JP2020091242A - Method for evaluating attention-deficit hyperactivity disorder and method for acquiring data for evaluating attention-deficit hyperactivity disorder - Google Patents

Abstract

To provide a method for evaluating the possibility that a subject is actually having or is going to have attention-deficit hyperactivity disorder (ADHD) with a high precision, and a method for acquiring data for evaluating the possibility that a subject is actually having or is going to have attention-deficit hyperactivity disorder (ADHD) with a high precision.SOLUTION: The present invention relates to a method for evaluating the possibility that a subject is actually having or is going to have attention-deficit hyperactivity disorder (ADHD), the method including an evaluation step of evaluating the possibility that the subject is actually having or is going to have ADHD on the basis of the amount of cytokine in a sample derived from the subject.SELECTED DRAWING: None

Description

The present invention provides a method for evaluating whether or not a subject to be evaluated suffers from attention deficit/hyperactivity disorder (ADHD) or has a possibility of developing, and a subject to be evaluated suffers from ADHD. The present invention relates to a method for obtaining data for evaluating whether or not a patient has or has a possibility of developing the disease.

Attention deficit/hyperactivity disorder (also referred to as attention deficit/hyperactivity disorder; hereinafter also referred to as “ADHD”) is characterized by symptoms such as impulsivity, hyperactivity, or inattention, and during childhood and subsequent life. It is a neurodevelopmental disorder that can cause significant disability. Overactivity is common in children with ADHD, but although it tends to disappear in adulthood, the majority of children with ADHD continue to have some carelessness throughout their lives.

Although ADHD has a genetic factor of 76%, it cannot be easily diagnosed with the genetic factor because the isolation is not sophisticated and the environmental factor of home is included.

ADHD develops before the age of 7 (before the age of 12 when using DSM-V), has continued for at least 6 months, and has been diagnosed when it interferes with normal life. Diagnosis is generally made by interview, as with many mental disorders. DSM-V is an evaluation scale for assisting diagnosis. The DSM-V does not use biological data such as MRI and blood tests as diagnostic items. Therefore, there are many misdiagnosis, and an objective index is needed.

Up to now, as a method other than the inquiry, a method of evaluating using a display (see, for example, Patent Literature 1), a method of using single nucleotide polymorphism as a marker (see, for example, Patent Literature 2), and a Pmch gene as a marker Various methods have been proposed, such as a method of utilizing (see, for example, Patent Document 3), a method of measuring peripheral skin temperature and a value indicating ADHD (see, for example, Patent Document 4).
However, there is a problem that a technology that can be clinically implemented has not been provided yet.

Further, until now, there is no cure for ADHD, and symptom management is performed by a combination of behavioral therapy and drug therapy. In drug therapy, central nervous system stimulant medications are widely used.
However, since drug therapy also carries the risk of adverse effects, it is ideal to avoid unnecessary administration and use only the minimum necessary amount.
Therefore, from the viewpoint of avoiding unnecessary medication, it is very important to accurately diagnose whether or not ADHD is involved.

Therefore, under the present circumstances, there is a strong demand for prompt provision of a new technique that can evaluate with high accuracy whether or not ADHD is affected or has a possibility of developing ADHD.

Japanese Patent Publication No. 2018-503410 JP, 2013-66454, A JP, 2010-4848, A JP, 2003-33355, A

It is an object of the present invention to solve the above-mentioned conventional problems and achieve the following objects. That is, the present invention provides an evaluation method capable of highly accurately evaluating whether or not ADHD is present or has the possibility of developing ADHD, and whether or not ADHD is involved or the possibility of developing ADHD. It is an object of the present invention to provide a method for acquiring data for highly accurately evaluating whether or not to have.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that the amount of a specific cytokine in a sample derived from a patient with ADHD is significantly higher than that in a sample derived from a healthy individual. I found that it was different.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> An evaluation step for evaluating whether or not suffering from attention deficit hyperactivity disorder (ADHD) or having a possibility of developing it, using the amount of cytokine in a sample derived from an evaluation subject as an index And a method for evaluating whether or not the subject to be evaluated has ADHD or has a possibility of developing ADHD.
<2> To evaluate whether or not the subject to be evaluated has ADHD or has a possibility of developing ADHD, which comprises a measurement step of measuring the amount of cytokine in a sample derived from the subject to be evaluated Is a method of acquiring the data of.

