WO2022211563A1 - Genetic polymorphism marker for determining wrinkle skin type and use thereof - Google Patents

Abstract

The present invention relates to: a composition for diagnosing whether a skin type that can easily form wrinkles is present, the composition comprising a probe capable of detecting a genetic polymorphism marker with a correlational significance with the degree of skin wrinkles, or an agent capable of amplifying the marker; a kit or a microarray comprising the composition; and a method for providing information on whether a skin type that can easily form wrinkles is present, the method using the genetic polymorphism marker or a combination of markers. Single nucleotide polymorphism markers of the present invention provide accurate information for the skin types of individuals, and, thus, it is possible to develop a personalized cosmetic product and a customized active ingredient.

Description

Genetic polymorphic marker for wrinkle skin type determination and use thereof

The present invention provides a composition for diagnosing whether a skin type is prone to wrinkling, comprising a genetic polymorphic marker having significance associated with the degree of skin wrinkle, a probe capable of detecting it or an agent capable of amplifying it, and the composition comprising the composition A kit or microarray for diagnosing whether a skin type exists, and a method for providing information on whether a skin type is easily wrinkled using the genetic polymorphism marker or a combination of markers.

The skin is the outermost layer of the human body and responds to the external environment to protect the body from harmful substances, sense different senses, and perform various functions such as thermoregulation, and the proper function of the skin is to protect the body from disease and maintain attractiveness. It is essential.

Skin wrinkles are one of the most representative skin aging characteristics, and changes in skin wrinkles can be influenced by environmental factors and factors such as age, gender, and genetic variation. According to the existing genome-wide association studies (GWAS) on skin wrinkles, it was found that genetic mutations located in genes functionally related to the biological pathway of wrinkles are significantly related to skin wrinkles, have. However, most of the existing GWAS studies have targeted Europeans, and there are few studies on Asian races, so the understanding of the genetic variation related to skin wrinkles in Asian races is low.

Since most of the existing GWAS studies rely on the visual judgment of the evaluator when defining the degree of skin wrinkling, there is a limit that how to define and subdivide the degree of skin wrinkle may differ depending on the evaluator. On the other hand, skin wrinkle evaluation using a professional skin diagnosis device can secure the reliability of the evaluation method in that it can quantify the degree of skin wrinkle by minimizing bias by the evaluator and applying the same standard to all evaluators. In addition, the evaluation method using a device has the advantage of being able to more easily reach a sample size sufficient to obtain a reasonable level of statistical power because it is easy to recruit and evaluate a larger number of subjects.

The discovery of skin-related genetic mutations and genes is expected to provide important information for understanding biological mechanisms and understanding and predicting individual skin characteristics. Genetic analysis research is moving from research that discovers genetic indicators related to diseases or phenotypes to researches that predict future diseases or phenotypes using the discovered genetic indicators. In particular, research on predicting facial shape transformation or skin aging through genetic analysis research on facial shape or skin characteristics is also underway (Kemp et al., Molecules. 2017 Feb 26;22(3), 2017).

The present inventors tried to establish a classification system for Korean skin characteristics through the construction of big data of genetic information and skin type information. As a result of diligent efforts to develop personalized active ingredients and contribute to the development of customized cosmetics for each skin characteristic through various product segmentation, specific single nucleotide polymorphism (SNP) markers that have a significant correlation with skin wrinkles were selected and based on genetic information The present invention was completed by confirming a method for diagnosing the degree of wrinkles.

One object of the present invention is to provide a single nucleotide polymorphism (SNP) marker for diagnosing whether a skin type is prone to wrinkles.

Another object of the present invention is to provide a composition for diagnosing whether a skin type is easy to generate wrinkles, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing whether a skin type is easy to generate wrinkles or an agent capable of amplifying it will do

Another object of the present invention is to provide a kit or microarray for diagnosing whether a skin type is easy to generate wrinkles comprising the composition.

Another object of the present invention is to provide a method of providing information on whether or not a skin type is prone to wrinkle formation, comprising the step of identifying a polymorphic site of the single nucleotide polymorphism marker.

It is possible to provide information on the degree of skin wrinkling of an individual through the genetic polymorphic markers having a significance associated with the degree of skin wrinkling of the present invention, and furthermore, it is possible to alleviate the degree of skin wrinkle according to the information of the genetic polymorphic markers observed in the individual. We will be able to develop custom ingredients or products that can be used.

Each description and embodiment disclosed in the present invention is applicable to each other description and embodiment as well. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

As one aspect for achieving the object of the present invention, the present invention provides a single nucleotide polymorphism (SNP) marker for diagnosing the degree of skin wrinkles.

In another aspect, the present invention provides a composition for diagnosing the degree of skin wrinkles, comprising a probe capable of detecting or amplifying a single nucleotide polymorphism (SNP) marker for diagnosing the degree of skin wrinkles.

The skin type of an individual may be classified according to the degree of skin wrinkle.

