Method for determining genetic predisposition to gray hair, method for evaluating effects of test substance in preventing, suppressing, and / or ameliorating gray hair, and screening method for agents for gray hair prevention, suppression, and / or amelioration

Abstract

To provide a method capable of determining genetic predisposition to gray hair with high accuracy.SOLUTION: The present invention is a method for determining genetic predisposition to gray hair, comprising a step of detecting an allele of a specific single nucleotide polymorphism (SNP) in a DNA-containing sample from a subject. The present invention also encompasses a method for determining genetic predisposition to gray hair, comprising the step of detecting an allele of a single nucleotide polymorphism (SNP) associated with at least one gene selected from the following group in a DNA-containing sample from a subject: PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP11-532F6.5, WARS2 P1, RP11-498 J9.2, CCND1, RP11-563 P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for determining a genetic predisposition to gray hair, a method for evaluating the effect of a test substance in preventing, suppressing and / or improving gray hair, and a method for screening an agent for preventing, suppressing and / or improving gray hair. [Background technology]

[0002] Hair is naturally colorless, but is colored by melanocytes (pigment cells) during the process of hair production. Therefore, gray hair is thought to occur when the function of melanocytes declines and hair is not properly colored. The role of melanocytes is to synthesize melanin within the cells and transfer it to surrounding cells, thereby controlling the color of skin and hair. Melanin synthesis takes place in intracellular organelles specific to melanocytes called melanosomes, and it is known that in many cases several melanosomes are packaged and transferred to keratinocytes and hair matrix cells.

[0003] The causes of the decline in the function of melanocytes include the effects of aging, poor circulation, nutritional deficiencies, lack of sleep, damage from ultraviolet rays, stress, etc., but it is generally known that there are individual differences in the susceptibility to graying of hair, and genetics are also thought to affect the occurrence of graying of hair. However, there are still many unknowns about the relationship between genetics and graying of hair.

[0004] In recent years, various methods for determining the likelihood of gray hair have been studied. For example, Patent Documents 1 to 3 (JP 2020-174547 A, JP 2007-517515 A, and JP 2005-532411 A) disclose methods for diagnosing and determining an individual's predisposition to gray hair by using specific genes or single nucleotide polymorphisms (SNPs) as markers. On the other hand, as a result of genome-wide association analysis of hair characteristics, specific single nucleotide polymorphisms (SNPs) associated with gray hair have been found, but it has also been reported that aging has a large effect on gray hair, and the effect of SNPs is less than 10% (Non-Patent Document 1: BMC Genomics, vol. 21, 538, 2020).

[0005] As research into the relationship between genetics and gray hair progresses, there is a demand for a method that can determine the genetic predisposition to gray hair with greater accuracy. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2020-174547 A [Patent Document 2] JP 2007-517515 A [Patent Document 3] JP 2005-532411 A [Non-patent literature]

[0007] [Non-Patent Document 1] BMC Genomics,vol.21,538,2020 Summary of the Invention [Problem to be solved by the invention]

[0008] In the above-mentioned circumstances, the present invention aims to provide a method for determining the genetic predisposition to gray hair with high accuracy. It also aims to provide a method for screening a substance (agent for preventing, inhibiting and / or improving gray hair) that has the effect of preventing, inhibiting and / or improving gray hair using the determination method. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the present inventors have found that a genetic predisposition to gray hair can be determined with high accuracy and in a simple manner by detecting an allele of a specific single nucleotide polymorphism (SNP).

[0010] [1] A method for determining a genetic predisposition to gray hair, comprising the step of detecting at least one single nucleotide polymorphism (SNP) allele selected from the group consisting of A1 to A51, B1 to B26, and C1 to C3 in the table below, from a DNA-containing sample of a subject.

[0011] [Table 1-1]

[0012] [Table 1-2] [2] The method for determining a genetic predisposition to gray hair described in [1], further comprising a step of determining that the subject has a genetic predisposition to gray hair if at least one of the bases of the detected allele is a risk allele. [3] For the DNA-containing samples of the subjects, the following were identified: PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, R 1. A method for determining a genetic predisposition to gray hair, comprising the step of detecting an allele of a single nucleotide polymorphism (SNP) associated with at least one gene selected from the group consisting of P11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2. [4] The method for determining a genetic predisposition to gray hair according to [3], further comprising a step of determining that the subject has a genetic predisposition to gray hair if at least one of the bases of the detected allele is a risk allele. [5] A method for evaluating the effect of a test substance on preventing, suppressing and / or improving hair graying, comprising a step of evaluating the regulatory activity of the test substance on the signal transduction mechanism mediated by Plexin A1 (PLXNA1) in cells that constitute skin tissue. [6] The evaluation method according to [5], wherein the signal transduction mechanism mediated by Plexin A1 (PLXNA1) is a Semaphorin 3A (SEMA3A)-Plexin A1 (PLXNA1) signal transduction mechanism. [7] The evaluation method described in [5] or [6], wherein the cells constituting the skin tissue are melanocytes. [8] The evaluation method described in [7], wherein the effect of the test substance in preventing, suppressing and / or improving gray hair is the effect of promoting melanin transport from melanocytes. [9] A method for screening an agent for preventing, suppressing and / or improving hair graying, comprising a step of evaluating the regulatory activity of a test substance on a signal transduction mechanism mediated by Plexin A1 (PLXNA1) in cells that constitute skin tissue.