ADVANTAGE OF THE INVENTION According to this invention, the said various problems in the past can be solved, the said objective can be achieved, and it can evaluate with high accuracy whether it has ADHD, or has a possibility of developing. It is possible to provide a possible evaluation method and a method for acquiring data for highly accurately evaluating whether or not ADHD is affected or the possibility of developing ADHD.

FIG. 1 is a graph showing the mean values of cytokine concentrations that were significantly different in saliva of healthy subjects and ADHD patients collected in Example 1. The solid line shows the cytokine concentration contained in saliva collected from the healthy subject group, and the broken line shows the cytokine concentration contained in saliva collected from the ADHD patient group.

(ADHD evaluation method)
A method for evaluating whether or not the subject of evaluation of the present invention suffers from attention deficit hyperactivity disorder (ADHD) or has a possibility of developing it (hereinafter, may be referred to as "ADHD evaluation method"). Includes at least an evaluation step and, if necessary, further includes other steps such as a measurement step.

ADHD is a neurodevelopmental disorder characterized by symptoms such as impulsivity, hyperactivity or inattention, which can cause significant disability in childhood and later life.

<Evaluation process>
In the evaluation step of the ADHD evaluation method, whether or not the evaluation target is suffering from ADHD using the amount of cytokine (hereinafter, also referred to as “concentration” in some cases) in the sample derived from the evaluation target as an index, or It is a step of evaluating whether or not there is a possibility of developing the disease.
In the present invention, whether or not the patient has ADHD includes the degree of ADHD symptoms.

The amount of cytokine in the sample derived from the subject to be evaluated may be measured in the measurement step described later or may be measured separately.
The unit of the data of the amount of the cytokine is not particularly limited and can be appropriately selected according to the purpose. For example, it can be obtained by adding, subtracting, multiplying, and multiplying a molar concentration, a weight concentration, and these concentrations by an arbitrary constant. Things are included.

-Evaluation target-
The subject to be evaluated is not particularly limited as long as it is a subject that needs to be evaluated whether or not it has ADHD or has a possibility of developing ADHD, and can be appropriately selected according to the purpose.
The type of the subject to be evaluated is not particularly limited and may be appropriately selected depending on the intended purpose, and may be human or animal other than human.
The sex to be evaluated is not particularly limited and may be appropriately selected depending on the purpose, and may be male or female.
The age to be evaluated is not particularly limited and can be appropriately selected according to the purpose. For example, when the subject to be evaluated is a human, it may be pre-adult or post-adult, but is preferably 20 years or younger, more preferably 15 years or younger, and particularly preferably 5 years or more and 15 years or less. ..

-Sample derived from evaluation target-
The sample derived from the evaluation subject is not particularly limited and may be appropriately selected depending on the purpose, for example, blood, serum, cerebrospinal fluid, saliva, pharyngeal swab, sweat, urine, tears, lymph, semen. , Ascites, breast milk, etc. These may be used alone or in combination of two or more.
Among these, blood and saliva are preferable, and saliva is more preferable because it can be obtained non-invasively.

The method for preparing the sample (hereinafter also referred to as “collection method”) is not particularly limited, and a known method can be appropriately selected.
For example, when the sample is blood, for example, a method of collecting blood using an ordinary syringe can be used.
When the sample is saliva, for example, a method of directly collecting saliva, a method of using a salivet or a commercially available collecting instrument, and the like can be mentioned.

The collection time of the sample derived from the evaluation subject (hereinafter, also referred to as “collection time zone”) is not particularly limited and can be appropriately selected according to the purpose. It is preferable that the time is about the same as the time when the sample is collected from the subject. In particular, when the sample is saliva, the amount of cytokine contained in saliva may vary greatly during the day, and therefore it is preferable to perform the sample at a certain time zone throughout the day.

The sample derived from the evaluation target may be subjected to a treatment such as dilution, if necessary.

