JP6963341B1 - How to determine the genetic predisposition of stains - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for accurately and easily determining a genetic predisposition of an individual stain. SOLUTION: In a DNA-containing sample collected from a subject, at least one single nucleotide polymorphism (SNP) of rs1001949, rs16910935, and rs72631692, or an allele of SNP having a linkage disequilibrium relationship with these SNPs is detected. Genetic predisposition that the subject is prone to darkening, increasing the number of stains, and / or increasing the area of the stain when the step and at least one of the detected allele bases is a risk allele. A method for determining the genetic predisposition of a stain, which comprises the step of determining that the person has. [Selection diagram] None

Description

The present invention relates to a method for determining the genetic predisposition of stains. More specifically, the present invention relates to a method for determining a genetic predisposition of a spot by detecting a single nucleotide polymorphism (SNP) associated with the type of appearance of the spot, and a kit used for the method.

The skin is an organ that encloses the entire human body, and is composed of three layers, the epidermis, the dermis, and the subcutaneous tissue, from the outside. The epidermis layer existing in the outermost layer is further divided into four layers: a basal layer, a spinous layer, a stratum granulosum, and a stratum corneum (Non-Patent Document 1). The epidermis layer plays an extremely important role as a biological defense mechanism, such as retaining water, stimulating from the outside such as ultraviolet rays, and preventing the invasion of foreign substances. Of these, melanocytes (pigment cells) existing in the basal layer protect against ultraviolet rays by producing melanin. Spots are caused by abnormalities in the development, proliferation, differentiation and melanin synthesis of melanocytes. Since spots have a great effect on the appearance of the face, prevention and improvement techniques, risk assessment techniques, etc. are required.

So far, as a method for evaluating the susceptibility to stains, a method using the blood C-peptide concentration and blood IGF-1 concentration of a subject as an index (Patent Document 1) and a single nucleotide in a gene correlated with stains have been used. Evaluation methods using polymorphism (SNP) as an index (Patent Documents 2 and 3) and the like are known.

On the other hand, there are various types of spots in which the causes and symptoms (darkness, number, size, site, contour, etc.) appear. For example, there are individual differences in the easiness of darkening of stains, the easiness of increasing the area, the easiness of increasing the number, etc., and it is thought that the mechanism and genetic factors that cause each, and the appropriate countermeasures are also different. .. However, little research has been done on SNPs that can predict the type of individual stain appearance.

Japanese Unexamined Patent Publication No. 2010-48610 JP-A-2018-78848 JP-A-2018-164451

Simpson CL, Patel DM, Green KJ. Deconstructing THE skin: cytoarchitectural determinants of epidermal morphogenesis. Nat Rev Mol Cell Biol 2011; 12: 565-80.

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

As a result of investigating SNPs that can predict the type of individual stain appearance in order to solve the above problems, the present inventors specifically indicate that rs1001949 and SNPs having a linkage disequilibrium relationship with them are prone to stain darkening. That rs16910935 and the SNP that has a linkage disequilibrium relationship can specifically predict the tendency of stains to thicken and the number of stains to increase, and that rs72631692 and the SNP that has a linkage disequilibrium relationship with it can predict the area of the stain. We have found that it is possible to specifically predict the susceptibility to increase in size, and have completed the present invention.

