JP2008079537A - Method for diagnosing depression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for evaluating readily, objectively and highly accurately the presence or absence of depression in a subject. <P>SOLUTION: The method for diagnosing the depression using RNA derived from the whole blood of the subject includes previously defining a gene expressed more in differential leukocyte count, as compared to that in the differential erythrocyte count in a microarray analysis that uses the only prescribed gene as object, correcting the center value of logarithmic expression ratios to a controlled sample to zero, and determining the presence or absence of the depression in the subject, by using the corrected logarithmic expression ratio. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for diagnosing depression. More specifically, by predefining a probe of a transcription product that is highly expressed in leukocytes and performing data correction using the specified probe, the subject's whole blood-derived RNA is used to affect depression. The present invention relates to a method for diagnosing depression characterized by determining the presence or absence of depression.

  Depression is a frequent disease with a lifetime prevalence of around 10%, and its frequency is expected to increase further in the future, driven by the stress of modern society. This disease is a serious disease that not only causes serious distress to the mental and physical health of the affected person, but also has a major impact on their social life, and often leads to suicide. It is estimated that the majority of suicides, more than 30,000 per year, were suffering from depression. It is also closely related to social issues such as neglect, job loss, withdrawal, and medical issues such as alcohol-related disabilities. Establishing a system for accurately diagnosing and promptly treating this disease is indispensable for improving people's lives and is an urgent need for society as a whole.

  Diagnosing depression, however, is never easy. The main symptoms of depression are depression, motivation, loss of interest and joy, loss of concentration and attention, self-evaluation and confidence, guilt and worthlessness, pessimism for the future, suicidal ideation, sleep Disability, loss of appetite. These symptoms have unique characteristics, which are different from the mood depression experienced by everyone, and are also different from the mental activity decline associated with exhaustion when suffering from physical illness. In order to understand the symptoms of depression, we will take a detailed medical history and ask how and when symptoms appearing in psychological behavior appear and what kind of problems are occurring in social and family life. However, it is mainly to check various symptoms from the patient's attitude at the time of consultation and the content of conversation. Family history, past medical history, physical health, growth history, life history, personality tendency, pre-illness social adaptation status, presence or absence of events that triggered the event, etc. are important reference items. In order to grasp these accurately, an interview for about an hour by a sufficiently skilled psychiatrist is required. Furthermore, it is confirmed that there are no major abnormalities in general physical condition and neurological condition, and diagnosis is made by excluding cerebral organ disease by electroencephalogram or brain image examination as necessary. It is common to confirm the diagnosis by comparing the findings with diagnostic criteria from the World Health Organization (WHO) and the American Psychiatric Association.

  A major problem with conventional methods for diagnosing depression is that skills required for diagnosis are required. Needless to say, sufficient knowledge and experience about depression is necessary, but there are many psychological, mental, and physical conditions that can cause depression if it does not fall into depression. Differential diagnosis with them is also essential. Therefore, there must be a psychiatrist with sufficient training for diagnosis. However, depression, a common disease with a lifetime prevalence of around 10%, often visits a primary care physician. Diagnosis of depression without objective test findings is not always easy for general doctors who are not familiar with psychiatric examinations. Depression is a medical disorder that requires treatment for the body (brain) including pharmacotherapy, and clinical psychology specialists such as clinical psychologists and mental health workers such as public health nurses It is difficult to diagnose alone.

  A major factor in the need for proficiency in the diagnosis of depression is the lack of a simple and objective symptom evaluation method. There is also a screening method using a self-filled questionnaire, but since it is filled in subjectively, it cannot distinguish between depression of mood and personal depression due to personality factors, environmental factors, or poor physical condition. The symptom evaluation scale used by doctors is often used to determine the severity, but this also requires an appropriate interview for the evaluation of each item, and is not a substitute for medical examination.

