WO2022075243A1

WO2022075243A1 - Schizophrenia biomarker

Info

Publication number: WO2022075243A1
Application number: PCT/JP2021/036560
Authority: WO
Inventors: 良郎五嶋; 宗孝野本
Original assignee: 公立大学法人横浜市立大学
Priority date: 2020-10-06
Filing date: 2021-10-04
Publication date: 2022-04-14

Abstract

Provided is a schizophrenia biomarker. A schizophrenia examination method including measuring the expression level of non-phosphorylated collapsin response mediator protein 2 (CRMP2) and/or phosphorylated CRMP2 in a sample derived from a subject.

Description

Schizophrenia biomarker

The present invention relates to a schizophrenia biomarker.

Schizophrenia (SCZ) is a psychiatric disorder characterized by impaired cognition, perception, emotions, and behavior. Diagnosis of schizophrenia is made by symptom-based scoring, and the development of quantitative biomarkers is awaited. Under such circumstances, Bader et al. Reported that a change was observed in one of the CRMP family molecules called CRMP1 (Non-Patent Document 1: Hum Mol Genet, 21, 4406-4418, 2012). However, the method of Bader et al. Had to adjust the lymphocytes, which was complicated.

An object of the present invention is to provide a biomarker for schizophrenia that can be detected by a simple method.

The present inventors may find that an increase in the expression level of non-phosphate Collapsin Response Mediator Protein 2 (CRMP2) in a blood sample and / or a decrease in the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio may be useful for diagnosing schizophrenia. It was found that there is, and the present invention was completed.

The gist of the present invention is as follows.
(1) A method for testing schizophrenia, which comprises measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from a subject.
(2) The test method according to (1), wherein an increase in the expression level of non-phosphorylated CRMP2 and / or a decrease in the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio indicates that the possibility or risk of developing schizophrenia is high.
(3) The inspection method according to (1) or (2), wherein the phosphorylated CRMP2 is CRMP2 phosphorylated with Ser522.
(4) The test method according to any one of (1) to (3), wherein the sample derived from the subject is a blood sample.
(5) The test method according to (4), wherein the blood sample is a peripheral blood mononuclear cell fraction.
(6) The method according to any one of (1) to (5), wherein the subject is under 40 years old.
(7) The method according to any one of (1) to (6), wherein the subject is under 30 years old.
(8) When the subject is under 30 years old, the possibility or risk of developing schizophrenia is that the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the subject's peripheral blood mononuclear cell fraction is 0.25 or less. The method according to any one of (1) to (7), which indicates that the value is high.
(9) A method for diagnosing schizophrenia in a subject.
Obtaining a sample from the subject,
b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject, and
c. The method comprising determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio.
(10) A method for diagnosing and treating schizophrenia in a subject.
Obtaining a sample from the subject,
b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject.
c. Determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio, and
d. The method described above comprising initiating treatment of the subject with schizophrenia if the likelihood or risk of developing schizophrenia is determined to be high.
(11) The expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the expression level of non-phosphorylated CRMP2 in the sample derived from a healthy person who has not developed schizophrenia, and / or the sample derived from the subject. Phosphorylated CRMP2: Non-phosphorylated CRMP2 ratio in a sample derived from a healthy person who has not developed schizophrenia is lower than the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the possibility or risk of developing schizophrenia. The method according to (9) or (10), wherein is determined to be high.
(12) Integration when the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the set value and / or the ratio of phosphorylated CRMP2: non-phosphorylated CRMP2 in the sample derived from the subject is lower than the set value. The method according to any one of (9) to (11), wherein it is determined that the possibility of developing schizophrenia or the risk of developing schizophrenia is high.
(13) Integration including a reagent capable of measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or a reagent capable of measuring the expression level of phosphorylated CRMP2 in a sample derived from a subject. A kit for testing for ataxia.

The present invention enables quantitative diagnosis of schizophrenia.

Increased CRMP2 and / or decreased pCRMP2: CRMP2 ratio may be useful in diagnosing schizophrenia.
This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2020-169396 and / or the drawings which are the basis of the priority of the present application.

The pCRMP2 to CRMP2 ratio of PBMC fractions in SCZ patients was significantly lower than that in unaffected control subjects. CRMP2 (pCRMP2) phosphorylated with serine 522 was examined by quantitative Western blot using certain well-certified antibodies. [A] Differences between cohorts under the age of 30 (* p <0.01) were the most prominent. [B] CRMP2 [left graph] and pCRMP2 [center graph] (using arbitrary values normalized to β-actin as an internal standard) were examined (aggregate) for non-affected subjects (aggregate). The pCRMP2: CRMP2 ratio [graph on the right] in the human PBMC fraction from patients with SCZ (S) compared to C) was significantly lower (p = 0.0051)). This is because the level of CRMP2 (denominator) is high (p = 0.0146), while pCRMP2 remains unchanged (p = 0.4373). Authentication of antibody sensitivity and specificity to CRMP2 and phosphorylated CRMP2 based on immunoblot analysis. [A] Demonstrated by immunoblot analysis of brain lysates from wild-type (wt), crmp1 ^-/- , crmp2 ^-/- , crmp1 ^-/- ; crmp2 ^--/- , and CRMP2S522A knock-in (crmp2 ^{ki / ki} ) mice Specificity of anti-CRMP2 monoclonal antibody (9F) [top panel] and anti-phosphorylated CRMP1 / 2 (S522) (pCRMP1 / 2) polyclonal antibody [bottom panel]. In the upper panel, a single band of 64 kDa was detected using 9F in brain lysates from wt, crmp1 ^-/- and crmp2 ^{ki / ki} mice, but not in crmp2 ^-/- mice. In the lower panel, a single band was detected with anti-pCRMP1 / 2 antibodies in the brain lysates from wt and crmp1 ^-/- , but in crmp1 ^-/- ; crmp2 ^-/- and crmp2 ^{ki / ki} mice. There wasn't. [B] A typical immunoblotting method using an anti-CRMP2 antibody against a human peripheral blood mononuclear cell (PBMC) fraction of a normal healthy control subject [left panel] was compared with a positive control brain lysate. In the right panel, it can be seen that the band is successfully blocked by the antigenic peptide of CRMP2. (See also Figure 3 for further validation of the antibodies used in this study). Further validation of the antibodies used in the immunoblotting in this study and evidence that the CRMP1 isoform is not associated with the phenomenon described in this study. The difference seen in SCZ and non-affected patients was also demonstrated by the use of antibodies against CRMP1 to be more important in the difference in the abundance of CRMP2 isoforms than in CRMP1 isoforms. In addition, none of the differences described in this study were related to CRMP1. This figure shows wild (WT) and crmp1 ^-/- ⁽ CRMP1 KO) or crmp2- (CRMP2 KO) mouse brain lysates, as well as human peripheral blood mononuclear cell (PBMC) fractions (specifically lymphocytes). ) Is shown in an immunoblot analysis using two different anti-CRMP1 antibodies (2E7G and 2C6G). Single bands were detected by 2E7G and 2C6G in WT mouse brain lysates, but not in CRMP1 knockout mouse brain lysates, indicating the specificity of these anti-CRMP1 antibodies. In this analysis, CRMP1 was not detected in human lymphocyte samples (a, b) using these two sensitive CRMP1 antibodies.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for testing schizophrenia, which comprises measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from a subject.