That is, the present invention includes the following inventions.
[1] A step of detecting one or more single nucleotide polymorphism (SNP) alleles of the following (a1) to (a4) and the bases of the detected alleles in the DNA-containing sample collected from the subject. A method for determining a genetic predisposition to the tendency of stains to be darkened, which comprises a step of determining that the subject is likely to be stained when at least one of them is a risk allele.
(a1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 1 (SNP: SNP specified by ID rs1001949)
(a2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1.
(a3) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935)
(a4) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5.
[2] The SNP having a chain imbalance coefficient r2 ≥ 0.8 with the SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1 of (a2) is a sequence. SNP at the 101st base of the base sequence shown in No. 2 (SNP: SNP specified by ID rs6484714), SNP at the 101st base of the base sequence shown in SEQ ID NO: 3 (SNP: ID rs10840590) SNP) or SNP (SNP: SNP specified by ID rs58077575) at the 101st base of the base sequence shown in SEQ ID NO: 4.
The SNP having a chain imbalance coefficient r2 ≥ 0.8 with the SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5 of (a4) is shown in SEQ ID NO: 6. SNP at the 101st base of the indicated base sequence (SNP: SNP specified by ID rs2900195), SNP at the 101st base of the base sequence shown in SEQ ID NO: 7 (SNP: SNP specified by ID rs2840322) There is a method according to [1].
[3] A step of detecting one or more single nucleotide polymorphism (SNP) alleles of the following (b1) to (b2) and the bases of the detected alleles in the DNA-containing sample collected from the subject. A method for determining a genetic predisposition to an increase in the number of stains, which comprises a step of determining that the number of stains in the subject is likely to increase when at least one of the alleles is a risk allele.
(b1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935)
(b2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5.
[4] The SNP having a chain imbalance coefficient r2 ≥ 0.8 with the SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5 in (b2) above is a sequence. Specified by SNP (SNP: SNP specified by ID rs2900195) at the 101st base of the base sequence shown in No. 6 or SNP (SNP: ID rs2840322) at the 101st base of the base sequence shown by SEQ ID NO: 7. SNP), the method according to [3].
[5] A step of detecting one or more single nucleotide polymorphism (SNP) alleles of the following (c1) to (c2) and the bases of the detected alleles in the DNA-containing sample collected from the subject. A method for determining a genetic predisposition for a tendency to increase the area of a stain, which comprises a step of determining that the area of the stain of the subject is likely to increase when at least one of the alleles is a risk allele.
(c1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692)
(c2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs72631692) at the 101st base of the base sequence shown in SEQ ID NO: 8.
[6] The SNP having a chain imbalance coefficient r2 ≥ 0.8 with the SNP (SNP: SNP specified by ID rs72631692) at the 101st base of the base sequence shown in SEQ ID NO: 8 of (c2) is a sequence. Specified by SNP (SNP: SNP specified by ID rs72631686) at the 101st base of the base sequence shown in No. 9 or SNP (SNP: ID rs7683971) at the 101st base of the base sequence shown by SEQ ID NO: 10. The method according to [5], which is SNP).
[7] A cosmetic having a stain-preventing and / or ameliorating effect according to the degree of genetic predisposition of the subject's stain based on the result determined by the method according to any one of [1] to [6]. And / or a method of providing cosmetics and / or food and drink, which provides the subject with food and drink.
[8] SNP at the 101st base of the base sequence shown in SEQ ID NO: 1 (SNP: SNP specified by ID rs1001949), SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: ID rs16910935) SNP specified by), SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692), or SNP having a chain imbalance coefficient r2 ≧ 0.8 with the SNP. SNP (SNP: SNP specified by ID rs1001949), SEQ ID NO: 5 at the 101st base of the base sequence shown in SEQ ID NO: 1 and / or a probe having a sequence containing 10 or more bases or a complementary sequence thereof. SNP at the 101st base of the base sequence shown in (SNP: SNP specified by ID rs16910935), SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692) Or, a kit for determining the genetic predisposition of a stain, which comprises a primer capable of amplifying a region containing an SNP having a chain imbalance coefficient r2 ≧ 0.8 with the SNP.

According to the method of the present invention, by detecting a single nucleotide polymorphism (SNP) allele contained in genome-derived DNA present in a biological sample of a subject, the tendency of the subject to darken the stain and the number of stains. It is possible to accurately and easily determine the easiness of increasing the number of stains or the easiness of increasing the area of stains. Therefore, based on this determination result, measures for prevention and improvement of stains can be taken at an early stage.

1. 1. Method for determining the genetic predisposition of stains The method for determining the genetic predisposition of stains of the present invention is a specific single nucleotide polymorphism (SNP) associated with the genetic predisposition of the type of appearance of an individual stain or linkage with the SNP. It uses unbalanced SNPs to predict what type of stains are likely to occur in the future, and the types of stains that appear are likely to be darkened and the number of stains is likely to increase. It also includes the tendency for the area of stains to increase.

