JP2023088553A - Method and Reagent for Determining Stress Reaction Tendency - Google Patents

Abstract

To provide a novel method for determining stress response tendencies by typing single nucleotide polymorphisms.SOLUTION: Provided is a method for determining stress response tendencies, characterized by associating the typing results of a single nucleotide polymorphism selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987, and rs1324092 or a single nucleotide polymorphism in linkage disequilibrium therewith with the stress response tendencies.SELECTED DRAWING: None

Description

Application for application of Article 30, Paragraph 2 of the Patent Law is filed Poster presentation at the American Society of Human Genetics (ASHG) 2021 virtual meeting (held online via a webinar (ASHG 2021 Virtual Meeting platform)) Date: October 18-22, 2021 American Society of Human Genetics (ASHG) 2021 Virtual Meeting Proceedings (PrgmNr 2109) https://eventpilotadmin. com/web/planner. php? id = ASHG21 Website publication date September 8, 2021

TECHNICAL FIELD The present invention relates to a method and reagent for determining stress reaction tendency.

In modern society, people are exposed to many stresses. If it is possible to predict in advance the tendencies of reactions to stress, it is useful because measures to prevent or relieve stress can be taken.

Certain types of stress are known to be influenced by genetic factors. For example, it is known that a single nucleotide polymorphism (SNP) near the BMP2 gene affects stress (e.g., Non-Patent Document 1), and it is known that the type of SNP can predict reactivity to stress. .

J Clin Psychiatry. 2016 Jan;77(1):e29-30.

An object of the present invention is to provide a novel method for determining stress response tendencies by typing single nucleotide polymorphisms. Another object of the present invention is to provide a screening method for a drug for treating or preventing stress response (inability to fall asleep) by measuring gene expression levels.

The present inventors have made intensive studies to solve the above problems. As a result, it was found that a single nucleotide polymorphism selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987 and rs1324092 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism is associated with stress reactions such as irritability and sleep disturbance. It was found that there is a significant correlation with the tendency, and it was found that typing these SNPs can determine the stress reaction tendency. In addition, the expression level of the MTRF1L gene is associated with sleep onset disorders, and by screening for compounds that reduce the expression level of the MTRF1L gene, it was found that candidate substances for the treatment or prevention of sleep onset disorders can be obtained, thereby completing the present invention. rice field.

That is, the gist of the present invention is as follows.
[1] Typing results of single nucleotide polymorphisms selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987 and rs1324092 or single nucleotide polymorphisms in linkage disequilibrium with said single nucleotide polymorphisms, and associating stress response tendencies A method for determining a stress reaction tendency, characterized in that the stress reaction tendency is determined by:
[2] The method of [1], wherein the stress reaction is irritability, and the single nucleotide polymorphism is rs35775177 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism.
[3] The method of [1], wherein the stress reaction is sleep stress, and the single nucleotide polymorphism is rs533737, rs10811987, or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism.
[4] The method of [1], wherein the stress reaction is anorexia, and the single nucleotide polymorphism is rs1779644 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism.
[5] The method of [1], wherein the stress reaction is overeating stress, and the single nucleotide polymorphism is rs1324092 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism.
[6] A stress reaction tendency determination system,
Means for inputting the typing results of single nucleotide polymorphisms in the test sample;
means for setting single nucleotide polymorphisms associated with stress response propensity;
Means for determining strength of stress reaction tendency based on the input typing result and the setting; and Means for displaying the determination result;
with
The SNP is a single nucleotide polymorphism selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987 and rs1324092 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism,
The setting of the single nucleotide polymorphisms related to the stress response tendency is G allele at rs35775177; G allele at rs1779644; T allele at rs10811987; A allele at rs533737; A corresponding allele of a single nucleotide polymorphism in a balanced relationship,
The determination means determines that the stress reaction tendency is strong when the typing result of the single nucleotide polymorphism corresponds to at least one setting of the single nucleotide polymorphism related to the stress reaction tendency, and when it does not correspond Judging that the stress reaction tendency is weak,
the determination system;
[7] A probe for determining stress reaction tendency, which comprises a polynucleotide having a sequence of 15 or more bases including the 51st base in the base sequence shown in any one of SEQ ID NOs: 1 to 5, or a complementary sequence thereof.
[8] A primer for determining stress reaction tendency, comprising a polynucleotide capable of amplifying a region containing the 51st base in the nucleotide sequence shown in any one of SEQ ID NOS: 1-5.
[9] adding a drug candidate substance to cells expressing the MTRF1L gene or a reporter gene linked to the promoter of the MTRF1L gene, measuring the expression level of the MTRF1L gene or the reporter gene, and a substance that reduces the expression level A method of screening for a preventive or therapeutic drug for sleep onset disorders, comprising the step of selecting