[10] The screening method described in [9], wherein the signal transduction mechanism mediated by Plexin A1 (PLXNA1) is a Semaphorin 3A (SEMA3A)-Plexin A1 (PLXNA1) signal transduction mechanism.

[11] The screening method described in [9] or

[10] , wherein the cells constituting the skin tissue are melanocytes.

[12] The screening method according to

[11] , wherein the agent for preventing, inhibiting and / or improving gray hair is an agent for promoting melanin transport from melanocytes. Effect of the Invention

[0013] According to the present invention, it is possible to provide a method for determining the genetic predisposition to gray hair with high accuracy. That is, according to the method of the present invention, by detecting the allele of a single nucleotide polymorphism (SNP) contained in the genome-derived DNA present in the biological sample of a subject, it is possible to determine with high accuracy and simply whether the subject has a genetic predisposition to gray hair (whether the subject is prone to gray hair). Based on the result of this determination, it is also possible to take measures for preventing or improving gray hair at an early stage. In addition, based on the information of the gene whose expression can be affected by SNP, it is also possible to search for a substance that has the effect of preventing, suppressing and / or improving gray hair. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1(a, b) shows the results of principal component analysis of genome-wide SNP data for subject samples. [Diagram 2] Figure 2 shows hair strand samples of six gray hair grades used in the gray hair questionnaire survey (from left to right: grades 1, 2, 3, 4, 5, and 6). [Diagram 3] FIG. 3 shows the number of SNPs associated with gray hair found by GWAS analysis of Traits A, B, and C. [Figure 4] FIG. 4 shows increased expression of P16INK4a mRNA (induction of senescence) in normal human epidermal melanocytes induced by X-ray irradiation. [Diagram 5] FIG. 5 shows increased expression of P21 mRNA (induction of senescence) in normal human epidermal melanocytes induced by X-ray irradiation. [Figure 6] FIG. 6 shows increased expression of PLXNA1 mRNA in X-ray-irradiated senescence-induced melanocytes. [Figure 7] FIG. 7 shows the dendrite outgrowth promoting effect of theophylline added to normal human epidermal melanocytes. [Figure 8] FIG. 8 shows the inhibitory effect of addition of Sema3A to normal human epidermal melanocytes on dendrite elongation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, a method for determining a genetic predisposition to gray hair, a method for evaluating the effect of a test substance on preventing, suppressing and / or improving gray hair, and a method for screening an agent for preventing, suppressing and / or improving gray hair will be described in detail.

[0016] [Terminology] The abbreviations for base sequences (nucleotide sequences), nucleic acids, etc. in this specification shall follow the IUPAC-IUB rules (IUPAC-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138, 9, 1984), "Guidelines for the preparation of specifications, etc. including base sequences or amino acid sequences" (edited by the Japan Patent Office), and conventional symbols in the relevant field.

[0017] In this specification, "DNA" includes not only double-stranded DNA but also each of the single-stranded DNAs, that is, the sense strand and the antisense strand, which constitute the double-stranded DNA. In this specification, "gene" includes double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand), single-stranded DNA (antisense strand) having a sequence complementary to the sense strand, and any of their fragments, unless otherwise specified. In addition, in this specification, "gene" refers to a regulatory region, a coding region, an exon, and an intron without distinction, unless otherwise specified. In addition, the "gene" or "DNA" includes not only "gene" or "DNA" represented by a specific base sequence, but also "gene" or "DNA" encoding its homologue, derivative, and mutant, to the extent that the biological function is equivalent to that of the protein encoded by these.

[0018] In this specification, "nucleotide", "oligonucleotide", and "polynucleotide" are synonymous with nucleic acid and include both DNA and RNA. The DNA includes cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, rRNA, and synthetic RNA. Furthermore, "nucleotide", "oligonucleotide", and "polynucleotide" may be double-stranded or single-stranded, and when a "nucleotide" (or "oligonucleotide", "polynucleotide") having a certain sequence is mentioned, it is intended to comprehensively mean a "nucleotide" (or "oligonucleotide", "polynucleotide") having a complementary sequence, unless otherwise specified. Furthermore, when the "nucleotide" (or "oligonucleotide", "polynucleotide") is RNA, the base symbol "T" shown in the sequence table is to be read as "U".