Schizophrenia is a mental illness of unknown cause that often occurs in adolescence. Although genetic predisposition to develop the disease is recognized, the onset is considered to be due to the interaction between environmental factors and genetic factors as well as genetic factors. Symptoms are characterized by peculiar thought disorders, inappropriate or dull feelings and motivation, autistic or bizarre lifestyles, and social and occupational dysfunction. Anomalous experiences such as hallucinations and delusions, ego disorders such as acts and affected experiences, and lack of insight are shown in the form of psychotic episodes. Although there are cognitive impairments such as proper selection, judgment, and execution of information, consciousness is clear and general intelligence is maintained. There are no clear physical findings. The course indicates either acute or chronic course, but most have a chronic course. It is the most frequent and important mental illness. The diagnosis of schizophrenia is made by the American Psychiatric Association, which describes in detail the characteristic symptoms and course of schizophrenia from a non-theoretical and descriptive standpoint. It is carried out based on the ICD-10 schizophrenia guidelines compiled by WHO. By accumulating recent genetic analysis of schizophrenia, brain image analysis, and morphological analysis of postmortem autopsy brain (Johnstone et al, Lancet 308, 924, 1976; Selemon et al, Biol Psychiatry 45, 17, 1999; Ripke et al, Nat Genet 45, 1150, 2013), neurodevelopmental disorders, and neural circuit abnormalities are becoming clear in the background (Murray & Lewis, Br Med J 295, 681, 1987; Weinberger, Arch Gen Psychiatry. 44, 660, 1987). The neurodevelopmental disorder hypothesis, which seeks the cause of such abnormalities in neurodevelopmental development, has been observed since the 1980s, when the enlargement of the lateral ventricle in schizophrenia correlates with premorbid adaptation and is also observed in the first episode of schizophrenia-like symptoms. (Weinberger et al, Arch Gen Psychiatry. 1982 Jul; 39 (7): 778-83.) As brain maturation and neural circuit formation processes become clear (Tessier-Lavigne M, Goodman CS. Science. 1996 Nov 15; 274 (5290): 1123-33.), Has evolved. Genetic studies have shown that neuregulin 1, DISC 1, and dysbindin, which are potential risk genes for schizophrenia, are more highly expressed in the fetal brain than in adults and are involved in the formation of synapses and neural circuits (Harrison). PJ, Weinberger DR. Mol Psychiatry. 2005 Jan; 10 (1): 40-68; image 5.). Glantz and Lewis (2000) also reported that Golgi's staining reduced the density of dendrite spines in pyramidal neurons in the third layer of the dorsolateral prefrontal cortex (Glantz LA, Lewis DA. Arch). Gen Psychiatry. 2000 Jan; 57 (1): 65-73.). It is known that even in the process of normal brain development, excess synapses are formed in nerve cells until the age of 1 year after birth, and then pruning is performed, and the thickness of the cerebral cortex becomes slightly thinner from puberty to adolescence. There is. Since the distribution of the site of decrease in cerebral cortex volume in schizophrenia shows almost the same pattern as normal (Sun et al, Schizophr Res. 2009 Mar; 108 (1-3): 85-92.), In schizophrenia. It is presumed that the synaptic pruning becomes excessive and the above-mentioned changes occur.

