Method for determining cold sensitivity and method for determining cold constitution type

Abstract

To clarify the genetic correlation in cold sensitivity and cold constitution, and to develop and provide a method for determining affected areas of cold sensitivity and cold constitution based on the obtained genetic information.SOLUTION: A comprehensive genome-wide SNPs analysis and a questionnaire survey related to coldness have been conducted, identifying SNPs associated with cold sensitivity and various cold constitution types. This provides a method for determining affected areas of cold sensitivity and cold constitution using these SNPs as biomarkers for cold sensitivity and the like.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for determining poor circulation, a method for determining the site of occurrence of poor circulation, and a kit or device used therefor. [Background technology]

[0002] Chilliness is the sensation of cold in the whole body or in a part of the body, even though there is no organic abnormality. In addition, sensitivity to cold refers to a constitution that produces cold, and sensitivity to cold refers to symptoms that cause physical pain due to cold and may interfere with daily life.

[0003] In general, the prevalence of chilliness is higher in women than in men. Therefore, chilliness has traditionally been considered one of the vague symptoms of menopausal disorders in women. However, in recent years, it has become clear that chilliness is more prevalent in young women (Non-Patent Document 1). Furthermore, it is known that women with chilliness not only experience the subjective symptom of chilliness, but also experience accompanying symptoms such as insomnia, fatigue, edema, and pain (Non-Patent Document 2). Therefore, it is currently believed that chilliness not only reduces women's QOL due to chilliness, but can also trigger the onset of other diseases, and understanding the actual situation and establishing a treatment method have become important issues.

[0004] Sensitivity to cold is thought to occur due to physiological causes such as vasomotor nerve disorder due to autonomic dysfunction, imbalance of female hormones due to decreased estrogen, and decreased thermoregulation function due to decreased muscle mass. On the other hand, it has been reported that more than 60% of mothers of women with sensitivity to cold also have sensitivity to cold, suggesting that sensitivity to cold is due to genetic background (Non-Patent Documents 3, 4). However, there have been few reports of genetic research on sensitivity to cold, and the influence of genetic factors on sensitivity to cold has not been clarified. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kimikazu Fujita et al., 2017, Sports and Health Science Research 39:19~27 [Non-Patent Document 2] Yoshino T., et al., 2019, Complementary Therapies in Medicine, 45: 7-13 [Non-Patent Document 3] Kondo, M. and Okamura, Y., 1987, Journal of the Japan Society of Obstetrics and Gynecology, 39(11):2000-2004. [Non-Patent Document 4] Arakawa K., et al., 2015, Jpn. J. Biometeor., 52(4):199-211 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention clarifies the genetic correlation between sensitivity to cold and cold symptoms, and develops a method for determining the site of onset of sensitivity to cold and cold symptoms based on the obtained genetic information, and also develops a kit or device for use in said determination method. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors conducted a questionnaire survey and SNP analysis on multiple adult female volunteers aged 20 to 60 years, and conducted a comprehensive analysis of the genetic association between cold and SNPs using the Genome Wide Association Study (GWAS). As a result, in a two-group comparison of the presence or absence of cold, multiple SNPs strongly associated with cold were found. Furthermore, in a two-group comparison between a group with no cold and each group with cold legs and hips, cold hands and feet, and cold whole body, multiple SNPs different for each type of cold were found. The present invention relates to cold and cold sensitivity based on the genetic information of subjects, which was developed based on the above analysis results, and provides the following.