[0019] In this specification, the term "complementary sequence" means that the sequence is in a base-complementary relationship (complementary strand, antisense strand) with a polynucleotide consisting of an arbitrary base sequence (sense strand). The complementary base sequence includes a base sequence that can hybridize with the target base sequence under stringent conditions in addition to a case where the complementary base sequence forms a completely complementary sequence with the target base sequence. Here, the stringent conditions can be exemplified by washing conditions of about "1xSSC, 0.1% SDS, 37°C", more stringent hybridization conditions can be exemplified by conditions of about "0.5xSSC, 0.1% SDS, 42°C", and even more stringent hybridization conditions can be exemplified by conditions of about "0.1xSSC, 0.1% SDS, 65°C". For example, a base sequence that can hybridize with the target base sequence under stringent conditions can be exemplified by a base sequence that has an identity of 90% or more, preferably 95% or more, with the target base sequence.

[0020] A "single nucleotide polymorphism (SNP)" generally refers to a state in which only one site in the nucleotide sequence of a gene differs, and the site of that difference. A polymorphism generally refers to two or more alleles that exist at a frequency of 1% or more in a population.

[0021] As used herein, "SNP" refers to an SNP registered in the SNP database (http: / / www.ncbi.nlm.nih.gov / SNP / ) of the National Center for Biotechnology Information (NCBI), a public database that is freely available to those skilled in the art, and can be identified by its reference number, the rs number.

[0022] As used herein, the term "allele" refers to each type having different bases that can be taken at a certain SNP site. In addition, 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 that are the above combinations, and a combination of the same alleles is called a homozygous type, and a combination of different alleles is called a heterozygous type. For example, there are three types of genotypes that are combinations of opposing alleles at the SNP specified by rs7903933 in (A1), namely, C / C type, C / G type, and G / G type.

[0023] [How to determine genetic predisposition to gray hair] The method of the present invention for determining a genetic predisposition to gray hair is a method for determining a genetic predisposition to gray hair, comprising a step of detecting an allele of at least one single nucleotide polymorphism (SNP) selected from the group consisting of A1 to A51, B1 to B26, and C1 to C3 in the table below, from a DNA-containing sample of a subject.

[0024] [Table 2-1]

[0025] [Table 2-2]

[0026] In the above table, A1 to A51, B1 to B26, and C1 to C3 indicate SNPs identified by each SNP:ID. The above table also indicates the chromosome number on which each SNP exists and its position on the chromosome.

[0027] In the present invention, "detecting an allele of a single nucleotide polymorphism (SNP)" means identifying the type of base of the allele of the SNP, and embodiments of "detecting an allele of a single nucleotide polymorphism (SNP)" include detecting one allele of the SNP, detecting both alleles of the SNP, and identifying the genotype of the SNP.

[0028] In the determination method of the present invention, when at least one of the bases of the detected allele is a risk allele, the subject is determined to have a genetic predisposition to gray hair (prone to gray hair). Specifically, in the single nucleotide polymorphisms (SNPs) A1 to A51, B1 to B26, and C1 to C3, if a risk allele is detected in at least one allele, it can be determined that the subject has a genetic predisposition to gray hair (prone to gray hair), for example, that the possibility of gray hair occurring inappropriately for age is high, and the possibility of gray hair becoming gray in the future is relatively high, and the degree of the genetic predisposition to gray hair (prone to gray hair) can be determined to be high, compared to when the risk allele is not detected.

[0029] In the method of the present invention, the race of the subject is not particularly limited, but is preferably Asian, more preferably East Asian (a person originating from Japan, South Korea, North Korea, China, Taiwan, or Mongolia), and even more preferably Japanese. In addition, the age and sex of the subject are not limited.

[0030] In the determination method of the present invention, the DNA-containing sample is not particularly limited as long as it is a biological sample collected from a subject and contains DNA, but the DNA is preferably genomic DNA. However, when the SNP to be detected is an SNP located in a region present in mRNA other than a non-transcriptional region such as a promoter or a region removed by RNA splicing such as an intron, a biological sample containing mRNA or total RNA may be used instead of genomic DNA. As the DNA-containing sample, for example, any body fluid, secretion, tissue, cell, culture of tissue or cell, etc. from which genomic DNA can be collected can be used, and specifically, the subject's saliva, blood, urine, sputum, pharyngeal swab, nasal swab, oral (inner cheek) mucosa swab, lacrimal gland secretion, sweat, hair, nails, skin, mucous membrane, tape strip peeled off after skin attachment, etc. are mentioned, but saliva is preferable from the viewpoint of ease and low invasiveness. The sample can be collected according to the method performed in general clinical examination, and prepared using known extraction and purification methods. In this case, a commercially available genomic DNA extraction kit may be used.