According to the present invention, it is possible to determine the tendency of stress reactions, predict the likelihood of stress reactions occurring in an individual, and take measures to prevent or relieve stress.
In addition, according to the present invention, candidate substances for therapeutic or preventive drugs for sleep onset disorders can be screened.

<1> Stress reaction tendency determination method The method of the present invention is a single nucleotide polymorphism (SNP) selected from rs35775177, rs1779644, rs533737, rs10811987 and rs1324092, or a typing result of an SNP in linkage disequilibrium with the single nucleotide polymorphism. It is characterized by determining the stress reaction tendency by associating the stress reaction tendency with the stress reaction tendency. In the present invention, "judgment" means presenting information about the tendency of stress reactions, for example, presenting information that a person tends to easily develop a stress reaction or tends not to easily develop a stress reaction. do.

Specific examples of stress response tendencies include irritability, loss of appetite, sleep disturbance, and overeating stress. Sleep disorders include difficulty falling asleep and excessive daytime sleepiness (deep sleep disorder). Overeating stress includes the behavior of stocking up on sweets and the like.

The method of the present invention can be applied to subjects of any race, but can be preferably applied to Asian subjects such as Japanese and Chinese, and European and American subjects.

A useful SNP for determining whether a person tends to feel irritated is rs35775177. where the rs number is the d of the National Center for Biotechnology Information
Accession numbers of the bSNP database (http//www.ncbi.nlm.nih.gov/projects/SNP/) are indicated.

rs35775177 is a SNP located in the linkage disequilibrium block near the BSG and HCN2 genes in the p13.3 region of human chromosome 19 of GRCh38 and is adenine (A)/ It means a polymorphism of guanine (G), and when this base is G, there is a strong tendency to feel irritated. In addition, when the genotype is taken into account in the analysis, the tendency to feel irritated is strong in the order of GG>GA>AA. Therefore, for subjects whose base is G, it is conceivable to propose healing products and supplements that relieve irritation and behaviors that actively encourage a change of mood.

Regarding rs35775177, a sequence of a total length of 101 bp including the SNP base and the 50 bp region before and after it is shown in SEQ ID NO:1. The 51st base in SEQ ID NO: 1 has a polymorphism, and when this base is A, it is determined that the stress reaction tendency is strong.

In the present invention, bases corresponding to the above bases are analyzed. "A base corresponding to the above base" means the corresponding base in the above region on a human chromosome. That is, "analyzing the base corresponding to the above base" includes analyzing the corresponding base in the above region even if the above sequence slightly changes at a position other than the SNP due to differences in race or the like. .

Further, the bases to be analyzed in the present invention are not limited to those described above, and polymorphisms of bases in linkage disequilibrium with the above bases may be analyzed. Here, "a base in linkage disequilibrium with the above base" refers to a base that preferably satisfies the relationship of r ² >0.8, more preferably r ² >0.9 with the above base. r ² is the linkage disequilibrium coefficient. Nucleotides in linkage disequilibrium can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja).

Examples of bases in linkage disequilibrium with rs35775177 include, specifically, , rs67945626, rs8259, rs4919812, rs4919864, rs12609751, rs12609750, rs8111125 , rs372293671, rs72618586, rs113534512, rs542257702, rs3814894.

A useful SNP for determining whether a person is prone to anorexia is rs1779644.

rs1779644 is a SNP located in the region between the PRPF38AP1 gene and the LINC00708 gene in the human chromosome 10 p14 region of GRCh38, and is a guanine (G)/thymine (T) at base 8191935 of GenBank Accession No. NC_000010.11. It means polymorphism, and when this base is G, there is a strong tendency to cause anorexia. In addition, when analyzed in consideration of genotype, there is a strong tendency to cause anorexia in the order of GG > GT > TT. Therefore, it is conceivable to propose supplements that improve appetite to subjects whose base is G.