The above-mentioned "linkage disequilibrium" means that two alleles are linked to each other more frequently than when they are inherited independently. A group of alleles exhibiting such linkage disequilibrium is called a haplotype. In the present invention, an SNP having a linkage disequilibrium coefficient r2 of 0.8 or more, preferably 0.9 or more, more preferably 0.95 or more, and most preferably 1 can be used. SNPs that are in linkage disequilibrium with a particular SNP include, for example, the Ensembl Genome Browser (https://asia.ensembl.org/index.html) and the HapMap database (http://www.hapmap.org/index.html. It can be identified using ja) and the like. Alternatively, DNA collected from a plurality of persons (usually about 20 to 40 persons) can be identified by sequence analysis with a sequencer and searching for SNPs having linkage disequilibrium.

The method for determining the susceptibility to darkening of stains, which is the first aspect of the present invention, is to use one or more single nucleotide polymorphisms (a1) to (a4) below for a DNA-containing sample collected from a subject. It includes a step of detecting a type (SNP) allele and a step of determining that the subject is likely to have dark spots when at least one of the bases of the detected allele is a risk allele.
(a1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 1 (SNP: SNP specified by ID rs1001949)
(a2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1.
(a3) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935)
(a4) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5.

Examples of SNPs having a linkage disequilibrium coefficient r2 ≥ 0.8 with the SNP specified by rs1001949 in (a2) above include rs6484714, rs11043121, rs11827022, rs11043122, rs11043119, rs1875776, rs10840589, rs10840590, rs7129423, rs11043123, rs10840591, rs11821418. , Rs11043127, rs11043116, rs952362, rs7112239, rs58077575, etc., and examples of SNPs having a linkage disequilibrium coefficient r2 ≥ 0.8 with the SNP specified by rs16910935 in (a4) above include rs2900195, rs1571804, rs2840322, etc. Be done.

The method for determining the genetic predisposition to increase the number of stains, which is the second aspect of the present invention, is one or 2 of the following (b1) to (b2) for a DNA-containing sample collected from a subject. Includes a step of detecting a single nucleotide polymorphism (SNP) allele of a species or more, and a step of determining that the number of stains on the subject is likely to increase when at least one of the bases of the detected allele is a risk allele. ..
(b1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935)
(b2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5.

Examples of the SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with the SNP specified by rs16910935 in (b2) above include rs2900195, rs1571804, and rs2840322.

The method for determining the genetic predisposition to increase the area of stains, which is the third aspect of the present invention, is one of the following (c1) to (c2) or one of the following (c1) to (c2) for a DNA-containing sample collected from a subject. A step of detecting two or more single nucleotide polymorphism (SNP) alleles and a step of determining that the area of the stain of the subject is likely to increase when at least one of the detected allele bases is a risk allele. including.
(c1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692)
(c2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs72631692) at the 101st base of the base sequence shown in SEQ ID NO: 8.

Examples of the SNP having a linkage disequilibrium coefficient r2 ≧ 0.8 with the SNP specified by rs72631692 in (c2) above include rs75801632, rs72631686, rs113023273, rs113775056, and rs7683971.

Table 1 shows SNPs related to the above-mentioned type of appearance of stains, and base sequences containing SNPs having a linkage disequilibrium coefficient r2 ≥ 0.8 with the SNPs (indicated by [], indicate SNPs).

As shown in the results of the examples described later, the SNPs of the above rs1001949, rs6484714, rs10840590, rs58077575, rs16910935, rs2900195, rs2840322, rs72631692, rs72631686, rs7683971 are the types of appearance of stains (easiness of stains, stains). It has been proved to be statistically related to the genetic predisposition of (easiness to increase the number of stains and the tendency to increase the area of stains). Although even one of the above SNPs can determine the genetic predisposition of the type of appearance of stains (the tendency of stains to darken, the number of stains to increase, or the area of stains to increase). By combining two or more types, the determination accuracy can be improved, and the patterns of the determination types can be increased.

Single nucleotide polymorphism (SNP, hereinafter may be referred to as "SNP") generally refers to a state in which the base sequence of a gene differs by only one site and the site thereof. In addition, the polymorphism generally refers to two or more alleles (alleles) that are present at a frequency of 1% or more in the population. The "SNP" in the present invention is an SNP database (http://www.ncbi.nlm) of the National Center for Biotechnology Information (NCBI), which is a public database freely available to those skilled in the art. It is an SNP registered in .nih.gov / SNP /) and can be specified by its reference number, rs number.