-Cytokine-
The cytokine is a general term for relatively small proteins (approximately 5 to 20 kDa) that play an important role in cell signaling.
The cytokine used in the evaluation step is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include chemokines such as interleukins; lymphokines; Hematopoietic factors such as colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO); epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) ), hepatocyte growth factor (HGF), transforming growth factor (TGF) and other cell growth factors; tumor necrosis factor (TNF-α), lymphotoxin (TNF-β) and other cytotoxic factors; leptin, TNF- Examples include adipokines such as α; neurotrophic factors such as nerve growth factor (NGF). These may be used alone or in combination of two or more.

Among the above-mentioned cytokines, interleukin-1 receptor antagonist protein (IL-1ra), interleukin-1β (IL-1β), interleukin-7 (IL-7), in that the accuracy of evaluation can be further increased. , Interleukin-8 (IL-8), interleukin-12 p70 (IL-12 p70), granulocyte colony stimulating factor (G-CSF), interferon-γ (IFN-γ), and vascular endothelial growth factor (VEGF). 1) or more selected from the group consisting of 2), 2 or more selected from the above group are more preferable, and 2 types of IL-1ra and VEGF are particularly preferable.

-Evaluation-
The evaluation method is not particularly limited and may be appropriately selected depending on the intended purpose, as long as it can be evaluated whether the evaluation subject has ADHD or has a possibility of developing ADHD. it can.

Examples of the evaluation method include a method including a process of comparing the amount of cytokine in a sample derived from the evaluation target with a reference value.

The reference value is not particularly limited and may be appropriately selected depending on the purpose. For example, (I) the amount of cytokine in a sample derived from a healthy individual (hereinafter, may be referred to as “normal value”). And (II) a value based on the amount of cytokine (hereinafter sometimes referred to as “disease value”) in a sample derived from an ADHD patient. These may be used alone or in combination of two or more.
The ADHD patient may be a typical ADHD patient or a mild, moderate or severe ADHD patient.

The value based on the above-mentioned (I) normal value is not particularly limited and may be appropriately selected depending on the purpose. For example, in the case of not overlooking a disease group as in the case of screening test, the highest priority is given. , Prioritizing sensitivity over specificity, and referring to the normal value, setting a threshold value (hereinafter, sometimes referred to as “cutoff value”) to the normal value or a value close to the normal value. Be done.

The value based on the (II) disease value is not particularly limited and may be appropriately selected depending on the purpose. For example, in the case of evaluating the degree of ADHD symptom, the disease value is referred to as a threshold value. Is set to the disease value or a value close to the disease value.

In the treatment, when the reference value is (I) a value based on a normal value, and the amount of cytokine in the sample derived from the evaluation target is less than the value based on the normal value, the evaluation target is ADHD. It can be assessed as being afflicted or having the potential to develop ADHD.
The healthy individual is preferably an individual of the same type as the evaluation target, and an individual of similar age.

Further, in the case where the reference value is a value based on (II) disease value, the evaluation target is ADHD when the amount of cytokine in the sample derived from the evaluation target is equal to or less than the value based on the disease value. Suffering from or having the potential to develop ADHD.
The ADHD patient is preferably an individual of the same type as the subject of evaluation and of a similar age.

The reference value may be set based on the amount of cytokine in the sample that is also measured when performing the method for evaluating ADHD, or may be set based on the amount of cytokine in the sample that has already been measured. You may use what was done.

The evaluation method also includes a method using an evaluation formula capable of evaluating whether or not ADHD is affected or whether or not ADHD is developed.

For example, by substituting the cytokine concentration data into an evaluation formula capable of evaluating whether or not ADHD is affected or has a possibility of developing based on the cytokine concentration data. A method of evaluating whether or not the subject to be evaluated has ADHD or has a possibility of developing ADHD. Here, the evaluation formula includes a variable into which the concentration value of the cytokine is substituted. The number of the variables is not particularly limited as long as it is at least one, and can be appropriately selected according to the purpose, but at least two is preferable from the viewpoint that the accuracy of evaluation can be further improved. That is, in the evaluation formula capable of evaluating whether or not ADHD is affected or the possibility of developing ADHD based on the data of the amount of two or more cytokines, It is preferable that the evaluation formula includes at least two variables into which the data of the amounts of the two or more kinds of cytokines are substituted, including the substitution of the amount data.
The cytokine is the same as that described in the above-Cytokine-section.