Regarding rs1779644, a sequence of a total length of 101 bp including the SNP base and the 50 bp region before and after it is shown in SEQ ID NO:2. The 51st base in SEQ ID NO: 2 has a polymorphism, and when this base is G, it is determined that there is a strong tendency to cause anorexia.

Specific examples of bases in linkage disequilibrium with rs1779644 include rs1779645, rs1779647, rs1796872, rs1779643, rs2489248, rs1361313, rs1416563, and rs35409436.

SNPs useful in determining whether a person is prone to sleep disorders include rs10811987 and rs533737. Among the sleep disorders, rs10811987 correlates with sleep disorders in which excessive sleepiness is felt during the day (deep sleep disorders), and rs533737 correlates with sleep onset disorders.

rs10811987 is a SNP located in the region between the RP11-321L2.1 gene and the RP11-321L2.2 gene in the human chromosome 9 p21.3 region of GRCh38, and is a cytosine at base 23871889 of GenBank Accession No. NC_000009.12. It means a polymorphism of (C)/thymine (T), and when this base is T, there is a strong tendency to develop deep sleep disorders. In addition, when analyzed in consideration of genotype, there is a strong tendency to develop deep sleep disorder in the order of TT > TC > CC. Therefore, it is conceivable to propose supplements or the like that improve deep sleep disorders to subjects whose base is T.

Regarding rs10811987, a sequence of a total length of 101 bp including the SNP base and the 50 bp region before and after it is shown in SEQ ID NO:3. The 51st base in SEQ ID NO: 3 has a polymorphism, and when this base is T, it is determined that there is a strong tendency to develop deep sleep disorders.

Nucleotides in linkage disequilibrium with rs10811987 include rs528394, rs1626477, rs2383296, rs10811988, rs10811993, rs1376138, rs1817037, rs10738686, rs2122537, rs10811998, rs10811990 , rs10811989, rs10738685, rs10966133, rs12683995, rs11515276, rs2027523, rs7044269, rs10812001, rs7875041 , rs10812002, rs10812003, rs10966149, rs10966151, rs10811991, rs7467812, rs10966119, rs527885, rs10966120, rs1528049, rs78134254, rs553556, rs70370 42, rs10966135, rs7858335.

rs533737 is a SNP located in the region between the VIP gene and the RP1-200K18.1 gene in the q25.2 region of human chromosome 6 of GRCh38, and is a guanine (G )/adenine (A) polymorphism, and when this base is A, there is a strong tendency to develop difficulty falling asleep. In addition, when analyzed in consideration of genotype, there is a strong tendency to develop sleep onset disorders in the order of AA > AG > GG. Therefore, for subjects whose base is A, it is conceivable to propose a supplement or the like that improves their difficulty falling asleep.

Regarding rs533737, a sequence of a total length of 101 bp including the SNP base and the 50 bp region before and after it is shown in SEQ ID NO:4. The 51st base in SEQ ID NO: 4 has a polymorphism, and when this base is A, it is determined that there is a strong tendency to develop sleep disorders.

Nucleotides in linkage disequilibrium with rs533737 include, specifically, 99558, rs9478358, rs7759363, rs9479402, rs76223855, rs9478361, rs200438260 , rs11340348, rs500841, rs500762, rs36085354, rs75284686, rs79213203, rs661461, rs641110.

A useful SNP for determining whether a person is prone to overeating stress is rs1324092. Overeating stress includes the behavior of stocking up on sweets and the like.

rs1324092 is a SNP located in the region between the KIAA1009 gene and the RP1-90L14.1 gene in the q14.3 region of human chromosome 6 of GRCh38, and is a guanine (G )/thymine (T) polymorphism, and when this base is G, there is a strong tendency to feel overeating stress. In addition, when analyzed in consideration of genotype, there is a strong tendency to feel overeating stress in the order of GG > GT > TT. Therefore, subjects whose base is G may be offered stress-relieving products or positively stimulating behaviors.

In addition, for rs1324092, a total of 101 bp including the SNP base and the 50 bp region before and after it
The length of the sequence is shown in SEQ ID NO:5. The 51st base in SEQ ID NO: 5 has a polymorphism, and when this base is G, it is determined that there is a strong tendency to be susceptible to overeating stress.

