JP2022152000A - Method for Determining Genetic Predisposition to Age Spots - Google Patents

Abstract

To provide accurate and simple means for determining individual genetic predisposition to age spots.SOLUTION: Provided is a method for determining a genetic predisposition to age spots comprising: detecting at least one single nucleotide polymorphism (SNP) of rs1001949, rs16910935, and rs72631692 or alleles of SNPs in linkage disequilibrium with these SNPs, for DNA-containing samples collected from subjects; and determining that the subject has a genetic predisposition to easily darken age spots, increase the number of age spots, and/or increase the area of age spots, when at least one of the bases of the detected allele is a risk allele.SELECTED DRAWING: None

Description

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

The skin is an organ that covers the entire human body, and is composed of three layers, the epidermis, the dermis, and the subcutaneous tissue, from the outside. The epidermal layer, which is the outermost layer, is further divided into four layers: the stratum basale, the stratum spinosum, the stratum granulosum, and the stratum corneum (Non-Patent Document 1). The epidermis layer plays an extremely important role as a biological defense mechanism, such as retention of moisture, prevention of external stimuli such as ultraviolet rays, and invasion of foreign substances. Of these, melanocytes (pigment cells) present in the stratum basale protect against ultraviolet rays by producing melanin. Age spots are caused by abnormal development, proliferation, differentiation and melanin synthesis of melanocytes. Since blemishes have a great effect on the appearance of the face, there is a demand for prevention and improvement techniques, risk evaluation techniques, and the like.

So far, as a method for evaluating the susceptibility of spots to occur, a method using the blood C-peptide concentration and the blood IGF-1 concentration of a subject as indices (Patent Document 1), and a single base in a gene correlated with spots 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 causes and symptoms (density, number, size, site, outline, etc.) of the blemishes. For example, there are individual differences in the susceptibility to dark spots, the susceptibility to large areas, and the susceptibility to increase in number, etc., and it is thought that the mechanisms, genetic factors, and appropriate countermeasures that cause them also differ. . However, little has been done to search for SNPs that can predict the type of individual blemish appearance.

Japanese Patent Application Laid-Open No. 2010-48610 JP 2018-78848 A JP 2018-164451 A

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 means for accurately and simply determining an individual's genetic predisposition to blemishes.