[0031] The method for detecting SNPs and determining the type of SNP (SNP typing) is not particularly limited, and can be performed by known methods, such as a method in which allele-specific primers are used, amplification is performed by PCR or the like, and the polymorphism of the amplified product is detected by fluorescence or luminescence, or a method in which a probe that can distinguish the type of base at the SNP site based on the presence or absence of hybridization is used. For example, methods well known to those skilled in the art include 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) hybridization 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), etc. In addition, commercially available reagents and kits for determining the type of SNP, such as TaqMan SNP Genotyping Assays (manufactured by Thermo Fisher Scientific), may be used.

[0032] The allele-specific primer is a primer that can be used in PCR for amplifying the SNP site, and may be an oligonucleotide 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 a primer is preferably 10 to 30 bases, more preferably 15 to 25 bases. The allele-specific primer may be an oligonucleotide that can initiate complementary strand synthesis toward the SNP site using the DNA of the region containing the single nucleotide polymorphism (SNP) sites A1 to A51, B1 to B26, and C1 to C3 as a template, and may be set at a position upstream or downstream of the SNP site.

[0033] The above-mentioned probe is preferably a probe that contains the above-mentioned SNP site and can distinguish the type of base at the SNP site based on the presence or absence of hybridization. Specifically, the probe has a sequence of at least 10 consecutive bases, preferably 15 consecutive bases or more, including the bases of the single nucleotide polymorphism (SNP) sites A1 to A51, B1 to B26, and C1 to C3, or a complementary sequence thereof. The length of the probe is preferably 15 to 40 bases, more preferably 20 to 35 bases. The probe may be labeled with a suitable labeling substance, and examples of the labeling substance include enzymes (peroxidase, β-galactosidase, alkaline phosphatase, etc.), fluorescent substances (FITC, RITC, Cy3, Cy5, etc.), luminescent substances (luminol, luminol derivatives, luciferin, lucigenin, etc.), radioisotopes (3H, 14C, 32P, 125I, 131I, etc.), biotin, digoxigenin, and polypeptides containing tag sequences. Alternatively, a quencher (quenching substance) that absorbs the fluorescent energy emitted by the fluorescent substance may be further bound to the vicinity of the fluorescent substance.

[0034] The oligonucleotides to be used as the primers or probes can be synthesized by methods known in the art for synthesizing oligonucleotides, such as the phosphoramidite method and the H-phosphonate method, using a commonly used automatic DNA synthesizer.

[0035] The above-mentioned single nucleotide polymorphisms (SNPs) A1 to A51, B1 to B26, and C1 to C3 are present in overlapping genes, or in the most upstream or downstream terminal positions of the SNPs, or the expression of which is known to be affected by the SNPs in the literature. These genes include PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, and LDHAP. The following genes were identified (see Examples): 5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2.

[0036] The method of the present invention is to detect the following genes in a DNA-containing sample from a subject: PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4 The present invention also includes a method for determining a genetic predisposition to gray hair, comprising a step of detecting an allele of a single nucleotide polymorphism (SNP) associated with at least one gene selected from the group consisting of RP11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2. In addition, this method preferably further comprises a step of determining that the subject has a genetic predisposition to gray hair when at least one of the bases of the detected allele is a risk allele. The above-mentioned genes in the determination method of the present invention are preferably PLXNA1, CHCHD6, TXNRD3, TERT, and EIF2A, and more preferably PLXNA1.

[0037] [Method for evaluating the effect of test substances on preventing, inhibiting and / or improving gray hair] The method of the present invention for evaluating the effect of a test substance on preventing, inhibiting and / or improving gray hair is to evaluate the following for the test substance: PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1 The method includes a step of evaluating the regulatory activity of the signal transduction mechanism mediated by at least one selected from the group consisting of RN7SKP120, AC009502.4, RP11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1, and RP5-1077H22.2. The regulatory activity of the signal transduction mechanism may be evaluated using cells having the signal transduction mechanism. As such cells, naturally occurring cells (preferably cells constituting skin tissue such as melanocytes) or cells into which the signal transduction mechanism has been artificially introduced may be used.

[0038] In the above step, in the presence of a test substance, PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP11- The evaluation may be performed using as an index the change in expression of at least one gene or protein selected from the group consisting of 532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1, and RP5-1077H22.2, or using as an index the inhibition of the signal transduction mechanism mediated by these genes or proteins. When the test substance suppresses the expression of the gene or protein in the cells or inhibits the signal transduction mechanism, it is evaluated as having the effect of preventing, suppressing, and / or improving gray hair.

[0039] In the present invention, PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP1 The expression level of at least one gene or protein selected from the group consisting of 1-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2 can be measured by any method known to those skilled in the art. The expression level of a gene refers to the amount of mRNA, which is a transcription product of the gene. The measurement of the amount of mRNA can be appropriately selected from methods known to those skilled in the art. For example, a gene amplification method (PCR method) using an oligonucleotide that hybridizes to the above gene as a primer, or a hybridization method using an oligonucleotide that hybridizes to the above gene as a probe can be used. Specifically, RT-PCR method, real-time RT-PCR method, microarray method, Northern blot method, dot blot method, RNase protection assay method, etc. can be mentioned. The expression level of a protein refers to the expression level or expression intensity of the protein in a cell, and can be analyzed by a standard method based on the amount of the protein produced, secreted, activity, etc. by the cell. When the protein is expressed on the cell surface, the expression intensity can be analyzed by FACS analysis using a specific antibody, etc.