[0008] (1) A method for determining in vitro a genetic predisposition to cold sensitivity in a subject, the method comprising: a cold sensitivity determining SNP detection step of detecting a single gene polymorphism (cold sensitivity determining SNP) associated with cold sensitivity from nucleic acid derived from the subject; and a cold sensitivity determination step of determining that the subject has a genetic predisposition to cold sensitivity when at least one of the bases in an allele of the detected cold sensitivity determining SNP is a risk allele. (2) The method according to (1), wherein the SNP for determining sensitivity to cold is any one of 1 to 11 in Table 1 below, and the risk allele is a base shown in Table 1, or a SNP that is in linkage disequilibrium with the risk allele of any one of the SNPs for determining sensitivity to cold, 1 to 11. [Table 1] (3) The method according to (2), wherein the linkage disequilibrium coefficient of the SNPs in the linkage disequilibrium relationship is 0.8 or more. (4) A method for determining in vitro a genetic predisposition to a type of sensitivity to cold in a subject, the method comprising: a step of detecting a type-of-sensitivity to cold determining SNP, which detects a single gene polymorphism (a type-of-sensitivity to cold determining SNP) based on the site of occurrence of sensitivity to cold from nucleic acid derived from the subject; and a step of determining that sensitivity to cold in the subject may occur in the site of that type of sensitivity to cold when at least one of the bases in the allele of the detected type-of-sensitivity to cold determining SNP is a risk allele. (5) The method according to (4), wherein the cold sensitivity type is any one of lower body type, peripheral limb type, and whole body type. (6) The method according to (5), wherein the SNP for determining the cold sensitivity type is any one of a to u in Table 2 below, and the risk allele is a base shown in Table 2 below, or the SNP for determining the cold sensitivity type is any one of a to u, and is in linkage disequilibrium with the risk allele shown in Table 2. [Table 2] (7) The method according to (6), wherein the linkage disequilibrium coefficient of the SNPs in the linkage disequilibrium relationship is 0.8 or more. (8) The method according to any one of (4) to (7), wherein the subject is determined to have a genetic predisposition to cold sensitivity by the determination method according to any one of (1) to (3).

[0009] (9) A kit for detecting a genetic predisposition to poor circulation, comprising a probe capable of specifically binding to a poor circulation determining SNP associated with poor circulation and / or an SNP in linkage disequilibrium with the poor circulation determining SNP and nucleotides adjacent thereto, and / or a primer set that specifically recognizes and amplifies the poor circulation determining SNP and / or an SNP in linkage disequilibrium with the poor circulation determining SNP and nucleotides adjacent thereto. (10) The kit according to (9), wherein the SNP for determining poor circulation is any one of 1 to 11 in Table 1 of (2). (11) A kit for determining a type of sensitivity to cold, comprising a probe capable of specifically binding to a SNP determining sensitivity to cold based on the site of onset of sensitivity to cold and / or an SNP in linkage disequilibrium with the SNP determining sensitivity to cold and nucleotides adjacent thereto, and / or a primer set that specifically recognizes and amplifies the SNP determining sensitivity to cold and / or an SNP in linkage disequilibrium with the SNP determining sensitivity to cold and nucleotides adjacent thereto. (12) The kit according to (11), wherein the SNP for determining a type of cold sensitivity is any one of a to u in Table 2 of (6). (13) The kit according to (11) or (12), further comprising the probe and / or primer set according to (9) or (19).

[0010] (14) A device for detecting a genetic predisposition to poor circulation, comprising a probe capable of specifically binding to a poor circulation determining SNP associated with poor circulation and / or an SNP in linkage disequilibrium with the poor circulation determining SNP and nucleotides adjacent thereto, and / or a primer set that specifically recognizes and amplifies the poor circulation determining SNP and / or an SNP in linkage disequilibrium with the poor circulation determining SNP and nucleotides adjacent thereto. (15) The device according to (14), wherein the SNP for determining poor circulation is any one of 1 to 11 in Table 1 of (2). (16) A device for determining a type of sensitivity to cold, comprising a probe capable of specifically binding to a SNP determining sensitivity to cold based on the site of onset of sensitivity to cold and / or an SNP in linkage disequilibrium with the SNP determining sensitivity to cold and nucleotides adjacent thereto, and / or a primer set that specifically recognizes and amplifies the SNP determining sensitivity to cold and / or an SNP in linkage disequilibrium with the SNP determining sensitivity to cold and nucleotides adjacent thereto. (17) The device according to (16), wherein the SNP for determining a type of cold sensitivity is any one of a to u in Table 2 of (6). (18) The device according to (16) or (17), further comprising the probe and / or primer set according to (14) or (15). (19) The device according to any one of (14) to (18), which is a device for measurement by a hybridization technique. (20) The device according to (19), wherein the hybridization technology is a nucleic acid array technology.