The evaluation formula is not particularly limited and may be appropriately selected depending on the purpose, for example, logistic regression formula, fractional formula, linear discriminant formula, multiple regression formula, formula created by support vector machine, Mahalanobis distance. The formulas created by the method, the formulas created by the canonical discriminant analysis, the formulas created by the decision tree, and the like. This makes it possible to further improve the reliability of information that can be used as a reference for knowing the ADHD state.

Further, the formula adopted as the evaluation formula means a form of a formula generally used in multivariate analysis, and for example, a fractional formula, multiple regression formula, multiple logistic regression formula, linear discriminant function, Mahalanobis distance, positive Quasi-discriminant functions, support vector machines, decision trees, formulas such as those represented by the sum of different forms of the formula, and the like. Here, a coefficient and a constant term are added to each variable in the multiple regression equation, the multiple logistic regression equation, and the canonical discriminant function, but the coefficient and the constant term are preferably real numbers, and more preferably , Any value that belongs to the range of 99% confidence intervals of the coefficient and the constant term obtained from the data to perform the various classifications is more preferable, and more preferably, the value obtained from the data to perform the various classifications. Any value may be used as long as it belongs to the range of the 95% confidence interval of the coefficient and the constant term. Further, the value of each coefficient and its confidence interval may be obtained by multiplying it by a real number, and the value of the constant term and its confidence interval may be obtained by adding, subtracting, multiplying, and dividing an arbitrary real constant. When using display formulas such as logistic regression, linear discriminant, multiple regression analysis, etc. as evaluation formulas, the linear conversion (addition of constants, multiplication of constants) and conversion of monotonic increase (decrease) of display formulas (eg logit conversion) are evaluation performance. Since this is not the same as before conversion but is equivalent to that before conversion, this display formula also includes the one after these conversions are performed.

For example, in the case of evaluation using the concentration data of two types of cytokines, the formula (concentration of cytokine A)+(concentration of cytokine B) can be mentioned. According to this, it is possible to quantify the evaluation of whether or not ADHD is involved or whether or not ADHD is likely to develop as a specific numerical value. In addition, weighting may be performed for each type of cytokine based on the results obtained by previously examining the correlation between various cytokines and ADHD patients. Furthermore, the value obtained by using (or the value of the concentration data of each cytokine to be evaluated)/(the average value of the concentration data of each cytokine of a healthy subject) (or a certain weighting to that value) is used. You may evaluate. Further, the cytokine concentration value may be corrected depending on age, sex, race, the presence or absence of ADHD genetic background, and the like. The degree of correction and the presence or absence of correction may be changed depending on the type of cytokine. The evaluation value obtained as described above is compared with the value of a general person (for example, the average value of Japanese people), and depending on the magnification, whether or not ADHD is present or there is a possibility of developing ADHD. You may evaluate whether or not.

The type of cytokine using the concentration data as a variable is not particularly limited and may be appropriately selected depending on the purpose. However, IL-1ra and IL-1β can be more accurately evaluated. , IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF are preferable, and two or more types are more preferable, and IL-1ra and VEGF are more preferable.
In extracting the cytokine, for example, an AUC (Area Under the Curve) of a ROC curve (Receiver Operating Characteristic curve) can be used. In the present invention, for example, the combination of cytokines that maximizes AUC can be obtained by the variable reduction method.

<Other processes>
The other steps in the ADHD evaluation method are appropriately selected depending on the intended purpose without any limitation, provided that the effects of the present invention are not impaired. Examples thereof include a measurement step and a sample preparation step.

<<Measuring process>>
The measurement step in the ADHD evaluation method is a step of measuring the amount of the cytokine in the sample derived from the evaluation target.

-Measurement-
The method for measuring the amount of the cytokine in the sample is not particularly limited, and a known method can be appropriately selected, and examples thereof include a method of measuring the amount of protein and a method of measuring the amount of mRNA. Be done. These may be used alone or in combination of two or more.
The amino acid sequence and nucleotide sequence of the cytokine can be easily obtained through public databases such as GenBank (NCBI).