Examples of bases in linkage disequilibrium with rs1324092 specifically include rs7767211, rs34053687, rs13200720, rs79549621, and rs59203163.

Stress reaction tendency can be determined by typing the above SNPs and correlating the obtained typing result with stress reaction tendency based on the above criteria.
Criteria for association of each SNP with stress response are summarized below.
1) rs35775177 has a strong tendency to feel irritated when the allele is G 2) rs1779644 has a strong tendency to cause loss of appetite when the allele is G 3) rs10811987 has a T allele Criterion that there is a strong tendency to develop deep sleep disorder in some cases 4) Criterion that if rs533737 is allele A, there is a strong tendency to develop sleep onset disorder 5) If rs1324092 is allele G, overeating stress is likely to occur Criteria for strong tendency

The method of the present invention may include a typing step of examining the SNP type (base). However, the step of examining the SNP type is not essential, since the stress response tendency can be determined based on the predetermined SNP type.
A method for examining the base type of SNPs will be described below.

The above SNPs may be analyzed individually, or a plurality of them may be analyzed collectively. For example, a plurality of the above SNPs may be analyzed collectively, or at least one of the above SNPs may be analyzed in combination with known SNPs associated with stress response tendencies or SNPs in linkage disequilibrium with the known SNPs. may Collective analysis of multiple SNPs associated with stress reaction tendencies improves the accuracy of stress reaction tendency testing. For any SNP, either strand of the double-stranded DNA may be analyzed. For example, each sequence may be analyzed on the sense strand or the antisense strand.

The sample used for SNP analysis is not particularly limited as long as it contains chromosomal DNA. Samples used for SNP analysis include, for example, body fluids such as blood, urine, and cerebrospinal fluid, cells such as uterine cervix and oral mucosa, body hair such as hair, and the like. Although these samples can be used directly for SNP analysis, it is preferable to isolate chromosomal DNA from these samples by a conventional method and use it for analysis.

SNP analysis can be performed by a normal genetic polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, invader method, and the like.

A sequence analysis can be performed by a normal method. Specifically, a sequence reaction is performed using a primer set at a position several dozen bases 5' to the base showing the polymorphism, and the type of base at the corresponding position is determined from the analysis results. can do. In addition, it is preferable to amplify the fragment containing the SNP site in advance by PCR or the like before the sequencing reaction.

In addition, SNP analysis can be performed by examining the presence or absence of amplification by PCR. For example, primers each having a sequence corresponding to a region containing a base exhibiting a polymorphism and having a 3' end corresponding to each polymorphism are prepared. PCR can be performed using each primer and the type of polymorphism can be determined by the presence or absence of an amplified product. Alternatively, an isothermal amplification method such as the LAMP method or a single-strand amplification method may be used.

It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization. That is, it is also possible to examine which base the SNP is by preparing probes corresponding to each base and examining which probe it hybridizes to.

By determining which base the SNP is in this way, it is possible to obtain data for examining stress response tendencies.
The results determined by the method of the present invention are provided to doctors and the like as necessary. Physicians and others provided with the results can take measures to prevent or alleviate stress. Also, the determination results may be provided to the subject, and supplements, programs, and the like for preventing or relieving stress may be proposed.

<2> Stress reaction tendency determination system The present invention also provides a stress reaction tendency determination system for determining whether or not a subject has a strong stress reaction tendency.
The system includes means for inputting SNP typing results in the test sample;
Means for designating SNPs associated with stress response propensity;
Means for judging the strength or weakness of the subject's stress reaction tendency based on the input typing result and pre-stored association setting between stress reaction tendency and SNP type; and means for displaying the judgment result. The determination system may further comprise means for typing SNPs in the test sample.

Specifically, the stress reaction tendency determination system is
Means for inputting results of single nucleotide polymorphism (SNP) typing in a test sample;
Means for designating SNPs associated with stress response propensity;
Means for determining strength of stress reaction tendency based on the input typing result and the setting; and Means for displaying the determination result;
with
The SNP is a SNP selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987 and rs1324092 or a SNP in linkage disequilibrium with the SNP;
The setting of SNPs related to the stress response tendency is G allele at rs35775177; G allele at rs1779644; T allele at rs10811987; A allele at rs533737; and G allele at rs1324092; is the corresponding allele of the polymorphism,
The determination means determines that the stress reaction tendency is strong when the SNP typing result corresponds to at least one setting of SNPs related to the stress reaction tendency, and the stress reaction tendency is weak when it does not correspond. I judge.