[0011] (21) A biomarker consisting of SNPs that determine sensitivity to cold to determine genetic predisposition to cold. (22) The biomarker according to (21), wherein the SNP for determining poor circulation is an SNP selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278, and rs4818919, or an SNP in linkage disequilibrium with any one of the SNPs selected from the group. (23) A biomarker consisting of SNPs that determine the type of sensitivity to cold to determine the type of sensitivity to cold. (24) The biomarker according to (23), wherein the cold sensitivity type is a lower body type, and the SNP for determining the cold sensitivity type is an SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006, and rs13376933, or an SNP in linkage disequilibrium with any one of the SNPs selected from the group. (25) The biomarker according to (23), wherein the cold sensitivity type is a peripheral limb type, and the SNP for determining the cold sensitivity type is an SNP selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186, and rs4818919, or an SNP in linkage disequilibrium with any one of the SNPs selected from the group. (26) The biomarker according to (23), wherein the type of poor circulation is systemic type, and the SNP for determining the type of poor circulation is an SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343, and rs6506395, or an SNP in linkage disequilibrium with any one of the SNPs selected from the group. Effect of the Invention

[0012] According to the method for determining poor circulation of the present invention, it is possible to determine in vitro whether or not a subject has a genetic predisposition to poor circulation.

[0013] According to the method for determining the site at which poor circulation develops in accordance with the present invention, it is possible to determine in vitro the genetic predisposition of a subject to develop poor circulation in which site of the body.

[0014] According to the kit or device for determining poor circulation of the present invention, it is possible to simply and easily detect and determine in vitro whether or not a subject suffers from poor circulation.

[0015] According to the kit or device for determining the site where poor circulation develops in the present invention, the site where poor circulation develops in the body of a subject can be detected and determined simply and easily in vitro. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows a Manhattan plot (A) and a QQ (quantile-quantile) plot (B) of the GWAS results regarding the presence or absence of chilliness. [Diagram 2] FIG. 1 shows a Manhattan plot (A) and a quantile-quantile (QQ) plot (B) of the GWAS results regarding the location of cold (legs and lower back). [Diagram 3] FIG. 1 shows a Manhattan plot (A) and a quantile-quantile (QQ) plot (B) of the GWAS results regarding the location of cold (hands and feet). [Figure 4] FIG. 1 shows a Manhattan plot (A) and a quantile-quantile (QQ) plot (B) of the GWAS results regarding the site of cold (whole body). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. How to determine if you have poor circulation 1-1. Overview The first aspect of the present invention is a method for determining poor circulation. The method of the present invention determines the genetic predisposition to poor circulation in a subject in vitro based on a biomarker, a SNP for determining poor circulation. According to the method of the present invention, it is possible to easily determine in vitro whether a subject has a genetic predisposition to poor circulation from nucleic acid derived from the subject.

[0018] 1-2.Definition of Terms As used herein, the following terms are defined. As described above, in this specification, "chilliness" refers to a subjective sensation of coldness in the whole body or a part of the body despite the absence of any organic abnormality. Note that in this specification, "chilliness" may be expressed not only in the above sense, but also as a term that encompasses a constitution (poor circulation) and symptoms (poor circulation) related to cold.

[0019] As used herein, the term "poor circulation" refers to a constitution that causes sensitivity to cold. As used herein, "sensitivity to cold" refers to a condition in which a person feels a sense of physical burden due to awareness of cold, the awareness is persistent, and recovery of the skin surface temperature after exposure to cold stress is slow, which can interfere with daily life.

[0020] In this specification, the term "type of cold sensitivity" refers to a type (form) classified based on the body part or condition where cold sensitivity develops. Three types are known: distal limb type, lower body type, and whole body type. The "proximal limb type" is a type where cold sensitivity develops in the hands and feet. The "lower body type" is a type where cold sensitivity develops in the lower body such as the legs and waist, and is sometimes expressed as upper heat and lower cold type when accompanied by hot flashes or flushing of the upper body. The "whole body type" is a type where cold sensitivity develops throughout the body. In this specification, the main focus is on the three types of distal limb type, lower body type, and whole body type based on the body part where cold sensitivity develops, without any limitation.