[0040] In the evaluation method of the present invention, as the cell artificially introduced with the signal transduction mechanism, a cell introduced with a plasmid in which a reporter gene is operably linked to the transcriptional regulatory region of each gene may be used, and the expression level of the reporter gene may be measured. As the reporter gene, a gene known to those skilled in the art, such as a GFP gene, a luciferase gene, or a β-glucuronidase gene, may be used.

[0041] As the above-mentioned genes in the evaluation method of the present invention, PLXNA1, CHCHD6, TXNRD3, TERT, and EIF2A are preferable, and PLXNA1 is more preferable.

[0042] As described above, the single nucleotide polymorphisms (SNPs) A1 to A51, B1 to B26, and C1 to C3, which have been confirmed to be associated with gray hair, are located in overlapping genes, or in the most upstream or downstream terminal positions of the SNPs, or the expression of which is known to be affected by the SNPs, are PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, F ARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2 were identified. Among these, PLXNA1 is a Semaphorin receptor, and it is known that Semaphorin3A binds to it. It is believed that by controlling the PLXNA1-Semaphorin3A signaling mechanism via PLXNA1 on melanocytes, it is possible to control melanin transport from melanocytes and prevent, suppress and / or improve hair graying.

[0043] If the test substance has the effect of inhibiting the semaphorin 3A (SEMA3A)-Plexin A1 (PLXNA1) signaling mechanism, for example, if the test substance inhibits the binding of semaphorin 3A (SEMA3A) and Plexin A1 (PLXNA1), inhibits the activity of semaphorin 3A (SEMA3A), inhibits the activity of Plexin A1, suppresses the expression of semaphorin 3A (SEMA3A) or Plexin A1 (PLXNA1) in cells that make up skin tissue, etc., the test substance is considered to have the effect of preventing, suppressing and / or improving hair graying.

[0044] From the above viewpoint, the evaluation method of the present invention is preferably a method for evaluating the effect of a test substance on preventing, suppressing, and / or improving gray hair, comprising a step of evaluating the regulatory activity of the PLXNA1-mediated signal transduction mechanism in cells constituting skin tissue. Moreover, the cells constituting the skin tissue are preferably melanocytes, and the signal transduction mechanism mediated by PLXNA1 is preferably the semaphorin 3A (SEMA3A)-plexin A1 (PLXNA1) signal transduction mechanism. Furthermore, the effect of preventing, suppressing, and / or improving gray hair is preferably an effect of promoting melanin transport from melanocytes.

[0045] The test substances may be mainly components that can be used in medicines, quasi-drugs, cosmetics, and / or food and beverages. For example, mixtures containing multiple compounds such as extracts of animal or plant tissues or microbial cultures, or preparations purified from them, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, low molecular weight compounds, etc., but are not limited thereto.

[0046] A substance determined to have the effect of preventing, inhibiting, and / or improving gray hair by the evaluation method of the present invention can be used as an ingredient in compositions for preventing, inhibiting, and / or improving gray hair, such as cosmetics, pharmaceuticals, quasi-drugs, foods and beverages.

[0047] [Method for screening agents for preventing, inhibiting and / or improving gray hair] The screening method for agents for preventing, inhibiting and / or improving gray hair of the present invention is directed to the screening of the following compounds for the test substance: PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, The method includes a step of evaluating the regulatory activity of the signal transduction mechanism mediated by at least one selected from the group consisting of RN7SKP120, AC009502.4, RP11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.2. The above-mentioned genes in the screening method of the present invention are preferably PLXNA1, CHCHD6, TXNRD3, TERT, EIF2A, and more preferably PLXNA1. The screening method of the present invention is a method for selecting a substance that can be effectively used as an agent for preventing, suppressing and / or improving gray hair by the above-mentioned method for evaluating the effect of preventing, suppressing and / or improving gray hair of the test substance. Therefore, the specific details can be applied to the explanation in the section on the method for evaluating the effect of the test substance on preventing, suppressing and / or improving gray hair, as described above.

