventional subjective diagnosis method.

  In addition, data on quantitative values of biological markers (such as nucleic acids and / or proteins) that can be used as indicators in the determination method of the present invention from patients who are clearly determined to have schizophrenia by any method. Can be collected, it is possible to make a definitive determination only by the determination method of the present invention. That is, whether or not the quantitative value of the expression level of the biological marker obtained by the measurement step of the present invention is within the range of the quantitative value of the expression level of the biological marker in a definite schizophrenic patient. It can also be determined in the determination step of the present invention.

  The subject of the present invention is to provide an objective determination (diagnosis) method of schizophrenia, and the individual extraction operation, amplification operation, and analysis operation specifically described in the present specification. It does not exist. Therefore, it should be noted that determination methods using operations other than the above operations also belong to the scope of the present invention.

  As described above, when the determination method according to the present invention is used, the expression level of a biological marker involved in schizophrenia in the test target organism (the expression level of the nucleic acid (gene) and / or the organism derived from the nucleic acid (gene)) It is possible to easily and accurately determine whether or not the expression level of a biological product (protein) is within a predetermined range. Then, by using the determination result as an index, it is possible to objectively diagnose whether or not the test target organism suffers from schizophrenia. Therefore, the present invention includes the following uses.

[2] Usage Since the determination method according to the present invention has the above-described advantages, various usages are included in the present invention. Hereinafter, as a method of using the determination method according to the present invention, a determination kit, a diagnosis method, a model animal determination method, and a screening method will be described in order.

[2-1] Determination Kit The determination kit according to the present invention may be any one for carrying out the determination method described in the above [1] column. Specific configurations, materials, equipment, and the like included in the determination kit are as follows. There is no particular limitation. Specifically, the thing for implementing each process of the said determination method should just be contained. For example, in the case of a collecting step of collecting a sample from a test target organism, a syringe or the like is used as a device for collecting blood. Moreover, in the case of a measurement process, what is necessary in order to implement the said measurement, for example, various reagents and laboratory instruments, are mentioned. In the case of the determination step, various arithmetic devices (for example, a computer or the like) necessary for performing the above determination may be mentioned.

  According to said structure, the determination method concerning this invention can be implemented simply and reliably. For this reason, determination (diagnosis) of schizophrenia can be performed using this determination method.

  In particular, when the measurement step includes a PCR step, PCR primers and various reagents (DNA polymerase) for amplifying the nucleic acid of any one of (a) to (c) described in the column [1] above. And PCR equipment such as nucleotides and thermal cyclers). In addition, when the biological marker is RNA and an RT-PCR step is included, a reagent for RT-PCR (such as reverse transcriptase) can be mentioned. Furthermore, when using the above-described “TaqMan probe method”, a TaqMan probe specific to a biological marker (such as a VLDLR gene or a fragment thereof) is required. That is, the oligonucleotide is complementary to at least a part of the base sequence of any one of the nucleic acids (a) to (c) described in the above [1] column, and the 5 ′ end is modified with a fluorescent substance. In addition, an oligonucleotide whose 3 ′ end is modified with a substance that quenches the fluorescence of the fluorescent substance is required.

  According to said structure, there exists an advantage that the measurement process including RT-PCR process can be simply performed using the TaqMan probe specific to the nucleic acid which codes VLDLR which is a biological marker.

[2-2] Diagnosis method The present invention also includes a method for diagnosing whether or not a test subject organism has schizophrenia, or whether or not the test subject organism may suffer from schizophrenia. included. The diagnosis method may use the above-described determination method or determination kit, and other specific configurations, conditions, and the like are not particularly limited. For example, it is determined whether or not the test target organism suffers from schizophrenia using the expression level of the biological marker in an organism not suffering from schizophrenia (a healthy organism) as an index. More specifically, when diagnosing a single nucleic acid and / or protein quantitative value as an index, an appropriate threshold is set with reference to the normal value, and if the threshold is above or below, Diagnosis is likely to be schizophrenia. That is, when the expression level of the biological marker increases in a schizophrenic patient, it may be determined that the possibility of schizophrenia is high if the expression level exceeds a set threshold.

  Therefore, according to the present diagnostic method, it is possible to easily and accurately diagnose whether the test target organism has schizophrenia or whether the test target organism may suffer from schizophrenia. . This diagnostic method can be divided into a diagnostic method for a subject (human) and a diagnostic method for a test animal (such as a laboratory animal). Needless to say, the usefulness of diagnostic methods for humans can be applied, for example, for the purpose of examining the presence or absence of legal responsibility or for other purposes. In addition, a diagnostic method for a test animal will be very useful, for example, in animal experiments such as when developing a therapeutic drug for schizophrenia.