Collapsin Response Mediator Protein 2 (CRMP2) (Protein, Dihydropyrimidinase-related protein 2; Gene, DPYSL2) was first identified as a molecule that transmits the intracellular signal of axonal repulsive factor semaphorin 3A (Sema3A). (Goshima et al, Nature. 1995 Aug 10; 376 (6540): 509-14.). The amino acid sequence of CRMP2 (DPYSL2) is Q16555 (https://www.uniprot.org/uniprot/Q16555) in UniProtKnowledgebase, and the base sequence and genetic information are in the NCBI database GeneID: 1808 (https: //). It is registered as www.ncbi.nlm.nih.gov/gene/1808) and can be known. CRMPs are cytoplasmic proteins, and five subtypes (CRMP1-5) have been identified so far. All of these show high levels of expression during the developmental stage of the brain, but each shows a specific expression distribution and time of expression. Of these, CRMP2 shows high expression levels in the sensory system, hippocampus, cerebral cortex, cerebellum, etc. even in the adult brain. CRMPs are homologous molecules of C. elegans Unc-33, and mutations in C. unc-33 cause axonal elongation and abnormal guidance in C. elegans neurons. CRMPs are phosphorylated downstream of Sema3A, where cyclin dependent kinase 5 (Cdk5) phosphorylates the 522nd Serin residue, thereby continuing to glycogen synthase kinase (GSK3b) Ser517, Thr514, Thr509 residues. It is a protein that undergoes two-step phosphorylation modification, and its regulation of phosphorylation plays an important role in the development and maturation of nerves. In addition to this phosphorylation modification, CRMP2 undergoes various post-translational modifications such as Rho / ROCK kinase (ROCK1), Fyn (FYN), Fes (FES) tyrosine kinase, and SUMOylation. Through phosphorylation modification, CRMP2 changes its interaction with molecules such as tuberin and calcium channels, and through this, it plays a role as a mediator of neuronutrient factors and other external signals, including Sema3A, as well as polarity and axon formation. It has been reported to be involved in various nerve functions and pathological conditions such as nerve cell migration, synaptogenesis, synaptic plasticity, and nerve disease (Nakamura et al, Front Cell Neurosci. 2020 Jun 23; 14: 188.). CRMP2 is phosphorylated by Cdk5 (cyclin-dependent kinase 5, CDK5) and GSK3b (GSK3b, glycogensynthase3b), which reduces the interaction with tuberin and suppresses protrusion elongation.

CRMP2 is highly expressed mainly in the central nervous system, especially in the cerebral spinal cord tissue during development. Among the CRMP family molecules, the expression level of CRMP2 is maintained even in the relatively mature stage, and its role in adults such as learning, memory, and cognitive function is presumed. The expression of CRMP2 is also expressed in cardiovascular system, immune system, and digestive system tissues, but its role is often unknown. CRMP2 undergoes various post-translational modifications, including phosphorylation, as described above. An important modification is phosphorylation modification by Cdk5, GSK3b, Rho / ROCK kinase at the C-terminus of CRMP2. As an example, Cdk5 phosphorylates CRMP2 with Ser522, which promotes phosphorylation modification of CRMP2 by GSK3β in Thr514 and Ser518. This two-step phosphorylation modification mode is called prime phosphorylation (Nakamura et al, Front Cell Neurosci. 2020 Jun 23; 14: 188.). In this case, phosphorylated CRMP2 has reduced interaction with tubulin and reduced cytoskeletal system function. On the other hand, when dephosphorylated and becomes non-phosphorylated, CRMP2 interacts with tubulin better and the formation of a cytoskeletal system occurs. From the viewpoint of promoting the formation of the cytoskeleton, the phosphorylated CRMP2 is sometimes referred to as an inactive form, and the dephosphorylated form is sometimes referred to as an active form. Other known physiological significance of CRMP2 post-translational modification is the interaction of CRMP2 with sodium and calcium channels and their activity regulation (Chew and Khana, Neuronal Signal. 2018; 2 (1): NS20170220.).

In the present invention, the phosphorylated CRMP2 is preferably CRMP2 phosphorylated by Ser522.

In the present invention, the subject is a mammal suspected of developing schizophrenia, but all mammals at risk of developing schizophrenia may be targeted. It is typically human. Humans are preferably under the age of 40, more preferably under the age of 30. Samples derived from the subject include cells, tissues, body fluids, etc. obtained from the subject, specifically, the subject's blood (for example, whole blood, serum, plasma, plasma exchange external fluid, etc.), brain tissue, saliva, tears, etc. Etc. can be exemplified. A blood sample such as whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample. The blood sample may be a peripheral blood mononuclear cell fraction.

In the method of the present invention, the expression of non-phosphorylated CRMP2 and / or phosphorylated CRMP2 in a sample derived from a subject may be measured by measuring the abundance of the above protein or a fragment thereof in the sample. The measuring means is not particularly limited, and a known method may be used. It is preferably measured at the protein level, but may be measured at the nucleic acid level.

In order to measure the expression of the protein at the protein level, it is advisable to use an antibody that specifically recognizes the protein. The antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by known methods, or commercially available ones may be used. In the examples described below, an anti-phosphorylated CRMP1 / 2 (S522) (p-S522-CRMP1 / 2) polyclonal antibody that recognizes CRMP2 phosphorylated by Ser522 was used. Typical methods include immunoassays such as an ELISA method and an immunochromatography method. Since the immunoassay method does not require a special device or technique and can easily and quickly detect and quantify the target protein, it can be preferably used for the measurement of the above protein in the present invention as well. Antibodies to the above proteins are known, and commercially available products also exist. Further, as described above, since the amino acid sequence of the above protein and the base sequence encoding these are also known, a specific antibody against each protein may be prepared by preparing a general hybridoma.

The immunoassay itself is well known in this field. Depending on the reaction format, there are a sandwich method, a competitive method, an agglutination method, a Western blotting method and the like, and based on the label, there are enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay and the like. In the present invention, any immunoassay method capable of quantitative detection may be used. Although not particularly limited, for example, a sandwich method such as sandwich ELISA can be preferably used. In the sandwich method, the antibody that binds to the target protein is immobilized and reacted with the sample. The target protein bound to the immobilized antibody is measured using a detection antibody labeled with an enzyme or the like. As the detection antibody, it is preferable to use an antibody that binds to the target protein at a site different from that of the immobilized antibody. The immobilized antibody and the detection antibody may be a polyclonal antibody or a monoclonal antibody, and an antigen-binding fragment of the antibody may be used. The target protein bound to the immobilized antibody is reacted with the detection antibody, washed, and then the amount of the bound detection antibody is measured by a signal from the substance labeled on the antibody. For example, when an antibody labeled with alkaline phosphatase is used as a detection antibody, the substrate of the enzyme may be added into the reaction system, and the amount of color development, fluorescence, and luminescence generated by the enzyme reaction may be measured by a corresponding device. Perform immunomeasurement on a standard sample containing a target protein with a known concentration, prepare a calibration curve plotting the relationship between the signal of the labeling substance and the concentration, and perform the same operation on a sample with an unknown target protein concentration. By applying the obtained signal measurement value to the calibration curve, the target protein in the sample can be quantified.