[0048] A preferred screening method for agents for preventing, inhibiting and / or improving gray hair of the present invention is a method including a step of evaluating the regulatory activity of a test substance on a signal transduction mechanism mediated by Plexin A1 (PLXNA1) in cells constituting skin tissue. The cells constituting the skin tissue are preferably melanocytes, and the signal transduction mechanism mediated by PLXNA1 is preferably the Semaphorin 3A (SEMA3A)-Plexin A1 (PLXNA1) signal transduction mechanism. Furthermore, the prevention, inhibition and / or improvement of gray hair is preferably achieved by promoting melanin transport from melanocytes. EXAMPLES

[0049] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0050] [Example 1: SNP measurement] Saliva provided by 2,201 Japanese male and female subjects (aged 18-84 years) was used as a sample to analyze SNPs of each subject. First, genomic DNA was extracted and purified from the sample using MagaZorb (registered trademark) DNA Mini-Prep Kit (manufactured by Promega). Next, SNP analysis was performed using Illumina's Infinium HTS Assay Kit. In detail, after DNA was amplified and fragmented, hybridization was performed between the fragmented DNA and the DNA on the beads on a microarray (Human OmniExpress-24+ BeadChip (manufactured by Illumina) and Infinium OmniExpress-24+ BeadChip (manufactured by Illumina)), and the DNA bound to the bead chip was fluorescently labeled and analyzed.

[0051] Sample-QC was performed on the 2,201 subjects. Sex inconsistency and low genotyping accuracy (missing call rate > 99.0%) samples were checked, but no samples were found to be excluded. To exclude relatives, PI_HAT (IBD estimates) was used as the IBD (identity by descent) evaluation value, and pairs showing a commonly used threshold of 0.1875 (a value halfway between second-degree and third-degree consanguinity) or higher were checked, but no samples were found to be excluded. Then, a principal component analysis (PLINK “-indep-pairwise 50 5 0.5”) was performed on the genome-wide SNP data to examine the presence of population structure. After plotting the data in the principal component space based on the first and second principal components obtained from the principal component analysis (Fig. 1a), 15 samples that fell outside the population cluster were excluded, and the principal component analysis was performed again on the excluded samples (Fig. 1b) to confirm that there were no samples that fell outside the population cluster. The breakdown of the samples from 2,186 individuals (male: 957 (43.8%) / female: 1,229 (56.2%)) after excluding the 15 samples that fell outside the population cluster is shown in Table 3 below. The SNPs after Sample-QC were subjected to the subsequent genotype imputation (a method of statistically estimating genotype information for sites not designed in the SNP array by referring to the reference panel).

[0052] [Table 3]

[0053] After sample-QC, 684,430 SNPs on autosomes were phased using Eagle v2.4.1 software with reference to East Asian samples from the 1KGP (International 1000 Genomes Project Whole Genome Reference Panel). Genotype imputation was performed on the genomic data after phasing using Minimac3. SNPs with low imputation accuracy were removed by adjusting the imputation Rsq value (excluding Rsq < 0.7) for all SNPs after genotype imputation.

[0054] Marker-QC was performed on the SNPs after genotype imputation. The call rate for each SNP was adjusted (excluding missing call rates > 0.01), the Hardy Weinberg equilibrium test threshold was adjusted (excluding p < 1E-06), and the minor allele frequency (MAF) was adjusted (excluding MAF < 0.05). Finally, a genome-wide association study (GWAS) was performed on the 4,347,442 SNPs after Marker-QC.

[0055] [Example 2: Questionnaire survey on gray hair] The subjects were given the following questionnaire regarding gray hair. The questionnaire items regarding gray hair were a question about "amount of gray hair," and participants were asked to select one of six options: "1. No gray hair (all black hair)," "2. Almost no gray hair, but a few gray hairs in places," "3. A few gray hairs overall," "4. Half of the total is gray," "5. Mostly gray hair," and "6. All gray hair," and were treated as an interval scale. When answering the questions, participants used the hair bundle sample (Figure 2) with the six gray hair grades listed above as the criterion for judgment (six gray hair grades: grades 1, 2, 3, 4, 5, and 6 from the left in Figure 2).

[0056] The summary of the survey results on gray hair for 2,186 people after Sample-QC was conducted is as follows:

[0057] For the questionnaire item "Do you currently have any gray hair?", the ratio of those with or without gray hair was 1,978 (90.5%) / 208 (9.5%), and when broken down by gender, the ratio of men with or without gray hair was 871 (91.0%) / 86 (9.0%), and the ratio of women with or without gray hair was 1,107 (90.1%) / 112 (9.9%). From the above, no significant gender differences were observed in the proportion of those with gray hair.

[0058] Phenotypic information was obtained from the subjects' responses to the above-mentioned questionnaire regarding canities. Cases / controls for each trait were defined based on the responses to the questionnaire regarding canities (Table 4).

[0059] [Table 4]

[0060] [Example 3: GWAS analysis] Regarding the association between genotype data and phenotype data, GWAS was conducted for five traits related to gray hair using PLINK version 1.9 (https: / / www.cog-genomics.org / plink / 1.9 / ) to verify the contribution of genetic factors to gray hair. Age, sex, and the first and second principal components of the top principal components were set as covariates for GWAS, and GWAS was conducted using a logistic regression model. Note that since gender factors were excluded from Trait B and C, the covariates were removed. As a result of the analysis of Trait A, B, and C, 80 gray hair-related SNPs were found for these three traits (Figure 3, Table 5-1, 2). A total of 51 gray hair-related SNPs (A1 to A51 in the table below) were found for Trait A, a total of 26 gray hair-related SNPs (B1 to B26 in the table below) were found for Trait B, and a total of 3 gray hair-related SNPs (C1 to C3 in the table below) were found for Trait C. In addition, Trait A and Trait B shared four hair-graying-related SNPs.

