JP2005080603A - Method for determining level of biological rhythm disturbance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique to surely and easily estimating biological rhythm disturbance, and provide a diagnostic criterion to determine the diagnostic and therapeutic measures for the disturbance level of a patient having biological rhythm disturbance. <P>SOLUTION: The level of biological rhythm disturbance is determined by using a temporal change in the expression quantity of a clock gene hPer2 as an index. Preferably, the human clock gene is collected from the human body fluid. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for determining the degree of a biological rhythm disorder.

Many organisms living on the earth have developed and maintained a biological clock that ticks in a 24-hour cycle in the body in order to adapt to environmental changes created by the rotation of the earth. For this reason, there are many endogenous biological rhythms governed by biological clocks. These are called circadian rhythms (circadian rhythms). The cycle that appears in a constant environment is called the free run cycle, which is approximately 25 hours in humans.

  Recently, the number of patients with sleep disorders due to this biological rhythm disorder is increasing. Such patients are designated as Circadian Rhythm Sleep Disorders (CRSD) in the International Classification of Sleep Disorders. This circadian rhythm disorder is desirable for sleep disorders caused by living on a schedule against the endogenous biological rhythm, such as night shift and rapid movement to a time zone, and sleep / wake schedule due to modulation of the endogenous biological rhythm itself It is divided into sleep disorders that deviate chronically from the time zone. Among the sleep disorders caused by the modulation of the latter endogenous biological rhythm itself, the sleep phase regression syndrome (DSPS) and the non-24-hour sleep / wake syndrome (Non-24) are particularly social problems.

The following documents are related to a method for examining a biological rhythm.
JP-A-6-217946 JP 2000-166877 A Ebisawa et al., Alleicvariants of human melatonin 1a receptor: function and prevalence in subjectswith circadian rhythm sleep disorders, Biochem Biophys Res Commun, 262,: 832-837 (1999) Okamura Hitoshi, Masumi Satoru, Ichikawa Naoyuki, Shun Yamaguchi, Food Gene Loop of Clock Gene, Progress in Neurological Research, 45: 755-762 (2001) Bjamason et al., Circadian expression of clock genes in human oral mucosa and skin: association withspecific cell-cycle phases, AmJPathol, 158: 1793-1801 (2001)

  It is thought that students / students who are not attending school as pediatric chronic fatigue syndrome with reduced function of learning, memory, cognition, and motivation are caused by the above DSPS or Non-24.

  Research on such biological rhythm disorders has been energetically pursued not only to elucidate disease states but also to the development of treatments. However, the true nature has not been fully elucidated, and the treatment methods are trial and error. The diagnostic method is also in the development stage and has not yet been established.

  Patent Document 1 discloses a method for measuring a biological rhythm from electrocardiogram measurement data. Patent Document 2 discloses a method for inspecting a biological rhythm by measuring a muscle movement of a repetitive rhythm exercise. Non-Patent Document 1 describes that melatonin is involved in biological rhythm. It has also been reported that the expression level of blood cortisol can be an indicator of biological rhythm. However, according to the study by the present inventors, although the expression level of cortisol is a temporary index of biological rhythm, it is difficult to clearly determine abnormalities in biological rhythm.

  Non-Patent Document 2 discloses a clock oscillation mechanism in which circadian rhythm is driven by a series of genes called clock genes. However, there is no mention of the relationship between clock gene expression and biological rhythm diseases.

  Non-Patent Document 3 reports that circadian rhythm expression was observed in clock genes hPer1, hBmall and hCry1 in the skin and oral mucosa of healthy subjects. However, it has not been known at all that the expression of these clock genes serves as an index for quantifying biological rhythm disorders. Furthermore, the behavior of body fluids, particularly blood, has not been investigated in humans.

The present invention has been made under such circumstances, and an object thereof is to provide a technique for accurately and simply evaluating a biological rhythm disorder. Another object of the present invention is to provide a diagnostic standard for diagnosing and treating a disorder in a patient with a biological rhythm disorder.

That is, the present invention is a method for determining the degree of rhythm disorder using the temporal change in the expression level of the clock gene hPer2 as an index.

The human clock gene is preferably collected from a human body fluid.

The body fluid is preferably blood.

  The gene is preferably mRNA.

It is normal that the temporal change in the expression level of the hPer2 gene, considered as a clock gene, forms a smooth curve with the highest part at 4-10 am and the lowest part at 2-8 pm It is preferable to determine the degree of failure based on the degree of dissociation therefrom.