As used herein, the term "polymorphism" refers to a case in which two or more alleles exist at one locus, and among polymorphic sites, only a single base differs from person to person. It is called (single nucleotide polymorphism, SNP). Preferred polymorphic markers have two or more alleles exhibiting an incidence of at least 1%, more specifically at least 10% or 20%, in a selected population. A 'genetic polymorphic marker' is usually two or more alleles at the same locus (base). It refers to a case in which alleles are observed, and generally, depending on the individual, a major allele/major allele, a major allele/minor allele, and a lower allele. The case of a minor allele/minor allele exists. In the present invention, it may be used interchangeably with "polymorphic marker", and may mean a base and a base region of a lower allele, or may be defined together with the number and base position of a chromosome, but is not limited thereto.

As used herein, the term "allele" refers to several types of one gene present at the same locus of homologous chromosomes. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles. In addition, it refers to a combination of two or more bases having the same chromosome number and base position, and the base is a higher allele with a higher frequency of occurrence in a specific group of individuals and a lower with lower frequency of occurrence than the upper allele contains minor alleles.

Specifically, the genetic polymorphism markers of the present invention are significantly related to skin wrinkles, and when one or more of the two alleles have a lower allele, the skin wrinkles are the major allele. allele) / can be said to be significant compared to individuals with a major allele. That is, in the case of a major allele/minor allele, and a minor allele/minor allele, the major allele/major allele ), it can be seen that the degree has higher or lower skin characteristics compared to the degree of skin wrinkles.

In the present invention, the term "rs_id" refers to rs-ID, an independent marker given to all SNPs initially registered by the NCBI, which has been accumulating SNP information since 1998. rs_id described in this table means a SNP marker, which is a polymorphic marker of the present invention.

As used herein, the term “skin type” refers to the skin type of an individual to be measured or diagnosed. The skin type that can be measured by the SNP of the present invention includes without limitation, but may specifically be a wrinkle. According to an embodiment of the present invention, the present inventors classified the subject's skin type into skin with few wrinkles or skin with many wrinkles. Since the single nucleotide polymorphism marker of the present invention can measure the exact skin type, it can also provide information on the changed skin type of the skin in contact with the active ingredient, so it is possible to provide personalized cosmetics, etc. It is not limited thereto.

In addition, for the purpose of the present invention, the term 'skin wrinkle' means that the elasticity of some parts of an individual's skin is lowered and the skin folds are observed. Due to the fold of the skin, the wrinkled area is shaded compared to the surrounding skin, so it is observed to be relatively dark compared to the surrounding skin. In addition, since most wrinkles have a linear shape, they may be defined as wrinkles when shaded areas are connected and observed over a certain length, but is not limited thereto.

The single nucleotide polymorphism marker may be one or more single nucleotide polymorphism markers selected from among the single nucleotide polymorphism markers shown in Tables 1 to 4. The single nucleotide polymorphism markers shown in Tables 1 to 4 may be used to determine the degree of correlation with the degree of skin wrinkles.

The degree of skin wrinkling of the single nucleotide polymorphism marker of the present invention was determined by measuring the frequency of each marker. Such significance is characterized by, but is not limited to, a p-value such as, but not limited to, a p-value of less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, or less than 0.000000001. Specifically, the p-value may be less than 0.01, more specifically, the p-value may be less than 0.001, and more specifically, it may be less than 0.0001, but is not limited thereto.

The single nucleotide polymorphism (SNP) marker of the present invention may be any one or more selected from the markers shown in Tables 1 to 4, but is not limited thereto. The single nucleotide polymorphism (SNP) marker may be one or more, and may be used as a combination of a number such as two or more, three or more, four or more that can determine wrinkles, but is not limited thereto.

The marker may be, but is not limited to, the SNP itself, or a polynucleotide consisting of 5-100 consecutive DNA sequences including the SNP position, or a polynucleotide consisting of a complementary sequence thereof.

Specifically, in one embodiment, the single nucleotide polymorphism marker may be any one or more selected from the markers shown in Table 1, but is not limited thereto.

A marker selected from among the markers shown in Table 1 may be described as follows.

For example, if the SNP ID is rs12695694, if Chr.Position (GRCh ver. 37) is described as "3:139000458" and Allele is C>G, this is the 139000458th base of human chromosome 3 is C or G, and a base located to the left of ">" of an allele may mean a major allele, and a base located to the right of an allele may mean a minor allele.