As used herein, the term "allele" refers to each type having different bases that can be taken at a certain SNP site. Further, as used herein, the term "genotype" refers to a combination of alleles that oppose each other at a certain SNP site. At a certain SNP site, there are three types of genotypes that are the combination, the combination of the same allele is called a homotype, and the combination of different alleles is called a heterotype. For example, there are three types of genotypes, C / C type, C / G type, and G / G type, which are combinations of alleles that oppose each other in SNP identified by rs1001949.

In the determination method of the present invention, "detecting a single nucleotide polymorphism (SNP) allele" means identifying the type of base of the SNP allele, and "detecting a single nucleotide polymorphism (SNP) allele". 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 bases of the detected allele is a risk allele, the subject tends to have dark spots (dark spots are likely to occur) and the number of spots is likely to increase (spots). (The number of stains tends to increase), or the area of stains tends to increase (large stains tend to occur). For example, in rs6484714, rs10840590, rs58077575, which are SNPs having a linkage disequilibrium r2 ≥ 0.8 relationship with rs1001949 or rs1001949, or rs2900195, rs2840322, which are SNPs having a linkage disequilibrium r2 ≥ 0.8 relationship with rs16910935 or rs16910935. In the above, if the risk allele shown in Table 2 below is detected in at least one of the alleles, it can be determined that the stain is likely to be darkened (dark stain is likely to be formed) as compared with the case where the risk allele is not detected. Further, in rs2900195 and rs2840322, which are SNPs having a linkage disequilibrium coefficient r2 ≥ 0.8 with rs16910935 or rs16910935, if the risk allele shown in Table 2 below is detected in at least one of the alleles, the risk allele is not detected. It can be determined that the number of stains is likely to increase (the number of stains is likely to increase) as compared with the case. Further, in rs72631686 and rs7683971 which are SNPs having a linkage disequilibrium coefficient r2 ≧ 0.8 with rs72631692 or rs72631692, if the risk allele shown in Table 2 below is detected in at least one allele, the risk allele is not detected. It can be determined that the area of the stain is likely to be large (large stain is likely to be formed) as compared with the case.

For example, in the case of SNP specified by rs1001949, the genotype of G / G is more likely to cause darker stains than the case of C / G type or C / C type (darker stains are more likely to occur). ), And that the genotype is C / G type is more likely to cause darker stains (darker stains are more likely to occur) than the case of C / C type. That is, it shows that the tendency of dark spots (the ease of dark spots) is higher in the order of G / G type, C / G type, and C / C type.

In the determination method of the present invention, the race of the subject is not particularly limited, but is preferably East Asian, and more preferably Japanese. Here, the East Asian means a person whose origin is any of the people of Japan, Korea, China, Taiwan and Mongolia.

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 detected SNP is located in a region existing in mRNA other than a non-transcription region such as a promoter or a region excluded by RNA splicing such as an intron. If it is a located SNP, 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, secretory fluid, tissue, cell, tissue or cell culture from which genomic DNA can be collected can be used, and specifically, the saliva, blood, etc. of the subject. Urine, sputum, pharyngeal swab, nasal swab, oral (inner cheek) mucosal swab, lacrimal gland secretion, sweat, hair, nails, skin, mucous membrane, tape strips peeled off after skin adhesion, etc. Saliva is preferred because of its minimal invasiveness. The sample can be collected according to a method performed in a general clinical test, and prepared by using a known extraction method and purification method. At that time, a commercially available genomic DNA extraction kit can be used.

The method for detecting SNP and determining the type of SNP (SNP typing) is not particularly limited, and for example, allele-specific primers (and probes) are used to amplify the polymorphism by PCR or the like, and the polymorphism of the amplified product is fluorescent or luminescent. It can be carried out 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 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) and the like can be mentioned. All of the above methods are well known to those skilled in the art, and reagents and kits for determining the type of SNP are also commercially available. For example, TaqMan SNP Genotyping Assays (manufactured by Thermo Fisher Scientific) or the like is used. Can be done.

