JP2022119613A - Method for determining genetic predisposition to stress sensitivity - Google Patents

Abstract

To provide means for accurately and easily determining genetic predisposition to individuals' stress sensitivity.SOLUTION: A method for determining a genetic predisposition to stress sensitivity comprises the steps of: detecting an allele at the 101st single nucleotide polymorphism (SNP) site of the nucleotide sequences shown in SEQ ID NOs 1 to 5 for a DNA-containing sample collected from a subject; and determining that the subject has a genetic predisposition to stress sensitivity when at least one of the detected allele bases is a risk allele.SELECTED DRAWING: None

Description

The present invention relates to a method for determining a genetic predisposition to stress sensitivity. More specifically, the present invention relates to a method for determining a genetic predisposition to stress susceptibility by detecting a single nucleotide polymorphism (SNP) associated with stress susceptibility, and a kit used for the method.

Stress is a state of tension that occurs in the body and mind when an organism receives external stimuli. Stress includes environmental factors such as noise and lighting, physical factors such as illness and injury, psychological factors such as anxiety and tension, and social factors such as busyness and heavy responsibility. The skin is considered to be a tissue susceptible to stress, and excessive stress causes disturbance of the autonomic nerves and hormone balance, resulting in various skin troubles. For example, it has been reported that stress causes secretion of nerve-related substance P in the skin, causing rough skin (Patent Document 1). It has also been clarified that skin pigmentation symptoms are caused not only by ultraviolet rays but also by stress (Non-Patent Document 1).

As methods for evaluating the stress state, a method using cortisol in blood collected from a subject as an index (Patent Document 2), a method using α-amylase activity in saliva collected from a subject as an index (Patent Document 3), and the like are known. It is However, these methods are intended to evaluate the current stress state of the subject, and not to evaluate the subject's inherent susceptibility to stress (genetic predisposition). On the other hand, genetic polymorphisms associated with noradrenaline transporters and serotonin transporters have been reported as genetic predispositions associated with neuropsychiatric disorders such as depression and anxious personality (Non-Patent Documents 2 and 3).

Japanese Patent Application Laid-Open No. 2005-185103 JP 2012-251857 A JP-A-2002-168860

Hiroko Kaminaga, Tatsuro Shinomiya, Journal of Nissinkai, 107(5):615-622, 1997. Kazuyuki I. et al, Positive Association between T-182C Polymorphism in the Norepinephrine Transporter Gene and Susceptibility to Major Depressive Disorder in a Japanese Population, 2004, Neuropsychobiology 50(4):301-4 K. P. Lesch et al., Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region, 1996, SCIENCE. vol. 274.5292.1527-1531

An object of the present invention is to provide a means for accurately and simply determining the genetic predisposition of an individual's susceptibility to stress.

The present inventors discovered a specific single nucleotide polymorphism (SNP) associated with a genetic predisposition to stress sensitivity, and completed the present invention.

That is, the present invention includes the following inventions.
[1] A step of detecting one or more of the following (a1) to (a5) single nucleotide polymorphism (SNP) alleles in a DNA-containing sample collected from a subject, and bases of the detected alleles is a risk allele, determining that the subject has a genetic predisposition to stress.
(a1) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs74548608)
(a2) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 2 (SNP identified by SNP: ID rs57001330)
(a3) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 3 (SNP identified by SNP: ID rs6950885)
(a4) SNP at the 101st base of the base sequence shown in SEQ ID NO: 4 (SNP identified by SNP: ID rs8092883)
(a5) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs2359433)
[2] Cosmetics and/or having stress preventive and/or ameliorating action according to the degree of genetic predisposition to stress susceptibility of the subject based on the results determined by the method described in [1] A method for providing cosmetics and/or food and drink, comprising providing food and drink to the subject.
[3] A probe having a sequence of 10 or more bases including the 101st base in the base sequence shown in any of SEQ ID NOS: 1-5, or a complementary sequence thereof, and/or any of SEQ ID NOS: 1-5 A kit for determining a genetic predisposition to stress susceptibility, comprising primers capable of amplifying a region containing the 101st base in the nucleotide sequence shown in .