Specific stress responses to each SNP are as described above, but alleles may be set for each of the strengths and weaknesses of stress response tendencies as described below.
1) rs35775177 has a strong tendency to feel irritated when the base is G, and a weak tendency to feel irritated when the base is G (difficult to feel irritated)
2) rs1779644 has a strong tendency to cause anorexia when the base is G, and a weak tendency to cause anorexia when the base is T (less likely to cause anorexia)
3) rs10811987 has a strong tendency to develop deep sleep disturbance when the base is T, and a weak tendency to develop deep sleep disturbance when the base is C (difficult to develop deep sleep disturbance)
4) rs533737 has a strong tendency to develop sleep onset disorders when the base is A, and a weak tendency to develop sleep onset disorders when the base is G (hard to develop sleep onset disorders)
5) rs1324092 has a strong tendency to cause overeating stress when the base is G, and a weak tendency to cause overeating stress when the base is T (hard to cause overeating stress)

The results obtained by the above SNP typing means are manually or automatically entered into a computer or the like by a means for entering the SNP typing results in the test sample. The input results are compared and/or collated with alleles having a strong stress reaction tendency preset by means for setting SNPs related to stress reaction tendency, and the strength of the stress reaction tendency is judged by the judgment means, Judgment results are displayed. These means can be, for example, a computer or a software program running on a computer.
The stress reaction tendency determination system may further include means for providing information for preventing or relieving stress based on the stress reaction tendency determination result. For example, when it is determined that the person tends to feel irritated, there is a means of providing information on supplements and activities for preventing or alleviating the irritated feeling. Such information may be set in advance in association with SNPs associated with stress reaction tendencies.

Note that SNP typing may be performed for only one SNP, and is preferably performed for two or more SNPs in consideration of improving the accuracy of determination. Therefore, it is preferable that multiple SNPs be set for SNPs associated with stress response tendencies.

<3> Test Reagent for Stress Reaction Tendencies The present invention also provides test reagents such as primers and probes for determining stress reaction tendencies. Examples of such probes include probes that contain the above SNP site and can determine the type of base at the SNP site based on the presence or absence of hybridization. Specifically, a sequence containing the 51st base of the base sequence shown in any one of SEQ ID NOS: 1 to 5, or a probe consisting of a polynucleotide having a length of 15 bases or more having a complementary sequence thereof, or linked to the base Examples include probes consisting of a polynucleotide having a length of 15 bases or more having a sequence containing bases in an unbalanced relationship or a complementary sequence thereof. In addition, the nucleotide sequences of the "nucleotides in a relationship of linkage disequilibrium with the relevant nucleotides" and the regions before and after them can be obtained from, for example, the dbSNP database of the National Center for Biotechnology Information (http//www.ncbi.nlm.nih.gov/projects /SNP/). The length of the polynucleotide as a probe is preferably 15-35 bases, more preferably 20-35 bases.

In addition, as the primer, a primer consisting of a polynucleotide that can be used for PCR for amplifying the SNP site, or a primer consisting of a polynucleotide that can be used for sequence analysis (sequencing) of the SNP site is used. mentioned. Specifically, a primer composed of a polynucleotide capable of amplifying or sequencing a region containing the 51st base of the nucleotide sequence shown in any of SEQ ID NOs: 1 to 5, or linkage disequilibrium with the base A primer consisting of a polynucleotide capable of amplifying or sequencing a region containing bases having a relationship of is mentioned. The length of the polynucleotide used as such a primer is preferably 15 to 50 bases, more preferably 15 to 35 bases, and even more preferably 20 to 35 bases.

As the primer for sequencing the SNP site, a primer consisting of a polynucleotide having a sequence upstream of the 5′ region of the base, preferably 30 to 100 bases upstream, or a 3′ region of the base, preferably 30 bases, is used. A primer consisting of a polynucleotide having a sequence complementary to a region ~100 bases downstream is exemplified. Primers used for determining polymorphisms based on the presence or absence of amplification by PCR include a primer comprising a polynucleotide having a sequence containing the above base and containing the above base on the 3′ side, and a complementary sequence of a sequence containing the above base. and a primer consisting of a polynucleotide containing a complementary base of the above-mentioned base on the 3′ side.