The results obtained by the above determination method serve as a useful index for the subject to select cosmetics and / or foods and drinks having a stain-preventing and / or ameliorating effect. For subjects who are determined to have a genetic predisposition, the number of stains is likely to increase (the number of stains is likely to increase), and the area of stains is likely to increase (large stains are likely to occur). Measures to prevent and / or improve stains can be recommended. Therefore, according to another aspect of the present invention, based on the results obtained by the above-mentioned determination method, it depends on the degree of genetic predisposition of the subject's stain (what type of stain is likely to occur in the future). Also provided is a method of providing a cosmetic and / or food or drink that provides the subject with a cosmetic and / or food or drink that has a stain-preventing and / or ameliorating effect. For example, the types of spots are typically senile pigmented spots, freckles, ADM (acquired dermal melanocytosis, bilateral nevus-like pigmented spots), chloasma, post-inflammatory hyperpigmentation, etc. There are mixed types of these, each of which is characterized by the density, number, size, site, and contour of the spots, and most of them occur and worsen due to ultraviolet rays, aging, hormonal imbalance, delayed turnover, etc. It is a thing. Therefore, by knowing the type of appearance of individual spots, cosmetics and / or foods and drinks containing ingredients having UV protection, whitening, anti-inflammatory, etc. according to the type of spots mentioned above can be used. By actively incorporating the above-mentioned stains, the stains can be effectively prevented and / or ameliorated.

2. Kit for determining the genetic predisposition of stains In the above SNP detection and typing methods, probes and primers corresponding to each method are used. Such probes and primers are also included in the scope of the present invention and can be provided as a kit.

Examples of the probe include the above-mentioned SNP site, and a probe capable of discriminating the type of base of the SNP site depending on the presence or absence of hybridization can be mentioned. Specifically, SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1 and SNP (SNP: SNP: SNP: SNP at the 101st base of the base sequence shown in SEQ ID NO: 5. SNP specified by ID rs16910935), SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692), or the relationship between the SNP and the chain imbalance coefficient r2 ≥ 0.8 Examples thereof include a probe having a sequence of 10 bases or more containing a certain SNP, preferably a sequence of 15 bases or more, or a complementary sequence thereof. The length of the probe is preferably 15-40 bases, more preferably 20-35 bases. The probe may be labeled with an appropriate labeling substance, and examples of the labeling substance include enzymes (peroxidase, β-galactosidase, alkaline phosphatase, etc.), fluorescent substances (FITC, RITC, Cy3, Cy5, etc.), and the like. Examples thereof include luminescent substances (luminol, luminol derivatives, luciferin, luciferin, etc.), radioactive isotopes ( ³ H, ¹⁴ C, ³² P, ¹²⁵ I, ¹³¹ I, etc.), biotin, digoxigenin, and polypeptides containing tag sequences. Alternatively, a quencher (quenching substance) that absorbs the fluorescence energy emitted by the fluorescent substance may be further bonded in the vicinity of the fluorescent substance.

In addition, the probe may be immobilized on a solid phase (DNA array). The DNA array can simultaneously detect hybridization for each probe by hybridizing sample DNA to a large number of probes arranged on the same plane and scanning the plane. Therefore, DNA arrays are useful for analyzing a large number of SNP sites at the same time. The oligonucleotides that serve as probes to be mounted on the array are usually synthesized in situ. For example, an in situ synthesis method of an oligonucleotide by a lithographic method (Thermo Fisher Scientific), an inkjet method (Agilent), a bead array method (Illumina), or the like is known.

Examples of the primer include a primer that can be used for PCR for amplifying the SNP site, and a primer that can be used for sequence analysis (sequencing) of the SNP site. Specifically, SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1 and SNP (SNP: SNP: SNP: SNP at the 101st base of the base sequence shown in SEQ ID NO: 5. SNP specified by ID rs16910935), SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692), or the relationship between the SNP and the chain imbalance coefficient r2 ≥ 0.8 Examples include primers that can amplify or sequence regions containing certain SNPs. The primer that can be used for PCR for amplifying the SNP site 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. Primers can be set upstream or downstream of the SNP site.

Those skilled in the art can design probes and primers according to the analysis method based on the nucleotide sequence information of the peripheral DNA region including the SNP site. Further, the oligonucleotide to be a probe and a primer can be obtained by using a DNA automatic synthesizer usually used by a method known in the art as a method for synthesizing an oligonucleotide, for example, a phosphoramidite method, an H-phosphonate method or the like. It is possible to synthesize.