[2-3] Determination method and screening method of model animal The determination method and determination kit of the present invention include a method for determining the usefulness of a model animal for schizophrenia excluding humans, and a drug using such a model animal. In screening, it can be applied to a method for determining the effectiveness of a drug. That is, when determining the usefulness of a model animal for schizophrenia, the test animal has schizophrenia based on the expression level of the biological marker (gene), as in the determination method and the diagnosis method. It is determined whether or not the animal has developed, and if the animal has developed schizophrenia, it can be determined that the animal is useful as a model animal for schizophrenia.

  In addition, the “test animal” referred to here includes mice, rats, and monkeys, but any animal other than a human can be a “test animal”. Since determination (diagnosis) of schizophrenia was more difficult in animals other than humans, it can be said that this method is extremely useful. Further, after administering a candidate substance for an anti-schizophrenia drug to such a model animal, the nucleic acid and / or protein of the biological marker is quantified as described above, and schizophrenia is cured or improved. For example, it can be determined that the candidate substance has an anti-schizophrenia action. That is, by using the determination method of the present invention, a candidate substance for an anti-schizophrenia drug can be easily and accurately screened. Note that the “candidate substance for anti-schizophrenia drug” referred to here may be any substance desired by the examiner.

  In addition, as shown in the Example mentioned later, since it is thought that the biological marker (VLDLR) utilized for this invention is not only schizophrenia but the etiology susceptibility gene with respect to major depression, above-mentioned this invention It can be said that the determination method, determination kit, diagnosis method, model animal determination method, and screening method according to the present invention can be similarly used for major depression. That is, “schizophrenia” in the above methods and kits can be read as “major depression” and used as a determination method, a determination kit, or the like for “major depression”.

  Hereinafter, examples will be shown, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

[Method and results]
Subjects were all untreated individuals. (i) Schizophrenia group consists of patients diagnosed with Schizophrenia, Schizophreniform disorder, Schizophaffective disorder (n = 20) and major depression in DSM-IV according to DSM-IV (American Psychiatric Association Diagnostic Manual 4th edition) Patients with pathological disorder (n = 20). In addition, (ii) the control group is a treatment group for each of the healthy persons (n = 20) who have no psychiatric disorder in their parents and siblings whose sex / age are matched, and psychiatric disorder.

The study contents were explained in writing to the above-mentioned subjects, and consent was obtained. After that, a specific experiment was conducted. Specifically, first, 20 ml of venous blood is collected in an EDTA blood collection tube, a mononuclear cell fraction containing lymphocytes is extracted by the Ficoll / Paque concentration gradient method, and total RNA is prepared. Real-time PCR method (RT) using ABI PRISM 7900HT (manufactured by Applied Biosystems Inc.) for each of the Lipoprotein Receptor family, which is known to modify the function of neural stem cells, and the IL-6 receptor family. -PCR method), mRNA expression levels were quantified and compared between groups. The target molecules (biological markers) to be amplified by PCR are shown below.
・ Lipoprotein receptor family: Apolipoprotein E receptor type 2, Very low density lipoprotein receptor (VLDLR), Megalin, Low density lipoprotein (LDL) receptor, LDL receptor-related protein
・ LI-6 receptor family: Ciliary neurotrophic factor, Leukemia inhibitory factor (LIF), LIF receptor
The primers and TaqMan probe used are shown below. Real-time PCR was performed under the following conditions. The primers are based on the mRNA sequence of VLDLR, Genbank accession #: NM_003383, primer A: 5'CTG CAG GGA CTG GAG TGA TGA 3 'on exon 7, primer B: 5' GCA on exon 9 Designed with GAT TCC TGG ATT TTG GCA 3 '.

  The TaqMan probe was designed on exon 8 as 5 'CGA GTG TGA CTG TGC AGC TGG GTT TGA 3'.

  Real-time PCR was performed using ABI PRISM 7900HT (Applied Biosystems Inc) Sequence Detection System under conditions of 55 cycles of 95 ° C. for 30 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 30 seconds.

  The result is shown in FIG. In the figure, Healthy represents a healthy person, Schizophrenia (not medicated) for schizophrenia (untreated group), Schizophrenia (medicated) for schizophrenia (treated group), and Depression (not medicated) for depression (untreated). Treatment group), Major Depression (medicated) represents a major depression condition (treatment group), and Bipolar Disorder (medicated) represents a patient in a condition (treatment group) that repeats “so” and “depression”.

  As shown in FIG. 1, among the above target molecules, the inventors of the present invention have a significant expression level of Very low density lipoprotein receptor (VLDLR) mRNA in untreated schizophrenia patients and major depression patients. I found that it has dropped. In addition, in healthy subjects and patients in the treatment group, a decrease in the VLDLR mRNA expression level was not observed.

  For this reason, the inventors of the present application have determined that VLDLR can be used as a biological marker common to schizophrenia and major depression. In addition, it has not yet been reported that VLDLR is a common etiological susceptibility gene for both of the above diseases, and this discovery can be said to be the first in the world.

  Therefore, it can be said that this factor is involved in the onset of schizophrenia and the like, and can be used as a biological marker at the onset of schizophrenia and the like. Moreover, it can also be used as a trait marker in differentiation from other psychiatric disorders and the course of treatment.

  As described above, according to the determination method according to the present invention, whether or not the test subject organism is schizophrenia can be easily and reliably determined, and therefore, for prevention, diagnosis, treatment, etc. of schizophrenia. Very useful. In other words, it can be used not only for academic utility but also for a very wide range of applications such as the pharmaceutical industry, medical equipment, and medical industry.

It is a figure which shows the result of the Example in this invention.

Claims (11)

A method of determining whether the expression level of a biological marker involved in schizophrenia in a test subject organism is within a predetermined range,
A measurement step of measuring the expression level of a biological marker involved in schizophrenia in a sample collected from a test subject organism to obtain a quantitative value;
A determination step of determining whether the quantitative value obtained by the measurement step is within the predetermined range,
The biological marker includes at least one substance selected from the following (a) to (e):
(A) a nucleic acid encoding an ultra-low density lipoprotein receptor (b) a nucleic acid having a base sequence complementary to the nucleic acid of (a) (c) a fragment of (a) or (b) (d) an ultra-low density lipo Fragment of protein receptor (e) (d)   The determination method according to claim 1, wherein the predetermined range is a range of an expression level of a biological marker involved in the schizophrenia in a healthy organism.   The determination method according to claim 1, wherein the sample is peripheral blood. The biological marker is mRNA of a very low density lipoprotein receptor,
The determination method according to claim 1, wherein the measurement step includes a reverse transcription polymerase chain reaction step.   In the reverse transcription polymerase chain reaction step, the oligonucleotide is complementary to at least a part of the base sequence of any of the nucleic acids (a) to (c), and the 5 ′ end is modified with a fluorescent substance. The method further comprises a step of adding an oligonucleotide whose 3 ′ end is modified with a substance that quenches the fluorescence of the fluorescent substance to the PCR reaction system. Judgment method.   The determination kit for implementing the method of any one of Claims 1-5. The determination kit according to claim 6, comprising at least one substance selected from the following (i) to (iii).
(I) an oligonucleotide complementary to at least a part of the base sequence of any one of the nucleic acids (a) to (c) above, wherein the 5 ′ end is modified with a fluorescent substance, and the 3 ′ end is Oligonucleotides modified with a substance that quenches the fluorescence of the fluorescent substance (ii) Primer for amplifying the base sequence of any one of the nucleic acids (a) to (c) (iii) Reagents for nucleic acid amplification   Using the determination method or determination kit according to any one of claims 1 to 7, whether or not the test target organism has schizophrenia, or the test target organism may suffer from schizophrenia A diagnostic method characterized by diagnosing whether or not.   The diagnostic method according to claim 8, wherein the test target organism is a human. A method for determining a model animal for determining whether a test animal is useful as a model animal for schizophrenia,
A diagnostic process for diagnosing whether or not the test animal suffers from schizophrenia by the determination method or determination kit according to any one of claims 1 to 7,
A determination process for determining that the test animal is useful as a model animal for schizophrenia, using as an index that the test animal is suffering from schizophrenia in the diagnosis process, Animal judgment method. A screening method for screening candidate substances for anti-schizophrenia drugs,
A process of supplying a test substance to a model animal of schizophrenia,
A process of diagnosing whether schizophrenia of the model animal has been cured or improved by the determination method or determination kit according to any one of claims 1 to 7,
And a step of determining that the test substance is a candidate substance for an anti-schizophrenia drug using, as an index, that the schizophrenia of the model animal is cured or improved.

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