In order to measure the expression of the above protein at the nucleic acid level, it is preferable to use a nucleic acid probe that can specifically hybridize with the mRNA of the above protein (when measuring by Northern blotting). Alternatively, at least one pair of nucleic acid primers capable of specifically amplifying the cDNA synthesized using the mRNA of the above protein as a template may be used (when measured by the RT-PCR method). Nucleic acid probes and nucleic acid primers can be designed based on the genetic information (described above) of the above proteins. A nucleic acid probe of about 15 to 1500 bases is usually suitable. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme or the like. As the nucleic acid primer, one having about 15 to 30 bases is usually suitable. Nucleic acid primers may be labeled with radioactive elements, fluorescent dyes, enzymes and the like.

If an increase in the expression level of non-phosphorylated CRMP2 and / or a decrease in the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio is confirmed, it is highly possible that the patient has schizophrenia or develops schizophrenia. It can be determined that the possibility (risk of onset) is high. Therefore, the present invention can assist in the diagnosis of schizophrenia. INDUSTRIAL APPLICABILITY The present invention can be used for evaluation of the possibility or risk of developing schizophrenia, differentiation of schizophrenia (distinguishing from other diseases), and the like.

As an example of the present invention, schizophrenia can be diagnosed according to the following criteria. When the expression level of non-phosphorylated CRMP2 in a blood sample (for example, plasma, serum) collected from a subject is measured, and a higher value is obtained in a blood sample collected from an unaffected person (for example, a healthy person). , Subjects are evaluated to have developed schizophrenia or have a high probability of developing schizophrenia (risk of developing schizophrenia). Alternatively, the expression levels of non-phosphorylated CRMP2 and phosphorylated CRMP2 in blood samples (eg, plasma, serum) collected from the subject were measured, and the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio was calculated, and this ratio was determined from the set value. If it is also low, the onset of schizophrenia can be suspected. This preset value can be appropriately set by those skilled in the art. For example, a 95% confidence interval of the quantitative value of a healthy person who has not developed schizophrenia can be used as a reference value, or a cutoff value can be set from the ROC curve. As one embodiment, when the age is less than 30 years old in FIG. 1A and the reference value for the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio is set to 0.25, the ratio in the measured value is equal to or less than the reference value. Can be determined to be more likely to have schizophrenia.

Furthermore, for non-phosphorylated CRMP2, expression in samples derived from subjects determined to be highly likely to have schizophrenia was measured once or multiple times at different times, and the expression level was close to the cutoff value or the reference value. If the level drops to a level, it is determined that the patient has recovered from schizophrenia by treatment, and if the level is high or does not decrease, it is determined that the patient has not recovered from schizophrenia by treatment or the recovery is insufficient. can do. In addition, the expression of non-phosphorylated CRMP2 and phosphorylated CRMP2 in samples derived from subjects judged to have a high possibility of schizophrenia was measured once or multiple times at different times, and phosphorylated CRMP2: non-phosphorylated. When the CRMP2 ratio rises to a level close to the cutoff value or the reference value, it is judged that the patient has recovered from schizophrenia by treatment, and when the level is low or does not rise, the patient has recovered from schizophrenia by treatment. It can be determined that there is no or insufficient recovery. The method of the present invention can be used not only for diagnosing schizophrenia, but also for prognostic examination and confirmation of therapeutic effect.

If it is determined that the subject is likely to have schizophrenia, drug treatment will be the basis, and lifestyle guidance, psychotherapy, and community care will be added to promote social recovery. The purpose of treatment is to reduce or suppress psychiatric symptoms and dysfunction, prevent recurrence, and reduce social and occupational activity restrictions and participation restrictions, that is, rehabilitation. Drug therapy is based on drug selection according to the symptoms. By using antipsychotic drugs, chlorpromazine and haloperidol, which have a strong effect of suppressing acute symptoms such as hallucinations and delusions, are used. Since these are potent dopamine D2 receptor antagonists, extrapyramidal symptoms such as Parkinson's disease-like symptoms can be problematic as side effects. On the other hand, these drugs generally do not improve, but even worsen, for negative symptoms such as cognitive impairment, withdrawal, and decreased motivation. For these symptoms, risperidone, olanzapine, lumateperon, iroperidone, zipracidone, lulacidone, lexarti, cariprazine, asenapine, which also have anti-serotonin action, in addition to anti-D2 receptor action, and pariperidone, which also has anti-α2 adrenergic receptor action. , Lybalvi, which is a combination of olanzapine and the opioid receptor antagonist Samiddlefan, and aripiprazole, which has the effect of promoting the release of dopamine and noradrenaline by presynaptic D2 receptor antagonist, are used. In addition, the anxiolytics lorazepam, paroxetine, the hypnotic solpitem, flunitrazepam, etc. are prescribed according to the psychological symptoms.

The present invention provides a method for diagnosing schizophrenia. That is, the present invention is a method for diagnosing schizophrenia in a subject.
Obtaining a sample from the subject,
b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject, and
c. Provided above are the methods comprising determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio.

The present invention also provides a method of treating schizophrenia in combination with the diagnosis of schizophrenia. That is, the present invention is a method for diagnosing and treating schizophrenia in a subject.
Obtaining a sample from the subject,
b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject.
c. Determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio, and
d. Provided above are methods that include initiating treatment for schizophrenia for a subject if the likelihood or risk of developing schizophrenia is determined to be high.