The kit of the present invention may contain at least the above-mentioned probe and oligonucleotide used as a primer. In addition, if necessary, the kit includes reagents for DNA extraction, reagents for PCR such as PCR buffer and DNA polymerase, reagents for detection such as stains and gels for electrophoresis, immobilization carriers, labeling substances, and the like. Substrate compounds used to detect labels, positive and negative standard samples, instructions describing how to use the kit, etc. can also be included. The reagent in the kit may be a solution or a freeze-dried product.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.

(Example 1)
In conducting this study, the consent form, genotyping, etc. were approved by the in-house ethics review committee. Samples were taken from subjects who received consent by signing the consent form, and genotyping and related analysis were performed.

(1) DNA sample Saliva was collected from 740 Japanese female subjects (mean age 53.6 years) with facial stains, and genomic DNA was extracted using the Maxwell RSC Stabilized Saliva DNA Kit (manufactured by Promega). A sample was obtained.

(2) Analyzed SNP
Among the SNPs registered in the SNP database (http://www.ncbi.nlm.nih.gov/SNP/), rs1001949, rs16910935, and rs72631692 were analyzed. Furthermore, SNPs with a linkage disequilibrium coefficient r2 ≥ 0.8 with each SNP of rs1001949, rs16910935, and rs72631692 in Japanese searched using the Ensembl Genome Browser (https://asia.ensembl.org/index.html) ( Of the table 3) below, rs6484714, rs10840590, rs58077575, rs2900195, rs2840322, rs72631686, and rs7683971 were also analyzed.

(3) Genotyping For the DNA sample obtained in (1), use TaqMan SNP Genotyping Assays (manufactured by Applied Biosystems) and SsoAdvanced Universal Probes Supermix (manufactured by Bio Rad), and use the CFX384 Touch Real-Time PCR Detection System. The genotype was determined by (Bio Rad).

(4) Phenotypic data (easy to darken stains, easy to increase area, easy to increase number)
The questionnaire item regarding stains is to answer one image that is close to you from the image that divides the density, area, and number of stains into 5 stages (in ascending order of 1 to 5 shows the severity). Was treated as an interval scale.

(5) Association analysis Regarding the relationship between the hereditary data and the appearance of stains, multiple regression analysis was performed with the phenotypic data as the objective variable, the number of minor alleles in the hereditary data, and the age as explanatory variables, and the minor The p value of the partial regression coefficient of the number of alleles was evaluated. Since 10 types of SNPs and 3 types of phenotypes were analyzed, the significance level was corrected by the Bonferroni method (0.05 / 10/3) to avoid the increase of type I errors, and the p value was 0.0016 (1.6E). -03) If it is less than -03), it is judged that there is a significant relevance.

(6) Results Table 4 shows the chromosome number, physical position, bases of alleles and minor alleles in which each SNP analyzed, phenotypes related to stains, bases of risk alleles for each phenotype, regression coefficients, and p-values. .. If the sign of the regression coefficient is positive, the minor allele is the risk allele, and if the sign of the regression coefficient is negative, the major allele is the risk allele. In the p-value column, each p-value is displayed in exponential format with a base of 10, and the numerical values before and after the sign E indicate the mantissa and exponent part when the p-value is displayed in exponential format, respectively. ..

As shown in Table 4, rs1001949 has a genetic predisposition to the tendency to darken stains, rs16910935 has a genetic predisposition to the tendency to darken stains and the number of stains to increase, and rs72631692 has a large area of stains. There is a significant relationship with the genetic predisposition of easiness, and it was confirmed as an SNP that can determine the easiness of darkening of stains, the easiness of darkening of stains and the easiness of increasing the number of stains, and the susceptibility of stains to grow. In addition, since the same results were obtained for SNPs that have a linkage disequilibrium relationship with each SNP, stains tend to become darker for SNPs that have a linkage disequilibrium relationship with rs1001949, rs16910935, and rs72631692. , It was found that it is possible to judge the easiness of darkening and increasing the number of stains, and the easiness of increasing the area of stains.

According to the method of the present invention, it is possible to accurately and easily determine whether or not a subject has a genetic predisposition to stains. Therefore, based on the judgment result, it is possible to provide the subject with customized cosmetics and supplements for prevention and improvement of age spots, and to provide counseling and advice on care methods for prevention and improvement of age spots.