According to the method of the present invention, by detecting alleles of single nucleotide polymorphisms (SNPs) contained in genome-derived DNA present in a biological sample of a subject, it is possible to determine whether the subject has a genetic predisposition to easily feel stress. can be determined accurately and simply. Therefore, based on this determination result, it is possible to quickly take measures to prevent or improve stress.

1. Method for determining genetic predisposition for susceptibility to stress In the method for determining a genetic predisposition for susceptibility to stress of the present invention, one of the following (a1) to (a5) is used for a DNA-containing sample collected from a subject. Alternatively, the step of detecting two or more single nucleotide polymorphism (SNP) alleles, and if at least one of the bases of the detected alleles is a risk allele, the subject has a genetic predisposition to easily feel stress. including the step of determining.
(a1) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs74548608)
(a2) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 2 (SNP identified by SNP: ID rs57001330)
(a3) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 3 (SNP identified by SNP: ID rs6950885)
(a4) SNP at the 101st base of the base sequence shown in SEQ ID NO: 4 (SNP identified by SNP: ID rs8092883)
(a5) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs2359433)

Table 1 shows the base sequences containing the above SNPs (a1) to (a5) (SNPs are shown in brackets).

As shown in the results of Examples described later, the above SNPs (a1) to (a5) were suggested to be statistically related to the genetic predisposition to stress sensitivity. Although one of the SNPs (a1) to (a5) above can be used to determine the genetic predisposition to stress, combining two or more SNPs can further increase the determination accuracy. For example, the combination of rs74548608 and rs57001330, the combination of rs74548608 and rs6950885, the combination of rs74548608 and rs8092883, the combination of rs74548608 and rs2359433 are preferred, and the combination of rs74548608 and rs57001330 is particularly preferred.

A single nucleotide polymorphism (SNP, hereinafter sometimes referred to as "SNP") generally refers to a state in which the nucleotide sequence of a gene differs at only one location and its location. In addition, polymorphism generally refers to two or more alleles present at a frequency of 1% or more in a population. "SNP" in the present invention refers to the SNP database (http://www.ncbi.nlm) of the US National Center for Biotechnology Information (NCBI), which is a publicly available database freely available to those skilled in the art. nih.gov/SNP/) and can be identified by its reference number, rs number.

As used herein, the term “allele” refers to each type having different bases that can be present at a certain SNP site. As used herein, the term "genotype" refers to a combination of opposing alleles at a certain SNP site. At a certain SNP site, there are three types of genotypes, which are the combinations. A combination of the same alleles is called a homotype, and a combination of different alleles is called a heterotype. For example, there are three genotypes, C/C type, C/G type, and G/G type, which are genotypes that are combinations of opposing alleles in the SNP identified by rs74548608 in (a1).

In the determination method of the present invention, "detecting an allele of a single nucleotide polymorphism (SNP)" means identifying the base type of the allele of the SNP, and "allele of a single nucleotide polymorphism (SNP) The aspect of "detecting" includes detecting one allele of the SNP, detecting both alleles of the SNP, and identifying the genotype of the SNP.

In the determination method of the present invention, when at least one of the detected allele bases is a risk allele, it is determined that the subject has a genetic predisposition to easily feel stress. Specifically, in the single nucleotide polymorphisms (SNPs) of (a1) to (a5), if the risk allele shown in Table 2 below is detected in at least one allele, it is compared with the case where the risk allele is not detected. As a result, it can be determined that the individual has a genetic predisposition to easily feel stress. For example, environmental factors such as noise and lighting, physical factors such as illness and injury, psychological factors such as anxiety and tension, and social factors such as busyness and heavy responsibility may cause physical and mental abnormalities and disorders. can be determined to be high.