In the diagnostic and therapeutic methods of the present invention, the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the expression level of non-phosphorylated CRMP2 in the sample derived from a healthy person who has not developed schizophrenia. / Or, if the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the sample derived from the subject is lower than the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the sample derived from a healthy person who has not developed schizophrenia, schizophrenia. It can be determined that the possibility of developing or the risk of developing the disease is high. Further, in the diagnostic method and the therapeutic method of the present invention, the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the set value, and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the sample derived from the subject is high. When it is lower than the set value, it can be determined that the possibility of developing schizophrenia or the risk of developing schizophrenia is high. The set values are described above.

The present invention includes a reagent capable of measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or a reagent capable of measuring the expression level of phosphorylated CRMP2 in a sample derived from a subject. Kits for testing for schizophrenia are also provided.

As an example, the kit of the present invention contains as a reagent an antibody capable of specifically recognizing non-phosphorylated CRMP2 and / or phosphorylated CRPM2. The antibody should be capable of recognizing CRMP2 phosphorylated by Ser522. The antibody may be either a monoclonal antibody or a polyclonal antibody. The antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane or a substrate. The kit may further include an instrument for collecting a sample derived from a subject, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or discrimination criteria for schizophrenia.

As another example, the kit of the present invention contains a nucleic acid probe that can specifically hybridize with CRMP2 mRNA as a reagent. The nucleic acid probe may be immobilized on the substrate. The kit also includes equipment for collecting biological samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, etc. May be good. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or discrimination criteria for schizophrenia.

As yet another example, the kit of the present invention contains at least one pair of nucleic acid primers as a reagent capable of specifically amplifying cDNA synthesized using mRNA of CRMP2 as a template. The kit also includes equipment for collecting subject-derived samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, etc. It is good to be. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or discrimination criteria for schizophrenia.

In addition, the kit of the present invention may include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction vessel, and the like.

The kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.

Hereinafter, the present invention will be described in more detail by way of examples.
[Example 1]
Schizophrenia (SCZ) is characterized by impaired cognition, perception, emotions, and behavior. Currently, there are few validated biomarkers for this neuropsychiatric disorder. This is because the underlying pathogenic mechanism that causes and / or promotes the disease is unknown. However, in symptomology, it is recognized that SCZ depends on synaptic connections on dendrite spines, a dysfunction of the neural network that is significantly mediated by processes partially driven by cytoskeletal regulation. It is increasing. "Collapsin Response Mediator Protein 2" (CRMP2) is a master regulator of axonal guidance, dendrite bifurcation, and spine formation. That is, it is a neural network modulator. The activity of this intracellular protein depends on its phosphorylated state. "Toggling" between inactive (phosphorylated) CRMP2 and active (non-phosphorylated) CRMP2 is physiological, but there is evidence that abnormal regulation of CRMP2 in neuropsychiatric disorders is involved. I'm starting. To date, it has been found that lithium-responsive (LiR) bipolar disorder (BPD) is uniquely characterized by an abnormally high phosphorylated inactive CRMP2 (pCRMP2) and an increased pCRMP2: CRMP2 ratio. There is. Since CRMP2 is also expressed in lymphocytes, we examined the CRMP2 in the peripheral blood of SCZ patients and healthy controls. In peripheral blood mononuclear cells (PBMC) in SCZ patients younger than 40 years, we observed the opposite of what was found in BPD. The level of non-phosphorylated active CRMP2 is higher than that of the non-affected age-matched control, but the level of phosphorylated inactive pCRMP2 remains unchanged from the control. High abundance of active CRMP2, but perhaps inadequate amounts of conflicting inactive pCRMP2 (as if delayed phosphorylation of the increased CRMP2 substrate) leads to a unique reduction in the pCRMP2: CRMP2 ratio. This suggests that the normal equilibrium between active and inactive CRMP2 is disrupted and that the function of the neural network (particularly dendritic spine) is also abnormal. These data indicate that measuring CRMP2 and pCRMP2 in peripheral blood is the basis for relatively rapid, minimally invasive, sensitive and specific auxiliary diagnostic tests for early SCZ (together with testing and imaging). It suggests that it may form. Increased CRMP2 and / or decreased pCRMP2: CRMP2 ratio is useful for diagnosis in newly developed young patients with suspected SCZ, especially when trying to rule out other mimicry disorders (eg, manic episodes in BPD). there is a possibility.

Introduction Introduction
Schizophrenia (SCZ) is an idiopathic psychiatric disorder with substantial morbidity and mortality, and enormous personal and social costs. It is characterized by impaired cognition, perception, emotion and behavior. The pathogenic mechanism by the underlying pathogenic molecule that causes and / or promotes the disease is unknown. However, it is known that there are important genetic components (1). Nonetheless, the concordance rate for disease between identical twins remains at 50%, and SCZ is likely to be multigenic, epigenetic, and multifactorial in its pathogenicity and expression. It suggests that.

The underlying pathophysiology of SCZ is not understood, but it is believed to be related to changes in the structure and function of the neural network of the brain (2-5). In brain development, neural circuits are formed through neurogenesis, differentiation, axonal guidance, dendrite development, synaptogenesis, and activity-dependent refinement of immature synapses (6). Because synapses are sites of neurotransmitter signaling, dendritic spine dysfunction may play an important pathogenic role in SCZ. In fact, numerous genetic linkages and related studies suggest that abnormalities in genes involved in synaptogenesis and / or maturation may be risk factors for SCZ (4, 6-8). The lack of reliable, pathogenic, and clinically accessible biomarkers hinders not only an understanding of the underlying etiological mechanism, but also the diagnosis of SCZ. Increasing the speed, sensitivity, and specificity of early SCZ diagnosis will facilitate systematic testing of genes across a more homogeneous patient population, improve clinical management, and accelerate the development of new treatment options. ..