[0021] As used herein, the term "genetic predisposition" refers to the possibility of contracting a particular disease due to genetic factors including mutations such as base substitutions.

[0022] "Genetic polymorphism" refers to the variation in the base sequence of an allele present at a homologous locus on the genome between individuals in a population of the same species. This particularly applies to mutations that occur at a frequency of 1% or more in the population. Genetic polymorphisms include SNPs (single-nucleotide polymorphisms), indels (insertion / deletion), STRPs (short tandem repeat polymorphisms), VNTRs (variable number of tandem repeats), and CNVs (copy number variations).

[0023] An "allele" is also called an allele and refers to an individual gene that constitutes a genetic polymorphism. If it is the same gene, it does not necessarily have to be present at a homologous locus, and may be present at another locus due to translocation or duplication. In this specification, an allele generally refers to one that is present congenitally on the genome, but may also include one that is acquired later through mutation. Furthermore, each allele is generally considered to be present in all cells that constitute an individual, but may be present only in some cells, tissues, or organs.

[0024] In this specification, the term "risk allele" refers to an allele that is strongly associated with sensitivity to cold or the site where sensitivity to cold occurs in the SNP for determining sensitivity to cold or the SNP for determining the type of sensitivity to cold described below. Therefore, an individual having at least one base of a risk allele in the genotype can be determined to be sensitive to cold or to develop sensitivity to cold in a site where sensitivity to cold is suspected.

[0025] A "single nucleotide polymorphism" (often abbreviated as "SNP" herein) refers to a gene polymorphism in which one nucleotide in a nucleotide sequence in an allele is replaced with a different nucleotide. In this specification, the SNP is an SNP registered in the SNP database (dbSNP; http: / / www.ncbi.nlm.nih.gov / SNP / ) of the National Center for Biotechnology Information (NCBI), which is a public database, and can be identified by its reference number, the rs number.

[0026] As used herein, the term "chilliness determining SNP" refers to a SNP that is strongly associated with chilliness and can be used to determine chilliness.

[0027] As used herein, the term "SNP determining type of sensitivity to cold" refers to a SNP that is associated with the site of occurrence of sensitivity to cold and can be used to determine the type of sensitivity to cold.

[0028] As used herein, the term "genotype" refers to a combination of alleles at a SNP site.

[0029] As used herein, the term "linkage disequilibrium" refers to a phenomenon in which alleles or genetic polymorphisms at multiple gene loci are inherited in a correlated manner rather than independently.

[0030] As used herein, the term "subject" refers to a human individual (living individual) that is used as a target in the method, kit, and device of the present invention. The subject may be of any age or sex. For example, East Asian or Southeast Asian women, including Japanese, aged 15 to 70 years or 20 to 60 years are suitable.

[0031] As used herein, the term "nucleic acid derived from a subject" refers to a nucleic acid that is derived from the subject and is contained in a biological sample from the subject.

[0032] As used herein, the term "biological sample" refers to a sample containing nucleic acid, preferably genomic DNA, derived from a subject, such as body fluids, cells, tissues (including nails, hair, epidermis, oral epithelium, etc.), and cell cultures.

[0033] As used herein, the term "body fluid" refers to a liquid biological sample that contains nucleic acid or cells that contain nucleic acid. Examples of body fluid include, but are not limited to, blood, cerebrospinal fluid (cerebrospinal fluid), lymphatic fluid, secretions, mucus, ascites, pleural fluid, sputum, nasal mucus, saliva, lacrimal gland secretions, semen, vaginal fluid, urine, pharyngeal swabs, nasal swabs, perineural fluid, sweat, and tissue or cell extracts.

[0034] As used herein, the term "natural nucleotide" refers to a nucleotide that is naturally occurring, i.e., exists in nature. Examples include ribonucleotides, which are known as building blocks of RNA, and deoxyribonucleotides, which are known as building blocks of DNA.