For example, in the case of the SNP identified by rs74548608 in (a1), the C/C genotype indicates that stress is more likely to be felt than the C/G or G/G genotype. , indicates that the C/G genotype is more susceptible to stress than the G/G genotype. In other words, it shows that C/C type, C/G type, and G/G type are more susceptible to stress in that order.

In the determination method of the present invention, the race of the subject is not particularly limited, but is preferably East Asian, more preferably Japanese. Here, East Asians refer to people of Japanese, Korean, Chinese, Taiwanese or Mongolian origin.

The DNA-containing sample used in the determination method of the present invention is not particularly limited as long as it is a biological sample containing DNA collected from a subject. The DNA contained in the DNA-containing sample is preferably genomic DNA, but the SNP to be detected is in a region present in mRNA other than a non-transcribed region such as a promoter and a region removed by RNA splicing such as an intron. When the SNP is located, a biological sample containing mRNA or total RNA may be used instead of genomic DNA. Examples of DNA-containing samples that can be used include any bodily fluids, secretions, tissues, cells, and cultures of tissues and cells from which genomic DNA can be collected. Urine, sputum, pharyngeal swab, nasal swab, oral (inner cheek) mucosal swab, lacrimal gland secretion, sweat, hair, nails, skin, mucous membranes, tape strips peeled off after adhering to skin, etc. And from the viewpoint of low invasiveness, saliva is preferred. The sample can be collected according to a method commonly used in clinical examinations and prepared using a known extraction method and purification method. At that time, a commercially available genomic DNA extraction kit can be used.

The method of SNP detection and SNP type determination (SNP typing) is not particularly limited. It can be performed by a known method such as a method of detecting by. For example, PCR-RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-strand conformation polymorphism) method, PCR-SSO (sequence specific oligonucleotide) method, direct sequencing method, ASO (Allele Specific Oligonucleotide) high Bridization method, ASP-PCR (Allele Specific Primer-PCR) method, Snapshot method, ARMS (Amplification Refracting Mutation System) method, TaqMan PCR method, Invader method, MALDI-TOF/MS method, RNase A cleavage method, DOL (Dye- Labeled Oligonucleotide Ligation) method, TDI (Template-directed Dye-terminator Incorporation), and the like. All of the above methods are well known to those skilled in the art, and reagents and kits for SNP type determination are commercially available. For example, TaqMan SNP Genotyping Assays (manufactured by Thermo Fisher Scientific) can be used. can be done.

The results obtained by the above determination methods are useful indicators for selecting cosmetics and/or foods and beverages that have stress-preventing and/or improving effects on subjects. Measures to prevent and/or ameliorate stress can be recommended to subjects determined to have it. Therefore, according to another aspect of the present invention, based on the results obtained by the above-described determination method, stress prevention and/or amelioration action according to the degree of genetic predisposition to the subject's susceptibility to stress is provided. A method for providing cosmetics and/or food and drink is also provided, comprising providing the subject with the cosmetics and/or food and drink. For example, chronic psychological stress is known to be involved in the onset and exacerbation of many skin troubles and skin diseases. Examples include dermatitis, alopecia areata, vitiligo vulgaris, and psoriasis vulgaris. Therefore, cosmetics and/or foods and beverages having stress preventive and/or ameliorating effects are also effective in preventing and/or ameliorating such skin troubles and skin diseases. For example, in the provision of cosmetics and/or foods and beverages, in addition to the provision of cosmetics and/or foods and beverages containing active ingredients and fragrance ingredients that have a stress relieving effect, psychological effects such as uplifting and relaxation effects are provided. It also includes makeup recommendations and makeup methods that are said to be available.

2. Kit for Determining Genetic Predisposition to Susceptibility to Stress Probes and primers suitable for each method are used in the above SNP detection and typing methods. Such probes and primers are also included in the scope of the present invention and can be provided as kits.