In order to solve the above problems, the present inventors examined SNPs that can predict the type of appearance of individual blemishes, and found that rs1001949 and SNPs in a linkage disequilibrium relationship with it are specific for darkening of blemishes. rs16910935 and SNPs in a linkage disequilibrium relationship with it can specifically predict the susceptibility to stain darkening and increase in number, rs72631692 and SNPs in a linkage disequilibrium relationship with it can predict the stain area The inventors have found that it is possible to specifically predict the tendency of the to increase, leading to the completion of 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 (a4) single nucleotide polymorphism (SNP) alleles in a DNA-containing sample collected from a subject; is a risk allele, a method for determining a genetic predisposition for susceptibility to darkening of blemishes, comprising the step of determining that the subject is likely to develop dark blemishes.
(a1) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs1001949)
(a2) A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs1001949)
(a3) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935)
(a4) SNP in the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935) and SNP having linkage disequilibrium coefficient r2≧0.8
[2] A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 of (a2) (SNP identified by SNP: ID rs1001949) is the sequence SNP at the 101st base of the nucleotide sequence shown in number 2 (SNP: SNP identified by ID rs6484714), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 3 (SNP: identified by ID rs10840590 SNP), or the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 4 (SNP identified by ID rs58077575),
The SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 of (a4) (SNP identified by SNP: ID rs16910935) and the SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 are in SEQ ID NO: 6 SNP at the 101st base of the base sequence shown (SNP: SNP identified by ID rs2900195), SNP at the 101st base of the base sequence shown in SEQ ID NO: 7 (SNP: SNP identified by ID rs2840322) There is, the method described in [1].
[3] A step of detecting one or more of the following (b1) to (b2) single nucleotide polymorphism (SNP) alleles in a DNA-containing sample collected from a subject; is a risk allele, a method for determining a genetic predisposition for a tendency to increase the number of blemishes, comprising the step of determining that the number of blemishes is likely to increase in the subject.
(b1) SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935)
(b2) SNP in the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935) and SNP having linkage disequilibrium coefficient r2≧0.8
[4] A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 in (b2) (SNP identified by SNP: ID rs16910935) is the sequence SNP at the 101st base of the nucleotide sequence shown in number 6 (SNP: SNP identified by ID rs2900195), or SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 7 (SNP: identified by ID rs2840322 SNP), the method according to [3].
[5] A step of detecting one or more of the following (c1) to (c2) single nucleotide polymorphism (SNP) alleles in a DNA-containing sample collected from a subject; is a risk allele, a method for determining a genetic predisposition for the susceptibility of the area of blemishes to increase, comprising the step of determining that the area of blemishes of the subject is likely to increase.
(c1) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP identified by SNP: ID rs72631692)
(c2) A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP identified by SNP: ID rs72631692)
[6] A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 of (c2) (SNP identified by SNP: ID rs72631692) is the sequence SNP at the 101st base of the nucleotide sequence shown in No. 9 (SNP identified by ID rs72631686) or SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 10 (SNP: identified by ID rs7683971 The method according to [5], which is SNP).
[7] A cosmetic having an effect of preventing and/or improving blemishes according to the degree of genetic predisposition to blemishes of the subject based on the results determined by the method according to any one of [1] to [6]. and/or a method of providing cosmetics and/or food and drink to the subject.
[8] SNP at the 101st base of the base sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs1001949), SNP at the 101st base of the base sequence shown in SEQ ID NO: 5 (SNP: ID rs16910935 SNP identified by ), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP identified by ID rs72631692), or SNP having a relationship with said SNP with a linkage disequilibrium coefficient r2 ≥ 0.8 or a probe having a sequence of 10 or more bases containing or a complementary sequence thereof, and / or a SNP at the 101st base of the base sequence shown in SEQ ID NO: 1 (SNP: SNP identified by ID rs1001949), SEQ ID NO: 5 SNP at the 101st base of the nucleotide sequence shown in (SNP: SNP identified by ID rs16910935), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP: SNP identified by ID rs72631692) Alternatively, a kit for determining genetic predisposition to age spots, comprising primers capable of amplifying a region containing an SNP having a relationship with said SNP with a linkage disequilibrium coefficient r2≧0.8.

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, the likelihood of dark spots in the subject and the number of spots It is possible to accurately and easily determine the susceptibility to an increase in blemishes or the susceptibility to an increase in the area of blemishes. Therefore, based on this determination result, it is possible to quickly take measures to prevent or improve blemishes.

1. Method for determining genetic predisposition to blemishes The method for determining genetic predisposition to blemishes of the present invention includes a specific single nucleotide polymorphism (SNP) associated with a genetic predisposition for the type of appearance of individual blemishes or a specific single nucleotide polymorphism (SNP) linked to the SNP. Using unbalanced SNPs, it predicts what types of blemishes are likely to occur in the future. and susceptibility to large areas of blemishes.

The above-mentioned "linkage disequilibrium" means that two alleles are inherited in linkage with 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 in linkage disequilibrium with specific SNPs can be found, for example, in the Ensembl Genome Browser (https://asia.ensembl.org/index.html) or the HapMap database (http://www.hapmap.org/index.html. ja) can be used for identification. Alternatively, it can be identified by performing sequence analysis on DNA collected from a plurality of people (usually about 20 to 40 people) and searching for SNPs in linkage disequilibrium.

According to the first aspect of the present invention, the method for determining the susceptibility to darkening of spots is performed by extracting one or more of the following (a1) to (a4) from a DNA-containing sample collected from a subject. The method includes a step of detecting a type (SNP) allele, and a step of determining that the subject's blemishes tend to become darker when at least one of the bases of the detected allele 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 rs1001949)
(a2) A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs1001949)
(a3) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935)
(a4) SNP in the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935) and SNP having linkage disequilibrium coefficient r2≧0.8

上記(a2)のrs1001949で特定されるSNPと連鎖不平衡係数r2≧0.8の関係にあるSNPとしては、rs6484714、rs11043121、rs11827022、rs11043122、rs11043119、rs1875776、rs10840589、rs10840590、rs7129423、rs11043123、rs10840591、rs11821418 , rs11043127, rs11043116, rs952362, rs7112239, rs58077575, etc. Examples of SNPs that have a linkage disequilibrium coefficient r2 ≥ 0.8 with the SNP identified in (a4) rs16910935 include rs2900195, rs1571804, rs2840322, etc. be done.