In one embodiment, the marker selected from Table 1 is a polynucleotide consisting of 5-100 consecutive DNA sequences including the 119633160 nucleotide, wherein the 119633160 nucleotide of the human chromosome 2 is G or T (rs7597233). nucleotides; a polynucleotide consisting of 5-100 consecutive DNA sequences including the 119632252 base, wherein the 119632252 base of human chromosome 2 is G or A (rs12612325); a polynucleotide consisting of 5-100 consecutive DNA sequences including base 119599377, wherein base 119599377 of human chromosome 2 is A or T (rs13005242); a polynucleotide consisting of 5-100 consecutive DNA sequences including the 119632724 base, wherein the 119632724 base of human chromosome 2 is G or A (rs12621139); a polynucleotide consisting of 5-100 contiguous DNA sequences including base 119628722, wherein base 119628722 of human chromosome 2 is G or T (rs1438842); a polynucleotide consisting of 5-100 consecutive DNA sequences including base 139000458, wherein base 139000458 of human chromosome 3 is C or G (rs12695694); a polynucleotide consisting of 5-100 contiguous DNA sequences including base 139002193, wherein base 139002193 of human chromosome 3 is A or T (rs9853827); a polynucleotide consisting of 5-100 contiguous DNA sequences comprising base 139006664, wherein base 139006664 of human chromosome 3 is C or G (rs6802174); a polynucleotide consisting of 5-100 consecutive DNA sequences including the 138997688 nucleotide, wherein the 138997688 nucleotide of human chromosome 3 is G or A (rs9866054); a polynucleotide consisting of 5-100 consecutive DNA sequences including the 139004920 nucleotide, wherein the 139004920 nucleotide of human chromosome 3 is G or A (rs10212419); a polynucleotide consisting of 5-100 contiguous DNA sequences including the 139021139 nucleotide, wherein the 139021139 nucleotide of human chromosome 3 is C or T (rs4894405); a polynucleotide consisting of 5-100 consecutive DNA sequences including the 146537655 base, wherein the 146537655 base of human chromosome 4 is A or G (rs117927610); a polynucleotide consisting of 5-100 contiguous DNA sequences including the 124239525 base, wherein the 124239525 base of human chromosome 4 is C or T (rs6832894); a polynucleotide consisting of 5-100 consecutive DNA sequences including the 20303599 base, wherein the 20303599 base of human chromosome 9 is C or T (rs7033540); a polynucleotide consisting of 5-100 contiguous DNA sequences comprising the 20308808 base of human chromosome 9, wherein the 20308808 base is C or A (rs57500604); and one or more polynucleotides selected from the group consisting of their complementary polynucleotides, but is not limited thereto. The above-described markers are only partially described in Table 1 as examples, and may be selected in the same manner as above in chromosomes at other positions.

As another embodiment, as in Table 1, any one or more of the single nucleotide polymorphism (SNP) markers shown in Table 2 may be selected, but the present invention is not limited thereto.

As another embodiment, as in Table 1, any one or more of the single nucleotide polymorphism (SNP) markers shown in Table 3 may be selected, but the present invention is not limited thereto.

As another embodiment, as in Table 1, any one or more of the single nucleotide polymorphism (SNP) markers shown in Table 4 may be selected, but the present invention is not limited thereto.

The single nucleotide polymorphism markers shown in Tables 2 to 4 may be interpreted and selected as described above, but are not limited thereto.

For the purpose of the present invention, the term 'skin wrinkle degree' refers to the result of measuring the ratio of the total measurement site by summing all the target areas when a linear area of a certain length or more is observed relatively dark compared to the surrounding skin due to skin wrinkles. mean, but not limited thereto. Specifically, it means determining whether wrinkles are easy to generate using any one or more single nucleotide polymorphism (SNP) markers selected from Tables 1 to 4. More specifically, a skin type that is easy to generate wrinkles refers to a skin type that has a high rate of generation of wrinkles per skin area due to rapid aging.

The allele of the present invention has the same number of chromosomes in each individual, and among them, a major allele and a minor allele of the SNP exist, and the base of the polymorphic site of the polymorphic marker is lower As the allele increases one by one, the upper allele may decrease one by one, and as one increases into the upper allele one by one, the lower allele may decrease by one. However, the range in which the lower allele and upper allele can increase and decrease is i) major allele/major allele, ii) major allele/minor allele ), iii) may be within three types of lower alleles/minor alleles, and the allele may decrease or increase within the range of the three types, but is not limited thereto. .

In addition, in the present invention, the marker is a marker capable of determining the degree of increase or decrease in the phenotype (wrinkle) as the base allele of the polymorphic site of the polymorphic marker increases one by one. More specifically, (1) a major allele/minor allele, and (2) a lower allele carrying one or more minor alleles among the two alleles (minor allele/in the case of minor allele), the degree of skin wrinkling is lower than that of those carrying the major allele/major allele, which is a common individual. It can be judged to have high or low skin characteristics.

For example, among the markers shown in Table 1, when the upper allele C at the 139000458th base of the individual's chromosome 3 and the lower allele is G (rs12695694), compared with those who have C / C, C / If you have G or G/G, the effect size is negative (-), so it can be judged that the under-eye wrinkles are small, and the upper allele G is the upper allele G at the 119633160th base of the individual's chromosome 2, and the lower allele is T (rs7597233), compared with those who have G/G, in the case of holding G/T or T/T, the effect size is positive (+), so it can be judged that there are many wrinkles under the eyes. not limited

More specifically, in the present invention, the marker is a marker capable of determining the degree of increase or decrease in the phenotype (wrinkle) as the bases of the polymorphic site of the polymorphic marker of the individual increase by one minor allele.