The method for measuring the amount of the protein of the cytokine is not particularly limited, and a known method can be appropriately selected. For example, measurement is performed by an immunological method using an antibody that specifically recognizes the cytokine. There is a method of doing so.

The immunological method is not particularly limited and may be appropriately selected from known methods. For example, antibody array, flow cytometric analysis, radioisotope immunoassay (RIA method), ELISA method (Methods in Methods). Enzymol.70:419-439 (1980)), dot blotting, immunonucleic acid assay, western blotting, immunohistostaining and the like.

Specific examples of the ELISA method include a direct method in which a target cytokine is immobilized on a microplate and a labeled antibody that recognizes the cytokine is allowed to act, an indirect method using a labeled secondary antibody that recognizes an anti-cytokine antibody, and an anti-cytokine. Sandwich method using immobilized antibody and labeled anti-cytokine antibody, competitive method in which anti-cytokine antibody is immobilized and enzyme-labeled antigen whose target protein and concentration are known in advance are reacted simultaneously in the same microplate, etc. Is mentioned.

The method for measuring the amount of mRNA is not particularly limited, and a known method can be appropriately selected, and examples thereof include PCR method, real-time PCR method, DNA array method, Northern blotting method and the like.

The amount of the cytokine in the sample can also be measured using a commercially available cytokine measurement kit.

<<Sample preparation process>>
The sample preparation step in the ADHD evaluation method is a step of preparing a sample derived from the evaluation target, and a known method can be appropriately selected according to the type of the sample.

The ADHD evaluation method of the present invention can be performed in combination with evaluation by other means such as an interview.

According to the ADHD evaluation method of the present invention, by using the amount of the cytokine in the sample derived from the evaluation subject as an index, whether the evaluation subject has ADHD or has a possibility of developing ADHD. Can be evaluated with high accuracy.

Therefore, the present invention comprises a method for diagnosing whether or not an evaluation subject has ADHD or has a possibility of developing ADHD, which includes at least the evaluation step and further includes the other steps as necessary. , Or a method of assisting in the diagnosis of whether the subject to be evaluated has ADHD or has the possibility of developing ADHD.
Further, the present invention is a biomarker for evaluating, diagnosing, or assisting in diagnosing whether an evaluation subject has ADHD or has a possibility of developing ADHD, which is a biomarker comprising a cytokine. It also concerns.

(Method of acquiring ADHD evaluation data)
A method for acquiring data for evaluating whether or not the evaluation subject of the present invention has ADHD or has a possibility of developing ADHD (hereinafter, referred to as "ADHD evaluation data acquisition method"). A) includes at least a measurement step, and further includes other steps as necessary.

<Measuring process>
The measurement step in the ADHD evaluation data acquisition method is a measurement step of measuring the amount of cytokine in a sample derived from an evaluation target, and is performed in the same manner as the measurement steps in the other steps of the ADHD evaluation method of the present invention described above. It can be carried out.

<Other processes>
The other steps in the ADHD evaluation data acquisition method are appropriately selected depending on the intended purpose without any limitation, provided that the effects of the present invention are not impaired. Examples thereof include a sample preparation step. ..

<<Sample preparation process>>
The sample preparation step in the ADHD evaluation data acquisition method is a step of preparing a sample derived from the evaluation target, and is performed in the same manner as the sample preparation step in the other steps of the ADHD evaluation method of the present invention described above. You can

The ADHD evaluation data acquisition method of the present invention may be performed in combination with the measurement of the amount of a substance other than the cytokine.

According to the ADHD evaluation data acquisition method of the present invention, it is possible to collect data that is useful for highly accurately evaluating whether or not one has ADHD or has a possibility of developing ADHD.

(ADHD evaluation, diagnosis, or diagnostic aid kit)
As described above, according to the ADHD evaluation method of the present invention, by using the amount of the cytokine in the sample derived from the evaluation subject as an index, whether the evaluation subject is suffering from ADHD or may develop the disease. Since it is possible to evaluate whether or not to have high accuracy with high accuracy, the present invention is for evaluating, diagnosing, or assisting diagnosis of whether or not a subject to be evaluated has ADHD or has a possibility of developing ADHD. The present invention also relates to a kit (hereinafter, sometimes referred to as "ADHD evaluation, diagnosis, or diagnosis aid kit").