In the present invention, the rhythm disorder particularly targets one or more symptoms selected from the group consisting of sleep disorder, school refusal, depression, and chronic fatigue.

  According to the present invention, it is possible to evaluate a biological rhythm disorder more accurately and simply than an index that can display other biological rhythms. Further, according to the evaluation method of the present invention, the degree of healing of a patient with a biological rhythm disorder can be clearly determined. This degree of disability cannot be diagnosed by the existing diagnostic criteria such as blood pressure, body temperature, in vivo hormones such as melatonin and cortisol, but it is more accurate by monitoring the clock gene of the present invention. It became possible to diagnose the degree of biological rhythm disorder.

Hereinafter, the present invention will be described in detail focusing on embodiments.
The method for judging the degree of the biological rhythm disorder of the present invention is preferably characterized in that the expression level of the clock gene hPer2 in the body fluid is used as an index.

The clock gene is a gene that plays a role in regulating biological rhythms in humans, and hPer1, hPer2, hPer3, hBmall, hCry1, hCry2, hDec1, and hDec2 have been known so far. In mice, these genes have similar base sequences.

  The clock gene hPer2 in humans has its nucleotide sequence registered as No (NM — 022817) in Genebank. The clock oscillator gene hPer2 controls its transcription by a protein encoded by itself as a negative factor. The oscillation cycle is determined by the magnitude of the time lag that occurs in the process from production of negative factors to suppression. On the other hand, there is a positive factor (Bmal1 and clock heterodimer) that promotes transcription of the negative factor, and the negative vibration that is translated periodically suppresses the positive factor, and the oscillation continues.

In the method for judging the degree of a biological rhythm disorder of the present invention, a clock gene is collected from a cell tissue or a body fluid. Among these, it is preferable to collect from a body fluid that can be easily collected. Examples of the body fluid include whole blood, plasma fluid, serum fluid, lymph fluid, cerebral spinal fluid, semen, saliva or milk. Among these body fluids, whole blood is preferable because it is easy to collect mRNA.

  In order to collect and quantify the clock gene hPer2 from body fluids, particularly blood, mRNA is first collected from blood. Separation of mRNA from blood may be performed by various techniques that are usually studied. For example, it can be collected using the total RNA kit shown in the following Examples.

  Next, in order to prevent DNA contamination with respect to the collected total RNA, the DNA is decomposed with DNase, and only the mRNA of the clock gene portion is collected and purified. This collection and purification can be performed using a commercially available RNA extraction kit or the like.

  Using reverse transcriptase for mRNA of the clock gene purified by the above method, cDNA is prepared. This method is also a known method, and reverse transcriptase can be easily obtained from a reagent company or the like.

  The clock gene cDNA collected by the above method is then cloned by PCR reaction. The PCR reaction method itself is also a known method, and cDNA is propagated by replicating with primers using cDNA as a template. In PCR, a DNA strand is usually replicated by making DNA single-stranded under conditions of high temperature, for example, 95 ° C.-15 seconds, and then reacting at low temperature, for example, 60 ° C. for 1 minute. This cycle is usually repeated about 50 times to grow DNA.

  The quantification of the clock gene hPer2 cloned by the PCR method can be determined, for example, by using β-actin as an internal standard, calculating the expression level of each gene by a calibration curve method, and comparing with the internal standard. Data may be obtained by performing rhythm analysis by using a single-cosinar method with a single period of 24 hours using co-signer analysis software (Co-signer) to obtain amplitude and vertex phase (acrophase). Alternatively, the highest density and the lowest density may be evaluated graphically.

  When the biological rhythm is normal, the expression level of the clock gene hPer2 is such that the temporal change in the expression level is highest at 4-10 am and lowest at 2-8 pm as shown in FIG. A smooth curve is formed. That is, the curve shows a sine curve with a period of about 24 hours.

When judging the presence or absence of a disorder in the biological rhythm, when the period of the expression curve of the clock gene deviates from 24 hours, when the time indicating the highest part and the lowest part is deviated, or when the level is not observed, it is not normal You may think that you have a biological rhythm disorder. Furthermore, if the curve of the expression level of the clock gene hPer2 shows a tendency to deviate from the normal curve even after some treatment, it is considered that the biological rhythm disorder is not completely cured. Thus, the degree of failure can be determined by the degree of dissociation from the normal curve.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. The measurement was performed by the following method.

(Measurement of melatonin concentration)
The blood melatonin concentration was measured by the RIA2 antibody method.
(Measurement of cortisol concentration)
The blood cortisol concentration was measured by the RIA solid phase method which is a known measurement method.