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

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

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

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

Table 1 shows the SNPs associated with the above types of appearance of blemishes, and the nucleotide sequences containing the SNPs having a relationship with the SNPs with a linkage disequilibrium coefficient r2≧0.8 (SNPs are shown in brackets).

As shown in the results of Examples described later, the above SNPs rs1001949, rs6484714, rs10840590, rs58077575, rs16910935, rs2900195, rs2840322, rs72631692, rs72631686, and rs7683971 indicate the type of appearance of blemishes (ease of darkening of blemishes, The tendency to increase the number of blemishes and the tendency to increase the area of blemishes) has been statistically proven to be related to the genetic predisposition. Although even one of the above SNPs can be used to determine the genetic predisposition of the type of appearance of blemishes (easily darkening of blemishes, susceptibility to increase in number of blemishes, or susceptibility to increase in area of blemishes). By combining two or more types, it is possible to improve the judgment accuracy and increase the patterns of judgment types.

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 a combination of opposing alleles in the SNP identified by rs1001949.

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 bases of the detected allele is a risk allele, the subject tends to have dark spots (easily develop dark spots) and the number of spots tends to increase (spots The number of spots tends to increase) or the area of spots tends to increase (large spots tend to form). For example, rs6484714, rs10840590, and rs58077575, which are SNPs in a relationship with rs1001949 or rs1001949 with a linkage disequilibrium coefficient r2 ≥ 0.8, or rs2900195, rs2840322, which are SNPs in a relationship with rs16910935 or rs16910935 with a linkage disequilibrium coefficient r2 ≥ 0.8 In the above, if the risk alleles shown in Table 2 below are detected in at least one allele, it can be determined that stains are more likely to become darker (deeper stains are more likely to occur) than when the risk alleles are not detected. In addition, in rs2900195 and rs2840322, which are SNPs having a relationship of 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 allele, the risk allele is not detected. It can be determined that the number of blemishes tends to increase (the number of blemishes tends to increase) compared to the case. In addition, in rs72631686 and rs7683971, which are SNPs having a relationship of 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 tends to increase (large stain tends to occur) compared to the case.

For example, in the case of the SNP identified by rs1001949, when the genotype is G/G type, dark spots are more likely to occur than when the genotype is C/G type or C/C type. ), indicating that the C/G genotype tends to cause darker spots (facilitates darker spots) than the C/C genotype. That is, it indicates that the susceptibility to darkening of stains (easiness of forming dark stains) is high 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, 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 method serve as useful indicators for subjects to select cosmetics and/or foods and beverages that have action to prevent and/or improve spots. easy to form), easy to increase the number of stains (easily increase the number of stains), and easy to increase the area of stains (easily create large stains). Measures to prevent and/or ameliorate blemishes can be recommended. Therefore, according to another aspect of the present invention, based on the results obtained by the above determination method, according to the degree of genetic predisposition to blemishes of the subject (what type of blemishes are likely to occur in the future) A method for providing cosmetics and/or foods and beverages is also provided, comprising providing the subjects with cosmetics and/or foods and beverages having action for preventing and/or improving age spots. For example, the types of spots typically include senile pigment spots, freckle spots, ADM (acquired dermal melanocytosis, bilateral nevus of Ota-like pigment spots), melasma, post-inflammatory pigmentation, There are these mixed types, each of which has its own characteristics in terms of density, number, size, location, and outline of the spots. In most cases, the occurrence and symptoms worsen due to ultraviolet rays, aging, hormonal imbalance, delay in turnover, etc. It is a thing. Therefore, by knowing the type of appearance of individual blemishes, it is possible to select cosmetics and/or foods and drinks containing ingredients having UV protection, whitening, anti-inflammatory effects, etc. according to the type of blemishes described above. By positively incorporating it, the above spots can be efficiently prevented and/or improved.

