JP5641471B2 - Schizophrenia marker and its use - Google Patents

Description

  The present invention relates to a schizophrenia marker useful for examination of schizophrenia. More specifically, the present invention relates to a biomarker that can be used to determine the possibility of developing schizophrenia, and a method for testing schizophrenia using the biomarker.

  Schizophrenia is a chronic / progressive mental illness that develops in adolescence and adolescence, with positive symptoms (such as hallucinations and delusions, poorly organized speech and behavior), negative symptoms (flattened emotions, poor thinking) The main symptoms are cognitive impairment (attention disorder, decreased working memory, executive dysfunction, etc.). Schizophrenia is thought to be a multifactorial disease involving genetic and environmental factors, but there are still many unclear points regarding the molecular biological mechanism of the pathogenesis of schizophrenia, resulting in biochemical consequences. No valid inspection method has been found.

  Diagnosis of schizophrenia is performed by symptomatology based on doctors' interviews and patient complaints, and diagnostic methods based on biochemical tests have not been established. Schizophrenia progresses several years after its onset, causing irreversible disability and permanent social functioning, but is treated because there is no useful diagnostic test The current situation is that there are many cases in which refractory intervention is delayed, resulting in intractable cases. Development of a test method useful for the diagnosis of schizophrenia is eagerly desired, but the disease is thought to develop due to abnormalities in the brain molecules, but it is extremely difficult to detect the molecular pathology of the brain. The development of is not progressing.

Arcos-Burgos M. et al., Am J Hum Genet 77,6 p937-44,2005 Lewis CM. Et al., Am J Hum Genet 73, 34-48, 2003 Badner JA. And Gershon ES., Mol Psychiatry 7, 405-11, 2002

  Results from twin studies and other studies have revealed that genetic factors are greatly involved in the development of schizophrenia. Until now, several candidate genes suggested to be involved in the development of schizophrenia have been identified by genetic analysis using schizophrenia patients and healthy subjects (for example, Non-Patent Documents 1 to 3). There are few clues to clarify the pathogenesis of these genes and schizophrenia, and the molecular mechanism of the disease has not yet been elucidated. As a result, the pathophysiology of this disease is still unclear, and the current situation is that no diagnostic method based on biochemical tests has been established. Then, this invention makes it a subject to provide a useful technique for the test | inspection of schizophrenia while providing the biomarker specific to schizophrenia. More specifically, an object of the present invention is to provide a biomarker useful for testing schizophrenia, a test method using the biomarker, a test reagent used for the test method, and the like.

Schizophrenia is thought to develop due to abnormal brain function, but it is not practical to use changes in protein expression in the brain for diagnosis. On the other hand, peripheral blood is frequently used for biochemical tests. The inventors focused on lymphocytes contained in peripheral blood. In other words, immortalization of lymphocytes collected from peripheral blood and use of lymphoblastoid cells minimizes the effects of state-dependent factors such as therapeutic drugs, concurrent physical diseases, and circadian variation at the time of blood collection I thought it was possible. Based on these points of interest, we decided to compare the protein expression profiles of the patient group and the healthy group. As a result of detailed examination, a plurality of proteins having significant differences in expression levels were identified. That is, the present inventors succeeded in identifying a protein that exhibits expression changes specific to schizophrenia. In addition, most of the identified proteins are also expressed in the brain, strongly suggesting that they may be deeply involved in the pathogenesis of schizophrenia. The present invention described below is mainly based on the above results.
[1] Annexin A5, microtubule-related protein RP / EB family member 1, EF-hand domain-containing protein D2, tubulin folding cofactor B, inorganic pyrophosphatase, glutaredoxin-3, plastin-2, heat shock Protein 90β, pyridoxal-dependent decarboxylase domain-containing protein 1, ubiquitin / carboxyl terminal hydrolase isozyme L1, adenine / phosphoribosyltransferase, cytoplasmic histidyl tRNA synthetase, uroporphyrinogen decarboxylase, heat shock 70kDa protein 4L , Trifunctional purine biosynthetic protein adenosine-3, trifunctional purine biosynthetic protein adenosine-3 short isoform, β-lactamase-like protein 2, putative deoxyribonuclease TATDN1, vacuolar protein sorting Linkage protein 35, interferon-derived GTP-binding protein Mx1, kinesin-like protein KIF11, elongation factor 1-γ, immunoglobulin mu chain C region, immunoglobulin mu heavy chain disease protein, immunoglobulin heavy chain V-III region BRO and immunoglobulin heavy A schizophrenia marker consisting of any protein molecule selected from the group consisting of chain V-III region GA.
[2] Annexin A5, microtubule-related protein RP / EB family member 1, EF-hand domain-containing protein D2, tubulin folding cofactor B, inorganic pyrophosphatase, glutaredoxin-3, plastin-2, heat shock Protein 90β, pyridoxal-dependent decarboxylase domain-containing protein 1, ubiquitin / carboxyl terminal hydrolase isozyme L1, adenine / phosphoribosyltransferase, cytoplasmic histidyl tRNA synthetase, uroporphyrinogen decarboxylase, heat shock 70kDa protein 4L , Trifunctional purine biosynthetic protein adenosine-3, trifunctional purine biosynthetic protein adenosine-3 short isoform, β-lactamase-like protein 2, putative deoxyribonuclease TATDN1, vacuolar protein sorting Linkage protein 35, interferon-derived GTP-binding protein Mx1, kinesin-like protein KIF11, elongation factor 1-γ, immunoglobulin mu chain C region, immunoglobulin mu heavy chain disease protein, immunoglobulin heavy chain V-III region BRO and immunoglobulin heavy A method for testing schizophrenia, wherein the level in a sample of one or more protein molecules selected from the group consisting of chain V-III region GA is used as an index.
[3] The schizophrenia test method according to [2], comprising the following steps (1) to (3):
(1) a step of preparing a subject-derived specimen;
(2) detecting the one or more protein molecules in the specimen; and (3) determining a current or future possibility of developing schizophrenia based on the detection result.
[4] Annexin A5, microtubule-related protein RP / EB family member 1, EF-hand domain-containing protein D2, tubulin folding cofactor B, inorganic pyrophosphatase, glutaredoxin-3, plastin-2 , Heat shock protein 90β, pyridoxal-dependent decarboxylase domain-containing protein 1, ubiquitin / carboxyl terminal hydrolase isozyme L1, adenine / phosphoribosyltransferase, cytoplasmic histidyl tRNA synthetase, uroporphyrinogen decarboxylase, heat shock About 70kDa protein 4L, trifunctional purine biosynthetic protein adenosine-3, trifunctional purine biosynthetic protein adenosine-3 short isoform, β-lactamase-like protein 2, and putative deoxyribonuclease TATDN1 In accordance with the standards of the high possibility of onset with a reference, or can not be detected with high possibility of onset and the detected value is lower,
Vacuolar protein sorting-related protein 35, interferon-derived GTP-binding protein Mx1, kinesin-like protein KIF11, elongation factor 1-γ, immunoglobulin mu chain C region, immunoglobulin mu heavy chain disease protein, immunoglobulin heavy chain V-III region BRO For the immunoglobulin heavy chain V-III region GA, the determination in step (3) is performed according to the criterion that the detection probability is high when the detection value is high, or the criterion that the detection probability is high when the detection value is high. The method according to [3].
[5] The schizophrenia test method according to [3] or [4], wherein the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value of the control sample.
[6] The determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value in a sample collected in the past from the same subject, [3] or [ 4] The schizophrenia test method according to [4].
[7] The schizophrenia testing method according to any one of [2] to [6], wherein the specimen is blood, plasma, serum, oral mucosa, nasal mucosa, or skin.
[8] The schizophrenia testing method according to any one of [2] to [6], wherein the specimen is a lymphoblastoid cell obtained by immortalizing blood lymphocytes collected from a subject.
[9] The schizophrenia testing method according to any one of [2] to [6], wherein the specimen is a blood cell collected from a subject.
[10] A schizophrenia test reagent comprising a substance exhibiting specific binding to the schizophrenia marker according to [1].
[11] The schizophrenia test reagent according to [10], wherein the substance is an antibody.
[12] A schizophrenia test kit comprising the schizophrenia test reagent according to [10] or [11].