The kit for evaluating, diagnosing, or assisting diagnosis of ADHD includes at least cytokine detection means, and may further include other components as necessary.

<Cytokine detection means>
The cytokine detection means is a group consisting of a target cytokine in the sample, preferably IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF. There is no particular limitation as long as it can detect one or more cytokines selected from the above, and it can be appropriately selected according to the purpose. In order to immunologically detect the cytokine, an antibody against the cytokine It is preferable to include a detection instrument having fixed.

The target for immobilizing the antibody against the cytokine is not particularly limited as long as it is a carrier which is a solid or insoluble material, and can be appropriately selected according to the purpose, for example, a reaction mixture by filtration, precipitation, magnetic separation or the like. And a carrier that can be separated from the above.

The shape of the carrier is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include beads, magnetic beads, thin films, microtubes, filters, plates, microplates, carbon nanotubes, and sensor chips. Be done. The flat carrier such as the thin film or plate may be provided with pits, grooves, filter bottoms, and the like.

<Other configurations>
The other configuration is not particularly limited, and a configuration included in a known diagnostic kit or the like can be appropriately selected according to the purpose. For example, a reagent used in an immunological assay, an instruction manual for the kit. And so on.

According to the kit for evaluating, diagnosing, or assisting diagnosis of ADHD, it is possible to evaluate, diagnose, or assist with diagnosis with high accuracy whether or not ADHD is affected or is likely to develop.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1)
Two groups of 20 ADHD patients (group of patients) and 20 healthy subjects (group of healthy subjects) were used as subjects. The patient group was collected before the healthy subject group, and the healthy subjects whose number, sex ratio, and average age were the same were collected later.
The saliva samples were collected twice after waking up for ADHD patients and once during the day for healthy subjects after the day. Cytokines (27 kinds) contained in the collected saliva were analyzed.

The subject background is shown in Table 1.

Details of a sample collection institution, an analyzer, a cytokine analysis kit, types of cytokines analyzed, and test methods are shown below.
・Sample Collection Organization: Shinshu University Hospital ・Analyzer: Bio-Plex (Bio-Rad Laboratories Co., Ltd.)
・Cytokine analysis kit:
Bio-Plex Pro Human Cytokine 27-Plex (Bio-Rad Laboratories Co., Ltd.)
・Types of cytokines analyzed:
IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p70, IL-13, IL -15, IL-17A, FGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, PDGF-BB, MIP-1β, RANTES, TNF-α, VEGF.
· Inspection method :
(1) Method of collecting measurement sample from saliva 1) Prepare two collection cups (Nippon Medical: model number MS-50) and enter the IDs (the two collection cups have the same ID).
2) Place a line with a collection volume of 150 μL in the collection cup.
3) Put the required amount (about 150 μL) of saliva in the two cups and store frozen at −80° C. within 2 hours after collection.

The concentrations of 27 kinds of cytokines were measured for the samples collected as described above by using the above-described analyzer and cytokine analysis kit.

Further, Mann-Whitney test was performed on the obtained analysis results using statistical analysis software (IBM SPSS Statistics, IBM Japan Ltd.).
When the measured concentration value of the cytokine is lower than the detection limit, the LOD value (Limit of Detection) is the concentration of the blank concentration plus the concentration fluctuation observed between the blanks, and the lower limit of measurement determined from the blank is determined. Meaning that, the LOD/2 value was used as a measured value only when the blank value+2×blank SD value) could be calculated. As an application condition, it was applied only when the LOD/2 value was lower than the lower limit of measurement of sample concentration and LLOQ (low concentration range of standard solution). When the LLOQ value was lower than the LOD/2 value, the LLOQ/2 value was used as the measurement value.
The LOD value was calculated using the coefficient of the standard curve regression equation obtained from the measured values of eight standard concentrations. However, when the LOD/2 value was higher than the thinnest density value (S8) of the standard, the S8 density value/2 was used as the measured value.