Claims (8)

In the DNA-containing sample collected from the subject, the step of detecting one or more single nucleotide polymorphism (SNP) alleles of the following (a1) to (a4) and at least one of the detected allele bases. A method for determining a genetic predisposition to the tendency of stains to be darkened, which comprises a step of determining that the subject is likely to be stained when one is a risk allele.
(a1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 1 (SNP: SNP specified by ID rs1001949)
(a2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1.
(a3) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935)
(a4) SNP having a linkage disequilibrium coefficient r2 = 1 with SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5. The SNP having a chain imbalance coefficient r2 ≥ 0.8 with the SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 of (a2) is shown in SEQ ID NO: 2. SNP at the 101st base of the base sequence shown (SNP: SNP specified by ID rs6484714), SNP at the 101st base of the base sequence shown in SEQ ID NO: 3 (SNP: SNP specified by ID rs10840590), Alternatively, it is an SNP (SNP: SNP specified by ID rs58077575) at the 101st base of the base sequence shown in SEQ ID NO: 4.
The SNP having a chain imbalance coefficient r2 = 1 with the SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 of (a4) is shown in SEQ ID NO: 6. SNP at the 101st base of the base sequence shown (SNP: SNP specified by ID rs2900195), SNP at the 101st base of the base sequence shown in SEQ ID NO: 7 (SNP: SNP specified by ID rs2840322) The method according to claim 1. In the DNA-containing sample collected from the subject, the step of detecting one or more single nucleotide polymorphism (SNP) alleles of the following (b1) to (b2) and at least one of the detected allele bases. A method for determining a genetic predisposition to an increase in the number of stains, which comprises a step of determining that the number of stains in the subject is likely to increase when one is a risk allele.
(b1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935)
(b2) SNP having a linkage disequilibrium coefficient r2 = 1 with SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5. The SNP having a chain imbalance coefficient r2 = 1 with the SNP (SNP: SNP specified by ID rs16910935) at the 101st base of the base sequence shown in SEQ ID NO: 5 of (b2) is shown in SEQ ID NO: 6. SNP at the 101st base of the indicated base sequence (SNP: SNP specified by ID rs2900195) or SNP at the 101st base of the base sequence shown in SEQ ID NO: 7 (SNP: SNP specified by ID rs2840322) The method according to claim 3. In the DNA-containing sample collected from the subject, the step of detecting one or more single nucleotide polymorphism (SNP) alleles of the following (c1) to (c2) and at least one of the detected allele bases. A method for determining a genetic predisposition for the tendency of a stain to grow, including a step of determining that the subject's spot area is likely to grow when one is a risk allele.
(c1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692)
(c2) SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 with SNP (SNP: SNP specified by ID rs72631692) at the 101st base of the base sequence shown in SEQ ID NO: 8. The SNP having a chain imbalance coefficient r2 ≥ 0.8 with the SNP (SNP: SNP specified by ID rs72631692) at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 of (c2) is shown in SEQ ID NO: 9. SNP at the 101st base of the indicated base sequence (SNP: SNP specified by ID rs72631686) or SNP at the 101st base of the base sequence shown in SEQ ID NO: 10 (SNP: SNP specified by ID rs7683971) The method according to claim 5. Based on the result determined by the method according to any one of claims 1 to 6, a cosmetic and / or a cosmetic having an effect of preventing and / or improving the stain according to the degree of the genetic predisposition of the stain of the subject. A method of providing cosmetics and / or food and drink, which provides food and drink to the subject. Specified by SNP (SNP: SNP specified by ID rs1001949) at the 101st base of the base sequence shown in SEQ ID NO: 1 and SNP (SNP: ID rs72631692) at the 101st base of the base sequence shown by SEQ ID NO: 8. SNP), or SNP having a chain imbalance coefficient r2 ≧ 0.8 with the SNP , SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: SNP specified by ID rs16910935), or the SNP. A probe having a sequence of 10 bases or more including an SNP having a chain imbalance coefficient r2 = 1 with the SNP , or a complementary sequence thereof, and / or an SNP at the 101st base of the base sequence shown in SEQ ID NO: 1. SNP: SNP specified by ID rs1001949) , SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP specified by ID rs72631692), or SNP and chain imbalance coefficient r2 ≥ 0.8 Includes related SNPs , SNPs at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNPs: SNPs specified by ID rs16910935), or SNPs that have a chain imbalance coefficient r2 = 1 with the SNPs . A kit for determining the genetic predisposition of stains, including primers capable of amplifying the region.