Outline of preparation method of lymphoblastoid cell line. The table | surface which shows the background of analysis object, and the days required for stocking. Outline of proteome analysis by two-dimensional differential electrophoresis (2D-DIGE). Summary of proteome analysis results. 20 spots were selected from a total of 1174 spots. As a result of mass spectrometry, the following 22 types of proteins were identified. (1) Annexin A5 (Annexin A5, gene symbol ANXA5); (2) Microtubule-associated protein RP / EB family member 1, gene symbol MAPRE1; (3) EF- EF-hand domain-containing protein D2, gene symbol EFHD2; (4) Tubulin folding cofactor B (gene symbol TBCB); (5) Inorganic pyrophosphatase ( Inorganic pyrophosphatase, gene symbol PPA1); (6) Glutaredoxin-3 (Glutaredoxin-3, gene symbol GLRX3); (7) Plastin-2 (Plastin-2, gene symbol LCP1); (8) Heat shock protein 90β (Heat shock protein HSP 90-beta (HSP 90), gene symbol HSP90AB1); (9) Pyridoxal-dependent decarboxylase domain-containing protein 1 Gene symbol PDXDC1); (10) Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), gene symbol UCHL1); (11) Adenine phosphoribosyltransferase (APRT) (12) Cytoplasmic histidyl-tRNA synthetase (cytoplasmic, gene symbol HARS); (13) Uroporphyrinogen decarboxylase (URO-D, UPD), gene Symbol UROD); (14) Heat shock 70 kDa protein 4L (Heat shock 70 kDa protein 4L, gene symbol HSPA4L); (15) Trifunctional purine biosynthetic protein adenosine-3, gene symbol GART ) And trifunctional purine biosynthetic protein adenosine-3 short isoform (ISOFORM SHORT of Trifu) nctional purine biosynthetic protein adenosine-3, gene symbol GART); (16) beta-lactamase-like protein 2, gene symbol LACTB2; (17) putative deoxyribonuclease TATDN1, putative deoxyribonuclease TATDN1, gene symbol TATDN1 (18) Vacuolar protein sorting-associated protein 35 (VPS35); (19) Interferon-induced GTP-binding protein Mx1 (gene symbol MX1); (20) Kinesin (21) Elongation factor 1-gamma (EF-1-gamma), gene symbol EEF1G); (22) Immunoglobulin mu chain C Region (Ig mu chain C region, gene symbol IGHM), immunoglobulin mu heavy chain disease protein (gene symbol IGHM), immune glob Down heavy chain V-III region BRO (Ig heavy chain V-III region BRO, gene symbols IGHM) and immunoglobulin heavy chain V-III region GA (Ig heavy chain V-III region GA, gene symbol IGHM) 2D-DIGE results (gel image). A table summarizing the 22 proteins identified. The protein name and the corresponding gene symbol are shown. By database search, the presence or absence of expression in human brain or mouse brain was also examined (+ represents expression, − represents no expression).

(First aspect of the present invention: schizophrenia marker)
The first aspect of the present invention relates to a biomarker for schizophrenia (hereinafter also referred to as “biomarker of the present invention”). The biomarker of the present invention is a useful index for evaluating the possibility of current or future onset of schizophrenia. “Current onset possibility” represents whether or not schizophrenia has developed at the time of examination or the probability of developing it. On the other hand, the “probability of future onset” represents the possibility (risk) of developing schizophrenia in the future. “Schizophrenia” is a general term for a group of mental disorders based on the division of mental functions. According to the World Health Organization (WHO) International Statistical Classification of Diseases and Related Health Issues (ICD), it is classified into delusional, destructive, tension, indistinguishable, post-schizophrenia depression, remnant, and simple. ing.