In addition to these primers and probes, the test reagents of the present invention may contain polymerases and buffers for PCR, hybridization reagents, and the like.

<4> Screening method for prophylactic or therapeutic drug for sleep onset disorder In the present invention, as described later, the risk allele (A) of rs533737 causes an increase in MTRF1L expression, and the possibility that the increase in MTRF1L expression affects sleep onset disorder. It was suggested that there is Therefore, candidate substances for preventive or therapeutic drugs for sleep onset disorders can be obtained by screening drugs using the activity of reducing MTRF1L expression as an indicator.

MTRF1L is a protein involved in translation in mitochondria (Mol.Cell.2007. Sep 7;27(5):745-57., Genes to Cells,2008.May;13(5):429-38.), Specific examples of the MTRF1L gene include the region from 152987362 to 153003439 registered in GenBank Accession No. NC_000006.12.

That is, the screening method of the present invention includes the steps of adding a test substance to cells expressing the MTRF1L gene or a reporter gene linked to the promoter of the MTRF1L gene, measuring the expression level of the MTRF1L gene or the reporter gene, and Screening methods for prophylactic or therapeutic agents for sleep onset disorders, which include the step of selecting a substance that reduces the expression level, may be mentioned.

For example, when the expression level of the MTRF1L gene or reporter gene is reduced in cells in the presence of the test substance compared to the absence of the test substance, the test substance is used as a candidate for a prophylactic or therapeutic drug for sleep onset disorder. material can be selected.

Examples of cells that express the MTRF1L gene include mammalian cells such as mice and humans. For example, tissue cells such as epithelial cells and fibroblasts, and cultured cells such as HeLA cells can be used. can.

When a reporter gene linked to the promoter of the MTRF1L gene is used, the promoter of the MTRF1L gene is preferably a region containing about 2 kbp upstream of the transcription initiation site, more preferably a region containing about 5 kbp upstream. The promoter sequence information is available from the upstream region of the MTRF1L gene from 152987362 to 153003439 in GenBank Accession No. NC_000006.12, respectively.

Examples of reporter genes include luciferase gene, GFP gene, chloramphenicol acetyltransferase gene and the like. These reporter genes are ligated to the promoter of the MTRF1L gene, incorporated into a plasmid used for gene introduction into mammalian cells, and transfected into cells by a conventional method such as lipofection.

A drug candidate substance is added to cells expressing the MTRF1L gene or cells into which the reporter gene has been introduced, and the expression level of the MTRF1L gene or the reporter gene is measured.

The drug candidates are not particularly limited, and may be, for example, low-molecular-weight synthetic compounds or compounds contained in natural products. Moreover, it may be a peptide or a nucleic acid. Individual candidate substances may be used for screening, but compound libraries containing these substances may also be used. By selecting a candidate substance that reduces the expression level of the MTRF1L gene or reporter gene (compared to when it is not added), it is possible to obtain a substance that can be used as a prophylactic or therapeutic drug for sleep onset disorders.

The expression level of the MTRF1L gene can be measured by methods such as RT-PCR, quantitative PCR, northern blotting, ELISA, Western blotting, in situ hybridization, and immunohistochemical staining. Although the expression level of the reporter gene depends on the type of reporter gene, it can be measured by fluorescence intensity, luminescence intensity, radioactivity intensity, and the like.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to the embodiments of the Examples below.

<Method>
Subjects are members of genetic testing MYCODE (DeNA Life Science) who have obtained written informed consent to participate in research conducted using genetic information and questionnaire information. Individual responses regarding stress reactions and eating behavior were obtained through a voluntary web questionnaire conducted on the MYCODE page.

Saliva was used as a sample for SNP analysis. DNA was extracted and purified from the samples using MagaZorb DNA Mini-Prep Kit (Promega).
Genotyping of SNPs was performed using Illumina's Human OmniExpress-24+ BeadChip and Infinium OmniExpress-24+ BeadChip. In this method, after DNA is amplified and fragmented, the fragmented DNA is hybridized with the DNA on the beads on a microarray (bead chip), and the DNA bound to the bead chip is fluorescently labeled and analyzed.

SNP quality controls included SNPs with a call rate <0.99, SNPs deviating from Hardy-Weinberg equilibrium (HWE) (p < 10-6), SNPs on sex chromosomes, and minor allele frequency of SNPs. SNPs with (MAF) less than 0.01 were excluded.