"Collapsin Response Mediator Protein 2" (CRMP2), also called "Dihydropyrimidinase-like 2" (DPYSL2), is a master regulator for axon guidance, dendritic branching, and spine formation. Therefore, it is a neural network modulator. It was initially identified as an intracellular molecule that mediates signaling of the repulsive axon guidance molecule Semaphorin 3A (9), but has since been much more in neural development and maintenance of adult nervous system homeostasis. It has been recognized as playing a major role. The CRMP family of proteins is currently known to be composed of five homologous cytoplasmic proteins from CRMP1 to CRMP5 (10, 11). CRMP2 actively binds to non-phosphorylated active cytoskeletal elements. Phosphorylation of CRMP2, a two-step process, inactivates CRMP2 and induces the release of cytoskeletal elements. Cdk5 first phosphorylates CRMP2 with Ser522 and primes glycogen synthase kinase 3β (GSK3β) to phosphorylate CRMP2 with Thr514 and Ser518 (12). We now know that "toggling" between inactive (phosphorylated) and active (non-phosphorylated) CRMP2 is a continuous physiological adaptive mechanism to prevent the germination of abnormal neurons. ing.

The process by which we become aware that proteins in the CRMP family play important roles in axonal guidance, dendrite patterning, synaptogenesis, etc. is crucial for network abnormalities at the heart of neuropsychiatric disorders. It has come to be recognized as a thing (10, 11, 13, 14). Phenotypic analysis of crmp1 and crmp2 gene-deficient mice (crmp1 ^- ^/ ^- and crmp2 ^- ^/ ^- , respectively) reveals that both of these transgenic animals exhibit behavioral abnormalities that are thought to model neuropsychiatric symptoms. rice field. For example, Crmp1 ^- ^/ ^- mice show context-sensitive and long-term memory impairment and show reduced prepulse inhibition (15). ^Crmp2- ^/ ^-Mice show hyperactivity, emotional behavioral disorders, decreased social interaction, mild contextual learning disabilities, and high susceptibility to methamphetamine (16, 17). We have recently shown that aberrant CRMP2 posttranslational regulation is involved at the core of the etiology of lithium-responsive (LiR) bipolar disorder (BPD) (14, 18). Specifically, the "set value" of the ratio of phosphorylated (inactive) CRMP2 to non-phosphorylated (active) is abnormally high in LiR human brains and patient-specific human-induced stem cell (hiPSC) -derived neurons. By reducing the level of phosphorylated CRMP2 (pCRMP2), its setting was inversely correlated with the CRMP2 pathway modulators, lithium and cortical dendrite spine density, which normalized dendritic lesions. Therefore, we regarded BPD as a disorder of post-translational regulation of molecules important for development, not as a disorder of the gene itself. Serving as a biomarker for this disease due to the specificity of increased pCRMP2: CRMP2 ratio to LiR BPD (unaffected controls, other psycho-neurological disorders, as well as elevated levels not seen in patients with lithium non-reactive (LiNR) BPD) A possibility was suggested (14). Interestingly, a series of recent genome-wide association studies have shown that BPD clusters with cognitive disorders such as SCZ rather than mood disorders (19). Given the role of CRMP2 as a master regulator of the modulation of the cytoskeleton and thus the neural network, it is strongly suspected that its activity state is characterized by abnormal synaptogenesis, neurite formation, and neural network activity. We investigated whether it could also serve as a potential biomarker for SCZ.

Knowing that CRMP2 is also expressed in lymphocytes, can these brain abnormalities be reflected in readily accessible peripheral blood mononuclear cells (PBMCs) and serve as the basis for practical clinical diagnostic markers for SCZ? I decided. The difference in blood was found to be remarkable. CRMP2 levels were elevated compared to a healthy age-matched control group. The ratio of pCRMP2: CRMP2 is abnormally low (based on an increase in the denominator). These findings were most pronounced in young patients with SCZ who were more likely to have the disease "purer" (ie, less contaminated with age-related disease and other comorbidities, early diagnosis and treatment. May be the most beneficial). These CRMP2 findings are in stark contrast to the LiRBPD situation, where the pCRMP2: CRMP2 ratio was always abnormally high and CRMP2 was always normal, distinguishing between the two states of undiagnosed young patients. May provide molecular and biochemical methods for, and may not be clear in the first presentation (manic episodes and schizophrenia "breaks" may resemble each other). These changes in the production of CRMP2 in SCZ not only provide rapid, sensitive, specific non-invasively accessible biomarkers and diagnostic support, but also underlie the neuropathological changes seen in SCZ. Begins to provide insights into certain molecular mechanisms.

Results Results.
CRMP2 was not significantly different from age-appropriate non-affected patients, but the pCRMP2: CRMP2 ratio was significantly higher in LiR BPD patients compared to non-affected controls (similarly, other psychology, including LiNR BPD). Compared to patients with neurological disorders). The denominator (CRMP2) remained unchanged, with pCRMP2 (numerator) being abnormally high in LiR BPD patients. Differences in CRMP2 between SCZ patients and unaffected controls were significant, as detected through this more accessible peripheral blood-derived cell type assay (pCRMP2: CRMP2 ratio, we In addition to making it a suspicious and informative metric).

We first established a detection system for CRMP2 and pCRMP2 in PBMC samples of living patients [Fig. 2 & 3]. To examine patients who are likely to have "pure" SCZ and to eliminate the entanglement of age-related disorders as well as polypharmacy (including non-psychiatric disorders), we may be associated with aging. The evaluation focused on SCZ patients <40 years of age, before intervention of comorbidities unrelated to a disease. The table summarizes the demographic information of this SCZ patient group and the non-affected age-matched control group. There was no significant difference between the two groups in that respect. As expected, years of education completion and career requirements pursued were lower in SCZ patients compared to age-matched non-affected controls.