As an example, when G, the minor allele of the 139000458th base of the individual's chromosome 3 among the markers shown in Table 1 increases (rs12695694), the effect size is negative (-), so it can be determined that there are few wrinkles under the eyes, If T, the minor allele of base 119633160 of chromosome 2 of the individual increases (rs7597233), the effect size is positive (+), so it can be determined that there are many wrinkles under the eyes, but is not limited thereto.

Specifically, when the single nucleotide polymorphism marker includes one or more of the following bases, which are minor alleles, it may be a number of wrinkles under the eyes, but is not limited thereto:

Base 119633160 of human chromosome 2 is T; Base 119632252 of human chromosome 2 is A; Base 119599377 of human chromosome 2 is T; Base 119632724 of human chromosome 2 is A; Base 119628722 of human chromosome 2 is T; Base 146537655 of human chromosome 4 is G; Base 124239525 of human chromosome 4 is T.

In addition, when the single nucleotide polymorphism marker includes one or more of the following bases, which are minor alleles, it may have fewer wrinkles under the eyes, but is not limited thereto:

Base 139000458 of human chromosome 3 is G; Base 139002193 of human chromosome 3 is T; Base 139006664 of human chromosome 3 is G; Base 138997688 of human chromosome 3 is A; The 139004920 base of human chromosome 3 is A; Base 139021139 of human chromosome 3 is T; The 20033599 base of human chromosome 9 is T; The 20308808 base of human chromosome 9 is A.

As another example, if the minor allele C of the 89110410 base of chromosome 12 of the individual among the markers shown in Table 3 increases (rs72620727), the effect size is negative (-), so it can be determined that the wrinkles around the eyes are small. In the case of an increase in A, the minor allele of the 16794418th base of chromosome 9 (rs12376135), the effect size is positive (+), so it can be determined that there are many wrinkles around the eyes, but is not limited thereto.

Specifically, when the single nucleotide polymorphism marker includes one or more of the following bases, which are minor alleles, it may have many lateral wrinkles, but is not limited thereto:

Base 16794418 of human chromosome 9 is A; Base 16795241 of human chromosome 9 is C; Base 16795790 of human chromosome 9 is C; Base 30435055 of human chromosome 14 is T; The 28232737 base of human chromosome 15 is G; The 28233207 base of human chromosome 15 is G; The 28233905 base of human chromosome 15 is T; The 28233913 base of human chromosome 15 is C.

In addition, when the single nucleotide polymorphism marker includes one or more of the following bases, which are minor alleles, the lateral wrinkles may be small, but the present invention is not limited thereto:

Base 89110410 of human chromosome 12 is C; Base 3548231 of human chromosome 19 is G; Base 3540539 of human chromosome 19 is C; The 3542983 base of human chromosome 19 is T; Base 3550926 of human chromosome 19 is G; Base 3537184 of human chromosome 19 is G.

The bases are described as examples only in Table 1 or Table 3, and although not specifically described, the markers involved in wrinkles can be derived from Table 2 or Table 4 in the same manner as above.

As used herein, the term "probe capable of detecting a marker for diagnosing the degree of skin wrinkle" refers to a composition capable of diagnosing the degree of skin wrinkle by identifying it through a hybridization reaction specifically with the polymorphic region of the gene as described above, The specific method of the gene analysis is not particularly limited, and any gene detection method known in the art to which the present invention pertains may be used. In addition, the term may be used interchangeably with the term 'for diagnosing whether a skin type is easy to generate wrinkles'.

As used herein, the term "agent capable of amplifying a marker for diagnosing the degree of skin wrinkle" refers to a composition capable of diagnosing the degree of skin wrinkle by identifying the polymorphic region of the gene as described above through amplification, specifically, the skin It refers to a primer capable of specifically amplifying the polynucleotide of a marker for diagnosing the degree of wrinkles. In addition, the term may be used interchangeably with the term 'for diagnosing whether a skin type is easy to generate wrinkles'.

The primers used for amplifying the polymorphic marker are prepared under suitable conditions (e.g., 4 different nucleoside triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and template-directing DNA under an appropriate temperature. Refers to a single-stranded oligonucleotide that can serve as a starting point for synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template.

As used herein, the term "primer" refers to a nucleotide sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and is a start for template strand copying. A short sequence that functions as a branch. Primers are capable of initiating DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature. By performing PCR amplification, the skin type can be predicted by the degree of production of the desired product. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.

The probes or primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, "encapsulation", substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

As another aspect, the present invention provides a kit for diagnosing the degree of skin wrinkles, comprising the composition for diagnosing the degree of skin wrinkles. The kit may be an RT-PCR kit or a DNA chip kit, but is not limited thereto.