Examples of probes include probes that contain the above SNP site and that can determine the type of base at the SNP site based on the presence or absence of hybridization. Specific examples include probes having a sequence of at least 10 consecutive bases, preferably 15 bases or more, including the 101st base of the nucleotide sequence shown in any of SEQ ID NOs: 1 to 5, or a complementary sequence thereof. The length of the probe is preferably 15-40 bases, more preferably 20-35 bases. In addition, the probe may be labeled with an appropriate labeling substance. Examples of labeling substances include enzymes (peroxidase, β-galactosidase, alkaline phosphatase, etc.), fluorescent substances (FITC, RITC, Cy3, Cy5, etc.), Luminescent substances (luminol, luminol derivatives, luciferin, lucigenin, etc.), radioactive isotopes ( ³ H, ¹⁴ C, ³² P, ¹²⁵ I, ¹³¹ I, etc.), biotin, digoxigenin, polypeptides containing tag sequences, and the like. Alternatively, a quencher (quenching substance) that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance.

Alternatively, the probes may be immobilized on a solid phase (DNA array). A DNA array allows sample DNA to hybridize to a large number of probes arranged on the same plane, and scans the plane to detect hybridization to each probe at the same time. Therefore, DNA arrays are useful for simultaneous analysis of many SNP sites. Oligonucleotides that serve as probes to be mounted on the array are usually synthesized in situ. For example, in situ synthesis of oligonucleotides by a lithography method (Thermo Fisher Scientific), an inkjet method (Agilent), a bead array method (Illumina), and the like are known.

Further, examples of primers include primers that can be used for PCR for amplifying the SNP site, and primers that can be used for sequence analysis (sequencing) of the SNP site. Specific examples include primers capable of amplifying or sequencing a region containing the 101st base of the nucleotide sequence shown in any one of SEQ ID NOs: 1-5. Primers that can be used in PCR for amplifying the SNP site are oligonucleotides that can initiate complementary strand synthesis toward the SNP site using the DNA of the region containing the SNP site as a template. , the length of such primers is preferably 10-30 bases, more preferably 15-25 bases. Primers can be set upstream or downstream of the SNP site in the nucleotide sequences shown in any of SEQ ID NOs: 1-5.

A person skilled in the art can design probes and primers according to the analysis method based on the nucleotide sequence information of the surrounding DNA region containing the SNP site. Oligonucleotides that serve as probes and primers are synthesized by methods known in the art as methods for synthesizing oligonucleotides, such as the phosphoramidite method and the H-phosphonate method, using a commonly used automatic DNA synthesizer. Synthesis is possible.

The kit of the present invention may contain at least the oligonucleotides used as the probes and primers described above. In addition, if necessary, the kit contains DNA extraction reagents, PCR reagents such as PCR buffer and DNA polymerase, detection reagents such as stains and electrophoresis gels, immobilization carriers, labeling substances, Substrate compounds used to detect the label, positive and negative standards, instructions describing how to use the kit, etc., can also be included. The reagent in the kit may be either a solution or a lyophilized product.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these.

(Example 1) Identification of single nucleotide polymorphisms (SNPs) associated with genetic predisposition to stress received. Samples were collected from the sample providers who gave consent by signing the consent form, and genotyping and association analysis were performed.

(1) DNA sample
Saliva was collected from 698 female subjects (Japanese, average age 55.1 years old), genomic DNA was extracted using Maxwell RSC Stabilized Saliva DNA Kit (manufactured by Promega) to obtain DNA samples.

(2) Analyzed SNPs
Among the SNPs registered in the SNP database (http://www.ncbi.nlm.nih.gov/SNP/), rs74548608, rs57001330, rs6950885, rs8092883, rs2359433, and rs2242446, a noradrenaline transporter-related SNP, were compared. were analyzed.

(3) Determination of genotype
The DNA sample obtained in (1) was subjected to CFX384 Touch Real-Time PCR Detection System (Bio Rad) using TaqMan SNP Genotyping Assays (Applied Biosystems) and SsoAdvanced Universal Probes Supermix (Bio Rad). genotypes of the SNPs to be analyzed above were determined.