The biomarker of the present invention comprises a protein molecule that has been correlated with schizophrenia. The relevant protein molecules are shown below. In parentheses are the gene symbol of each molecule and the corresponding sequence number. For convenience of explanation, in the following explanation, each protein molecule is represented using a gene symbol.
Annexin A5 (ANXA5, SEQ ID NO: 1);
Microtubule-associated protein RP / EB family member 1 (MAPRE1, SEQ ID NO: 2);
EF-hand domain containing protein D2 (EFHD2, SEQ ID NO: 3);
Tubulin folding cofactor B (TBCB, SEQ ID NO: 4);
Inorganic pyrophosphatase (PPA1, SEQ ID NO: 5);
Glutaredoxin-3 (GLRX3, SEQ ID NO: 6);
Plastin-2 (LCP1, SEQ ID NO: 7);
Heat shock protein 90β (HSP90AB1, SEQ ID NO: 8);
Pyridoxal-dependent decarboxylase domain-containing protein 1 (PDXDC1, SEQ ID NO: 9);
Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1, SEQ ID NO: 10);
Adenine phosphoribosyltransferase (APRT, SEQ ID NO: 11);
Cytoplasmic histidyl tRNA synthetase (HARS, SEQ ID NO: 12);
Uroporphyrinogen decarboxylase (UROD, SEQ ID NO: 13);
Heat shock 70 kDa protein 4L (HSPA4L, SEQ ID NO: 14);
Trifunctional purine biosynthetic protein adenosine-3 (GART, SEQ ID NO: 15);
Trifunctional purine biosynthetic protein adenosine-3 short isoform (GART, SEQ ID NO: 16);
β-lactamase-like protein 2 (LACTB2, SEQ ID NO: 17);
Putative deoxyribonuclease TATDN1 (TATDN1, SEQ ID NO: 18);
Vacuolar protein sorting-related protein 35 (VPS35, SEQ ID NO: 19);
Interferon-induced GTP-binding protein Mx1 (MX1, SEQ ID NO: 20);
Kinesin-like protein KIF11 (KIF11, SEQ ID NO: 21);
Elongation factor 1-γ (EEF1G, SEQ ID NO: 22);
Immunoglobulin mu chain C region (IGHM, SEQ ID NO: 23);
An immunoglobulin mu heavy chain disease protein (IGHM, SEQ ID NO: 24);
Immunoglobulin heavy chain V-III region BRO (IGHM, SEQ ID NO: 25);
Immunoglobulin heavy chain V-III region GA (IGHM, SEQ ID NO: 26)

(Second aspect of the present invention: Schizophrenia examination method)
The second aspect of the present invention relates to the use of the biomarker of the present invention, and provides a method for examining the possibility of current or future onset of schizophrenia (hereinafter also referred to as “test method of the present invention”). The test method of the present invention is useful as a means for determining whether schizophrenia is presently occurring or as a means for determining the possibility of developing schizophrenia in the future. The test method of the present invention provides useful information for diagnosing schizophrenia. According to the test method of the present invention, it is possible to easily and objectively determine the possibility of developing schizophrenia.

  By the way, the time delay between the onset of psychosis such as schizophrenia and the start of pharmacotherapy is called the duration of untreated psychosis (DUP). It is known that the longer the DUP, the worse the clinical outcome. On the other hand, several years after the onset is an important period that affects the prognosis, and is called a critical period. Our study group reported that the longer the DUP, the smaller the volume of the temporal plane that is part of the left upper temporal gyrus (Takahashi et al .: Psychiatry Res Neuroimaging 154: 209-219, 2007). This finding shows that brain morphology changes progress during the early stages of treatment (critical phase), and early intervention that not only shortens DUP but also improves neurobiological changes in the critical phase is extremely important. Suggest that there is. The test method of the present invention can also be used for screening for schizophrenia and is useful for early detection and early treatment of schizophrenia.

  In the test method of the present invention, the level of the biomarker of the present invention in a specimen derived from a subject is used as an index. Here, “level” typically means “amount” or “concentration”. However, the term “level” is also used to indicate whether or not a molecule to be detected can be detected (ie, the presence or absence of an apparent presence) in accordance with common practice and common technical knowledge. Preferably, two or more types of biomarkers selected from the above 26 types (22 types as the number of genes) are used in combination as an index to obtain a test result. Basically, the more types of biomarkers used in combination, the better the inspection accuracy and reliability. Therefore, preferably 5 or more types, more preferably 10 or more types, and even more preferably 15 or more types of biomarkers are used in combination. Note that the number and type of biomarkers to be employed may be determined in consideration of the required accuracy and the ease of inspection. For the same type of protein (that is, the above two proteins specified by the gene symbol GART and the above four proteins specified by the gene symbol IGHM), in principle, any one of them may be adopted. .

  Here, ANXA5, MAPRE1, EFHD2, TBCB, PPA1, UCHL1, APRT, VPS35, MX1 and KIF11, which showed particularly low p-values in statistical analysis using samples of patients and healthy subjects, are particularly useful Can be evaluated. Therefore, in a preferred embodiment, the level of one or more protein molecules selected from these is used as an index. Also noteworthy are UCHL1, HSPA4L, VPS35, MX1, KIF11 and IGHM, which have found significant differences in expression levels between the patient group and the healthy group. Therefore, in another aspect, the level of one or more protein molecules selected from these is used as an index. On the other hand, ANXA5, MAPRE1, EFHD2, TBCB, PPA1, GLRX3, HSP90AB1, PDXDC1, UCHL1, APRT, HARS, UROD, HSPA4L, GART, LACTB2, TATDN1, VPS35, MX1, KIF11 and EEF1G, which are also expressed in the brain, This is particularly useful because it is likely to be deeply involved in the pathogenesis of schizophrenia. Therefore, in a preferred embodiment, the level of one or more protein molecules selected from these is used as an index.

In the inspection method of the present invention, the following steps are performed.
(1) Step of preparing a specimen derived from a subject (2) Step of detecting the one or more protein molecules (that is, one or more adopted biomarkers) in the specimen (3) Detection result Determining the current or future likelihood of developing schizophrenia based on

  In step (1), a specimen derived from the subject is prepared. As the specimen, blood, plasma, serum, oral mucosa, nasal mucosa, skin, etc. of the subject can be used. Preferably, a lymphoblastoid cell obtained by immortalizing blood lymphocytes collected from a subject is used as a specimen. “Lymphocyte-like cells” are cells similar to cells before differentiation into mature lymphocytes, and can be prepared by immortalizing lymphocytes isolated from blood with Epstein-Barr virus. It can be said that the established lymphoblastoid cells show a protein expression profile more reflecting the characteristics and traits of the disease than fresh blood cells. Therefore, if lymphoblastoid cells are used as specimens, the expression profile that should be originally shown (that is, the expression profile that should be detected by the present method) is the state of the subject at the time of collection that is an element other than the disease ( It is possible to prevent the state-dependent factor) from being masked. As a result, a highly reliable test result more reflecting the characteristics and traits of the disease can be obtained. On the other hand, blood cells collected from the subject may be used as the specimen. In this embodiment, since the operation of stocking or the like is not necessary, a simpler inspection is possible. Lymphocytes, B cells or T cells separated from blood cells may be used as the specimen. In addition, you may decide to use the cell after a subculture.