[0061] [Table 5-1]

[0062] [Table 5-2]

[0063] For the SNPs in Tables 5-1 and 5-2 obtained from Trait A (white hair vs. black hair), Trait B (white hair vs. black hair: females), and Trait C (white hair vs. black hair: males), target genes thought to be related to white hair were discovered using SNP Nexus (https: / / www.snp-nexus.org / v4 / ) (Tables 6-1 and 6-2). Specifically, the gene names (Overlapped_gene) with which the variants of the SNP overlap were analyzed. If the SNP did not overlap with any gene, the gene names (Nearest_upstream_gene) at the terminal position closest upstream of the SNP and the gene names (Nearest_downstream_gene) at the terminal position closest downstream of the SNP were analyzed. As shown in the table below, the genes suggested to be associated with gray hair are PLXNA1, CHCHD6, TERT, TUSC1, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP11-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1, and RP5-1077H22.2.

[0064] [Table 6-1]

[0065] [Table 6-2]

[0066] Target genes thought to be related to gene expression were discovered using FUMA version 1.5.3 (https: / / fuma.ctglab.nl / ) by comparing SNPs of Trait A (white hair vs. black hair) and Trait B (white hair vs. black hair: women) (Table 7). Specifically, Expression quantitative trait loci (eQTL) (a statistical method for investigating the association between gene expression level (amount of mRNA) and genotype of gene locus (SNP, etc.)) were analyzed. In this analysis, the target tissue was set to GTEx v8 Skin. PLXNA1, CHCHD6, TXNRD3, TERT, and EIF2A, shown in the table below, are genes whose expression changes in skin tissue and were considered to be target genes related to white hair. In particular, PLXNA1 is a Semaphorin receptor and is known to bind to Semaphorin3A. It is strongly suggested that controlling PLXNA1-Semaphorin3A signaling in melanocytes may make it possible to prevent, suppress, and / or ameliorate gray hair.

[0067] [Table 7]

[0068] [Test Example 1: Evaluation of PLXNA1 mRNA expression in X-ray irradiated aging-induced melanocytes] 2.8 × 10 normal human epidermal melanocytes (NHEM) (Lifeline Cell Technology; product number 01179) 5 The cells were seeded in a T25 flask at 16.5 cells / flask, cultured in medium (Lifeline; product number LL-0039) for 2 days, and then irradiated with 10 Gy of X-rays. Three days after irradiation, the cells were passaged once at a 1:3 dilution, and four days later, the cells were seeded in a 12-well plate. After culturing for 2 days, RNA was extracted from the cells. cDNA was then prepared using ReverTra AceTM qPCR RTMater Mix with gDNA Remover (TOYOBO; product number FSQ-301), and the target gene (P16 INK4aPrimers (table below) corresponding to the 5'-nucleotide polynucleotides P16, P21, and PLXNA1 were mixed with THUNDERBIRDTM SYBR (registered trademark) qPCR Mix (TOYOBO; product number QPS-201) to perform real-time qPCR using a QuantStudio 7 Flex (Thermo Fisher). The mRNA expression levels were analyzed from the Ct values ​​using the ΔΔCt method. Values ​​are shown as mean ± standard deviation. Statistical processing was evaluated using the Student's test, and a risk level of less than 5% was considered statistically significant. The results are shown in Figure 4 (P16 INK4a ), FIG. 5 (P21) and FIG. 6 (PLXNA1).

[0069] The primers used in the above tests are as shown in the table below.

[0070] [Table 8]

[0071] As shown in Figures 4 and 5, X-ray irradiation of normal human epidermal melanocytes increased the aging marker P16 INK4a It was confirmed that the expression of PLXNA1 and P21 increased. As shown in Figure 6, it was also confirmed that PLXNA1 is expressed in normal human epidermal melanocytes, and that the expression increased when aging was induced by X-ray irradiation. This result suggests that the expression level of PLXNA1 increases with age (senescence).

[0072] [Test Example 2: Evaluation of melanocyte dendrite elongation] Normal human epidermal melanocytes (NHEM) (Lifeline Cell Technology; product number 01179) were seeded in a 96-well plate at 1000 cells / well and cultured for 24 hours in medium (Lifeline; product number LL-0039) containing POLARIC (PMC; product number AK12) diluted 3 / 20000. Next, medium containing 50 mM theophylline or 100 ng / mL Sema3A was added at a 10-fold dilution (final concentration 5 mM theophylline, 10 ng / mL Sema3A). After 48 hours of culture, a cell nucleus stain (Hoechst33342 (DOJINDO; product number H342)) diluted 1000-fold was added, and 30 minutes later, the nuclei and cell dendrite lengths were counted using an imaging device to obtain the dendrite length per cell number. Values ​​are shown as mean ± standard deviation. Statistical analysis was performed using the Student's test, and a significance level of less than 5% was considered to be statistically significant. The results are shown in Figure 7 (theophylline) and Figure 8 (Sema3A).