There was a significant difference between SCZ patients and non-affected controls not only in the level of CRMP2 but also in the resulting pCRMP2: CRMP2 ratio [Fig. 1]. CRMP2 levels were significantly higher in PBMC in SCZ patients. Most notably, this was the case in SCZ patients younger than 30 years (* p <0.01). On the other hand, the phosphorylation of CRMP2 did not increase at the same time as the increase in the CRMP2 substrate, and therefore the denominator (CRMP2) increased based on the increase in its abundance, so that in the SCZ group (compared to the unaffected control group). pCRMP2: The ratio of CRMP2 was low [Fig. 1]. Excessive presence of non-phosphorylated active CRMP2 (compared to the equilibrium between normally present active and non-phosphorylated CRMP2) results in abnormal neural network function, especially in dendritic spines. It is expected to be. In particular, the lower pCRMP2: CRMP2 ratio in SCZ dramatically contrasted with the higher ratio (14,18) we previously reported in LiRBPD patients, increasing the potential usefulness of this diagnostic aid.

In short, for diagnostic purposes, excessive amounts of active non-phosphorylated CRMP2 in the blood may distinguish SCZ patients from unaffected control subjects (and also patients with other pseudopsychiatric disorders such as BPD). expensive).

Discussion
In this study, the literature states that the symptoms of this condition depend on dysfunction of the neural network, which is markedly mediated by synaptic connections on dendrite spines, i.e., partly driven by cytoskeletal regulation. Provides evidence of ancillary diagnostic biomarkers of SCZ derived from the increasing implications. CRMP2 is a master cytoskeletal regulator and, therefore, a neural network modulator. Phosphorylation of CRMP2 alters the binding affinity for specific subsets of cytoskeletons and related proteins such as tubulin, RhoA, and filamine-A (11, 20, 21). These changes in phosphorylation state affect the regulation of cytoskeletal dynamics. The "togling" between the inactive (phosphorylated) CRMP2 and the active (non-phosphorylated) CRMP2 is physiological (14, 18). However, imbalances between active and inactive CRMP2 (one too much) can cause abnormalities in synaptogenesis, synaptic maturation, and synaptic transmission (11, 22).

As mentioned above, CRMP2 is also present in lymphocytes. In fact, studies have shown similarities between receptor expression and transduction processes in cells and lymphocytes from the nervous system (23). Using our PBMC assay, we found that blood levels of CRMP2 were higher compared to age-matched non-affected controls. In addition, since the phosphorylated form of CRMP2 was almost the same as that of the control, the net effect of increasing the denominator while maintaining a constant numerator was that the ratio of pCRMP2: CRMP2 in the SCZ group was lower than that in the control group. .. These differences were most pronounced in the youngest group of SCZ patients. In SCZ patients younger than 30 years, the pCRMP2: CRMP2 ratio was lower than in control patients, up to the p <0.01 significance level.

Exact separation between low pCRMP2: CRMP2 ratios in young patients with SCZ and normal ratios in age-matched non-affected controls (as shown in Figure 1A) is easily and repeatedly accessible as PBMCs. Assaying informative cell types can rapidly measure CRMP2 and pCRMP2 in PBMC of patients with suspected SCZ (of course, in conjunction with psychiatric and neurobehavioral tests and medication tests). It suggests that it may serve as the basis for minimally invasive assisted diagnostic trials. Having such ancillary tests helps clinicians differentiate SCZ from other conditions that may be similar, including other behavioral problems, personality disorders, or systemic disorders. Will. For example, these values may help clinicians distinguish between SCZ psychotic and manic episodes relatively quickly in BPD patients. We will talk about the normal CRMP2 value and / or the increase in the pCRMP2: CRMP2 ratio later. Increased CRMP2 levels and / or decreased ratios will facilitate the diagnosis of SCZ in newly developed young patients. In addition, CRMP2 levels and ratios are SCZ-related behaviors because pCRMP2 begins to increase with age, especially with the development of Alzheimer's disease, when followed over time and over time in the same patient. May help distinguish between aging and age-related behaviors. In fact, in one patient, if the patient is already elderly and has many other comorbidities (24, 25), we nail the use of this biomarker for the first time. The specificity of the pCRMP2: CRMP2 ratio is likely to be worse in the older group.

In conclusion, in peripheral blood monocytes of SCZ patients, the levels of master cytoskeletal modulator and neural network determinant, CRMP2 are higher than in non-affected age-matched controls. In contrast to other psychiatric disorders, including those involving CRMP2 (eg, LiRBPD), higher active CRMP2 levels with constant inactive pCRMP2 levels are non-affected controls with a pCRMP2: CRMP2 ratio. It results in a unique reduction that is lower than that of the subject. This disruption in the normal equilibrium between the active and inactive CRMP2 can lead to an imbalance in the neural network as well. Since these brain changes can be detected in the peripheral blood of young patients (probably early, probably not yet diagnosed), testing these ratios is when trying to determine the etiology of a patient's abnormal behavior. There is an interesting possibility (in combination with examination and imaging) that it may ultimately serve as ancillary, relatively rapid, minimally invasive, sensitive and specific diagnostic aids.

Given that CRMP2 appears to be involved in the pathology of SCZ in some way (in fact, some have been considered risk genes (26)), the dynamic regulation of the cytoskeleton and neural network Further research may lead to greater insight into the molecular mechanisms underlying the neuropathological changes seen in SCZ, as well as new therapeutic interventions.

Methods
[Patients from whom PBMC was collected]
The table summarizes the demographics of SCZ patients and healthy control patients (range 18-40 years) who collected PBMCs. Such data include age and gender, family history, smoking history, drinking history, education, background and medication history. The subjects were inpatients or outpatients at Yokohama City University Hospital, Yokohama Medical Center, and Yokohama Maioka Hospital (belonging to Yokohama City University). This study was approved by the ethics review board of each hospital. Each SCZ patient was diagnosed by at least two psychiatrists based on the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Illness (DSM-IV). The severity of symptoms was assessed using PANSS (Positive and Negative Symptom Scale). Volunteers were enrolled by general open call for conforming non-affected controls. Psychiatrists interviewed each volunteer to rule out mental illness. Informed consent was obtained from all participants.