The kit of the present invention can diagnose the skin type by amplifying the SNP polymorphic marker, which is a marker for skin type diagnosis, or by checking the expression level of the SNP polymorphic marker and the mRNA expression level. As a specific example, the kit for measuring the mRNA expression level of the marker for skin type diagnosis in the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for the gene of a skin type diagnostic marker, the RT-PCR kit includes a test tube or other suitable container, reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs) , enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, and the like. In addition, a primer pair specific for a gene used as a quantitative control may be included. Also specifically, the kit of the present invention may be a kit for diagnosing a skin type including essential elements necessary for performing a DNA chip. A DNA chip kit is a method in which nucleic acid species are attached in a gridd array to a generally flat solid support plate, typically a glass surface no larger than a microscope slide, and the nucleic acids are uniformly arranged on the chip surface, so that the DNA chip It is a tool that allows multiple hybridization reactions between the nucleic acids on the chip surface and the complementary nucleic acids contained in the solution treated on the chip surface to enable massively parallel analysis.

As another aspect, the present invention provides a microarray for diagnosing the degree of skin wrinkles, comprising the composition for diagnosing the degree of skin wrinkles.

The microarray may include DNA or RNA polynucleotides. The microarray consists of a conventional microarray except that the polynucleotide of the present invention is included in the probe polynucleotide.

Methods for preparing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide refers to a hybridizable polynucleotide, and refers to an oligonucleotide capable of sequence-specific binding to a complementary strand of a nucleic acid. The probe of the present invention is an allele-specific probe. A polymorphic site exists in nucleic acid fragments derived from two members of the same species, so that it hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member. . In this case, the hybridization conditions should be sufficiently stringent to hybridize only one of the alleles by showing a significant difference in the hybridization intensity between alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used in a method for diagnosing a skin type by detecting an allele. The diagnostic methods include detection methods based on hybridization of nucleic acids, such as Southern blotting, and may be provided in a form previously bound to a substrate of a DNA chip in a method using a DNA chip. The hybridization may be usually performed under stringent conditions, for example, a salt concentration of 1M or less and a temperature of 25° C. or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30° C. may be suitable for allele-specific probe hybridization.

The process of immobilizing the probe polynucleotide related to the skin diagnosis of the present invention on a substrate can also be easily prepared using this conventional technique. In addition, hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is, for example, labeling a nucleic acid sample with a label capable of generating a detectable signal including a fluorescent substance, for example, a substance such as Cy3 and Cy5, and then hybridizing on a microarray and generating from the labeling material. The hybridization result can be detected by detecting the signal to be used.

In another aspect, the present invention provides a method comprising: (a) amplifying the polymorphic site of the single nucleotide polymorphism marker in DNA obtained from a sample isolated from an individual or hybridizing it with a probe; and (b) identifying the base of the amplified or hybridized polymorphic site of step (a).

As used herein, the term “subject” refers to a subject for diagnosing the degree of skin wrinkles. DNA may be obtained from the sample, such as hair, urine, blood, various body fluids, isolated tissues, isolated cells, or samples such as saliva, but is not limited thereto.

Any method known to those skilled in the art can be used for the method of obtaining genomic DNA in step (a).

Any method known to those skilled in the art may be used for amplifying the polymorphic site of the single nucleotide polymorphism marker or hybridizing with a probe from the DNA obtained in step (a). For example, it can be obtained by amplifying a target nucleic acid through PCR and purifying it. Others include ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)) and self-maintaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)) and nucleic acid-based sequence amplification (NASBA) can be used.

Among the methods, determining the base of the polymorphic site in step (b) includes sequencing analysis, microarray hybridization, allele specific PCR, and dynamic allele-specific hybridization. , DASH), PCR extension assay, SSCP, PCR-RFLP assay or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. Sequenom's MassARRAY system), mini-sequencing method, Bio- Plex systems (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion Probe array technologies (eg, Affymetrix GeneChip), and BeadArray Technologies (eg, Illumina GoldenGate and Infinium assays). . One or more alleles in polymorphic markers can be identified, including microsatellites, SNPs, or other types of polymorphic markers, by the above methods or other methods available to those skilled in the art. Determination of the base of such a polymorphic site can be specifically performed through a SNP chip.

The method is to additionally (c) determine that there are many or few wrinkles when the base of the amplified or hybridized polymorphic site contains one or more bases that are minor alleles according to the single nucleotide polymorphism marker. can, but is not limited thereto. In addition, (c) additionally (c) if the base of the amplified or hybridized polymorphic site includes one or more bases that are major alleles according to the single nucleotide polymorphism marker, it may be determined that there are many or few wrinkles. However, it is not limited thereto.

As used herein, the term "SNP chip" refers to one of the DNA microarrays that can check each base of hundreds of thousands of SNPs at once.

The TaqMan method comprises the steps of (1) designing and manufacturing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dye and VIC dye (Applied Biosystems); (3) using the DNA as a template, and performing PCR using the primers and probes; (4) after the PCR reaction is completed, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; and (5) determining the genotype of the polynucleotides of step (1) from the analysis result.

In the above, the sequencing analysis may use a conventional method for determining the nucleotide sequence, and may be performed using an automated gene analyzer. In addition, allele-specific PCR refers to a PCR method of amplifying a DNA fragment in which the SNP is located with a primer set including a primer designed with the 3' end of the base in which the SNP is located. The principle of the method is, for example, when a specific base is substituted from A to G, a primer containing A as a 3' end base and a reverse primer capable of amplifying a DNA fragment of an appropriate size are designed to perform PCR In the case of performing the reaction, when the base at the SNP position is A, the amplification reaction is normally performed and a band at the desired position is observed, and when the base is substituted with G, the primer can bind to the template DNA, but 3 ' This is taking advantage of the fact that the amplification reaction is not performed properly due to the inability to perform complementary binding at the end. DASH may be performed by a conventional method, and specifically may be performed by a method by Prince et al.