[0073] Addition of theophylline, a drug that promotes melanin biosynthesis, to normal human epidermal melanocytes promoted dendrite outgrowth (Fig. 7), whereas addition of Sema3A, a ligand for PLXNA1, inhibited dendrite outgrowth (Fig. 8). These results suggest that activation of PLXNA1-mediated signal transduction inhibits melanocyte dendrite outgrowth.

[0074] The results of Test Examples 1 and 2 above suggest that as the expression level of PLXNA1 increases with aging, signal transduction mediated by PLXNA1 is activated, and melanocyte dendritic extension is inhibited, thereby inhibiting melanin transport and causing gray hair. Therefore, it was thought that gray hair could be inhibited by reducing the expression level of PLXNA1 and controlling (inhibiting) the signal transduction mechanism mediated by PLXNA1. [Industrial Applicability]

[0075] According to the present invention, it is possible to provide a method for determining the genetic predisposition to gray hair with high accuracy. That is, according to the method of the present invention, by detecting the allele of a single nucleotide polymorphism (SNP) contained in the genome-derived DNA present in a biological sample of a subject, it is possible to determine with high accuracy and simply whether the subject has a genetic predisposition to gray hair (whether the subject is prone to gray hair). Based on the determination result, it is also possible to take measures for preventing or improving gray hair at an early stage. In addition, by using this determination method, it is also possible to search for substances that have the effect of preventing, suppressing and / or improving gray hair.

Claims

1. A method for determining a genetic predisposition to gray hair, comprising the step of detecting an allele of at least one single nucleotide polymorphism (SNP) selected from the group consisting of A1 to A51, B1 to B26, and C1 to C3 in the table below, from a DNA-containing sample of a subject. 【Table 9-1】 【Table 9-2】

2. The method for determining a genetic predisposition to gray hair according to claim 1, further comprising a step of determining that the subject has a genetic predisposition to gray hair if at least one of the bases of the detected allele is a risk allele.

3. For the DNA-containing samples of the subjects, PLXNA1, CHCHD6, TXNRD3, TERT, TUSC1, EIF2A, BRE, HTR4, ANKRD55, AL033381.1, MKLN1, FARSBP1, LDHAP5, AP000439.3, DYNC2H1, TSC22D2, RP1-309H15.2, AC090505.1, RN7SKP120, AC009502.4, RP1 A method for determining a genetic predisposition to canities, comprising the step of detecting an allele of a single nucleotide polymorphism (SNP) associated with at least one gene selected from the group consisting of 1-532F6.5, WARS2P1, RP11-498J9.2, CCND1, RP11-563P16.1, SERP1, MIR4457, RP1-209A6.1, XXYLT1, AC008281.1 and RP5-1077H22.

2.

4. The method for determining a genetic predisposition to gray hair according to claim 3, further comprising a step of determining that the subject has a genetic predisposition to gray hair if at least one of the bases of the detected allele is a risk allele.

5. A method for evaluating the effect of a test substance on preventing, suppressing and / or improving hair graying, comprising a step of evaluating the regulatory activity of the test substance on the signal transduction mechanism mediated by Plexin A1 (PLXNA1) in cells constituting skin tissue.

6. The evaluation method according to claim 5, wherein the signal transduction mechanism mediated by Plexin A1 (PLXNA1) is a semaphorin 3A (SEMA3A)-Plexin A1 (PLXNA1) signal transduction mechanism.

7. The evaluation method according to claim 5 or 6, wherein the cells constituting the skin tissue are melanocytes.

8. The evaluation method according to claim 7 , wherein the effect of the test substance in preventing, suppressing and / or improving gray hair is an effect of promoting melanin transport from melanocytes.

9. A method for screening for an agent for preventing, suppressing and / or improving gray hair, comprising a step of evaluating the regulatory activity of a test substance on a signal transduction mechanism mediated by Plexin A1 (PLXNA1) in cells constituting skin tissue.

10. The screening method according to claim 9, wherein the signal transduction mechanism mediated by Plexin A1 (PLXNA1) is a semaphorin 3A (SEMA3A)-Plexin A1 (PLXNA1) signal transduction mechanism.

11. The method of claim 9 or 10, wherein the cells constituting the skin tissue are melanocytes.

12. The screening method according to claim 11, wherein the agent for preventing, inhibiting and / or improving gray hair is an agent for promoting melanin transport from melanocytes.

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