[Blood sampling and lymphocyte separation]
PBMCs were isolated from all blood samples taken using mononuclear cell preparation tubes (BD Vacutainer CPT: Becton-Dickinson, NJ, USA). Whole blood specimens were centrifuged for 30 minutes (1500 g, 25 ° C) and the PBMC layer was collected in a 15 ml tube. These samples were then washed with phosphate buffered saline minus (PBS) and centrifuged at 427 g for 10 minutes at 4 ° C. three times. After discarding the supernatant, the pellet was resuspended in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and ampicillin.

The resuspended sample was sown on an uncoated Petri dish and cultured at 37 ° C for 2 days in a CO2 incubator. A supernatant fraction containing non-adhesive lymphocytes was collected and transferred to a 15 ml test tube. The sample was centrifuged at 200 g for 5 minutes at 4 ° C. After discarding the supernatant, the pellet was resuspended in 1 ml PBS and transferred to a 1.5 ml tube. After centrifugation at 200 g for 5 minutes at 4 ° C, the supernatant was discarded, the pellet was frozen in liquid nitrogen and stored at -80 ° C until use. CRMP2 and pCRMP2 levels were then evaluated in these samples as described below.

[antibody]
Anti-human CRMP2 monoclonal antibody (9F) was generated by injecting Balb / c mice into the C-terminal region of human CRMP2 (amino acids 486-528) (27).
An anti-phosphorylated CRMP1 / 2 (S522) (p-S522-CRMP1 / 2) polyclonal antibody that recognizes phosphorylated CRMP2 with Ser522 was produced in rabbits as previously reported (12). The specificity of the two antibodies was verified by peptide blocking experiments in immunoblot analysis of human PMBC samples as previously reported (12, 27, 28). (See Figures 2 and 3)

[Immunoblot analysis]
Sample pellets of 50 μl immunoprecipitated IP buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 1% Nonidet P-40, 50 μM ρ-APMSF [ρ-amidinophenylmethanesulfonyl fluoride] D]) was homogenized and centrifuged at 15,000 g at 4 ° C for 20 minutes. Next, sample the anti-β-actin mouse monoclonal (A5316, Sigma-Aldrich, MO, USA) antibody with anti-CRMP2 (9F), anti-phosphorylated CRMP1 / 2, and signal enhancer HIKARI (Nacalai Tesque, Kyoto, Japan). Used for immunoblot analysis by. Actin (2.0 mg / ml) and rat brain lysate (0.5 mg / ml) were used as load controls. Purified CRMP2 was used as a standard, and the CRMP2 concentration in the lymphocyte lysate was calculated by immunoblotting using ImageJ. Relative amounts of CRMP2 and its Ser522 phosphorylated CRMP2 (p-S522-CRMP2) were estimated using β-actin as an internal standard. (See Figures 2 and 3)

[Statistical analysis of data from peripheral blood samples]
Statistical analysis was performed using IBM SPSS Statistics 22 (IMB analysis). For the Mann-Whitney U test, the statistical significance was p <0.05. Regression analysis was performed for the examination between continuous variables, and the significance of the correlation coefficient was p <0.05.

[Test approval]
The clinical trial implementation plans at Yokohama City University Hospital, Yokohama Medical Center, and Yokohama Maioka Hospital were approved by the ethical review board of each hospital. All animal treatments are performed according to the facility guidelines outlined by the Animal Care and Use of Laboratory Animals (National Academies Press, 2011) of the Graduate School of Medicine, Yokohama City University. bottom.

table
Demographic information of patients analyzed for PBMC

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All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

The present invention can assist in the diagnosis of schizophrenia.

Claims

A method for testing schizophrenia, which comprises measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from a subject.
The test method according to claim 1, wherein an increase in the expression level of non-phosphorylated CRMP2 and / or a decrease in the phosphorylated CRMP2: ratio of non-phosphorylated CRMP2 indicates that the possibility or risk of developing schizophrenia is high.
The inspection method according to claim 1 or 2, wherein the phosphorylated CRMP2 is CRMP2 phosphorylated with Ser522.
The test method according to any one of claims 1 to 3, wherein the sample derived from the subject is a blood sample.
The test method according to claim 4, wherein the blood sample is a peripheral blood mononuclear cell fraction.
The method according to any one of claims 1 to 5, wherein the subject is under 40 years old.
The method according to any one of claims 1 to 6, wherein the subject is under 30 years old.
If the subject is under the age of 30, a phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the subject's peripheral blood mononuclear cell fraction of 0.25 or less is likely or at high risk of developing schizophrenia. The method according to any one of claims 1 to 7.
A method for diagnosing schizophrenia in subjects
Obtaining a sample from the subject,
b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject, and
c. The method comprising determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio.
A method of diagnosing and treating schizophrenia in a subject.
Obtaining a sample from the subject,
b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject.
c. Determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio, and
d. The method described above comprising initiating treatment of the subject with schizophrenia if the likelihood or risk of developing schizophrenia is determined to be high.
The expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the expression level of non-phosphorylated CRMP2 in the sample derived from a healthy person who has not developed schizophrenia, and / or phosphorylation in the sample derived from the subject. When the ratio of CRMP2: non-phosphorylated CRMP2 is lower than the ratio of phosphorylated CRMP2: non-phosphorylated CRMP2 in a sample derived from a healthy person who has not developed schizophrenia, the possibility or risk of developing schizophrenia is high. The method according to claim 9 or 10.
Schizophrenia when the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the set value and / or when the ratio of phosphorylated CRMP2: non-phosphorylated CRMP2 in the sample derived from the subject is lower than the set value. The method according to any one of claims 9 to 11, which determines that the possibility of onset or the risk of onset is high.
Of schizophrenia, including a reagent capable of measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or a reagent capable of measuring the expression level of phosphorylated CRMP2 in a sample derived from a subject. Kit for inspection.