  2.5 ml of venous blood collected every 4 hours from the subject was stored in a PAXgene blood RNA tube (Qiagen).

  Total RNA was purified from total blood using the PAXgene Blood RNA kit (Qiagen) according to the protocol. At this time, the total RNA was subjected to DNase treatment using an RNase-free DNase set (Qiagen).

  From the purified total RNA, reverse transcription reaction was performed using Lever Traace-α- (manufactured by Toyobo Co., Ltd.) to prepare cDNA. The reverse transcription reaction was performed using 25 μl-10 minutes, 42 ° C.-50 minutes, 95 ° Performed in 5 minutes.

  Real-time PCR is performed using ABIPRISM7700 sequence detector (Applied Biosystems) using the target cDNA specific template (Invitrogen) and Tacman probe (Applied Biosystems). It was. The PCR reaction was performed in a total volume of 25 μl containing 1 μl cDNA, 1 μM PCR primer, 1 μM Tacman probe (Applied Biosystems) and 12.5 μl Tacman universal master mix (Applied Biosystems). PCR conditions were 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, followed by 50 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. Primer and probe design was set from the sequence of the target gene. The sequence is shown in Table 1. Human β-actin was used as an internal standard. The gene expression level was determined by calculating the expression level of each gene by the calibration curve method and calculating the relative ratio to the internal standard. The primer sequences shown this time are not limited to those shown here.

  The data was analyzed by rhythm analysis using a single-cosinar method with a 24-hour single cycle using a co-signer analysis software (Co-signer) to determine the amplitude and the apex phase.

(Case)
Patient: 16 years old Female diagnosis: Non-24-hour sleep / wake syndrome (non-24)
Medical history: I was unable to get up in the morning since I was in fifth grade, and I was absent from school two or three times a week. Day and night were completely reversed during the summer vacation of the first grader in junior high school. Daytime sleepiness, general malaise, headache, stomach pain, and depression became severe.

(Treatment)
Hospitalized, it was performed and the light irradiation for irradiating the wakeup halo, melatonin, the time therapy oral after meals three times a day methyl B _12. In addition, exercise therapy and administration of fluvoxamine were performed. Administration was continued after discharge.

(Treatment result)
The sleep phase was advanced by the start of treatment, and the sleep / wake rhythm with a 24-hour period was maintained during hospitalization. However, despite the continued medication after discharge, the sleep phase retreated and returned to the non-24 sleep / wake pattern. The sleeping hours before and after hospitalization are shown in FIG.

(Evaluation)
In Example 2, patient blood was collected every 4 hours and the amount of hPer2 in the blood was quantified. The expression level of hPer2 before and after treatment is shown in FIG.

(Comparative Example 1)
In Example 2, it carried out like Example 2 except paying attention to hPer1 as an evaluation means. The results are shown in FIG.

(Comparative Example 2)
In Example 2, it carried out similarly to Example 2 except paying attention to hPer3 as an evaluation means. The results are shown in FIG.

(Comparative Example 3)
In Example 2, the evaluation was performed in the same manner as in Example 2 except that attention was paid to hClock. The results are shown in FIG.

(Comparative Example 4)
In Example 2, it carried out like Example 2 except paying attention to hBmal1 as an evaluation means. The results are shown in FIG.

(Comparative Example 5)
In Example 2, it carried out like Example 2 except paying attention to melatonin as an evaluation means. The results are shown in FIG.

(Comparative Example 6)
In Example 2, it carried out like Example 2 except paying attention to cortisol as an evaluation means. The results are shown in FIG.

The expression pattern of hPer2 was compared at the start of treatment and after hospitalization, but both were not similar to the healthy person pattern, and as described above, the pattern was clearly different. In addition, the value after the treatment of the existing test items, melatonin and cortisol, is similar to that of a healthy person and has not yet been detected as a biological rhythm disorder.

(Case)
Patient: 15 years old Female diagnosis: Sleep phase regression syndrome (DSPS)
Medical history: I was bullied when I was in the second year of junior high school, and I was unable to attend school.

(Treatment)
Hospitalized, it was performed and the light irradiation for irradiating the wakeup halo, melatonin, the time therapy oral after meals three times a day methyl B _12. In addition, exercise therapy paroxetine was also administered. Administration was not performed after discharge. The sleeping hours before and after hospitalization are shown in FIG.

(Treatment result)
The start of treatment advanced the sleep phase and improved the sleep / wake pattern desired by the patient. I was able to maintain that rhythm even after discharge.