  Several inspection methods have been tried so far to aim at an objective index. In depression, there is a functional modulation of the monoamine system in the brain, and it is known that the modulation has a considerable influence on the neuroendocrine system, neuroimmune system, and autonomic nervous system through psychosomatic correlation. In particular, an attempt to accurately grasp mild adrenal cortex hormone secretion, which is one of neuroendocrine abnormalities, by dexamethasone suppression test and apply it to the diagnosis of depression was studied energetically after the 1980s. Due to the complexity of taking test drugs and the limitations of sensitivity and specificity, clinical application has not been achieved. In the research stage, other neuroendocrine systems, neuroimmune systems, autonomic nervous systems, circadian rhythms and sleep organization abnormalities have also been reported. Recently, changes in cerebral blood flow and monoamine receptors in the brain have been pointed out, but in both cases, problems remain in sensitivity and reproducibility. In any case, it can be said that it is difficult to evaluate complicated mental illness such as depression by the method of measuring limited factors. In addition, conventional examinations require enormous time and labor to perform and evaluate, and in view of simplicity, it is difficult to hope for application to daily medical care.

  On the other hand, catecholamine hypothesis, indoleamine hypothesis, GABA hypothesis, glutamine hypothesis, dopamine hypothesis, neurogenesis hypothesis, etc. have been proposed as the etiology of depression. Many contradictions have been pointed out to these hypotheses, and no conclusion has been reached so far. Linkage studies and related studies using molecular genetic techniques and susceptibility regions of chromosomes have also been searched by linkage analysis, but predisposition (biological characteristics) is formed by the interaction of multiple genes like depression. In addition, it is extremely difficult to analyze pathogenic genes in diseases caused by environmental factors such as stress. From gene analysis so far, as functional candidate genes related to depression, serotonin transporter, serotonin 1A / 2C receptor, dopamine D2 / D3 receptor, dopamine transporter, tyrosine hydroxylase, tryptophan hydroxylase, Genes such as monoamine oxidase and ATPase have been reported. For example, regarding Na, K-ATPase and psychiatric disorders, a relationship with depression (for example, see Non-Patent Document 1) and mood dysfunction (for example, see Non-Patent Document 2) has been pointed out. In addition, it has been reported that improvement in symptoms by the depression drug carbamazepine correlates with an increase in erythrocyte Na, K-ATPase activity (see, for example, Non-Patent Document 3). However, on the other hand, there are negative follow-up tests for these.

  In addition, sampling techniques are a major problem in the field of clinical research targeting humans. Human whole blood (peripheral blood), which is the object of analysis by the present inventors, has long been used as a non-invasive and easily available sample in medical examinations and medical settings. In the field of gene expression analysis, including DNA chips, RNAs extracted from various organs are often measured in laboratory animal research, but in human research, whole blood is used. Often the extracted RNA is the target of measurement. Sometimes leukocytes are separated by antibody bead method, centrifugation method, filtering method, etc., and RNA extracted from the leukocytes is used as a measurement target. However, the leukocyte separation process is complicated and the leukocyte is stimulated to stimulate gene expression. There is a risk of changes in quantity. On the other hand, problems with whole blood RNA have been pointed out. Whole blood-derived RNA contains a large amount of reticulocyte-derived globin RNA, and it has been pointed out that this is a factor that reduces measurement accuracy in gene expression analysis (see Non-Patent Document 4).

Depress Anxiety 1997; 5: p53-65 J. Basic Clin Physiol Pharmacol 2000; 11 (4): p375-394 Neuropsychobiology 1999; 40 (3): p134-139 Physiol. Genomics 2004; 19: p247-254

  In the result of gene expression analysis using a DNA chip for a whole blood sample, an M-A plot having a shape as shown in FIG. 1 is often observed. The MA plot is a plot often used to show the results of gene expression analysis of DNA chips.The horizontal axis is the log-converted value of the geometric mean of Cy5 and Cy3, and the vertical axis is the log-converted value of the ratio of Cy5 and Cy3. is there. For example, in the gene expression analysis results for samples other than whole blood-derived RNA, such as liver-derived RNA and kidney-derived RNA, the M-A plot having the shape as shown in FIG. 1 is not observed.

  The present inventors pay attention to this phenomenon, separate red blood cells and white blood cells from whole blood, perform gene expression analysis using this, and a group of probes that are highly expressed in red blood cells when whole blood is used as a reference, and Each of the probe groups that was highly expressed in leukocytes was extracted. Then, in the MA plot of the gene expression analysis result using the RNA derived from whole blood, the distribution of these probe groups is analyzed (FIG. 2), and the distribution of erythrocytes and leukocytes in the MA plot are analyzed. We found that there are two distributions of origin distribution. In other words, both the Cy5 sample and Cy3 sample, which are comparatively analyzed, contain both erythrocyte-derived RNA and leukocyte-derived RNA, and the more the mixture ratio differs, the more the right end of the MA plot breaks. It was concluded that the measurement accuracy was reduced because an expression fluctuation ratio itself was affected by the mixture ratio.