On the other hand, PCR extension analysis first amplifies a DNA fragment containing a base in which the single nucleotide polymorphism is located with a pair of primers, and then inactivates all nucleotides added to the reaction by dephosphorylating, and here a SNP-specific extension primer; A primer extension reaction is performed by adding a dNTP mixture, dideoxynucleotide, reaction buffer and DNA polymerase. In this case, the extension primer uses the 3' end of the base immediately adjacent to the 5' direction of the base at which the SNP is located, the dNTP mixture excludes a nucleic acid having the same base as the dideoxynucleotide, and the dideoxynucleotide represents the SNP It is selected from one of the base types. For example, when there is a substitution from A to G, when a mixture of dGTP, dCTP and dTTP and ddATP are added to the reaction, the primer is extended by DNA polymerase at the base where the substitution has taken place, and after a few bases, A The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution does not occur, since the extension reaction is terminated at that position, it is possible to determine the type of base representing the SNP by comparing the lengths of the extended primers.

In this case, as a detection method, when the extension primer or dideoxynucleotide is fluorescently labeled, the SNP can be detected by detecting fluorescence using a gene analyzer (eg, ABI's Model 3700, etc.) used for general sequencing. In the case of using a non-labeled extension primer and dideoxynucleotide, the SNP can be detected by measuring the molecular weight using a matrix assisted laser desorption ionization-time of flight (MALDI-TOF) technique.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

The purpose of this study was to discover genomic regions (gene mutations) in which differences in the degree (number) of facial skin wrinkles according to genetic information were found in Koreans (females). The present invention is to discover genomic regions (gene mutations) related to wrinkles, and utilizes a microarray genotyping chip (product of Illumina) that can screen the full-length level of the genome without selecting candidate genes in advance.

In order to evaluate the degree of wrinkles in the present invention, an image-based specialized skin diagnosis device (PIE's Janus3) was used. was used after correction.

In order to confirm the correlation between the genomic region (gene mutation) and the degree of skin wrinkles (number), correlation significance and genetic effect were quantified using linear regression analysis.

Example 1: Characterization of skin and gene extraction

In order to derive genetic polymorphic markers that can explain the general degree of skin wrinkles, healthy Korean women in their 20s and 70s were recruited. In addition, for skin measurement, all analysis subjects washed their faces with cleansing or soap and waited for 30 minutes without applying any products so that the skin could adapt to the measurement environment, and then the degree of skin wrinkles was evaluated. A specialized device (PIE's Janus3) was used (measurement and analysis were performed according to the device manufacturer's manual). Specifically, after wearing a headband and putting a black cloth over clothes, the subject fixed their face on the forehead and chin rests built into the device, and maintained the posture for measurement with eyes closed, using the camera built into the Janus 3 device. A high-resolution image of the facial part of the subject to be analyzed was taken. The program provided by Janus 3 itself defines the outer lateral area of the eye and the area under the eye based on both eye areas, and extracts and quantifies all wrinkle areas within the area. In particular, by targeting the area around the eyes where wrinkles occur a lot, the analysis was divided into wrinkles under the eyes and wrinkles around the eyes.

The degree of skin wrinkle in a general individual (having a major allele/major allele) was measured on 11,079 analysis subjects in which two alleles were observed at the same gene location (base). It means the average of wrinkles under the eyes or the sides of the eyes, and the value was derived as the ratio of areas observed over a certain length that were relatively dark compared to the surrounding skin within the analysis area.

Gene collection was done through saliva collection, and for effective gene collection, all analysis subjects were prohibited from ingesting any food including water from 30 minutes before collection.

Among the above subjects, ① Pregnant, lactating, or planning to become pregnant within 6 months, ② If a skin exfoliant containing steroids has been used for more than 1 month for the treatment of skin diseases, ③ If 6 months have not elapsed since participating in the same test ⑥ If you have sensitive or hypersensitive skin, ⑤ If there are skin abnormalities such as spots, acne, erythema, telangiectasia, etc. In case of use, ⑦ If the test site has undergone treatment (dermabrasion, botox, other skin care) or planned within 6 months, ⑧ If you have a chronic wasting disease (asthma, diabetes, high blood pressure, etc.), ⑨ If you have atopic dermatitis, ⑩ Other cases judged to be difficult due to the judgment of the main investigator were excluded from the subjects.

Example 2: Genotyping according to skin characteristics

For gene extraction from saliva for gene analysis, human genomic DNA was extracted using QIAamp mini prep kit (QIAGEN), and the quality was absorbance (OD 260/280) or 1.7, concentration 50ng/ul, 1x TAE 1% agarose It was confirmed through the band test through the gel, and genetic analysis was performed only on cases that passed the quality.