(Evaluation)
As in Example 2, the patient's blood was collected every 4 hours, and the amount of hPer2 in the blood was quantified. FIG. 11 shows the expression level of hPer2 before and after treatment.

(Comparative Example 7)
In Example 3, it carried out similarly to Example 3 except paying attention to hPer1 as an evaluation means. The results are shown in FIG.

(Comparative Example 8)
In Example 3, it carried out similarly to Example 3 except paying attention to hPer3 as an evaluation means. The results are shown in FIG.

(Comparative Example 9)
In Example 3, the evaluation was performed in the same manner as in Example 3 except that attention was paid to hClock. The results are shown in FIG.

(Comparative Example 10)
In Example 3, it carried out like Example 3 except paying attention to hBmal1 as an evaluation means. The results are shown in FIG.

(Comparative Example 11)
In Example 3, it carried out like Example 3 except paying attention to melatonin as an evaluation means. The results are shown in FIG.

(Comparative Example 12)
In Example 3, it carried out similarly to Example 3 except paying attention to cortisol as an evaluation means. The results are shown in FIG.

As shown in the above examples, as a result of comparing the expression pattern of hPer2 at the start of treatment and after hospitalization treatment, the expression pattern of hPer2 that was abnormal before treatment was similar to the pattern of healthy people by treatment. became. On the other hand, the values of melatonin and cortisol, which are existing test items, are similar to those of a healthy person even before treatment and have not yet detected a biological rhythm disorder. Moreover, it is almost the same as that of a healthy person after treatment, and no change is observed. That is, it has been shown that a biological rhythm disorder that cannot be distinguished from a healthy person can be diagnosed by detecting a clock gene pattern with the existing diagnostic method, and that a change in therapeutic effect can be evaluated. This suggests that clock gene pattern detection is useful in the diagnosis of patients with biological rhythm disorders and provides sufficient information for creating a treatment plan.

  In combination with existing methods in biological rhythm diagnosis, a more accurate rhythm diagnosis becomes possible, and therefore, it is expected to provide a useful diagnostic method for a disease considered to be caused by a biological rhythm disorder.

FIG. 1 is a graph showing changes over time in the amount of hPer2 gene in healthy individuals. FIG. 2 is a graph showing changes in a patient's sleep time zone in Example 2. FIG. 3 is a graph showing changes over time in the amount of hPer2 gene in Example 2. FIG. 4 is a graph showing changes over time in the amount of hPer1 gene in Comparative Example 1. FIG. 5 is a graph showing changes over time in the amount of hPer3 gene in Comparative Example 2. FIG. 6 is a graph showing changes over time in the amount of hClock gene in Comparative Example 3. FIG. 7 is a graph showing the change over time in the amount of hBmall gene in Comparative Example 4. FIG. 8 is a graph showing changes over time in the amount of melatonin in Comparative Example 5. FIG. 9 is a graph showing changes over time in the amount of cortisol in Comparative Example 6. FIG. 10 is a graph showing changes in the sleeping hours of patients in Example 3. FIG. 11 is a graph showing changes over time in the amount of hPer2 gene in Example 3. FIG. 12 is a graph showing changes over time in the amount of hPer1 gene in Comparative Example 7. FIG. 13 is a graph showing changes over time in the amount of hPer3 gene in Comparative Example 8. FIG. 14 is a graph showing changes over time in the amount of hClock gene in Comparative Example 9. FIG. 15 is a graph showing changes over time in the amount of hBmall gene in Comparative Example 10. FIG. 16 is a graph showing changes over time in the amount of melatonin in Comparative Example 11. FIG. 17 is a graph showing the change over time in the amount of cortisol in Comparative Example 12.

Claims (6)

A method for judging the degree of a biological rhythm disorder, using as an index a temporal change in the expression level of the clock gene hPer2. The method for judging the degree of a biological rhythm disorder according to claim 1, wherein the human clock gene is collected from a human body fluid.   The method according to claim 2, wherein the body fluid is blood.   The method according to claim 1, wherein the gene is mRNA. The time course of the expression level of the clock gene is normal to form a smooth curve with the highest part at 4-10 am and the lowest part at 2-8 pm. The method for judging the degree of a biological rhythm disorder according to any one of claims 1 to 4, wherein the degree of the disorder is judged. The biological rhythm disorder according to claim 1, wherein the biological rhythm disorder is one or more symptoms selected from the group consisting of sleep disorder, school refusal, depression, and chronic fatigue. How to judge the degree of