  In order to objectively evaluate the presence or absence of depression, the present inventors focused on leukocytes that express many receptors for stress-related factors. Problems to be solved in the gene expression analysis used were clarified.

  One of the means for solving this problem is a method of separating specific blood cells from whole blood. However, the separation process is complicated, and the change in gene expression due to the stimulation of blood cells during the separation process. There are disadvantages such as possible. Therefore, the present inventors performed gene expression analysis of a whole blood sample without going through the process of blood cell separation, and corrected the data for the red blood cell-derived RNA by performing data correction on the probe that is highly expressed in white blood cells in the data analysis process. The impact has been reduced and this issue has been solved.

  That is, the present invention is a method for diagnosing depression using RNA derived from whole blood of a subject, and is expressed more in the leukocyte fraction than in the erythrocyte fraction in gene analysis using a nucleic acid-immobilized sample. In this case, the median logarithmic expression ratio with respect to the control sample is corrected to 0 for only the specified genes, and the corrected log expression ratio is used to determine whether the subject is suffering from depression. The present invention relates to a method for diagnosing depression characterized by determining the presence or absence. The gene analysis using the nucleic acid-immobilized sample includes all nucleic acid-immobilized samples capable of comprehensively analyzing genes such as microarrays (arrays) and chips.

  In the above method, the presence or absence of depression is determined using an evaluation index calculated by averaging the log expression ratio after correction.

  In one embodiment, as the evaluation index, an evaluation index calculated by averaging the logarithmic expression ratios after correction of the gene group described in Table 1 can be used.

  In another embodiment, the 95th percentile of the absolute value of the logarithmic expression ratio after correction of the gene group described in Table 1 can be used as the evaluation index. In this case, for example, if the 95th percentile of the absolute value of the logarithmic expression ratio is 1.15 or more, it can be determined that the patient is suffering from depression.

  The logarithmic expression ratio (variation) that serves as an evaluation index in the diagnostic method of the present invention is a phenomenon caused by changes in the number and activity of neutrophils in the whole blood of a subject. This is because the number and activity of neutrophils in the whole blood of the subject and the composition ratio of each white blood cell including neutrophils, monocytes, B lymphocytes, T lymphocytes, and NK cells in the whole blood of the subject. It is shown that the presence or absence of depression in the subject can be determined by evaluating.

  The present invention also provides a method for evaluating the efficacy of an antidepressant using the aforementioned method for diagnosing depression. For example, whole blood collected before and after administration of an antidepressant in a subject, the logarithmic expression ratio is corrected according to the diagnostic method, and the obtained evaluation index, or the presence or absence (degree) of depression determined from the evaluation index Based on this, the efficacy of the antidepressant in the subject can be evaluated.

  The present invention is also a system for carrying out the above-described method for diagnosing depression, comprising means for obtaining gene expression data in whole blood-derived RNA of a subject using a nucleic acid-immobilized sample, and the nucleic acid-immobilized sample. In gene analysis using, the genes that are expressed more in the leukocyte fraction than in the red blood cell fraction are specified in advance, and the median logarithmic expression ratio for the control sample is corrected to 0 for only the specified gene And a means for determining the presence or absence of depression in the subject using the corrected logarithmic expression ratio. A diagnostic system for depression is provided.

  Furthermore, the present invention preliminarily defines and prescribes genes that are expressed more in the leukocyte fraction than in the erythrocyte fraction in gene analysis using a nucleic acid-immobilized sample using RNA derived from whole blood of a subject. Provided is a gene expression data correction method characterized by correcting the median logarithmic expression ratio with respect to a control sample to 0 for only genes. A conceptual diagram of the depression diagnosis system is shown in FIG.

  According to the present invention, it is possible to easily and objectively and accurately diagnose the presence or absence of depression by gene expression analysis of messenger RNA obtained from only a few cc of whole blood of a subject. Thus, it is possible to easily carry out diagnosis of depression, which has been difficult in the past. The method of the present invention, as compared with the conventional method of measuring a limited factor, grasps biological functions from a large number of RNA expression levels, so that it can be used as a diagnostic method for complex diseases such as depression. It is also suitable in principle and has great utility.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.