  The subject is not particularly limited. That is, the possibility of the present or future development of schizophrenia (that is, the possibility of developing schizophrenia, the degree of possibility of developing schizophrenia, the possibility of developing schizophrenia in the future) The present invention can be widely applied to those who need to determine the degree of sex. For example, when the present invention is applied to a patient diagnosed with schizophrenia through a doctor's inquiry or the like, whether or not the diagnosis is appropriate can be determined based on an objective index of protein expression level. That is, according to the inspection method of the present invention, information that assists or supports the conventional diagnosis can be obtained. The information is useful for determining a more appropriate treatment policy, and promotes improvement of the treatment effect and improvement of the patient's QOL (Quality of Life). On the other hand, the present invention can be used for monitoring the diseased state to prevent intractable disease, seriousness, recurrence and the like.

  Those who are estimated to have a high risk of suffering from schizophrenia based on their family background (high-risk individuals) are also suitable subjects. Applying the present invention before symptom of schizophrenia appears in such a subject enables prevention or delay of onset or early treatment intervention. The present invention is also useful for the purpose of identifying those who have a high risk of suffering from schizophrenia. Such identification enables a reduction in the possibility of onset (possibility of morbidity) due to, for example, preventive measures or improvement of lifestyle habits. A person who cannot determine whether or not it is schizophrenia by a conventional diagnosis, such as a person who has no subjective symptoms, is also a suitable subject of the present invention. In addition, you may decide to implement this invention as one item of a health check.

  In step (2), a biomarker in the specimen is detected. It is not essential to strictly quantify the level of the biomarker. That is, the level of the biomarker may be detected to the extent that the possibility of developing schizophrenia can be determined in the subsequent step (3). For example, detection can be performed so that it can be determined whether or not the level of the biomarker in the sample exceeds a predetermined reference value.

  The method for detecting the biomarker is not particularly limited, but preferably an immunological technique is used. According to immunological techniques, rapid and sensitive detection is possible. Also, the operation is simple. In measurement by an immunological technique, a substance having specific binding property to the biomarker to be used is used. As the substance, an antibody is usually used, but any substance can be used as long as it has a specific binding property to the biomarker and can measure the binding amount. In addition, not only a commercially available antibody but the antibody newly prepared using the immunological method, the phage display method, the ribosome display method etc. may be used.

  Examples of the measurement method include latex agglutination method, fluorescence immunoassay method (FIA method), enzyme immunoassay method (EIA method), radioimmunoassay method (RIA method), and Western blot method. Preferable measurement methods include FIA method and EIA method (including ELISA method). According to these methods, it is possible to detect with high sensitivity, quick and simple. In the FIA method, a fluorescently labeled antibody is used, and an antigen-antibody complex (immune complex) is detected using fluorescence as a signal. On the other hand, in the EIA method, an enzyme-labeled antibody is used, and an immune complex is detected using color development or luminescence based on an enzyme reaction as a signal.

  The ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantitativeness, simple operation, and suitability for simultaneous processing of multiple samples. An example of a specific operation method when using the ELISA method is shown below. First, an anti-biomarker antibody is immobilized on an insoluble support. Specifically, for example, the surface of the microplate is sensitized (coated) with an anti-biomarker monoclonal antibody. The specimen is brought into contact with the antibody thus immobilized. As a result of this operation, an immune complex is formed if an antigen to the immobilized anti-biomarker antibody (protein molecule that is a biomarker) is present in the specimen. After removing non-specific binding components by washing operation, an immune complex is labeled by adding an antibody conjugated with an enzyme, and then a color is developed by reacting the substrate of the enzyme. Then, immune complexes are detected using the color development amount as an index. The details of the ELISA method are described in many books and papers, and can be referred to when setting the experimental procedure and experimental conditions of each method. In addition, you may decide to use not only a non-competitive method but the competing method (The method of making it compete with an antigen added with a test substance). Further, a method of directly detecting a biomarker in a specimen with a labeled antibody may be employed, or a sandwich method may be employed. In the sandwich method, two types of antibodies having different epitopes (capture antibody and detection antibody) are used.

  Means capable of simultaneously detecting a large number of specimens such as a protein array and a protein chip may be used. For example, a target biomarker-specific antibody is used as the probe.

  In step (3), the current or future possibility of developing schizophrenia is determined based on the detection result. In order to enable accurate determination, it is preferable to perform the determination after comparing the detection value obtained in step (2) with the detection value of the control sample (control). Determination of the possibility of onset may be qualitative or quantitative. It should be noted that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.

  In the present invention, in principle, the following criteria are adopted for each biomarker. Needless to say, the criterion “the detection probability is high when the detection value is low” is synonymous with the criterion “the detection probability is low when the detection value is high” (the same applies to other criteria).

  Biomarkers that adopt the criteria of “high probability of onset if detection value is low” or “high probability of onset if not detected”: ANXA5, MAPRE1, EFHD2, TBCB, PPA1, GLRX3, LCP1, HSP90AB1, PDXDC1, UCHL1, APRT, HARS, UROD, HSPA4L, GART (trifunctional purine biosynthetic protein adenosine-3 or trifunctional purine biosynthetic protein adenosine-3 short isoform), LACTB2, TATDN1

  Biomarkers that adopt the criteria of “High detection value means high probability of onset” or “High probability of detection detectable”: VPS35, MX1, KIF11, EEF1G, IGHM (immunoglobulin mu chain C Region, immunoglobulin mu heavy chain disease protein, immunoglobulin heavy chain V-III region BRO or immunoglobulin heavy chain V-III region GA)

  Specific examples of qualitative judgment and quantitative judgment are shown below. For convenience of explanation, biomarkers (for example, VPS35 and IGHM) whose increased expression level correlates with the onset of schizophrenia, and biomarkers (for example, UCHL1 and HSPA4L) whose decreased expression level correlates with the onset of schizophrenia. As an example, two types of biomarkers were used. In other embodiments in which the number of biomarkers used is different, the determination criteria can be set according to this example.