<Analysis 1>
In the patient group, the concentration of cytokine in saliva collected at the first time (after waking up), and the concentration of cytokine in saliva collected at the second time (daytime), and the concentration of cytokine in saliva collected in the healthy group Were compared. In addition, since the saliva of the second time (daytime) could not be collected from one person in the patient group, the number of samples collected in the second time (daytime) of the patient group was 19 specimens.

-Analysis result 1-
The concentrations of cytokines in saliva collected at the first time (after waking up) of the patient group were IL-13, G-CSF, IP-10, RANTES, except IL-15 and GM-CSF for which data could not be obtained. In addition, in cytokines other than VEGF, it was detected at a concentration higher than the concentration of cytokine in saliva collected in the healthy group.
On the other hand, the concentration of cytokines in saliva collected in the second time (daytime) of the patient group is lower than that of saliva collected after waking up, and tends to approach the average value of the healthy group. Was recognized.
Therefore, in the following analysis, in the patient group, the saliva sample collected the second time (daytime) was used.

<Analysis 2>
Statistical analysis by the Mann-Whitney Test was performed on the salivary cytokine concentration collected in the second time (daytime) of the patient group and the salivary cytokine concentration of the healthy subject group. The results are shown in Table 2.
Among the 27 cytokines analyzed, 11 cytokines (IL-2, IL-4, IL-5, IL-15, IL-17A, FGF, GM-CSF, MCP-1, PDGF-BB, MIP-1β, RANTES) were excluded from the analysis.

-Analysis result 2-
As a result of Analysis 2, regarding the cytokine concentration in saliva of the patient and the healthy subject, some significant differences were observed between the two (Table 2).
The presence or absence of a significant difference in sex and age was also analyzed for all cytokine types, but no significant difference in sex and age was observed.
From these results, among cytokines contained in saliva collected from patients, IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF It was shown that, regardless of sex and age, the concentrations of those cytokines contained in saliva collected from healthy subjects were significantly lower. A graph showing the average concentration of cytokines contained in saliva having a significant difference is shown in FIG. 1 (solid line: healthy subject group, broken line: ADHD patient group).

<Analysis 3>
As a result of analysis 2, the 8 cytokines that were significantly different between the patient group and the healthy group were further modeled by logistic regression analysis using the stepwise method, and the determination ability was verified by ROC analysis. went.

-Analysis result 3-
As a result of analysis 3, among the eight cytokines, the combination of IL-1ra and VEGF has an AUC (Area Under Curve) of 0.914 (sensitivity: 0.722, specificity: 1.000) by ROC analysis. And came closest to 1. The AUC calculated from the combination of other cytokines was lower than the AUC calculated from the combination of the IL-1ra and VEGF.
The AUC of IL-1β is 0.753, the AUC of IL-1ra is 0.708, the AUC of IL-7 is 0.717, the AUC of IL-8 is 0.821, and the AUC of IL-12 p70 is 0. .746, G-CSF had an AUC of 0.764, IFN-γ had an AUC of 0.756, and VEGF had an AUC of 0.808.

From the above results, regarding the concentration of cytokines in saliva, in ADHD patients, the cytokines (IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-) whose concentrations are lower than those in healthy subjects are obtained. CSF, IFN-γ, VEGF).
Furthermore, it was shown that the combination of IL-1ra and VEGF has a large difference, particularly between the patient group and the healthy subject group.
Therefore, it has been found that whether or not ADHD is affected or the possibility of being affected can be evaluated by using the amount of cytokine contained in the sample derived from the evaluation subject as an index.