[Example 1]
1. Patients and healthy subjects The target patient is the untreated depression patient who visited the University of Tokushima Medical Hospital Psychiatry Neurology between November 2002 and June 2005 for the development of this diagnostic method. It was assumed that consent to participate in the study was obtained in writing. This study was approved by the University of Tokushima Medical School Ethics Committee. The diagnosis was consistent with the depression episode of ICD-10 (International Classification of Diseases 10th edition). Excluded were those with serious physical complications and those taking medications for physical diseases. Healthy individuals selected healthy individuals of matching sex and age for each patient.

  The 18 depressed patients who obtained the pre-treatment samples were 5 males and 13 females, aged 23 to 68 years (average 39.1 years).

2. Gene expression analysis From a patient, 5 cc of blood was collected using a PAXgene Blood RNA System (Qiagen), and total RNA was extracted. Blood was collected from 10 am to 1 pm on an empty stomach, and a doctor or nurse collected blood from the elbow vein at rest. Total RNA yield was 5-15 micrograms.

  Next, 0.5 micrograms of the extracted total RNA was taken out and oligo (dT) 24 primer added with a T7 promoter sequence was annealed, and first strand DNA synthesis was performed first. Next, Second strand DNA having a T7 promoter sequence was synthesized using this First strand DNA as a template. Finally, cRNA was synthesized with T7 RNA polymerase using Second strand DNA as a template, and Cy5-CTP or Cy3-CTP was incorporated into the strand to synthesize fluorescently labeled cRNA.

  After mixing equal amounts of two types of cRNAs for comparative analysis, hybridization was performed at 60 ° C. for 18 hours over a Whole Human Genome Microarray (manufactured by Agilent). After washing, the fluorescence intensity of each spot was measured with a scanner (Agilent Scanner made by Agilent), and the ratio of the expression level of each gene in the Cy5 sample and Cy3 sample was determined. The analysis sample combinations were 18 sets for the patient-universal control combination and 18 sets for the healthy subject-universal control, for a total of 36 sets. The universal control was prepared from a sample of 20 healthy subjects different from the 18 healthy subjects.

  On the other hand, with the cooperation of a healthy person, whole blood sample (PAXgene), red blood cell separation sample (concentration gradient separation method), red blood cell separation sample (filtering method), white blood cell separation sample (concentration gradient separation method), white blood cell separation sample (filtering) Method) was prepared, and total RNA was extracted from each sample. Each blood cell separation sample was labeled with Cy5, the whole blood sample was labeled with Cy3, and the analysis combination was performed as a blood cell separation sample-whole blood combination.

3. 3. Data analysis 3.1 Definition of probes that are highly expressed in leukocytes First, the probes that are highly expressed in leukocytes were defined using four analyzes of blood cell separation samples. Clustering was performed on 21390 probes that had 10 or more signal intensities of Cy5 and Cy3 in all four experiments as data analysis targets (FIG. 3). Using a clustering tree, probes that are highly expressed in leukocytes were selected and 8411 probes were defined.

3.2 Correction of whole blood data with probes that are highly expressed in leukocytes 18 cases of patient-universal comparison, 18 pairs of healthy subject-universal comparison, 36 sets of expression data, defined in 3.1 Data correction using the 8411 probe (Global Normalization: Calculate the median of Log (Ratio) and Log (Ratio) for all probes, and adjust each log so that the median of corrected Log (Ratio) becomes 0 (A correction method for subtracting the median of Log (Ratio) from (Ratio)).

3.3 Evaluation index of corrected data Focusing on the distribution of log (Ratio) of each experiment for 36 sets of expression data that was global normalized using the specified 8411 probe, 18 sets of patient-universal comparison On the other hand, it was found that the variation of Log (Ratio) was small in 18 groups of normal subjects-universal comparison (FIG. 4), and this was used as a diagnostic index for depression. The 95th percentile of Log (Ratio) was adopted as an index representing the degree of variation of Log (Ratio). Furthermore, 10 probes each having a large difference between the average value of LogRatio of 18 patients and the average value of LogRatio of 18 healthy subjects were extracted (Table 1). The 20 probes listed in Table 1 are considered to be suitable probe sets for calculating an evaluation index for depression.