Using 492,209 autosomal SNPs after SNP quality control, subject quality control was performed on the 61,728 subjects who responded to the web questionnaire. Exclusion of one subject of a closely related pair with a proportion identical by descent (IBD) > 0.1875, call rate
rate) was 0.95 or less, and subjects whose self-reported sex and genotype-determined sex did not match were excluded.
Furthermore, to identify outliers who do not belong to the Japanese cluster, 1000 Genomes
Project phase 3 (1KGP) East Asian samples (N = 504) were used to perform principal component analysis. Subjects who deviated significantly from the human (JPT) cluster were excluded. In the end, 55,551 were left.

492,209 SNPs after SNP quality control were phased using Eagle followed by genotype imputation using minimac3. 1000 as reference panel
Individuals derived from Japanese (JPT; N=104) data from the Genomes Project phase 3 (1KGP; N=2,504) were used.

Five SNPs (rs35775177, rs1779644, rs533737, rs10811987 and rs1324092) met the imputation quality control accuracy criterion (Rsq ≥ 0.7). Furthermore, there was no difference in allele frequency from the 8.3KJPT sample of the Tohoku Medical Megabank Organization, the largest public database of genetic polymorphisms in the Japanese population (p ≥ 0.05, Chi-Square
test), confirming high quality data.

Using up to 5,164,043 SNPs on autosomes with a MAF of 0.05 or higher, a genome-wide association study (GWAS) of each phenotype was performed on the number of subjects for whom each phenotype data was obtained by web questionnaire. ) was implemented. Using PLINK, the correlation between each SNP and phenotype was analyzed by logistic regression analysis assuming an additive model for minor alleles. As covariates, we designated sex, age, and the first and second principal components based on principal component analysis of genetic information.

Stress reaction_Irritation As for feeling irritability, there were 3 items: "I feel angry", "Inwardly irritated", and "Irritated". We took a questionnaire in four stages, calculated the integrated score from these three items, and performed logistic regression analysis by converting the lower score to 10% as a case and converting the others into control binary data. .
A total of 5,163,271 SNPs were analyzed in 7,351 cases vs. 23,811 controls among a total of 31,162 individuals.

Stress reaction_decreased appetite A questionnaire was given on the 4 levels of decreased appetite: rarely, occasionally, often, and almost always. As a result, logistic regression analysis was performed by converting the other data into control binary data.
A total of 5,163,271 SNPs were analyzed in 1,354 cases vs. 29,841 controls among a total of 31,195 individuals.

Stress reaction_sleep complaint_excessive daytime sleepiness (deep sleep disorder)
Regarding deep sleep disorder, we took a questionnaire with "Yes" and "No" to the question "I am attacked by intense daytime sleepiness", and performed a logistic regression analysis by converting Yes to case and No to control binary data.
A total of 5,163,271 SNPs were analyzed in 7,637 cases vs. 22,785 controls among a total of 30,422 individuals.

Stress reaction_sleep complaints_disorder to fall asleep Regarding sleep onset problems, we took a questionnaire with "Yes" and "No" to the question "I can not fall asleep easily even if I put it in the futon", and converted it into binary data with Yes as case and No as control. A transformed logistic regression analysis was performed.
A total of 5,163,271 SNPs were analyzed in 5,431 cases vs. 24,991 controls among a total of 30,422 individuals.

Stress reaction _ Behavior of stocking up on sweets, etc. Concerning overeating stress, we took a questionnaire with "Yes" and "No" to the question "I keep stocking up on sweets, etc." A transformed logistic regression analysis was performed.
A total of 5,163,271 SNPs were analyzed in 10,177 cases vs. 16,562 controls among a total of 26,739 individuals.