(4) Phenotypic data (sensitivity to stress)
Questionnaire items related to the susceptibility to feeling stress consisted of the following three options: "1. No,""2.Sometimes," and "3. Strong." One answer was given and treated as an interval scale.

(5) Association analysis Regarding the relationship between genotype data and susceptibility to stress, multiple regression analysis was performed using the phenotype data as the objective variable, the number of minor alleles in the genotype data, and age as explanatory variables. The p-value of the partial regression coefficient for the number of alleles was evaluated. Since 6 SNPs were analyzed, the significance level was corrected by the Bonferroni method to avoid increasing the type I error, and p-values less than 8.3E-03 were considered significant associations. did.

(6) Results Table 3 below shows the chromosomal numbers, physical positions, bases of alleles and risk alleles, regression coefficients, and p-values where each analyzed SNP exists. In the p-value column, each p-value is displayed in exponential format with a base of 10, and the numbers before and after the symbol E indicate the mantissa and exponent of the p-value in exponential format. .

As a result, rs74548608, rs57001330, rs6950885, rs8092883, and rs2359433 were found to be significantly related to genetic predisposition to stress sensitivity, and could be identified as SNPs capable of determining stress sensitivity. On the other hand, rs2242446, which was used as a comparative example, was not found to be related to susceptibility to stress.

(Example 2) Multivariate analysis using single nucleotide polymorphisms (SNPs) associated with genetic predisposition to susceptibility to stress For the SNPs identified in Example 1, accuracy of determination of genetic predisposition to susceptibility to stress We examined combinations of SNPs that can increase Specifically, the coefficient of determination R2 was calculated in multiple regression analysis using the phenotypic data as the objective variable and the number of risk alleles in the genotypic data of the two SNPs as the explanatory variable, and the determination accuracy was compared. The results are shown in Table 4 below.

As shown in Table 4, when two SNPs are selected from the identified SNPs, the combination of rs74548608 and any one of the other four SNPs is an index showing the accuracy of the multiple regression equation. The coefficient of determination R2 increased and was maximized by the combination of rs74548608 and rs57001330. From the above, it is possible to more effectively improve the judgment accuracy by combining rs74548608 and any one of rs57001330, rs6950885, rs8092883, and rs2359433, especially by combining rs74548608 and rs57001330.

The method of the present invention makes it possible to accurately and simply determine whether a sample provider has a genetic predisposition to being easily stressed. Therefore, based on the judgment results, it is possible to provide sample providers with customized cosmetics and supplements for stress prevention and improvement, and to provide counseling and advice on care methods for stress prevention and improvement. .

Claims (3)

A step of detecting one or more of the following (a1) to (a5) single nucleotide polymorphism (SNP) alleles in a DNA-containing sample collected from a subject, and at least one of the bases of the detected allele determining that the subject has a genetic predisposition to stress if one is a risk allele.
(a1) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs74548608)
(a2) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 2 (SNP identified by SNP: ID rs57001330)
(a3) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 3 (SNP identified by SNP: ID rs6950885)
(a4) SNP at the 101st base of the base sequence shown in SEQ ID NO: 4 (SNP identified by SNP: ID rs8092883)
(a5) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs2359433) Based on the results determined by the method according to claim 1, cosmetics and/or foods and drinks having stress preventive and/or ameliorating effects according to the degree of the subject's genetic predisposition to stress susceptibility. A method for providing cosmetics and/or food and drink to the subject. A probe having a sequence of 10 or more bases including the 101st base in the base sequence shown in any of SEQ ID NOS: 1-5, or a complementary sequence thereof, and/or shown in any of SEQ ID NOS: 1-5 A kit for determining a genetic predisposition to stress sensitivity, comprising primers capable of amplifying a region containing the 101st base in a nucleotide sequence.