(Example 1 of qualitative judgment)
When the detection value of biomarker 1 (level in sample) is higher than the reference value and the detection value of biomarker 2 (level in sample) is lower than the reference value, it is determined that “the likelihood of onset is high”, and the reference When the detection value (in-specimen level) of biomarker 1 is lower than the value and the detection value (in-specimen level) of biomarker 2 is higher than the reference value, it is determined that “the possibility of onset is low”.
(Example 2 of qualitative judgment)
When biomarker 1 can be detected (reactivity is recognized) and biomarker 2 cannot be detected (reactivity is not recognized), it is determined that the “probability of onset is high”, biomarker 1 cannot be detected, and biomarker When marker 2 can be detected, it is determined that “the possibility of onset is low”.

(Example of quantitative judgment)
As shown below, the possibility of onset (%) is set in advance for each range of detection values, and the possibility of onset (%) is determined from the detection values.
Detection value of biomarker 1 is a to b, detection value of biomarker 2 is a ′ to b ′: probability of onset is 10% or less detection value b to c of biomarker 1, detection value b ′ to biomarker 2 c ′: The probability of onset is 10% to 30%
Detection values c to d of biomarker 1 and detection values c ′ to d ′ of biomarker 2: the likelihood of onset is 30% to 50%
Detection values de to e of biomarker 1 and detection values d ′ to e ′ of biomarker 2: probability of onset is 50% to 70%
Detection values e to f of biomarker 1 and detection values e ′ to f ′ of biomarker 2: the probability of onset is 70% to 90%

When two or more types of biomarkers are used in combination, the following determination method (1) or (2) may be employed.
(1) When all of the biomarkers included in the combination are positive (cutoff value or higher), it is determined as positive (for example, 50% or more likely to develop), and otherwise the case is negative (for example, 50% likely to develop) Less than).
(2) Even if one of the biomarkers included in the combination is positive (cutoff value or higher), it is determined as positive (for example, 50% or more likelihood of onset), and other cases are negative (for example, 50% of possibility of onset) Less than).

  Usually, diagnosis (detection) sensitivity and diagnosis (detection) specificity vary depending on the type and number of biomarkers to be combined. Therefore, an optimal combination of biomarkers may be selected according to the purpose. For example, combinations with high diagnostic sensitivity are suitable for screening tests. In contrast, a combination with a high diagnostic specificity is suitable for a test that requires a more reliable determination (for example, a secondary test or a tertiary test). By combining determination methods with different balances of diagnostic sensitivity and diagnostic specificity, it is possible to improve efficiency and improve accuracy or reliability. For example, after narrowing down positive subjects using a combination of biomarkers giving high diagnostic sensitivity (primary test, screening test), a final determination is made using a combination of biomarkers giving high diagnostic specificity (secondary Inspection). Not only such a two-step determination but also a three-step or higher determination can be performed.

  The number of determination categories, the level of the biomarker associated with each determination category, and the determination results can be arbitrarily set through preliminary experiments or the like without being limited to the above example. For example, the “threshold” when determining the likelihood of onset using a predetermined threshold as a boundary, and the “biomarker level range” related to the category related to the high or low possibility of onset are statistical values using a large number of specimens. It can be determined by analysis. In the case of analyzing using statistical processing, it is generally effective to set a high risk group and a low risk group. For example, the high-risk group includes a group of schizophrenic patients and families with many schizophrenic patients, and the low-risk group includes, for example, healthy groups and families without schizophrenic patients This group is applicable.

  In one embodiment of the present invention, for the same subject, the level of a biomarker measured at a certain time point is compared with the level of a biomarker measured in the past, Or examine the degree of increase or decrease. The resulting data regarding changes in the level of biomarker is useful information for monitoring changes in the likelihood of developing schizophrenia. That is, based on the change in the biomarker level, it can be determined that the possibility of onset has increased or decreased or has not changed between the previous test and the current test. If such evaluation is performed in parallel with the treatment of schizophrenia, not only can the therapeutic effect be confirmed, but also the signs of recurrence of schizophrenia can be grasped in advance. This makes it possible to determine a more appropriate treatment policy. Thus, the present invention can make a great contribution to maximizing the therapeutic effect and improving the patient's QOL (quality of life).

(Third Aspect of the Present Invention: Reagent and Kit for Examining Possibility of Developing Schizophrenia)
The present invention further provides reagents and kits for examining the possibility of developing schizophrenia. The reagent of the present invention comprises a substance (hereinafter referred to as “binding molecule”) that exhibits specific binding to the biomarker of the present invention. Examples of binding molecules include antibodies, nucleic acid aptamers and peptide aptamers that specifically recognize biomarkers. The type and origin of the binding molecule are not particularly limited as long as it has specific binding properties for the biomarker employed. In the case of an antibody, any of a polyclonal antibody, an oligoclonal antibody (a mixture of several to several tens of antibodies), and a monoclonal antibody may be used. As a polyclonal antibody or an oligoclonal antibody, an anti-serum-derived IgG fraction obtained by animal immunization, or an affinity-purified antibody using an antigen can be used. The anti-MIP1α antibody may be an antibody fragment such as Fab, Fab ′, F (ab ′) ₂ , scFv, or dsFv antibody.

  The binding molecule may be prepared by a conventional method. If a commercial item is available, the commercial item may be used. For example, antibodies can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (biomarker or a part thereof) is prepared, and an animal such as a rabbit is immunized using the antigen. An antigen can be obtained by purifying a biological sample. A recombinant antigen can also be used. A recombinant antigen can be obtained by, for example, introducing a gene encoding a biomarker (which may be a part of a gene) into a suitable host using a vector and expressing the gene in a recombinant cell obtained. Can be prepared.

  In order to enhance the immunity-inducing action, an antigen bound with a carrier protein may be used. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen in which MIP1α (or a part thereof) is expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

  Immunization is repeated as necessary, and blood is collected when the antibody titer sufficiently increases, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

  On the other hand, a monoclonal antibody can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

  Various modifications can be made to the obtained antibody on condition that the specific binding property to the biomarker is maintained. Such a modified antibody may be used as the reagent of the present invention.

  If a labeled antibody is used as a specific binding molecule, it is possible to directly detect the amount of bound antibody using the amount of label as an index. Therefore, a simpler inspection method can be constructed. On the other hand, in addition to the necessity of preparing an antibody to which a labeling substance is bound, there is a problem that detection sensitivity is generally lowered. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having a specific binding property to an antibody specific to the biomarker employed. For example, when an antibody specific for a biomarker is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as a secondary antibody. Labeled secondary antibodies that can be used against various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd., Cosmo Bio Co., Ltd., etc.) and are appropriate for the reagents of the present invention. Can be appropriately selected and used.

Examples of labeling substances include peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-D-galactosidase, enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase, fluorescein isothiocyanate (FITC), Fluorescent materials such as tetramethylrhodamine isothiocyanate (TRITC) and europium, chemiluminescent materials such as luminol, isoluminol and acridinium derivatives, coenzymes such as NAD, biotin, and radioactive materials such as ¹³¹ I and ¹²⁵ I.

  In one embodiment, the reagent of the present invention is immobilized on the basis of its use. The insoluble support used for the solid phase is not particularly limited. For example, a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass can be used. The antibody can be supported on the insoluble support by physical adsorption or chemical adsorption.

  The kit of the present invention contains the reagent of the present invention as a main component. Other reagents (buffers, blocking reagents, enzyme substrates, coloring reagents, etc.) and / or devices or instruments (containers, reaction devices, fluorescent readers, etc.) used in performing the test method may be included in the kit. Good. Moreover, it is preferable to include the protein molecule which is a biomarker, or its fragment as a standard sample in a kit. Usually, an instruction manual is attached to the kit of the present invention.

  This study aimed to identify schizophrenia-related molecules. Regarding schizophrenia, proteome analysis using the postmortem brain has been performed in the past, but there is a problem that the possibility of being affected by postmortem changes and therapeutic drugs cannot be denied. In this study, we decided to establish a lymphoblastoid cell line from the peripheral blood of schizophrenic patients and to comprehensively analyze protein expression changes by two-dimensional electrophoresis. By using lymphoblastoid cell lines, it is considered possible to minimize the influence of state-dependent factors such as therapeutic drugs, concurrent physical diseases, and circadian variation at the time of blood collection.

1. Establishment of lymphoblastoid cell line (Fig. 1)
The background of the schizophrenia patient (schizophrenia; SCZ) and the healthy subject (control; CON) to be analyzed is shown in FIG. Ficoll-Paque PLUS (GE Healthcare, Piscataway, NJ, USA) diluted with 5 mL of physiological saline (saline; SAL; Otsuka Pharmaceutical Co., Ltd., Tokyo) to 5 mL of peripheral blood of schizophrenic patients and healthy individuals Overlaid in a 15 mL tube (15 mL Conical Tube; BD Falcon, San Jose, CA, USA) to which 3.5 mL of the solution was added, 670 × g (1900 rpm; KN-70; Kubota Corporation, Tokyo) at room temperature, Centrifuge for 30 minutes. After centrifugation, after removing the upper layer, the intermediate layer containing lymphocytes was collected, suspended in 10 mL of physiological saline (Otsuka Pharmaceutical Co., Ltd.), 418 × g (1500 rpm; KN-70), 15 at room temperature. Centrifuge for minutes. The supernatant was removed, 5 mL of liquid basal medium was suspended in the white sediment, suspended, and centrifuged at 418 × g (1500 rpm; KN-70) at room temperature for 15 minutes. The supernatant was removed, and the resulting lymphocyte cells were suspended in 5 mL of a blasting medium and cultured at 37 ° C. under 5% CO ₂ .

  The composition of the liquid basal medium was as follows: RPMI1640 medium (sodium bicarbonate, L-glutamine (+); Invitrogen, Carlsbad, CA, USA) with penicillin-streptomycin solution (10,000 U / 10,000 μg / mL, GIBCO ) And tyrosine tartrate (Sigma-Aldrich, St. Louis, MO, USA) were added to give penicillin 50 μg / ml, streptomycin 50 μg / ml, and tyrosine 50 mg / mL, respectively. For subculture, 20% FBS medium and 10% FBS medium prepared by adding fetal bovine serum (FBS; Invitrogen, Lot # 1299344) to 20% and 10% in liquid basal medium are prepared. And used. The blasting medium is 4 mL of 20% FBS medium, 1 mL of Epstein-Barr virus (EBV) filtrate and cyclosporin A (CyA; Sandymun injection); Novartis Pharmaceuticals, New York , NY, USA) was added to 2 μg / mL.

  For subculture, the medium was changed twice a week. The first two weeks were cultured in 20% FBS medium, and then cultured in 10% FBS medium. The cells were confirmed to form colonies (after about 4 weeks) and frozen.

  For freezing, the culture solution was suspended and collected in a 50 mL tube (50 mL Conical Tube; BD Falcon), and centrifuged at room temperature at 225 × g (1100 rpm; KN-70) for 5 minutes. Remove the supernatant, add 10 mL of Cell Banker 1 (Toji Field, Tokyo), suspend, and then add 1 mL of cell suspension to each 2 mL cryogenic vial (gamma-ray sterilized, inner cap; BD Falcon). After dispensing, it was stored frozen in liquid nitrogen.

2. Protein extraction and protein purification from lymphoblastoid cell lines Lymphoblastoid cell lines from gender and age-matched schizophrenia patients and healthy individuals cryopreserved in liquid nitrogen at room temperature The mixture was centrifuged at 225 × g (1100 rpm; KN-70) for 5 minutes. The supernatant was removed, 10 mL of 20% FBS medium was added and suspended, and the mixture was centrifuged at room temperature at 225 × g (1100 rpm; KN-70) for 5 minutes. This operation was repeated once again to remove the stock solution (Cell Banker 1), and then culture was started with 20% FBS medium. For subculture, the medium was changed twice a week and cultured in 20% FBS medium for the first 7 days, and then cultured for 14 days in 10% FBS medium. During the passage, the culture conditions between the samples were standardized by adjusting the number of cells so that the culture date and time and the cell concentration were 3 × 10 ⁵ cells / mL. In this way, proteins were extracted from cells that had been subcultured for 21 days under the same culture conditions. After centrifuging 30 mL of the cell suspension at room temperature at 190 × g (1000 rpm; KN-70; Kubota Corporation, Tokyo) for 5 minutes, 10 mL of phosphate buffer saline (PBS) Wash 3 times [room temperature, 190 × g (1000 rpm; KN-70), centrifuge for 5 minutes], remove the supernatant, add 1 mL of PBS to the white sediment, and 1372 × g (3800 rpm at 4 ° C). himac CT13R; Hitachi Koki Co., Ltd., Tokyo), centrifuged for 5 minutes, and added reagents to disrupt the cells. Incubate for 30 minutes under ice-cooling, centrifuge at 16060 xg (13000 rpm) at 4 ° C for 20 minutes, collect the supernatant, and purify the protein using methanol (Kanto Chemical, Tokyo) and acetone (Kanto Chemical). (Methanol-acetone precipitation method) and stored at −80 ° C. until used for analysis.

3. Comparative analysis of protein expression by 2D-DIGE method (Figure 3)
The purified protein was directly compared for expression change by 2D-DIGE method. Internal standard prepared as an equal mixture of all samples to be analyzed by fluorescently labeling proteins from schizophrenia patients and healthy controls samples with Cy3 or Cy5 (CyDye DIGE Fluor minimal dyes; GE Healthcare, Piscataway, NJ, USA) (internal standard; IS) was fluorescently labeled with Cy2 (CyDye DIGE Fluor minimal dyes; GE Healthcare). After mixing 30 μg of the protein labeled with Cy3, Cy5 and Cy2 and applying it to a dry strip gel (24 cm Immobiline DryStrip pH 3-10 NL; GE Healthcare), the isoelectric point is the first-dimensional electrophoresis. Electrophoresis (isoelectric focusing; IEF) was performed. Ettan IPGphor 3 IEF system (Ettan IPGphor 3 Isoelectric Focusing Unit; GE Healthcare) was used for IEF. IEF was performed at 20 ° C., and the electrophoresis conditions were 12 hours after re-swelling of the dry strip gel, 30 V-2 hours, 100 V-1 hours, 200 V-5 minutes, then 8 hours 30 The voltage was raised to 8000 V by applying a minute gradient, and 8000 V-7 hours and 30 minutes were performed. In the second dimension, SDS-PAGE (sodium dodecyl sulfate-polyaclylamide gel electophoresis) was performed using Ettan DALTsix (GE Healthcare). Use 10% polyacrylamide gel (24 x 20 x 0.1 cm), run at 30 mA at 10 mA / gel, 80 V, 1 W / gel for 1 hour, 12 mA / gel, 150 V, 2 W / gel Electrophoresis was performed until the tip of the gel reached the end of the gel (15-17 hours). Using a fluorescent image analyzer (Typhoon Trio; GE Healthcare), the electrophoretic images of the respective proteins labeled with Cy3, Cy5 and Cy2 were visualized. The detection of protein spots in the gel and the analysis of the difference between the gels were carried out by comparison of expression levels using two-dimensional electrophoresis analysis software (PDQuest Advanced Version 8.0; Bio-Rad, Hercules, CA, USA). Considering the presence of specific protein expression in patients with schizophrenia, all protein spots present on individual electrophoretic images were analyzed. By calculating the expression level of each protein spot based on the internal standard gel, the difference in expression intensity between the gels was corrected, and the expression level of the protein spot between the schizophrenia patient group and the healthy subject group was compared.

  Among 1174 spots, 20 spots showed a significant difference in expression level (p <0.05) (FIGS. 4 and 5). We identified these protein spots with different expression by Peptide mass fingerprinting (PMF) method.

4). Protein identification by PMF method 2. Apply an equivalent amount of 100 μg of the internal standard sample prepared in step 2 to a dry strip gel (24 cm Immobiline DryStrip pH 3-10 NL; GE Healthcare), perform two-dimensional electrophoresis in the same way, and perform silver staining. To visualize the protein spots. After electrophoresis, the gel was immersed in a fixative [30% methanol (Kanto Chemical) / 10% acetic acid (Kanto Chemical) solution] overnight. After washing with 20% ethanol (Kanto Chemical) solution for 40 minutes, it was washed with sterilized purified water (MilliQ) three times for 5 minutes each. Immerse in a sensitizer [0.02% sodium thiosulfate (Sigma-Aldrich, St. Louis, MO, USA) solution] for 2 minutes, wash with sterilized purified water (MilliQ) three times for 30 minutes, and then add a staining solution [0.2% AgNO ₃ (Sigma-Aldrich) solution] for 25 minutes. After washing with sterilized purified water (MilliQ) three times for 1 minute each, the coloring solution [3% Na ₂ CO ₃ (Sigma-Aldrich) / 0.001% sodium thiosulfate (Sigma-Aldrich) / 0.05 v / v% formalin (Wako , Osaka) solution] for about 2 minutes and in stop solution [1.4% Na ₂ -EDTA (Sigma-Aldrich) solution] for 10 minutes. Candidate protein spots were cut out one by one and desilvered with 0.02% sodium thiosulfate / 0.2% potassium ferricyanide solution. After dehydration with acetonitrile, it was dried using Speed Vac (VC-36R; TAITEC, Saitama), 10 mM DTT solution [100 mM dithiothreitol (DTT, Wako): 100 mM NH ₄ HCO ₃ = 1: 9 solution] and 100 mM Cysteine residues were alkylated with iodoacetoamide solution [iodoacetoamide (Wako) 18 mg added to 1 mL of 100 mM NH ₄ HCO ₃ ]. After dehydration with acetonitrile, it was dried using Speed Vac (VC-36R), and trypsin digestion was performed overnight using trypsin (Trypsin Gold, Mass Spectrometry Grade; Promega, WI, USA) diluted 50 times. . Peptide extraction with 50% acetonitrile containing 0.1% Trifluoroacetic acid (TFA), mass spectrometer [HPLC: MAGIC2002 (Michrom Bioresources, Inc., CA, USA), high-throughput LC / MS autosampler: HTC-PAL ( CTC Analytics AG, Switzerland), Ion trap MS: LCQ Advantage (Thermo Fisher Scientific Ltd., MA, USA)]. Based on the obtained MS / MS data, the protein was identified by collating it with the database of Swiss-Prot and TrEMBL using protein identification software (MASCOT; Matrix Science, MA, USA) (FIG. 5). The presence or absence of expression in the brain was also confirmed (FIG. 5).

  The identification result is shown in FIG. From 20 spots, 22 types of proteins (a single group of proteins encoded by the same gene were grouped together) were identified. In the schizophrenic patient group, 5 proteins were found to be up-regulated (the most up-regulated were about 1.8 times the expression level), conversely, 17 proteins were down-regulated (the most down-expressed were about 0.5 times) Expression level). The p-values of ANXA5, MAPRE1, EFHD2, TBCB, PPA1, UCHL1, APRT, VPS35, MX1 and KIF11 are particularly low, suggesting a strong correlation with schizophrenia. In addition, UCHL1, HSPA4L, VPS35, MX1, KIF11 and IGHM are remarkably different in expression levels between the patient group and the healthy group, suggesting deep involvement in the pathogenesis of schizophrenia.

  As described above, we succeeded in identifying proteins (22 genes) that show schizophrenia-specific expression changes. These proteins are expected to be involved in the pathogenesis of schizophrenia, and can be said to be useful as an index of the onset possibility of schizophrenia. Most of the identified proteins (except LCP1 and IGHM) are also expressed in the brain (FIG. 6). Proteins that are recognized in the brain in this way are likely to be deeply involved in the pathogenesis of schizophrenia and are considered to be particularly useful.

  The test method of the present invention makes it possible to easily and objectively determine the possibility of developing schizophrenia. The test method of the present invention is useful as a means for determining whether or not schizophrenia has developed. It is also useful as a means for grasping the possibility of developing in the future. Early detection and early treatment using the test method of the present invention is expected to prevent schizophrenia from becoming intractable, serious (progression of disease state), and recurrence.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (11)

Annexin A 5 or Ranaru, schizophrenia marker. Characterized by using the analyte levels about the annexin A 5 as an index, a schizophrenia tests, the following steps (1) to (3), i.e.,
(1) a step of preparing a subject-derived specimen;
(2) detecting annexin A5 in the specimen; and
(3) determining a current onset possibility of schizophrenia based on the detection result;
A method for testing schizophrenia, wherein the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value of the control sample . Criteria to be high possibility of onset detection detection value is low, or in accordance with the standards of the high possibility of onset can not be detected, it is determined step (3) The method of claim 2. In the step (2), in addition to annexin A5, microtubule-related protein RP / EB family member 1, EF-hand domain-containing protein D2, tubulin folding cofactor B, inorganic pyrophosphatase, glutaredoxin-3, Plastin-2, heat shock protein 90β, pyridoxal-dependent decarboxylase domain-containing protein 1, ubiquitin / carboxyl terminal hydrolase isozyme L1, adenine / phosphoribosyltransferase, cytosolic histidyl tRNA synthetase, uroporphyrinogen decarboxylase , Heat shock 70kDa protein 4L, trifunctional purine biosynthetic protein adenosine-3, trifunctional purine biosynthetic protein adenosine-3 short isoform, beta-lactamase-like protein 2, putative deoxyribonuclease ZETADN1, vacuolar protein sorting-related protein 35, interferon-induced GTP-binding protein Mx1, kinesin-like protein KIF11, elongation factor 1-γ, immunoglobulin mu chain C region, immunoglobulin mu heavy chain disease protein, immunoglobulin heavy chain V- Detecting one or more protein molecules selected from the group consisting of III region BRO and immunoglobulin heavy chain V-III region GA;
In the step (3), the current onset possibility of schizophrenia is determined based on the detection result of annexin A5 and the detection result of the protein molecule.
The schizophrenia test method according to claim 2. Microtubule-related protein RP / EB family member 1, EF-hand domain-containing protein D2, tubulin folding cofactor B, inorganic pyrophosphatase, glutaredoxin-3, plastin-2, heat shock protein 90β, pyridoxal-dependent Decarboxylase domain-containing protein 1, ubiquitin / carboxyl terminal hydrolase isozyme L1, adenine / phosphoribosyltransferase, cytoplasmic histidyl tRNA synthetase, uroporphyrinogen decarboxylase, heat shock 70kDa protein 4L, trifunctional purine Synthetic protein adenosine-3, trifunctional purine biosynthetic protein adenosine-3 short isoform, β-lactamase-like protein 2, and putative deoxyribonuclease TATDN1 may develop at low detection values According to the criteria of high or the probability of onset if not detected,
Vacuolar protein sorting-related protein 35, interferon-derived GTP-binding protein Mx1, kinesin-like protein KIF11, elongation factor 1-γ, immunoglobulin mu chain C region, immunoglobulin mu heavy chain disease protein, immunoglobulin heavy chain V-III region BRO For the immunoglobulin heavy chain V-III region GA, the determination in step (3) is performed according to the criterion that the detection probability is high when the detection value is high, or the criterion that the detection probability is high when the detection value is high. The method according to claim 4. The schizophrenia test method according to any one of claims 2 to 5 , wherein the specimen is blood, plasma or serum. The schizophrenia test method according to any one of claims 2 to 5 , wherein the specimen is a lymphoblastoid cell obtained by immortalizing blood lymphocytes collected from a subject. The schizophrenia testing method according to any one of claims 2 to 5 , wherein the specimen is a blood cell collected from a subject.   A schizophrenia test reagent comprising a substance exhibiting specific binding to the schizophrenia marker according to claim 1. The schizophrenia test reagent according to claim 9 , wherein the substance is an antibody. The kit for a schizophrenia test containing the schizophrenia test reagent of Claim 9 or 10 .

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