2. Kit for Determining Genetic Predisposition to Age Spots 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. Specifically, the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by ID rs1001949), the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP: SNP identified by ID rs16910935), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP: SNP identified by ID rs72631692), or a relationship with the SNP with a linkage disequilibrium coefficient r2 ≥ 0.8 A probe having a sequence of 10 bases or more, preferably a sequence of 15 bases or more containing a certain SNP, or a complementary sequence thereof is included. 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 which include enzymes (peroxidase, β-galactosidase, alkaline phosphatase, etc.), fluorescent substances (FITC, RITC, Cy3, Cy5, etc.), Examples include 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. Specifically, the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by ID rs1001949), the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP: SNP identified by ID rs16910935), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP: SNP identified by ID rs72631692), or a relationship with the SNP with a linkage disequilibrium coefficient r2 ≥ 0.8 Examples include primers capable of amplifying or sequencing a region containing a certain SNP. 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.

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)
In conducting this study, the consent form, genotyping, etc. were approved by the company's ethics review committee. Samples were collected from subjects who gave consent by signing a consent form, and genotyping and association analysis were performed.

(1) DNA sample Saliva was collected from 740 Japanese female subjects (average age 53.6 years old) with spots on the face, and genomic DNA was extracted using the Maxwell RSC Stabilized Saliva DNA Kit (manufactured by Promega). Got a sample.

(2) Analyzed SNPs
Of the SNPs registered in the SNP database (http://www.ncbi.nlm.nih.gov/SNP/), rs1001949, rs16910935, and rs72631692 were analyzed. Furthermore, SNPs ( Of Table 3) below, rs6484714, rs10840590, rs58077575, rs2900195, rs2840322, rs72631686, and rs7683971 were also analyzed.

(3) Determination of genotype The DNA samples obtained in (1) were analyzed using TaqMan SNP Genotyping Assays (manufactured by Applied Biosystems) and SsoAdvanced Universal Probes Supermix (manufactured by Bio Rad) using the CFX384 Touch Real-Time PCR Detection System. (manufactured by Bio Rad) was used to determine the genotype.

(4) Phenotypic data (easiness of dark spots, tendency of area to increase, tendency of number to increase)
In the questionnaire items related to blemishes, respondents were asked to answer one image that was similar to their current self from images divided into 5 levels of darkness, area, and number of blemishes (the degree of severity is expressed in ascending order from 1 to 5). was treated as an interval scale.

(5) Association analysis Regarding the relationship between genotype data and the appearance of blemishes, 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 10 SNPs and 3 phenotypes were analyzed, the significance level was adjusted by the Bonferroni method to avoid increasing the type I error (0.05/10/3), with a p-value of 0.0016 (1.6E -03) was judged to have significant relevance.

(6) Results Table 4 shows the chromosomal number, physical position, allele and minor allele bases, blemish-related phenotypes, risk allele bases for each phenotype, regression coefficients, and p-values in which each analyzed SNP exists. . When the sign of the regression coefficient is positive, the minor allele is the risk allele, and when 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 numbers before and after the symbol E indicate the mantissa and exponent of the p-value in exponential format. .

As shown in Table 4, rs1001949 is a genetic predisposition to dark spots, rs16910935 is a genetic predisposition to dark spots and increases in number, and rs72631692 is a genetic predisposition to dark spots. There was a significant relationship with the genetic predisposition to susceptibility, and these SNPs were confirmed to be capable of determining the susceptibility to darkening of blemishes, the susceptibility to darkening of blemishes, the susceptibility to increasing the number of blemishes, and the susceptibility to large blemishes, respectively. In addition, similar results were obtained for SNPs in a linkage disequilibrium relationship with each SNP. , the susceptibility to darkening of blemishes, the susceptibility to increase in the number of blemishes, and the susceptibility to increase in the area of blemishes can be determined.

According to the method of the present invention, it is possible to accurately and simply determine whether or not a subject has a genetic predisposition to blemishes. Therefore, based on the determination results, it is possible to provide the subject with customized cosmetics and supplements for the prevention and improvement of blemishes, and to provide counseling and advice on care methods for the prevention and improvement of blemishes.