Consideration stress reaction _ frustration
The 19p13.3 region, associated with irritability, is located downstream of the BSG gene and upstream of the HCN2 gene.
Among the SNPs in linkage disequilibrium with the lead SNP; rs35775177, there was a missense mutation SNP of the HCN2 gene; P=1.75E-06). The functional alteration of HCN2 by rs113534512 is unknown.
HCN2 is involved in the regulation of resting membrane potential and plays an important role in neuronal excitability (Ludwig A, et al., Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. EMBO J 22: 216-224). (2003).
In addition, HCN2 mutations have been reported to be involved in febrile convulsions and generalized epilepsy (Online Mendelian Inheritance in Man (OMIM), *613542, https://www.omim.org/).
“Irritation has been studied as a symptom of dementia in particular. is considered to be involved in the enhancement of , the decrease of the serotonin system in the frontal lobe cortex, and the enhancement of the dopamine and noradrenaline systems (Takahashi Satoshi, Geriatric Psychiatry Journal 2011;22:115-20.)”
A loss-of-function mutation in HCN2 has been reported to increase neuronal excitability in patients with idiopathic generalized epilepsy (Jacopo C DiFrancesco, et al., Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy, J Neurosci. 2011. 31(48):17327-37.)
From the above, it is speculated that having the G allele of rs35775177 leads to decreased HCN2 function and increases the possibility of feeling irritated.

Stress response_sleep complaints_inability to fall asleep An association was observed in 6q25.2 (p = 2.3E-10) as a region associated with insomnia.
An eQTL search (GTEx Analysis Release V8) of the lead SNP (rs533737) suggested that it regulates the expression level of MTRF1L in many tissues.
Knockdown of MTRF1L reportedly increased mitochondrial production of reactive oxygen species (ROS) (Soleimanpour-Lichaei, HR et al., mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG. Molec. Cell 27: 745-757, 2007).
From the above, it is speculated that having the rs533737 G allele reduces the expression of MTRF1L and enhances the mitochondrial ROS production ability in the steady state, which works to protect against sleep onset disorders. be.
MTRF1L has been reported to be associated with circadian rhythms, which are closely related to sleep, from GWAS targeting Westerners (Youna Hu, et al., GWAS of 89,283 individuals identifies genetic variants associated with self-reporting of being a morning person. Nat Commun.7: 10448. 2016.), supporting the MTRF1L-mediated association of rs533737 with sleep onset disorders.

Claims (9)

By associating the typing result of a single nucleotide polymorphism selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987 and rs1324092 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism with the stress response tendency, stress A method for judging a stress reaction tendency, comprising judging a reaction tendency. 2. The method of claim 1, wherein the stress response is irritability and the single nucleotide polymorphism is rs35775177 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism. 2. The method of claim 1, wherein said stress response is sleep stress and said single nucleotide polymorphism is rs533737, rs10811987 or a single nucleotide polymorphism in linkage disequilibrium with said single nucleotide polymorphism. 2. The method of claim 1, wherein the stress response is anorexia and the single nucleotide polymorphism is rs1779644 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism. 2. The method of claim 1, wherein the stress response is overeating stress and the single nucleotide polymorphism is rs1324092 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism. A stress reaction tendency determination system,
Means for inputting the typing results of single nucleotide polymorphisms in the test sample;
means for setting single nucleotide polymorphisms associated with stress response propensity;
Means for determining strength of stress reaction tendency based on the input typing result and the setting; and Means for displaying the determination result;
with
The single nucleotide polymorphism is a single nucleotide polymorphism selected from the group consisting of rs35775177, rs1779644, rs533737, rs10811987 and rs1324092 or a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism,
The setting of the single nucleotide polymorphisms related to the stress response tendency is G allele at rs35775177; G allele at rs1779644; T allele at rs10811987; A allele at rs533737; A corresponding allele of a single nucleotide polymorphism in a balanced relationship,
The determination means determines that the stress reaction tendency is strong when the typing result of the single nucleotide polymorphism corresponds to at least one setting of the single nucleotide polymorphism related to the stress reaction tendency, and when it does not correspond Judging that the stress reaction tendency is weak,
the determination system; A probe for determining stress reaction tendency, comprising a polynucleotide having a sequence of 15 or more bases including the 51st base in the base sequence shown in any one of SEQ ID NOs: 1 to 5, or a complementary sequence thereof. A primer for determining stress response tendency, comprising a polynucleotide capable of amplifying a region containing the 51st base in the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 5. adding a drug candidate substance to cells expressing the MTRF1L gene or a reporter gene linked to the promoter of the MTRF1L gene, measuring the expression level of the MTRF1L gene or the reporter gene, and selecting a substance that reduces the expression level A method of screening for a preventive or therapeutic drug for sleep onset disorders, comprising the steps of: