WO2016117582A1

WO2016117582A1 - Marker for determining mental illness using mirna

Info

Publication number: WO2016117582A1
Application number: PCT/JP2016/051504
Authority: WO
Inventors: 功太郎服部; 浩功刀; 後藤　雄一; 新一 ▲高▼坂
Original assignee: 国立研究開発法人国立精神・神経医療研究センター
Priority date: 2015-01-20
Filing date: 2016-01-20
Publication date: 2016-07-28
Also published as: JPWO2016117582A1

Abstract

Developed and provided is a marker for determining mental illness with which it is possible to easily and accurately determine the presence of schizophrenia, depression, or bipolar disorder in a subject. Provided is a marker for determining mental illness from groups comprising miRNA that contains a base sequence shown by SEQ ID NOS: 1-193, the miRNA being contained in cerebrospinal fluid.

Description

Psychiatric disorder determination marker using miRNA

The present invention relates to a biomarker for determining the presence / absence of psychiatric schizophrenia, bipolar disorder or depression using a sample derived from a subject, and a mental disease determination method using the biomarker.

In modern society, mental disorders such as schizophrenia, depression, and bipolar disorder (Mental Disorder) have become a major social problem. For example, according to the 2011 survey by the Ministry of Health, Labor and Welfare in Japan, the number of inpatients with mental illness in Japan is about 23% of the total number of inpatients in Japan. When the number of outpatients and the number of potential patients are added, the number of patients with mental illness is expected to reach an enormous number in Japan. Since these mental illnesses are common in the working age group, not only the cost of medical expenses is increased, but also the social impact such as economic loss and social loss due to suicide is immeasurable.

In general, mental diseases have many symptoms that are close to each other, and each disease has a wide variety of symptoms, so the definition of each disease is not clear. For this reason, accurate diagnosis is often extremely difficult even for specialists.

Currently, diagnosis of psychiatric disorders is usually based on information such as symptoms, signs, and progress obtained through interviews with the person or family, and Diagnostic Statistical Manual of Mental Disorders (DSM) defined by the American Psychiatric Association It is done according to the diagnostic criteria. However, since this diagnostic criterion does not use an objective test, the diagnosis results often do not match between the doctors making the diagnosis. According to a large U.S. survey, only 38.4% of 5639 people diagnosed with depression based on DSM diagnosis in general practice were rediagnosed as depressive disorder in a structured diagnostic interview (Non-Patent Document 4). The structured diagnostic interview is a manual interview method. By setting in advance the format of the question content, re-question, etc., the difference in diagnosis by the interviewer can be reduced. In fact, if a structured diagnostic interview is used, a relatively high degree of coincidence can be obtained (Non-Patent Document 3). However, while interview times of one to two hours or more are required, the clinical benefits are not commensurate with them, so it is rarely done outside of research.

Also, since DSM diagnosis is determined for mental disorders, it cannot be said that it necessarily reflects biological pathology. In fact, there are many cases in which a DSM diagnosis is mistakenly diagnosed as a mental illness even if the cause is clearly a cause other than the brain. For example, at least 10% of patients diagnosed with depression are said to have side effects of hypothyroidism and anti-inflammatory drugs (Non-patent Document 1). It has also been reported that patients diagnosed with schizophrenia include anti-NMDA receptor antibody encephalitis (Non-patent Document 2).

By the way, the current antidepressant has not been sufficiently effective except in some severe cases (Non-patent Document 5). In addition, the effects of schizophrenia drugs are limited to some symptoms such as hallucinations and delusions. Thus, one of the reasons for the limited effect of therapeutic agents for mental illness is that the classification of mental illness is not well established. For example, there are multiple pathologies of mental illness diagnosed as depression, but currently prescribed antidepressants have a therapeutic effect only in some pathologies. Therefore, the development of essential treatments for mental illness requires the development of new classifications based on biological evidence and appropriate diagnostic techniques.

From the above background, biomarkers have recently attracted attention as a new diagnostic method based on biological evidence. A biomarker is a substance such as a nucleic acid or protein produced by a cell in response to the onset of a specific disease, and the presence, severity, or prognosis of an abundance or variation in body fluids or tissues. Therefore, it can be a discriminator for disease determination and treatment guideline determination.

In the field of mental illness, biomarker searches for schizophrenia, depression, or bipolar disorder have been conducted so far, and various candidate molecules such as cytokines and neurotrophic factors have been proposed. However, most of them do not have a sufficient detection effect, and practical biomarkers for mental illness have not been developed yet (Non-Patent Documents 6 and 7).

In conventional diagnosis of mental illness, there are problems with the diagnostic criteria themselves and the samples used for the search.

The problem of diagnostic criteria is that, when searching for biomarkers, candidate molecules are selected based only on the significant difference between the mean values of the patient group and the healthy group. Mental illness is a syndrome, and it is likely that there are multiple conditions in the syndrome. Therefore, even if a substance can be a useful biomarker for some pathological conditions, if the ratio of the pathological condition in all cases is small, it may be buried and excluded by a significant difference test.

Also, as a sample problem, blood or postmortem brain was used as a biomarker search sample. Blood directly contacts various organs such as the brain, intestines, liver, and muscles. On the other hand, a blood-brain barrier exists between the brain and other organs, and exchange of substances in the blood is restricted. Therefore, in the biomarker search using blood, particularly peripheral blood, it is highly likely that it is difficult to detect changes in substances characteristic of the brain due to the influence of other organs. That is, there is a problem that blood is noisy and hardly reflects the state of the brain. The postmortem brain is superior in that it uses the target organ itself, which is thought to be the cause of mental illness, and maintains the three-dimensional structure of the brain, but unlike animal experiments that can be removed immediately after death, the human brain is the cause of death. And changes due to postmortem changes are likely to make it difficult to detect changes in important candidate molecules such as neurotransmitters and signal molecules. Furthermore, in neuropathology and brain imaging research, no foci specific to each mental disorder have been found so far, and unlike Parkinson's disease, the superiority of maintaining the three-dimensional structure of the brain is poor.

Therefore, the present invention provides a highly practical psychiatric disease determination marker that can solve the above-mentioned problems and can easily and accurately determine a psychiatric disease such as schizophrenia, depression, or bipolar disorder in a subject. The task is to do.

Another object of the present invention is to provide a psychiatric disease determination marker that can further determine the subtype and / or severity of schizophrenia, depression, or bipolar disorder in a subject.

Furthermore, an object of the present invention is to provide a determination assisting method for accurately detecting the presence or absence of each mental disorder in a subject using the mental disorder determination marker.

In view of the above problems, the present inventors selected cerebrospinal fluid (CSF) as a sample used for biomarker search. Cerebrospinal fluid is a colorless and transparent fluid that exists only around the brain and spinal cord, separated from the other organs by the dura mater, and by the blood-brain barrier and the blood-cerebrospinal fluid barrier . Recent studies have shown that the majority of cerebrospinal fluid exudes from the parenchyma of the brain and that there is virtually no barrier between brain cells and cerebrospinal fluid (Wang C, et al., 2012, Cerebrospinal Fluid: Physiology, biomarker and methodology. In: V. S, Dolezal, T., editor. Cerebrospinal Fluid: Functions, Composition and Disorders. New York: Nova Spp. ).

Here, conventionally, proteins and mRNAs have been mainly used as disease determination markers. Although proteins can be measured in cerebrospinal fluid, there is a problem that when drug discovery is performed, compounds that inhibit the function must be searched simultaneously. In addition, mRNA markers are scarcely present in cerebrospinal fluid and are thought to be poorly correlated with central nervous system expression. Therefore, the present inventors decided to use miRNA instead of protein or mRNA as a psychiatric disorder determination marker. As described later, miRNA may be involved in pathological conditions through the regulation of gene expression, and its function can be easily inhibited by artificially synthesized antisense nucleic acid molecules (Weiler J et al., Gene). Ther. 13: 496-502, 2006).

Furthermore, as a statistical method for selecting biomarkers to solve the problem of diagnostic criteria, not only a method based on the average difference between the healthy group and the disease group, which has been frequently used, but also a cut-off value is set. A method for selecting cases showing abnormal values in each disease and a method for diagnosing the severity in case disease groups were newly added.

As a result, the present inventors were able to select 193 miRNAs as psychiatric disease determination markers. The present invention is based on the above results and provides the following.
(1) A psychiatric disease determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 193.
(2) The psychiatric disorder determination marker according to (1), wherein the psychiatric disorder is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 45, and the psychiatric disorder is schizophrenia.
(3) The psychiatric disease determination marker according to (2), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 34, and can subdivide schizophrenia into subtypes.
(4) The psychiatric disorder determination marker according to (2), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 5 and 40 to 45 and can identify the severity of schizophrenia.
(5) The psychiatric disease determination marker according to (1), which is selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 46 to 142, and the psychiatric disease is depression.
(6) The psychiatric disease determination marker according to (5), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 46 to 113 and can subdivide depression into subtypes.
(7) The psychiatric disorder according to (5), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 49, 80, 81, 83 and 131 to 142, and can identify the severity of depression Judgment marker.
(8) The psychiatric disease determination marker according to (1), wherein the psychiatric disease is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 176, and the mental illness is bipolar disorder.
(9) The psychiatric disease determination marker according to (8), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 157 and can subdivide bipolar disorder into subtypes.
(10) The psychiatric disease determination marker according to (1), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 177 to 187, and the psychiatric disorder is schizophrenia or depression.
(11) The psychiatric disease determination marker according to (1), which is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 188 to 191 and the mental illness is depression or bipolar disorder.
(12) The psychiatric disorder determination marker according to (1), wherein the psychiatric disorder is schizophrenia, depression, or bipolar disorder, comprising miRNA comprising the nucleotide sequence represented by SEQ ID NO: 192 or 193.
(13) A method for determining a psychiatric disorder, wherein the psychiatric disorder is selected from the group consisting of miRNAs containing the base sequences represented by SEQ ID NOs: 1 to 193, which are contained per unit amount of samples collected from subjects and healthy subjects. A measurement step of measuring the amount of the marker to obtain the measurement value, a comparison determination step of comparing the measurement value of the subject and the healthy subject obtained in the measurement step, and determining a mental illness in the subject based on the result Said method comprising.
(14) The method according to (13), wherein schizophrenia is determined by a psychiatric disorder determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 45.
(15) A subtype of schizophrenia is determined based on a measured value of a psychiatric disorder determination marker selected from the group consisting of miRNAs containing the base sequences represented by SEQ ID NOs: 1 to 34 and a predetermined cutoff value. 14) A method.
(16) The method according to (13), wherein depression is determined by a psychiatric disease determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 46 to 142.
(17) The depression subtype is further determined based on a measured value selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 46 to 113 and a predetermined cutoff value, Method.
(18) The method according to (13), wherein bipolar disorder is determined by a psychiatric disorder determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 176.
(19) Further determining a subtype of bipolar disorder based on a measured value of a psychiatric disease determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 157 and a predetermined cutoff value, The method according to (13).
(20) schizophrenia or depression is determined based on a measured value of a psychiatric disorder determination marker selected from the group consisting of miRNAs containing the base sequences represented by SEQ ID NOs: 177 to 187 and a predetermined cutoff value; The method according to 13).
(21) Depression or bipolar disorder is determined based on a measured value of a psychiatric disease determination marker selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 188 to 191 and a predetermined cutoff value ( The method according to 13).
(22) The method according to (13), wherein schizophrenia, depression or bipolar disorder is determined from a psychiatric disorder determination marker comprising miRNA comprising the base sequence represented by SEQ ID NO: 192 or 193.
(23) Determining the severity of schizophrenia based on the measured value of a marker for determining a psychiatric disorder selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 5 and 40 to 45 obtained in the measurement step The method according to (14), further comprising a severity determination step.
(24) Depression based on a measured value of a marker for determining a psychiatric disorder selected from the group consisting of miRNAs comprising the base sequences represented by SEQ ID NOs: 49, 80, 81, 83 and 131 to 142 obtained in the measurement step The method according to (16), further comprising a severity determination step of determining the severity of.
(25) The method according to any one of (13) to (24), wherein two or more different psychiatric disease determination marker amounts are measured in the measurement step.
(26) The method according to any one of (13) to (25), wherein the sample is cerebrospinal fluid.

This specification includes the disclosure of Japanese Patent Application No. 2015-008710, which is the basis of the priority of this application.

According to the psychiatric disease determination marker of the present invention, a biomarker capable of easily and accurately determining a psychiatric disease such as schizophrenia, depression, or bipolar disorder in a subject can be provided. This psychiatric disorder determination marker can be used to identify morbidity such as schizophrenia, depression, or bipolar disorder in a subject, identification of subtypes, and / or identification of severity.

According to the mental illness determination method of the present invention, there is provided a method for accurately determining morbidity, subtype identification, and / or severity identification of each mental illness in a subject using the mental illness determination marker. can do.

It is a conceptual diagram which shows the classification group of the mental disease determination marker of this invention. The distribution diagram of the measured value of the depression mental disorder determination marker hsa-miR-3620-5p in healthy individuals and patients with various mental disorders is shown. A horizontal straight line in each plot group indicates an average value in the plot group (the same applies to other drawings). The broken line (95%) is a cutoff value corresponding to the 95th percentile in the healthy group. The relationship between the measured value of hsa-miR-1908, which is a psychiatric disease determination marker for determining the severity of depression, and the evaluation value of the depression evaluation scale HAM-D is shown. The distribution diagram of the measured value of hsa-miR-3180, a marker for determining mental disorders for depression / bipolar disorder, in healthy individuals and patients with various mental disorders is shown. The broken line (95%) is a cutoff value corresponding to the 95th percentile in the healthy group. It is a figure which shows the measured value distribution of the mental disease determination marker hsa-miR-4749-5p for schizophrenia / depression / bipolar disorder in a healthy subject and each psychiatric patient. The cutoff value was the 5th percentile in the healthy group.

1. Psychiatric disease determination marker 1-1. Outline | summary The 1st aspect of this invention is a psychiatric disorder determination marker. The mental disease determination marker of the present invention is miRNA, and can be used as a biomarker for determining mental disease in a subject by measuring the amount contained in a sample derived from the subject.

1-2. Definitions Terms used in this specification are defined below.

“Psychiatric disorder” is a general term for extrinsic, psychogenic, or intrinsic brain functional or organic disorders in a broad sense. However, unless otherwise specified, the term “psychiatric disorder” in the present specification refers to FIG. 1 belonging to three syndromes classified as schizophrenia, depression, or bipolar disorder in the diagnostic category of conventional psychopathology. Means a narrowly defined mental illness.

As used herein, “determining (determining) a mental illness” refers to identifying a morbidity of a psychiatric disorder in a subject, identifying a subtype of the psychiatric disorder, and / or identifying the severity of the afflicted mental disorder. . In addition, since the mental illness determination of the present invention is determined based on the abundance of the mental illness determination marker in the sample, it does not require a person directly engaged in medical treatment such as a doctor, and only a person skilled in the art having sample analysis technology. Can be implemented.

In the present specification, the “mental disease determination marker” is composed of miRNA, and is selected from the group consisting of miRNA comprising the base sequences shown in the respective sequence numbers in Tables 1 to 6 described later.

“MiRNA (microRNA)” is a single-stranded non-coding RNA having a length of 21 to 23 bases existing in a cell. miRNA is transcribed from the genome in a precursor form called pri-miRNA, and then processed into a precursor called pre-miRNA by an endonuclease called Drosha in the nucleus. Furthermore, it is cleaved and processed by the action of an endonuclease called Dicer outside the nucleus and becomes an intermediate double-stranded miRNA (miRNA / miRNA ^* ) consisting of miRNA and miRNA ^* (miRNA star). miRNA / miRNA ^* is incorporated into the RNA-induced silencing complex (RISC), a protein factor complex, and finally miRNA, one of the RNA strands, functions as mature miRNA (mature miRNA) (Bartel DP, 2004, Cell, 116: 281-297, Kawamata T., et al., 2009, Nat Struct Mol Biol., 16 (9): 953-960). It is known that mature miRNAs repressively regulate target gene expression by binding to target gene mRNA in RISC and inhibiting its translation.

Thus, pri-miRNA, pre-miRNA, miRNA / miRNA ^* , and mature miRNA can be present in the cell.

1-3. Configuration The mental disease determination marker of the present invention consists of miRNA containing any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 193.

The miRNA of the present invention is a broad miRNA including any of pri-miRNA, pre-miRNA, miRNA / miRNA ^* , and mature miRNA. Particularly preferred is mature miRNA.

The “base sequence shown in SEQ ID NO: X” (X is an arbitrary integer from 1 to 193) is a base sequence constituting a mature miRNA. Therefore, in the present specification, the “miRNA containing the base sequence represented by SEQ ID NO: X” is an miRNA containing a mature miRNA consisting of the base sequence represented by SEQ ID NO: X. For example, the miRNA comprising the base sequence shown in SEQ ID NO: 1 includes a mature miRNA consisting of the base sequence shown in SEQ ID NO: 1, a pri-miRNA that is a precursor thereof, a pre-miRNA that is a precursor thereof, and its Intermediate miRNA / miRNA ^* is included.

The miRNA containing the nucleotide sequence represented by SEQ ID NOs: 1 to 193 can be a biomarker that can determine any one or more mental diseases such as schizophrenia, depression, or bipolar disorder, that is, a mental disease determination marker.

The psychiatric disease determination markers for schizophrenia, depression, or bipolar disorder can be roughly divided into groups 1 to 7 shown in FIG. That is, a first group marker for determining schizophrenia, a second group marker for determining depression, a third group marker for determining bipolar disorder, a fourth group marker for determining schizophrenia or depression, schizophrenia Group 5 markers for determining illness or bipolar disorder, Group 6 markers for determining depression or bipolar disorder, and Group 7 markers for determining schizophrenia, depression or bipolar disorder. The mental disease determination marker of the present invention can be classified into any of the 1st to 4th group, 6th group and 7th group markers excluding the 5th group marker. Hereinafter, the mental disease determination marker belonging to each group will be described in detail.

(1) Group for determining schizophrenia Table 1 shows markers for determining mental disorders belonging to Group 1.

The psychiatric disorder determination marker belonging to this group is a biomarker for determining schizophrenia. Specifically, it is a psychiatric disorder determination marker that consists of miRNA containing the base sequence represented by any of SEQ ID NOs: 1 to 45 and belongs to the section indicated by group 1 in FIG. By using this group of psychiatric disease determination markers, it is possible to identify schizophrenia morbidity, identify schizophrenia subtypes, and / or identify the severity of schizophrenia in a subject.

In the psychiatric disorder determination marker belonging to this group, miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 1 to 39 can differentiate morbidity of schizophrenia in a subject.

In the present specification, “differentiating (performing)” means determining the high possibility of suffering a specific mental illness in a subject. Therefore, the subject is highly likely to suffer from schizophrenia by using one or more schizophrenia disease differential markers selected from the group consisting of miRNAs comprising the nucleotide sequences shown in SEQ ID NOs: 1 to 39 Can be identified.

In the present specification, the “subject” refers to a human individual that is subjected to the method of the second aspect described later and is a target for which a mental illness is to be determined.

The miRNA containing the nucleotide sequences shown in SEQ ID NOs: 35 to 39 in the psychiatric disorder determination marker belonging to this group is based on the significant difference in the abundance of the miRNA contained in the cerebrospinal fluid sample between schizophrenia patients and healthy subjects. Isolated biomarker.

As used herein, “healthy person” refers to a human individual in a healthy state. “Healthy state” as used herein means a healthy mental state not affected by any of the above mental disorders. In other words, a healthy person in the present specification is a human individual who does not have a mental illness, preferably a healthy human individual who does not have any disease or abnormality. There are no particular restrictions on the physical conditions such as gender, age, height, and weight, and the number of healthy persons used in this embodiment, but it is the same as the subject and has the same physical conditions such as age, height, and weight. Or it is preferable that it is an approximation. In this specification, a group of a plurality of healthy persons is referred to as a “healthy person group”.

In this specification, “significant” means statistically significant. Specifically, when the difference between the measured values of the subject and the healthy person is statistically processed, it means that there is a significant difference between the two. For example, the risk rate (significance level) may be less than 5%, 1%, 0.3%, 0.2%, or 0.1%. The test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t-test or covariate analysis of variance can be used.

In Table 1, miRNAs having a risk rate of less than 5% (p <0.05) in the t-test are indicated with a “◯” mark as a mental disease determination marker having a difference from normal.

In the psychiatric disorder determination marker belonging to this group, the miRNA comprising the nucleotide sequence represented by SEQ ID NOs: 1 to 34 is a psychiatric illness determination marker capable of subdividing schizophrenia into subtypes in addition to the symptom diagnosis It is.

In the present specification, the “subtype” is a subordinate classification group of each mental illness classified based on a biological basis using the mental illness determination marker of the present invention. Examples include schizophrenia subtypes belonging to group 1, depression subtypes belonging to group 2, and bipolar disorder subtypes belonging to group 3. Such a subtype is classified, for example, as “(mental marker name) (determination marker molecule name) subtype”. Specifically, subjects who are determined to be schizophrenia based on the hsa-miR-6510-5p marker represented by SEQ ID NO: 1 should be identified as “hsa-miR-6510-5p subtype of schizophrenia”. Can do.

In the present specification, a psychiatric disorder determination marker that can identify a subtype of each psychiatric disorder (specifically, miRNA containing the nucleotide sequence represented by SEQ ID NOs: 1-34 for schizophrenia subtype, depression subtype described later) MiRNA containing the nucleotide sequence shown in SEQ ID NOs: 46 to 113 and miRNA containing the nucleotide sequence shown in SEQ ID NOs: 143 to 157 for bipolar disorder subtypes) are included in samples derived from cerebrospinal fluid of healthy subjects The biomarker isolated by the cutoff value set based on the measured value of the psychiatric disorder determination marker.

In this specification, the “cut-off value” refers to a boundary value for classifying a quantitative result into positive and negative. Here, positive means that there is a high possibility of suffering from a mental illness, and negative means that there is a high possibility that the person is not affected. The method for setting the cutoff value is not particularly limited. For example, measurement values obtained from a group of healthy subjects can be classified by percentile, and the percentile value used for the classification can be used as a cutoff value. Specifically, when classifying a subject's measurement value positive or negative by the 95th percentile of the measurement value obtained from a group of healthy subjects, the 95th percentile is a cut-off value. In addition, it can be set based on the calculation method described in Akobeng AKActa, 2007, Paediatr, 96: 644-647.

When using the percentile value as a cut-off value in this specification, use either the 5th percentile or the 95th percentile, positive if less than 5th percentile or greater than 95th percentile, greater than 5th percentile, or less than 95th percentile Negative. For example, if the measured value of the hsa-miR-6510-5p marker in a subject shows a value less than the 5th percentile of the healthy group, the subject may be suffering from schizophrenia. It is high and can be subdivided into hsa-miR-6510-5p subtypes of schizophrenia.

As mentioned above, general mental illness has been diagnosed by the results of interviews with the person or family, progress status, structured diagnostic interviews, etc. There is a problem that a large difference occurs in the pharmacological effect of (for example, an antidepressant). One reason for this is that the category of mental illness diagnosed by the conventional method is too large, and there are several pathological conditions (subtypes referred to in this specification) within the same mental illness. It is possible that only a pharmacological effect was observed. By using the mental illness determination marker of the present invention, which can subdivide each mental illness into subtypes, not only follows the conventional hypothetical mental illness diagnostic criteria but also reflects the molecular pathology of each psychiatric illness. Classification with higher objectivity based on the basis is possible.

In addition, the use of the psychiatric disorder determination marker of the present invention enables optimal treatment according to each subtype that may exist in the same psychiatric disorder. The above psychiatric disease markers that can be subdivided into subtypes have high psychiatric disease specificity and can accurately determine schizophrenia, bipolar disorder, or depression, and are not newly classified by conventional methods. Schizophrenia / depression (discussed below) and depression / bipolar disorder (discussed below), which are various mental illness groups, can be determined with high accuracy.

Table 1 shows the 5th percentile of healthy subjects as a cut-off value, and miRNAs that deviate from that value and show abnormal values below the 5th percentile are shown as mental disease determination markers that can be subdivided into subtypes. Specifically, miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 1 to 34 have a cutoff value of the 5th percentile of healthy individuals, and if the value is less than that value, in addition to the symptomatic diagnosis of schizophrenia, schizophrenia Can be subdivided into subtypes.

In addition, the measured values of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 5 and 40 to 45 in the psychiatric disease determination markers belonging to this group are correlated with the schizophrenia evaluation scale. Therefore, the severity of schizophrenia can be identified.

In this specification, “severity” refers to the degree of symptoms based on an evaluation scale for each mental disorder. As an evaluation scale for schizophrenia, PANSS (Positive) and Negative Syndrome Scale) may be used. The miRNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 40 to 45 is a psychiatric disorder determination marker that can identify the severity of schizophrenia that correlates with r> 0.5 in Pearson correlation analysis using PANSS as an evaluation scale.

In the psychiatric disorder determination marker capable of identifying the severity of schizophrenia, the miRNA containing the nucleotide sequence represented by SEQ ID NO: 5 is not only for the severity of schizophrenia but also for the identification and subtype of schizophrenia disease. It is a psychiatric disease marker that can be identified.

(2) Group for determining depression Table 2 shows the markers for determining mental disorders belonging to Group 2.

The mental disease determination marker belonging to this group is a marker that can determine depression. Specifically, it refers to a miRNA selected from the group consisting of miRNAs comprising the base sequences represented by any of SEQ ID NOs: 46 to 142, which are markers belonging to the compartments represented by group 2 in FIG. Using this group of psychiatric disease determination markers, it is possible to identify the morbidity of depression, identify the subtype of depression, and / or identify the severity of depression in the subject.

In Table 2, miRNAs showing p <0.05 in the t-test are indicated with a “◯” mark as a psychiatric disorder determination marker having a difference from normal.

In the psychiatric disorder determination marker belonging to this group, the miRNA containing the nucleotide sequence represented by SEQ ID NOs: 46 to 130 can distinguish between depression in a subject. Among these, miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 46, 48, 50, 56, 62, 79, 82, 85, 94, and 114 to 130 are samples derived from cerebrospinal fluid between depressed patients and healthy individuals. Is a biomarker isolated on the basis of a significant difference in the abundance of the miRNA contained in.

In addition, miRNAs containing the nucleotide sequences shown in SEQ ID NOs: 46 to 113 are biomarkers that can subdivide depression into subtypes. In Table 2, the 95th percentile and the 5th percentile of healthy subjects are shown as cut-off values, and miRNAs showing abnormal values larger than the 95th percentile or less than 5th percentile are shown as psychiatric disease determination markers that can be subdivided into subtypes.

For example, the miRNA containing the base sequences shown in SEQ ID NOs: 46 to 111 has a 95th percentile of healthy subjects as a cut-off value, and if it is larger than that value, it is added to the diagnosis of depression, and depression is subtyped. Can be subdivided. On the other hand, the miRNA containing the base sequence represented by SEQ ID NO: 112 or 113 has a cut-off value of the 5th percentile of healthy individuals, and if it is less than that value, it is added to the diagnosis of depression, and depression is subtyped. Can be subdivided.

Since the measured value of miRNA containing the nucleotide sequences represented by SEQ ID NOs: 49, 80, 81, 83 and 131 to 142 in the mental disease determination marker belonging to this group shows a correlation with the evaluation scale HAM-D for depression, The severity of depression can be determined. Specifically, the miRNA is a psychiatric disease determination marker capable of identifying the severity of depression that correlates with r> 0.5 in the Pearson correlation function using HAM-D as an evaluation scale. Here, the mental disease determination markers represented by SEQ ID NOs: 49, 80, 81, and 83 are also mental disease determination markers for identifying a subtype of depression.

(3) Group for determining bipolar disorder Table 3 shows markers for determining mental disorders belonging to Group 3.

The mental disease determination marker belonging to this group is a marker that can determine bipolar disorder. Specifically, it refers to a miRNA selected from the group consisting of miRNAs comprising the base sequences represented by any of SEQ ID NOs: 143 to 176, which are markers belonging to the compartments represented by group 3 in FIG. With the use of this group of psychiatric disease determination markers, it is possible to identify the presence of bipolar disorder in a subject and / or to identify a subtype of bipolar disorder.

In the psychiatric disorder determination marker belonging to this group, the miRNA containing the nucleotide sequence represented by SEQ ID NOs: 143 to 176 can distinguish the presence of bipolar disorder in a subject. Among these, miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 158 to 176 were isolated based on a significant difference in the abundance of the miRNAs contained in cerebrospinal fluid-derived samples between bipolar disorder patients and healthy subjects. It is a biomarker.

In Table 3, miRNAs showing p <0.05 in t-test are indicated with “◯” as a mental disease determination marker having a difference from normal.

In addition, miRNAs containing the base sequences represented by SEQ ID NOs: 143 to 157 are biomarkers that can subdivide bipolar disorder into subtypes. In Table 3, miRNAs showing normal values of 95th percentile and 5th percentile of healthy subjects as cut-off values, and abnormal values larger than 95th percentile or less than 5th percentile are shown as psychiatric disease determination markers that can be subdivided into subtypes.

For example, the miRNA containing the base sequence represented by SEQ ID NO: 143 has the 95th percentile of a healthy person as a cut-off value. Can be subdivided. On the other hand, miRNAs containing the nucleotide sequences shown in SEQ ID NOs: 144 to 157 have a cutoff value of the 5th percentile of healthy subjects. Can be subdivided into types.

(4) Group for determining schizophrenia / depression Table 4 shows the markers for determining mental disorders belonging to Group 4.

The mental disease determination marker belonging to this group is a marker that can determine schizophrenia or depression. The term “schizophrenia or depression” as used herein (often referred to as “schizophrenia / depression” in this specification) is a mental disorder of either schizophrenia or depression in the conventional classification method. A psychiatric disorder that has been classified as, and has the characteristics of both schizophrenia and depression according to the determination method of the present invention based on a biological basis using a biomarker. A new group of mental disorders that cannot be classified. Specifically, it refers to a miRNA comprising a base sequence represented by any of SEQ ID NOs: 177 to 187, which is a marker belonging to the section represented by group 4 in FIG. Use of this group of mental illness determination markers can distinguish that a subject is likely to have schizophrenia / depression.

The miRNA containing the nucleotide sequence shown in SEQ ID NOs: 177 to 187 is also a biomarker that can subdivide schizophrenia / depression into subtypes. These can be subdivided into subtypes in addition to the classification of schizophrenia / depression when the 5th percentile of healthy subjects is cut off and shows an abnormal value below the 5th percentile.

(5) Group for determining depression / bipolar disorder Table 5 shows markers for determining mental disorders belonging to Group 6.

The mental disease determination marker belonging to this group is a marker that can determine depression or bipolar disorder. As used herein, “depression or bipolar disorder” (herein often referred to as “depression / bipolar disorder”) is a mental disorder of either depression or bipolar disorder in the conventional taxonomy. A psychiatric disorder that has been classified as, and has characteristics of both psychiatric disorders of depression and bipolar disorder according to the determination method of the present invention based on a biological basis using a biomarker. A new group of mental disorders that cannot be classified. Specifically, it refers to a miRNA comprising the nucleotide sequences represented by SEQ ID NOs: 188 to 191 which are markers belonging to the compartments represented by group 6 in FIG. Use of this group of mental illness determination markers can distinguish that a subject is likely to be suffering from depression / bipolar disorder.

The miRNA containing the nucleotide sequences represented by SEQ ID NOs: 188 to 191 is also a biomarker that can subdivide depression / bipolar disorder into subtypes. If the 95th percentile of a healthy person has a cut-off value and shows an abnormal value greater than that value, it can be subdivided into subtypes in addition to the diagnosis of depression / bipolar disorder.

(6) Group for determining schizophrenia, depression, or bipolar disorder Table 6 shows the markers for determining mental disorders belonging to Group 7.

The mental disease determination marker belonging to this group is a marker that can determine schizophrenia, depression, or bipolar disorder. As used herein, “schizophrenia, depression or bipolar disorder” (herein often referred to as “schizophrenia / depression / bipolar disorder”) refers to schizophrenia in the conventional taxonomy, A mental illness classified as a mental illness of either depression or bipolar disorder, which has characteristics of three psychiatric disorders according to the determination method of the present invention based on a biological basis using a biomarker Mental illness group. Specifically, it refers to a miRNA that is a marker belonging to the compartments shown in group 7 in FIG. 1 and includes the base sequence shown in SEQ ID NO: 191 or 192. By using the mental disease determination marker of this group, it can be determined that the subject is highly likely to suffer from schizophrenia / depression / bipolar mental disease.

The miRNA containing the base sequence represented by SEQ ID NO: 191 or 192 is also a biomarker that can subdivide schizophrenia / depression / bipolar disorder into subtypes. These should be subdivided into subtypes in addition to the classification of schizophrenia / depression / bipolar disorder when the 5th percentile of healthy individuals is cut off and the abnormal value is less than the 5th percentile. Can do.

2. 2. Mental disease determination method 2-1. Outline | summary The 2nd aspect of this invention is a method of assisting mental disease determination. The present invention can biologically determine the presence or absence of a mental illness in a subject without using a doctor using the mental illness determination marker described in the first aspect.

2-2. Determination method 2-2-1. Step This determination method includes a measurement step and a comparison determination step as essential steps. Moreover, a severity determination process is included as a selection process. Hereinafter, each step will be specifically described.

(1) Measurement process The “measurement process” is to measure and measure the amount of the psychiatric disorder determination marker according to the first aspect contained in a unit amount of a sample collected from a subject and a healthy person. It is a process of obtaining a value.

As used herein, “sample” refers to a biological sample that may contain miRNA that is a psychiatric disease determination marker. For example, bodily fluids, cells (including cell extracts), or RNA recovered therefrom, in particular miRNAs. “Body fluid” refers to a liquid biological sample collected directly from a subject. For example, cerebrospinal fluid, blood (including liquid blood components such as serum, plasma and interstitial fluid), or lymph. In the method of this embodiment, a preferred sample is cerebrospinal fluid or blood, or RNA recovered therefrom, particularly miRNA.

“The sample collected” refers to a sample collected from each of the subject and the healthy subject. The collecting method is not particularly limited as long as it is a known method. For example, when the sample is cerebrospinal fluid, it may be collected by lumbar puncture. Lumbar puncture is less invasive because it can reduce pain or less by using a commercially available local anesthetic in advance, and can reduce side effects by using an atraumatic needle, This method is suitable for collecting cerebrospinal fluid. If the sample is blood or lymph, a known blood collection method may be followed. Specifically, in the case of peripheral blood, it may be collected by injection into a peripheral vein or the like. The sample can be used immediately after collection in the determination method of the present invention, but immediately after collection, it can be ice-cooled, and the supernatant obtained by centrifugation can be thawed and used. Good. Further, if necessary, dilution or concentration, or a blood coagulation inhibitor such as heparin can be added.

The “unit amount” is a predetermined unit of volume or weight, and examples thereof include microliter, milliliter, microgram, milligram, gram and the like.

In the present specification, the “measured value” is a value indicating the amount of the psychiatric disorder determination marker measured in this step. The measured value may be an absolute value such as volume or weight, or a relative value such as concentration, ionic strength, absorbance or fluorescence intensity.

In this step, the amount of the psychiatric disease determination marker contained in each of the sample derived from the subject (referred to as “subject sample”) and the sample derived from the healthy subject sample (referred to as “normal subject sample”) is measured.

The mental disease determination marker to be measured by this method may be selected according to the mental disease to be determined. For example, when determining whether or not a subject suffers from schizophrenia / depression / bipolar disorder, the mental disease determination marker belonging to Group 7 described in the first aspect may be selected. Moreover, what is necessary is just to select the mental disease determination marker which belongs to 4 groups as described in a 1st aspect, when determining whether a test subject is suffering from schizophrenia / depression. When determining whether or not a subject is suffering from depression / bipolar disorder, the mental disease determination marker belonging to Group 6 described in the first aspect may be selected. Moreover, what is necessary is just to select the mental disease determination marker which belongs to 1 group as described in a 1st aspect, when determining whether a test subject suffers from schizophrenia. When determining whether or not the subject suffers from depression, the mental disease determination marker belonging to Group 2 described in the first aspect may be selected. And when determining whether a test subject suffers from bipolar disorder, what is necessary is just to select the mental disease determination marker which belongs to 3 groups as described in a 1st aspect.

The method for measuring a psychiatric disorder determination marker is not particularly limited as long as it is a known nucleic acid quantification method. Examples thereof include a nucleic acid amplification method, a hybridization method, and an RNase protection method.

“Nucleic acid amplification method” refers to a method of amplifying a specific region of a target nucleic acid with a nucleic acid polymerase using a forward / reverse primer set. For example, PCR method (including RT-PCR method), NASBA method, ICAN method, LAMP (registered trademark) method (including RT-LAMP method) can be mentioned. The PCR method is preferred. In the present invention, since the psychiatric disorder determination marker is miRNA, a nucleic acid amplification method via reverse transcription reaction (RT reaction), for example, RT-PCR method or RT-LAMP method is usually employed. In particular, in the present invention, since it is necessary to measure the amount of a psychiatric disorder determination marker present in a sample such as a body fluid, it is preferable to use a quantitative nucleic acid amplification method such as a real-time RT-PCR method. However, the nucleic acid to be detected in the present invention is a miRNA having a mature form of about 20 bases. Therefore, the target nucleic acid is too short for normal quantitative nucleic acid amplification and cannot be amplified properly. Therefore, amplification may be performed using a miRNA amplification kit or a special primer commercially available from each manufacturer. One example is Applied Biosystems TaqMan MicroRNA Assays Kit, which is commercially available from Life Technologies (currently Thermo Fisher Fisher Scientific). Use of each miRNA-specific Looped RT primer included in this kit is useful because it enables efficient amplification after reverse transcription of the target mature miRNA. The Looped RT primer self-forms a hairpin structure with several bases protruding at the 3 'terminal part having a sequence complementary to the 3' terminal region of the target mature miRNA. After the protruding 3 'terminal portion anneals with the 3' terminal region of the target miRNA, the target miRNA is extended with RTase as a template. Thereafter, by performing normal real-time PCR using the extension product as a template, the target miRNA can be specifically amplified.

The reaction conditions for real-time PCR are generally based on known PCR methods, the base length of the nucleic acid fragment to be amplified and the amount of template nucleic acid, the base length and Tm value of the primer to be used, and the optimal reaction of the nucleic acid polymerase to be used. Since it fluctuates depending on temperature, optimum pH, etc., it may be determined appropriately according to these conditions. For example, a denaturation reaction is usually performed at 94 to 95 ° C. for 5 seconds to 1 minute, an annealing reaction is performed at 50 to 70 ° C. for 10 seconds to 1 minute, and an extension reaction is performed at 68 to 72 ° C. for 30 seconds to 3 minutes. One cycle can be repeated for about 15 to 40 cycles, and finally an extension reaction can be performed at 68 to 72 ° C. for 30 seconds to 10 minutes. When using a kit commercially available from the manufacturer, the protocol attached to the kit may be used in principle.

The nucleic acid polymerase used in real-time PCR is a DNA polymerase, particularly a heat resistant DNA polymerase. Various types of such nucleic acid polymerases are commercially available, and they can also be used. For example, Taq DNA polymerase attached to Applied Biosystems TaqMan MicroRNA Assays Kit (Life Technologies (currently Thermo Fisher Fisher Scientific)) can be mentioned. In particular, such a commercially available kit is useful because a buffer optimized for the activity of the attached DNA polymerase is attached.

The “hybridization method” is a method in which a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of a target nucleic acid to be detected is used as a probe, and base pairing between the nucleic acid and the probe is used. This is a method for detecting and quantifying a target nucleic acid or a fragment thereof. Several methods with different detection means are known as hybridization methods. In the present invention, since the target nucleic acid is miRNA, for example, Northern hybridization method (Northern blot hybridization method), RNA microarray method The surface plasmon resonance method or the quartz crystal microbalance method is preferable.

The “Northern hybridization method” is the most common method for analyzing gene expression. RNA prepared from a sample is separated by electrophoresis on agarose gel or polyacrylamide gel under denaturing conditions, and transferred to a filter (blotting). ), And then detecting the RNA using a probe having a base sequence specific to the target RNA. By labeling the probe with an appropriate marker such as a fluorescent dye or a radioisotope, for example, a chemirumi (chemiluminescence) imaging analyzer (for example, Light Capture; Atto), a scintillation counter, an imaging analyzer (for example, FUJIFILM) : The target RNA can also be quantified using a measuring device such as BAS series. Northern hybridization is a well-known technique in the field, such as Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New See York.

“RNA microarray method” is a method in which DNA microarray method is applied to RNA. A nucleic acid fragment complementary to all or part of the base sequence of a target nucleic acid on a substrate is placed as a probe in a high density in a small spot and solid-phased. This is a method for detecting and quantifying a nucleic acid hybridized to a spot by fluorescence or the like. Detection and quantification can be achieved by detecting and measuring fluorescence based on hybridization of a target nucleic acid or the like with a microplate reader or a scanner. The RNA microarray method is also a well-known technique in the art. For example, see DNA microarray method (DNA microarray and latest PCR method (2000) Masaaki Muramatsu, supervised by Hiroyuki Nami, Shujunsha).

"Surface plasmon resonance (SPR) method" means that the intensity of reflected light is significantly attenuated at a specific incident angle (resonance angle) when the incident angle of the laser beam applied to the metal thin film is changed. This is a method for detecting and quantifying the adsorbate on the surface of the metal thin film with high sensitivity using the phenomenon. In the present invention, for example, after immobilizing a nucleic acid probe having a sequence complementary to the base sequence of the target miRNA on the surface of the metal thin film and blocking the other metal thin film surface portion, the sample was collected from the subject as a sample. The target miRNA can be detected and quantified from the difference in the measured values before and after the sample flow by forming a base pairing between the target miRNA and the nucleic acid probe by circulating blood on the surface of the metal thin film. Detection and quantification by the surface plasmon resonance method can be performed using, for example, an SPR sensor commercially available from Biacore. This technique is well known in the art. See, for example, Kazuhiro Nagata and Hiroshi Handa, real-time analysis experiment of biological material interactions, Springer Fairlake Tokyo, Tokyo, and 2000.

The "quartz crystal microbalance (QCM) method" uses the phenomenon that the resonance frequency of a crystal resonator decreases according to its mass when a substance is adsorbed on the electrode surface attached to the crystal resonator. This is a mass measurement method that quantitatively captures a very small amount of adsorbate based on the amount of change in resonance frequency. The detection and quantification by this method were also collected from a subject using a commercially available QCM sensor as in the SPR method, for example, a nucleic acid probe having a sequence complementary to the base sequence of the target miRNA immobilized on the electrode surface. Target miRNA can be detected and quantified by base pairing with target miRNA in blood. This technique is well known in the art, for example, J. Christopher Love, LAEstroff, JKKriebel, RGNuzzo, GMWhitesides (2005) Self-Assembled Monolayers of a Form of Nanotechnology, Chemical Review, 105: 1103-1169 See Toyotomi Moriizumi, Takamichi Nakamoto, (1997) Sakai Sensor Engineering, Shoshodo.

The probe used in the hybridization method may be a nucleic acid fragment having a base sequence complementary to all or part of the base sequence represented by SEQ ID NOs: 1 to 193. The base length of the probe is 8 bases or more, preferably 10 bases or more, more preferably 12 bases or more, still more preferably 15 bases or more, or less than the full length. The nucleic acid constituting the probe is not particularly limited. Usually, DNA that can be synthesized at low cost and has high stability may be used, but if necessary, all or part of PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate-type DNA, phosphorothioate-type It may also include chemically modified nucleic acids such as DNA and 2′-O-methyl RNA, pseudo-nucleic acids, or combinations thereof. Probes used in the hybridization method include fluorescent dyes (for example, fluorescamine and derivatives thereof, rhodamine and derivatives thereof, FITC, cy3, cy5, FAM, HEX, VIC), quencher substances (TAMRA, DABCYL, BHQ-1, Modification or labeling with BHQ-2 or BHQ-3), biotin or (strept) avidin, modifying substances such as magnetic beads, or radioisotopes (eg, ³² P, ³³ P, ³⁵ S) Can do. Hybridization is preferably performed under stringent conditions in order to exclude non-target nucleic acids that hybridize nonspecifically. High stringent conditions at low salt concentration and high temperature are more preferable. For conditions of highly stringent hybridization, see Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

The “RNA protection method” is a method in which a target RNA and probe are hybridized using a probe having a base sequence complementary to the target RNA, and then the hybridized RNA that is free from degradation by RNase treatment is electrophoresed. In this method, the target RNA is detected and quantified by separating and detecting the target RNA. The method of separation and detection by electrophoresis is basically the same as the hybridization method.

In addition, the measurement value of the healthy person in the measurement step may be determined by measuring the amount of each psychiatric disease determination marker in a sample obtained from a healthy person or a group of healthy persons in advance, and using it as a database. Good.

In this step, in order to correct the measured values of the subject and the healthy person, a known miRNA expected to have no quantitative difference in the sample per unit amount may be further measured as an endogenous control. Examples of such endogenous control miRNA include hsa-miR-93-5p.

(2) Comparison determination process The “comparison determination process” is a process of determining the morbidity of a mental illness in a subject based on the measurement value obtained in the measurement process.

The comparison of the measured values is a cut-off obtained between the measured values of the subject obtained in the measuring step and the measured values of the healthy person or the healthy person group, or based on the measured values of the subject and the healthy person group. Do by value. At this time, the measurement value of the subject and the healthy subject can be corrected using the measurement value of the endogenous control miRNA.

Judgment is made when the measured value of the subject is significantly higher or lower than the measured value of the healthy subject or the group of healthy subjects, or when the subject is classified as positive by the cut-off value, the subject is determined to have the mental disease selected in the measuring step. It is determined that there is a high possibility of having a mental illness that can be determined with a marker. For example, when a mental disease determination marker including a base sequence represented by SEQ ID NOs: 35 to 39 belonging to one group described in the first aspect is selected in the measurement step, the measured value in the subject is more than the measured value of the healthy subject group When significantly higher, it can be determined that the subject is likely to have schizophrenia. In addition, when a psychiatric disease determination marker including a base sequence represented by SEQ ID NOs: 1 to 34 belonging to one group described in the first aspect is selected in the measurement step, the measurement value is previously determined based on the measurement value of the healthy subject group. If the subject is classified as positive according to the determined cut-off value, it can be determined that the subject is likely to have schizophrenia. On the other hand, if the measured value is not significantly high, or if the measured value is classified as negative by the cut-off value, it is determined that the subject is likely not suffering from schizophrenia.

In addition, a psychiatric disease determination marker comprising the base sequences represented by SEQ ID NOs: 46, 48, 50, 56, 62, 79, 82, 85, 94, and 114 to 130 belonging to Group 2 described in the first aspect was used. If the measurement value of the subject is significantly higher than the measurement value of the healthy person, the subject is determined to be highly likely to suffer from depression. In addition, when a psychiatric disease determination marker including the nucleotide sequence represented by SEQ ID NOs: 46 to 113 belonging to Group 2 is used, the marker is positively classified according to a predetermined cutoff value based on the measurement value of the healthy group The subject is determined to have a high probability of suffering from depression.

When the mental disease determination marker comprising the base sequence represented by SEQ ID NOs: 158 to 176 belonging to Group 3 described in the first aspect is used, if the measured value of the subject is significantly higher than the measured value of the healthy subject, the subject Determine that they are more likely to have bipolar disorder. In addition, when a psychiatric disease determination marker including the nucleotide sequence represented by SEQ ID NOs: 143 to 157 belonging to Group 3 is used, the marker is positively classified according to a predetermined cutoff value based on the measurement value of the healthy group The subject is determined to be more likely to have bipolar disorder.

When used as a psychiatric disease determination marker comprising the base sequences represented by SEQ ID NOs: 177 to 187 belonging to the four groups described in the first aspect, a cutoff value (5th percentile) determined in advance based on the measurement values of the healthy group ), The subject is determined to have a high probability of suffering from schizophrenia / depression.

In the case of using a psychiatric disorder determination marker including the base sequence represented by SEQ ID NOs: 188 to 191 belonging to group 6 described in the first aspect, a cutoff value (95th percentile) determined in advance based on the measurement value of the healthy group ), If the subject exhibits an abnormal value greater than the 95th percentile, it is determined that the subject is likely to have depression / bipolar disorder.

When the mental disease determination marker including the nucleotide sequence represented by SEQ ID NO: 192 or 193 belonging to Group 7 according to the first aspect is used, the measurement value of the subject is a predetermined cut based on the measurement value of the healthy group If the subject is classified positive by an off-value (5th percentile), ie, shows an abnormal value below the 5th percentile, the subject is likely to have schizophrenia / depression / bipolar disorder To do.

(3) Severity determination step The “severity determination step” is a step selected when identifying the severity of a mental illness.

In this process, it is assumed that the mental illness is identified and specified in the comparison and determination process. That is, the severity determination step is a supplementary step of further identifying the severity of the mental illness on the assumption that the subject suffers from schizophrenia, bipolar disorder, or depression. This process is performed after the comparison determination process in principle, but can be performed simultaneously with the comparison determination process.

A mental disease determination marker (severity identification marker) that can identify the severity of a mental illness indicates that the measured value in the sample of the marker correlates with a predetermined evaluation scale of each mental illness. Therefore, a calibration curve indicating the relationship between the measured value of each severity identification marker and the evaluation scale may be created in advance. From the measured value of the severity identification marker in the subject and the calibration curve, the severity of the mental illness in the subject can be easily identified. For example, when the measured value of the mental illness determination marker and the value of the evaluation scale are in a positive proportional relationship, it can be determined that the severity of the mental illness is high if the measured value is high.

In this step, when the severity of schizophrenia is identified, miRNAs containing the nucleotide sequences shown in SEQ ID NOs: 5 and 40 to 45 are used as psychiatric disorder determination markers. The measurement value of the marker is correlated with PANSS. In addition, when identifying the severity of depression, miRNA selected from the group consisting of miRNAs containing base sequences represented by SEQ ID NOs: 49, 80, 81, 83, and 131 to 142 are used. The measured values of these psychiatric disease determination markers are correlated with HAM-D.

2-2-2. Combination In the determination method of this aspect, any one of the mental disease determination markers described in the first aspect may be used in the measurement step, but two or more may be combined and measured. By measuring in combination with a plurality of markers for determining mental illness, the accuracy of determining mental illness can be further enhanced.

The combination of psychiatric disorder determination markers may be between different groups. For example, by selecting one psychiatric disease determination marker belonging to each group described in the first aspect and measuring them in combination, it is determined whether or not the subject is afflicted with mental illness and If so, you can classify which group is affected.

In addition, the combination of psychiatric disorder determination markers may be from the same group. Thereby, it becomes possible to increase the accuracy of the subject suffering from the mental illness belonging to the group and to determine the severity. For example, there is a case where hsa-miR-6510-5p of SEQ ID NO: 1, hsa-miR-4753-5p of SEQ ID NO: 35 and hsa-miR-3925-5p of SEQ ID NO: 40 are selected from one group. Hsa-miR-6510-5p and hsa-miR-4753-5p were used to verify whether the subject was schizophrenic or not, and if it was schizophrenia, its severity was assessed using hsa-miR-3925-5p Can be measured.

<Isolation of a marker for determining mental illness>
1. Sample collection and sample collection The sample provider for isolating the psychiatric disorder determination marker of the present invention is the National Psychiatric and Neurological Research Center (Tokyo, Japan) website, free paper advertisement, or center hospital. We recruited with recruitment posters posted inside. As a result, more than 300 donors were obtained. Table 7 shows sample information of a total of 106 patients, 30 patients with schizophrenia, 30 patients with depression, 16 patients with bipolar disorder, and 30 healthy individuals as controls.

In the table, N indicates the total number of each mental illness and healthy person, and DF indicates the number of patients not taking the drug (drug free). These 106 cerebrospinal fluids were used as specimens in this example. Patients were diagnosed in accordance with DSM-IV (Diagnostic and Statistical, Manual, of Mental, Disorders, 4th, Ed.) From interviews and medical history of two or more psychiatrists. Healthy subjects excluded mental illnesses using the simplified structured interview method (MINI). The subjects explained the purpose of the study, the flow of specimens, the frequency of side effects, etc., and consent was obtained in writing. The study has been approved by the National Center for Psychiatry and Neurology Research Ethics Committee. The subjects are all unrelated Japanese.

The method of collecting cerebrospinal fluid from the subject was performed according to the conventional method. First, after neurological examinations including fundus examination, local anesthesia is performed between the 3rd and 4th lumbar vertebrae and the 4th and 5th lumbar vertebrae, and lumbar puncture is performed with an atraumatic needle (22G, Unisys, UNIEVER puncture needle) And about 10 mL of cerebrospinal fluid was collected. After the start of collection, the first approximately 2 mL is used for general specimen testing (cell count, total protein amount, sugar, etc.), and the next approximately 8 mL is used for isolating psychiatric disease markers (Sumitomo Bakelite, Proteo). Save 15 mL). After collection, the cerebrospinal fluid was immediately ice-cooled and centrifuged at 4000 g for 15 minutes at 4 ° C. Thereafter, 0.5 mL of the supernatant was dispensed into a low protein adsorption tube and stored in an ultra-low temperature bath at −80 ° C. until use.

2. Analysis of miRNA in cerebrospinal fluid For analysis of miRNA in cerebrospinal fluid, a contract analysis service provided by Toray Industries, Inc. using 3D-Gene miRNA Oligo chip was used. 3D-Gene can analyze more than 2000 miRNAs registered in miRBase (http://www.mirbase.org/) using a microarray developed by the company (Konishi et al., 2012, British Journal of Cancer, 106: 740-747; Hisaoka et al., 2012, Genes Chromosomes Cancer, 50: 137-145).

MiRNA was extracted from 500 μL of cerebrospinal fluid of each case. Samples were once divided into 250 μL, and RNA was extracted from each using 3D-Gene RNA extraction reagent from liquid sample from Toray Industries, Inc., and mixed to obtain an RNA sample of one case. RNA was labeled with Hy5 and hybridized with 3D-Gene miRNA Oligo chip chip at 32 ° C for 16 hours.

In the analysis, a value 2 × standard deviation higher than the background was used. Furthermore, statistical analysis was performed on the molecules for which values were obtained in 27 or more of 30 healthy subjects as controls. In the statistical analysis, the psychiatric disease determination marker of the present invention was isolated based on the following three criteria.

(1) Regarding the measured value of each molecule, the normal value was determined as the 5th to 95th percentile from the data of 30 healthy subjects, and the molecule showing abnormal value in approximately 20% or more of each disease was selected as a marker for determining mental illness .

(2) Regarding the measured value of each molecule, a molecule having a large difference in the average value between each disease group and the healthy subject group (effective amount Cohen's D was approximately 0.6 or more) was selected as a psychiatric disease determination marker.

(3) Regarding the measured value of each molecule, a molecule whose correlation with symptom evaluation in each disease group was approximately 0.5 or more was selected as a psychiatric disease determination marker using Pearson's product moment correlation coefficient.

3. Results In accordance with the above criteria, 193 mature miRNAs consisting of the nucleotide sequences represented by SEQ ID NOs: 1 to 193, which are extremely useful as biomarkers for determining mental illness, were selected. The groups shown in Tables 1 to 6 are obtained by classifying each miRNA with respect to its attributes, which correspond to the groups 1 to 4, 6 and 7 in FIG. Representative data showing the characteristics of biomarkers for determining mental disorders belonging to each group are illustrated in FIGS.

FIG. 2 is a diagram showing a measured value distribution and a cut-off value of hsa-miR-3620-5p represented by SEQ ID NO: 65 belonging to Group 2. The cutoff value was the 95th percentile in the healthy group. Eight out of 30 patients with depression were more positive than the cutoff value. On the other hand, in the schizophrenia patient group (0 of 30 cases) and the bipolar disorder patient group (2 cases of 16 cases), most of them were negative below the cut-off value. Therefore, some of the mental disease determination markers belonging to the two groups, such as hsa-miR-3620-5p, become a mental disease determination marker for depression by setting a cut-off value. It has also been shown to be a psychiatric disease determination marker for determining type. Mental illness determination markers having the same characteristics as above were also confirmed for psychiatric illness determination markers for schizophrenia belonging to Group 1 and some of psychiatric disease determination markers for bipolar disorder belonging to Group 3.

FIG. 3 is a diagram showing the relationship between the measured value of depression by hsa-miR-1908 indicated by SEQ ID NO: 131 belonging to Group 2 and the evaluated value by the evaluation scale HAM-D. As shown in this figure, there is a correlation between the hsa-miR-1908 concentration and the symptom evaluation scale HAM-D, and the lower the hsa-miR-1908 concentration, the more severe the symptoms. Therefore, the severity of depression can be determined by using hsa-miR-1908 as a marker for determining mental disorders of depression. Mental illness determination markers having the same characteristics as above were also confirmed for psychiatric illness determination markers for schizophrenia belonging to Group 1 and some of psychiatric disease determination markers for bipolar disorder belonging to Group 3.

FIG. 4 is a diagram showing the measured value distribution and cut-off value of hsa-miR-3180 represented by SEQ ID NO: 190 belonging to Group 6. The cutoff value was the 95th percentile in the healthy group. Eight out of 30 patients with depression and 4 out of 16 bipolar disorder were more positive than the cut-off value. On the other hand, most of the patients with schizophrenia (1 of 30) were negative below the cut-off value. Therefore, a mental disease determination marker belonging to 6 groups such as hsa-miR-3180 becomes a mental disease determination marker for depression / bipolar disorder by setting a cut-off value, and depression / bipolar disorder. It has also been shown to be a psychiatric disorder determination marker for determining subtypes of Mental illness determination markers showing similar characteristics were also confirmed for schizophrenia / depression mental illness determination markers belonging to Group 4.

FIG. 5 is a diagram showing a measured value distribution and a cut-off value of hsa-miR-4749-5p represented by SEQ ID NO: 193 belonging to Group 7. The cutoff value was the 5th percentile in the healthy group. With this marker, 12 of 30 patients with schizophrenia, 7 of 30 patients with depression, and 5 of 16 patients with bipolar disorder were positive below the cut-off value.

Therefore, mental disease determination markers belonging to 7 groups such as hsa-miR-4749-5p become mental disease determination markers for schizophrenia / depression / bipolar disorder by setting a cut-off value, It has also been shown to be a psychiatric disease marker for determining subtypes of schizophrenia / depression / bipolar disorder.

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

Claims

A psychiatric disorder determination marker selected from the group consisting of miRNAs containing the base sequences represented by SEQ ID NOs: 1 to 193.
2. The psychiatric disorder determination marker according to claim 1, wherein the psychiatric disorder is selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 1 to 45, and the psychiatric disorder is schizophrenia.
The psychiatric disorder determination marker according to claim 2, wherein the psychiatric disorder determination marker is selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 1 to 34, and can subdivide schizophrenia into subtypes.
3. The psychiatric disorder determination marker according to claim 2, wherein the psychiatric disorder determination marker is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 5 and 40 to 45, and can identify the severity of schizophrenia.
The psychiatric disorder determination marker according to claim 1, wherein the psychiatric disorder determination marker is selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 46 to 142, and the psychiatric disorder is depression.
The psychiatric disease determination marker according to claim 5, which is selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 46 to 113 and can subdivide depression into subtypes.
6. The psychiatric disorder determination marker according to claim 5, wherein the marker is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 49, 80, 81, 83 and 131 to 142, and the severity of depression can be identified.
2. The psychiatric disease determination marker according to claim 1, wherein the psychiatric disease determination marker is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 176, and the psychiatric disorder is bipolar disorder.
The psychiatric disease determination marker according to claim 8, which is selected from the group consisting of miRNAs containing the base sequences represented by SEQ ID NOs: 143 to 157 and can subdivide bipolar disorder into subtypes.
2. The psychiatric disorder determination marker according to claim 1, wherein the psychiatric disorder determination marker is selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 177 to 187, and the psychiatric disorder is schizophrenia or depression.
The psychiatric disease determination marker according to claim 1, wherein the psychiatric disease determination marker is selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 188 to 191 and the mental illness is depression or bipolar disorder.
The psychiatric disease determination marker according to claim 1, wherein the psychiatric disease is miRNA comprising the nucleotide sequence represented by SEQ ID NO: 192 or 193, and the psychiatric disease is schizophrenia, depression, or bipolar disorder.
A method for determining mental illness,
The amount of a psychiatric disease determination marker selected from the group consisting of miRNAs comprising a base sequence represented by SEQ ID NOs: 1 to 193, contained per unit amount of a sample collected from a subject and a healthy person, is measured and the measured value is obtained. Measuring process to obtain,
The said method including the comparison determination process which compares the measured value of the test subject obtained by the said measurement process, and a healthy person, and determines the mental disease in a test subject based on the result.
The method according to claim 13, wherein schizophrenia is determined by a psychiatric disorder determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 45.
15. The subtype of schizophrenia is determined based on a measured value of a psychiatric disorder determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 1-34 and a predetermined cut-off value The method described.
The method according to claim 13, wherein depression is determined by a psychiatric disorder determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 46 to 142.
The method according to claim 16, wherein the subtype of depression is further determined based on a measurement value selected from the group consisting of miRNAs containing the nucleotide sequences represented by SEQ ID NOs: 46 to 113 and a predetermined cutoff value.
The method according to claim 13, wherein bipolar disorder is determined by a psychiatric disorder determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 176.
The bipolar disorder subtype is further determined based on a measured value of a psychiatric disease determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 143 to 157 and a predetermined cut-off value. The method described in 1.
The schizophrenia or depression is determined on the basis of a measured value of a psychiatric disorder determination marker selected from the group consisting of miRNAs containing the base sequences represented by SEQ ID NOs: 177 to 187 and a predetermined cut-off value. The method described.
The depression or bipolar disorder is determined on the basis of a measured value of a psychiatric disease determination marker selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 188 to 191 and a predetermined cutoff value, The method described.
The method according to claim 13, wherein schizophrenia, depression or bipolar disorder is determined from a psychiatric disorder determination marker comprising miRNA comprising the base sequence represented by SEQ ID NO: 192 or 193.
Severity that determines the severity of schizophrenia based on the measured value of the amount of a marker for determining a psychiatric disorder selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 5 and 40 to 45 obtained in the measurement step The method of claim 14, further comprising a determining step.
The severity of depression based on the measured value of the amount of marker for determination of mental illness selected from the group consisting of miRNAs comprising the nucleotide sequences represented by SEQ ID NOs: 49, 80, 81, 83 and 131 to 142 obtained in the measurement step The method according to claim 16, further comprising a severity determination step of determining.
The method according to any one of claims 13 to 24, wherein two or more different psychiatric disease determination marker amounts are measured in the measurement step.
The method according to any one of claims 13 to 25, wherein the sample is cerebrospinal fluid.