3.4 Sensitivity and specificity of depression diagnosis
For each of the 36 subjects, calculate the diagnostic index described in 3.3, set the threshold to 1.15, and determine whether each subject is a depressed patient or a healthy person based on the magnitude relationship between the diagnostic index and the threshold A determination was made (FIG. 5). When the sensitivity and specificity were calculated based on the judgment results, the sensitivity was 94.4% (17/18) and the specificity was 83.3% (15/18)%.

3.5 Relevance between depression and white blood cell types Next, separate monocytes, neutrophils, T cells, and B cells from the white blood cell fraction using antibody magnetic beads, and extract total RNA from each. 2. Gene expression analysis was performed by the method described in 1. As a Cy3 sample at this time, a universal control prepared by mixing total RNA derived from each blood cell was prepared and used. In the data of 18 depressive patients, the expression analysis result in each blood cell was evaluated for the top 300 probes that expressed higher than 18 healthy subjects. The expression was increased in neutrophils and expressed in T cells and B cells. Decreased (FIG. 6). Therefore, the distribution of Log (Ratio) observed in the expression analysis results of patients with depression is a phenomenon caused by an increase or increase in neutrophils and a decrease or decrease in activity of T cells and B cells. The distribution of Log (Ratio) adopted as an evaluation index for depression is a comprehensive analysis of the number and activity of various leukocytes such as monocytes, neutrophils, T cells, B cells, and NK cells in leukocytes. It was thought that it was evaluated.

  The method of the present invention is useful as an objective and simple diagnostic method for depression in clinical settings.

FIG. 1 shows an M-A plot of gene expression analysis results for whole blood-derived RNA. FIG. 2 shows the distribution of erythrocyte-derived RNA and leukocyte-derived RNA in the M-A plot of gene expression analysis results for whole blood-derived RNA. FIG. 3 shows the clustering of blood cell separation sample 4 analysis. FIG. 4 shows the patient-universal comparison MA plot after data correction and the healthy subject-universal comparison MA plot. FIG. 5 shows a graph of the depression diagnosis index (95th percentile of Log (Ratio)) for 36 subjects. FIG. 6 shows 11 analysis clustering using whole leukocytes, monocytes, neutrophils, T cells, and B cells. FIG. 7 shows a conceptual diagram of the depression diagnosis system.

Claims (8)

  A method for diagnosing depression using RNA derived from whole blood of a subject, and pre-defining genes that are expressed more in the leukocyte fraction than in the erythrocyte fraction in gene analysis using nucleic acid-immobilized samples. , Targeting only the specified genes, correcting the median log expression ratio relative to the control sample to 0, and determining the presence or absence of depression in the subject using the corrected log expression ratio A diagnostic method of depression characterized.   The method of diagnosing depression according to claim 1, wherein the presence or absence of depression is determined using an evaluation index calculated by averaging the log expression ratio after correction.   The depression according to claim 1, wherein the presence or absence of depression is determined using an evaluation index calculated by averaging the logarithmic expression ratios after correction of the gene group described in Table 1. How to diagnose the disease.   The method for diagnosing depression according to claim 1, wherein the presence or absence of depression is determined using the 95th percentile of the absolute value of the logarithmic expression ratio after correction of the gene group described in Table 1 as an evaluation index. .   5. The method for diagnosing depression according to claim 4, wherein if the 95th percentile of the absolute value of the logarithmic expression ratio is 1.15 or more, it is determined that the patient is suffering from depression.   A method for evaluating the efficacy of an antidepressant using the method for diagnosing depression according to any one of claims 1 to 5.   A system for carrying out the method for diagnosing depression according to any one of claims 1 to 5, comprising means for obtaining gene expression data in RNA derived from whole blood of a subject using a nucleic acid-immobilized sample, and the nucleic acid In gene analysis using solid-phased samples, genes that are expressed more in the leukocyte fraction than in the erythrocyte fraction are specified in advance, and the median logarithmic expression ratio for the control sample is only for the specified gene. And a means for determining the presence or absence of depression in the subject using a logarithmic expression ratio after correction, and a diagnostic system for depression.   In gene analysis using nucleic acid-immobilized samples using RNA derived from whole blood of subjects, genes that are expressed more in the leukocyte fraction than in the erythrocyte fraction are specified in advance, and only the specified genes are targeted. A method for correcting gene expression data, wherein the median of the logarithmic expression ratio relative to a control sample is corrected to zero.

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