Gene analysis was performed using Illumina's microarray genotyping chip, and specifically, the genes of the subjects were analyzed using the global screening array product of the same company.

Illumina's microarray genotyping chip gene analysis experiment was performed according to the provided manual, and using the provided reagents, genomic DNA amplification, DNA fragmentation, precipitation, hybridization, and staining were performed. , washing, coating, and scanning were performed.

The microarray genotyping chip on which the experiment was completed was scanned using the iScan Control Software (Illumina), and when the scan was completed, the idat file was automatically created and data quality management (sample call rate 98%, marker call) was performed using the GenomeStudio (Illumina) program. rate 98%) and genetic information was confirmed.

In this experiment, only data that passed data quality control after genetic analysis were used.

Example 3: Derivation of significant genetic polymorphism markers associated with skin wrinkle degree using genotyping analysis for each skin type

Linear regression analysis was performed using the polymorphic markers of the analytes to derive genetic polymorphic markers that had correlational significance with the degree of skin wrinkling. For the analysis, PLINK v 1.90 and SNP & Variation suite (Golden Helix, Inc., Bozeman, Montana, USA) program was used.

Imputation analysis was performed using the Beagle v 5.1 program to secure information on additional genetic polymorphism markers using the genetic polymorphism markers identified through the experiment. The information registered in the data database, 1000 Genomes project, was used.

Imputation is a statistical technique for inferring unanalyzed genetic polymorphic marker information based on experimentally obtained genetic polymorphic marker information.

For quality control of the polymorphic markers of the gene to be analyzed, only those that exceed the criteria of minor allele frequency or 0.01 and Hardy-Weinberg equilibrium or 0.000001 were used for each polymorphic marker.

The significance of the genetic polymorphic markers correlated with the degree of skin wrinkling was evaluated through linear regression analysis F-statistics, and the criterion was set as P-value < 0.00000005. In addition, when 0.00000005 ≤ P-value < 0.0001, which is a somewhat relaxed criterion, genetic polymorphism markers showing a weak level of association with the degree of skin wrinkles were additionally selected.

In order to minimize external effects that may affect the degree of skin wrinkling and derive effects according to genetic information, the degree of skin wrinkles is corrected with information on age or BMI or lifestyle (drinking, smoking, eating habits, sleeping habits, etc.) It can be used for analysis (eg, performing linear regression analysis).

A very large number of genetic polymorphism markers related to skin wrinkle degree were derived, and it was confirmed that the polymorphic markers were mainly located on chromosomes 2, 3, 4, 9, 12, 14, 15, and 19 in the human genome.

A list of SNP markers significantly related to skin wrinkles is shown in Tables 1 to 4 below. Specifically, Tables 1 and 2 relate to polymorphic markers related to the degree of under-eye wrinkles. Those with a significance level (P) less than 5.00E-8 are shown in Table 1, and those with a significance level (P) of 5.00E-8 or more and less than 1.00E-4 are Table 2 shown in In addition, Tables 3 and 4 relate to polymorphic markers related to the degree of wrinkles around the eyes. Those with a significance level (P) less than 5.00E-8 are shown in Table 3, and those with a significance level (P) of 5.00E-8 or more and less than 1.00E-4 are shown in Table 4. indicated.

Genetic polymorphic markers associated with the degree of under-eye wrinkles (P < 5.00E-8)

	SNP 1)	Gene 1)	Chr:Position 1)	Allele 2)	MAF 3)	P -value 4)	Effect size (β) 5)	Type 6)
One	rs12695694	MRPS22	3:139000458	C>G	0.40	1.30E-10	-1.27	imputed
2	rs9853827	MRPS22	3:139002193	A>T	0.40	1.57E-10	-1.26	imputed
3	rs6802174	MRPS22	3:139006664	C>G	0.40	1.58E-10	-1.26	imputed
4	rs9866054	MRPS22	3:138997688	G>A	0.40	1.59E-10	-1.26	imputed
5	rs10212419	MRPS22	3:139004920	G>A	0.40	2.51E-10	-1.25	imputed
6	rs7597233	-	2:119633160	G>T	0.35	5.95E-10	1.26	imputed
7	rs4894405	MRPS22	3:139021139	C>T	0.40	1.02E-09	-1.22	genotyped
8	rs12612325	-	2:119632252	G>A	0.36	1.34E-09	1.23	imputed
9	rs13005242	-	2:119599377	A>T	0.37	1.80E-09	1.22	imputed
10	rs12621139	-	2:119632724	G>A	0.36	2.03E-09	1.22	imputed
11	rs1438842	-	2:119628722	G>T	0.36	3.66E-09	1.19	imputed
12	rs117927610	-	4:146537655	A>G	0.03	7.48E-09	3.46	imputed
13	rs6832894	SPATA5	4:124239525	C>T	0.40	4.06E-08	1.09	genotyped
14	rs7033540	-	9:20303599	C>T	0.30	4.36E-08	-1.17	imputed
15	rs57500604	-	9:20308808	C>A	0.31	4.73E-08	-1.16	imputed