Claims (8)

A step of detecting one or more of the following (a1) to (a4) 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 A method for determining a genetic predisposition for susceptibility to darkening of blemishes, comprising the step of determining that the test subject is likely to develop dark blemishes when 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 rs1001949)
(a2) A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by SNP: ID rs1001949)
(a3) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935)
(a4) SNP in the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935) and SNP having linkage disequilibrium coefficient r2≧0.8 The SNP in the 101st base of the base sequence shown in SEQ ID NO: 1 of (a2) (SNP identified by SNP: ID rs1001949) and the SNP having a relationship of linkage disequilibrium coefficient r2 ≥ 0.8 is SEQ ID NO: 2 SNP at the 101st base of the indicated base sequence (SNP: SNP identified by ID rs6484714), SNP at the 101st base of the base sequence shown in SEQ ID NO: 3 (SNP: SNP identified by ID rs10840590), or a SNP (SNP identified by SNP: ID rs58077575) at the 101st base of the nucleotide sequence shown in SEQ ID NO: 4,
The SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 of (a4) (SNP identified by SNP: ID rs16910935) and the SNP having a linkage disequilibrium coefficient r2 ≥ 0.8 are in SEQ ID NO: 6 SNP at the 101st base of the base sequence shown (SNP: SNP identified by ID rs2900195), SNP at the 101st base of the base sequence shown in SEQ ID NO: 7 (SNP: SNP identified by ID rs2840322) 2. The method of claim 1, wherein there is A step of detecting one or more of the following (b1) to (b2) 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 A method for determining a genetic predisposition for a tendency to increase the number of blemishes, comprising the step of determining that the number of blemishes in a 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 identified by SNP: ID rs16910935)
(b2) SNP in the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP identified by SNP: ID rs16910935) and SNP having linkage disequilibrium coefficient r2≧0.8 The SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 of (b2) (SNP identified by SNP: ID rs16910935) and the SNP having a linkage disequilibrium coefficient r2 ≧ 0.8 are in SEQ ID NO: 6 SNP at the 101st base of the indicated base sequence (SNP: SNP identified by ID rs2900195) or SNP at the 101st base of the base sequence shown in SEQ ID NO: 7 (SNP: identified by ID rs2840322) 4. The method of claim 3, wherein A step of detecting one or more of the following (c1) to (c2) 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 A method for determining a genetic predisposition for the susceptibility of the area of blemishes to increase, comprising the step of determining that the area of blemishes of the subject is likely to increase when one is a risk allele.
(c1) SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP identified by SNP: ID rs72631692)
(c2) A SNP having a linkage disequilibrium coefficient r2≧0.8 with the SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP identified by SNP: ID rs72631692) The SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 of (c2) (SNP identified by SNP: ID rs72631692) and the SNP having a relationship of linkage disequilibrium coefficient r2 ≥ 0.8 is SEQ ID NO: 9 SNP at the 101st base of the indicated base sequence (SNP: SNP identified by ID rs72631686) or SNP at the 101st base of the base sequence shown in SEQ ID NO: 10 (SNP: SNP identified by ID rs7683971) 6. The method of claim 5, wherein Based on the results determined by the method according to any one of claims 1 to 6, a cosmetic and/or having an effect of preventing and/or improving blemishes according to the degree of genetic predisposition to blemishes of the subject. A method for providing cosmetics and/or food and drink, comprising providing food and drink to the subject. SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by ID rs1001949), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 5 (SNP: identified by ID rs16910935 SNP), SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 8 (SNP identified by SNP: ID rs72631692), or SNP having a relationship with the SNP with a linkage disequilibrium coefficient r2 ≧ 0.810 A probe having a sequence of more than one base, or a complementary sequence thereof, and/or an SNP at the 101st base of the nucleotide sequence shown in SEQ ID NO: 1 (SNP identified by ID rs1001949), shown in SEQ ID NO: 5 SNP at the 101st base of the base sequence (SNP: SNP identified by ID rs16910935), SNP at the 101st base of the base sequence shown in SEQ ID NO: 8 (SNP: SNP identified by ID rs72631692), or A kit for determining a genetic predisposition to age spots, containing primers capable of amplifying a region containing an SNP having a relationship of linkage disequilibrium coefficient r2≧0.8 with the SNP.