Examples of aspects of the present invention include the following.
<1> An evaluation step for evaluating whether or not suffering from attention deficit hyperactivity disorder (ADHD) or having a possibility of developing it, using the amount of cytokine in a sample derived from an evaluation subject as an index And a method for evaluating whether or not the subject to be evaluated has ADHD or has a possibility of developing ADHD.
<2> The method according to <1>, wherein the evaluation method further includes a measurement step of measuring the amount of cytokine in the sample derived from the evaluation target.
<3> The evaluation step includes a process of comparing the amount of cytokine in the sample derived from the evaluation target with a reference value,
The reference value is at least one of a value based on the amount of cytokine in a sample derived from a healthy individual and a value based on the amount of cytokine in a sample derived from an ADHD patient,
When the amount of cytokine in the sample derived from the subject of evaluation is less than the value based on the amount of cytokine in the sample derived from the healthy individual, and when the amount is equal to or less than the value based on the amount of cytokine in the sample derived from the ADHD patient The method according to any one of <1> to <2>, which is a step of evaluating that the subject to be evaluated has or is likely to develop ADHD in at least either case.
<4> The cytokine is one or more cytokines selected from the group consisting of IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF. The method according to any one of <1> to <3> above.
<5> In the evaluation step, it is possible to evaluate whether the patient has ADHD or has a possibility of developing ADHD based on the data of the amount of the cytokine. The method according to any one of <1> to <2>, including substituting data, and the evaluation formula includes at least one variable into which the data on the amount of the cytokine is substituted. ..
<6> Two or more cytokines selected from the group consisting of IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF. And
In the evaluation step, the evaluation formula capable of evaluating whether or not ADHD is afflicted or has a possibility of developing ADHD based on the data of the amount of the two or more kinds of cytokines. The method according to <5>, wherein the evaluation formula includes at least two variables into which the data of the amounts of the two or more types of cytokines are substituted. Is.
<7> The method according to any one of <1> to <6>, wherein the sample derived from the evaluation target is saliva.
<8> The method according to any one of <1> to <7>, wherein the cytokine is IL-1ra and VEGF.
<9> To evaluate whether the evaluation subject has ADHD or has a possibility of developing ADHD, comprising a measurement step of measuring the amount of cytokine in a sample derived from the evaluation subject Is a method of acquiring the data of.
<10> The cytokine is one or more cytokines selected from the group consisting of IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF. The method according to <9> above.
<11> The method according to any one of <9> to <10>, wherein the sample derived from the evaluation target is saliva.
<12> The method according to any one of <9> to <11>, wherein the cytokine is IL-1ra and VEGF.

Claims (11)

The method comprises an evaluation step of evaluating whether or not suffering from attention deficit hyperactivity disorder (ADHD) or having a possibility of developing it, using the amount of cytokine in a sample derived from an evaluation subject as an index. A method for evaluating whether or not a subject to be evaluated has ADHD or has a possibility of developing ADHD. The method according to claim 1, wherein the evaluation method further includes a measurement step of measuring the amount of cytokine in a sample derived from the evaluation target. The evaluation step includes a process of comparing the amount of cytokine in a sample derived from the evaluation target with a reference value,
The reference value is at least one of a value based on the amount of cytokine in a sample derived from a healthy individual and a value based on the amount of cytokine in a sample derived from an ADHD patient,
When the amount of cytokine in the sample derived from the subject of evaluation is less than the value based on the amount of cytokine in the sample derived from the healthy individual, and when the amount is equal to or less than the value based on the amount of cytokine in the sample derived from the ADHD patient The method according to any one of claims 1 to 2, wherein in at least one of the cases, it is a step of evaluating that the subject to be evaluated is suffering from ADHD or has a possibility of developing ADHD. The cytokine is one or more cytokines selected from the group consisting of IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF. Item 4. The method according to any one of Items 1 to 3. The evaluation step substitutes the data on the amount of the cytokine into an evaluation formula capable of evaluating whether or not the patient has ADHD or has the possibility of developing ADHD based on the data on the amount of the cytokine. The method according to claim 1, wherein the evaluation formula includes at least one variable into which the data on the amount of the cytokine is substituted. The method according to claim 1, wherein the sample derived from the evaluation target is saliva. The method according to claim 1, wherein the cytokines are IL-1ra and VEGF. Data for evaluating whether or not the evaluation subject suffers from ADHD or has a possibility of developing ADHD, which comprises a measurement step of measuring the amount of cytokine in a sample derived from the evaluation subject. How to get. The cytokine is one or more cytokines selected from the group consisting of IL-1ra, IL-1β, IL-7, IL-8, IL-12 p70, G-CSF, IFN-γ, and VEGF. Item 8. The method according to Item 8. The method according to claim 8, wherein the sample derived from the evaluation target is saliva. The method according to any one of claims 8 to 10, wherein the cytokines are IL-1ra and VEGF.