1) National Institutes of Health (NIH) ID, you can check the sequence on the website

2) Meaning of (major allele) > (minor allele)

3) minor allele frequency = (2mm + Mm)/2(MM + Mm + mm)

4) Statistical significance of phenotypic differences for three genotypes (M/M, M/m, m/m) (M: major allele, m: minor allele)

5) As the minor allele increases one by one, the degree of increase or decrease of the phenotype (under-eye wrinkles) (-: reduction of under-eye wrinkles, +: increase of under-eye wrinkles), wrinkle degree = ratio of area where wrinkles occur to the total area, However, intensity weighting and threshold value applied to the wrinkle area, using commercially available equipment (Janus3, PIE, Korea)

6) Analyze using Beagle v5.1

Genetic polymorphism markers associated with the degree of lateral folds (P < 5.00E-8)

	SNP 1)	Gene 1)	Chr:Position 1)	Allele 2)	MAF 3)	P -value 4)	Effect size (β) 5)	Type 6)
One	rs72620727	-	12:89110410	A>C	0.39	1.55E-13	-1.22	genotyped
2	rs2240751	MFSD12	19:3548231	A>G	0.34	3.95E-12	-1.19	genotyped
3	rs12984831	MFSD12	19:3540539	G>C	0.32	1.06E-11	-1.18	imputed
4	rs12608592	MFSD12	19:3542983	C>T	0.32	2.00E-11	-1.17	imputed
5	rs12376135	BNC2	9:16794418	C>A	0.42	9.53E-10	1.00	imputed
6	rs75555084	MFSD12	19:3550926	T>G	0.30	2.82E-09	-1.05	imputed
7	rs10810635	BNC2	9:16795241	T>C	0.42	3.00E-09	0.97	imputed
8	rs16935073	BNC2	9:16795790	A>C	0.43	1.16E-08	0.93	genotyped
9	rs112244493	OCA2	15:28232737	T>G	0.38	1.52E-08	0.94	imputed
10	rs77733715	FZR1	19:3537184	A>G	0.36	1.65E-08	-0.95	imputed
11	rs12442916	OCA2	15:28233207	A>G	0.39	1.92E-08	0.93	imputed
12	rs10775262	OCA2	15:28233905	C>T	0.37	3.16E-08	0.92	imputed
13	rs10775263	OCA2	15:28233913	T>C	0.37	3.16E-08	0.92	imputed
14	rs34470885	PRKD1	14:30435055	C>T	0.29	3.23E-08	0.98	imputed

1) National Institutes of Health (NIH) ID, you can check the sequence on the website

2) Meaning (major allele) or (minor allele)

3) minor allele frequency = (2mm + Mm)/2(MM + Mm + mm)

4) Statistical significance of phenotypic differences for three genotypes (M/M, M/m, m/m) (M: major allele, m: minor allele)

5) As the minor allele increases one by one, the degree of increase or decrease in the phenotype (wrinkle next to the eye) (-: decrease in wrinkles around the eyes, +: increase in wrinkles around the eyes), the degree of wrinkles = the ratio of the area where the wrinkles occur to the total area, However, intensity weighting and threshold value applied to the wrinkle area, using commercially available equipment (Janus3, PIE, Korea)

6) Analyze using Beagle v5.1

As a result, genetic mutations mainly related to skin wrinkles were identified in a total of 8 genomic regions (chromosomes 2, 3, 4, 9, 12, 14, 15, 19 =).

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (8)

1. A composition for diagnosing whether a skin type is easy to generate wrinkles, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing whether any one or more wrinkle-generating skin types selected from Tables 1 to 4 or an agent capable of amplifying it .
2. The method according to claim 1, wherein the single nucleotide polymorphism marker for diagnosing whether the skin type is prone to wrinkles is a polynucleotide corresponding to one or more single nucleotide polymorphism markers selected from any one of Tables 1 to 4; and one or more polynucleotides selected from the group consisting of their complementary polynucleotides.
3. A kit for diagnosing whether the skin type is prone to wrinkles, comprising the composition of claim 1 or 2.
4. The kit for diagnosing skin wrinkles according to claim 3, wherein the kit is an RT-PCR kit or a DNA chip kit.
5. A microarray for diagnosing whether a skin type is easy to generate wrinkles, comprising the single nucleotide polymorphism (SNP) marker for diagnosing whether the skin type is easy to generate wrinkles according to claim 1 .
6. (a) amplifying the polymorphic site of the single nucleotide polymorphism marker for diagnosing the degree of skin wrinkles according to claim 1 in DNA obtained from a sample isolated from an individual or hybridizing it with a probe; and

   (b) A method of providing information on whether a skin type is prone to wrinkling, comprising the step of confirming the base of the amplified or hybridized polymorphic site of step (a).
7. The method of claim 6, wherein the sample is hair, urine, blood, various body fluids, isolated tissues, isolated cells, or saliva.
8. The method of claim 6 , wherein the amplification and confirmation of the polymorphic site uses a SNP chip.