[0035] As used herein, the term "non-natural nucleotide" refers to a nucleotide that does not exist in nature and has properties and / or structures similar to those of the natural nucleotide. Specifically, the term refers to an artificially constructed or artificially chemically modified nucleotide, or a nucleotide containing a non-natural nucleoside or a non-natural base. The artificially constructed nucleotide includes, for example, peptide nucleic acid (PNA), peptide nucleic acid (PHONA) having a phosphate group, bridged nucleic acid (BNA / LNA: bridged nucleic acid / locked nucleic acid), morpholino nucleic acid, etc. The artificially chemically modified nucleotide includes, for example, chemically modified nucleic acid and nucleic acid analogues such as methylphosphonate type DNA / RNA, phosphorothioate type DNA / RNA, phosphoramidate type DNA / RNA, and 2'-O-methyl type DNA / RNA.

[0036] In this specification, the term "nucleic acid array", also referred to as "microarray" or "nucleic acid chip", refers to a high-density integrated array in which different nucleic acids are arranged and fixed on a substrate. By applying photolithography, inkjet printing, and microfabrication technology in semiconductor manufacturing, a large number of nucleic acids can be densely integrated on a chip, allowing for parallel analysis of a huge variety and amount of nucleic acids. A nucleic acid array in which DNA fragments are arranged on a substrate is called a DNA microarray or DNA chip.

[0037] 1-3. Method The method for assessing poor circulation of the present invention includes a step of detecting SNPs for poor circulation and a step of assessing poor circulation as essential steps, and a step of extracting nucleic acid as an optional step. Each step will be described below.

[0038] 1-3-1. Nucleic acid extraction process The "nucleic acid extraction step" is a step of extracting nucleic acid from a biological sample derived from a subject. This step is a selection step in the method of the present invention, and may be performed as necessary.

[0039] There is no restriction on the type of biological sample used in this step, but body fluids or oral epithelium are preferred because they can be collected at any time, are easy to obtain, and are less invasive to the subject. Among the body fluids, saliva, pharyngeal swabs, and nasal swabs are particularly suitable. If 1 mL or more of a biological sample is required, blood (whole blood) or lymph is preferred.

[0040] The biological sample may be collected by a known method, and is not limited thereto. For example, saliva may be collected directly. For pharyngeal swabs, nasal swabs, or oral epithelium, a predetermined site may be swabbed using a cotton swab or the like. In the case of blood, for example, blood collected directly by injection from a vein or the like, blood collected whole blood subjected to anticoagulation treatment by adding heparin or the like, blood separated as plasma or serum, blood once stored in a refrigerator or freezer, or any other blood may be used as a sample. It may be collected using a commercially available kit. The collected biological sample may be purified by a known method as necessary, or may be stored at a low temperature, for example, below freezing point such as -20°C, until this step is carried out.

[0041] In this step, nucleic acid is extracted from the biological sample. The nucleic acid extracted in this step is preferably DNA, particularly genomic DNA, but is not limited thereto. For example, if the SNP to be detected is present in mature mRNA, it may be RNA, particularly mRNA or total RNA. The method for extracting nucleic acid from a biological sample may be prepared according to a conventional method in the field according to the type of nucleic acid. For example, it can be extracted according to the nucleic acid extraction method described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. In addition, kits for extracting nucleic acid from biological samples are commercially available from life science manufacturers, and these may be used. When using a kit, the specific extraction method may be performed according to or in accordance with the attached protocol.

[0042] 1-3-2. SNP detection process for determining sensitivity to cold The "step of detecting SNPs that determine sensitivity to cold" is a step of detecting SNPs that determine sensitivity to cold in vitro from nucleic acid derived from a subject.

[0043] The SNPs for determining poor circulation to be detected in this step are not limited to, but may be any one or more SNPs selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278, and rs4818919, or any one of the SNPs and any of the SNPs 1 to 11 for determining poor circulation that are in linkage disequilibrium with the base of the risk allele shown in Table 1. The linkage disequilibrium relationship may be 0.8 or more, 0.85 or more, 0.9 or more, 0.95 or more, or 1.0 in terms of linkage disequilibrium coefficient.

[0044] "Detecting a SNP for determining sensitivity to cold" refers to identifying the base of an allele in a SNP for determining sensitivity to cold. The detection may be one allele of the opposing allele of the SNP for determining sensitivity to cold, or both alleles. In the step of determining sensitivity to cold described below, sensitivity to cold is determined based on whether or not at least one allele in the SNP for determining sensitivity to cold is a risk allele. Therefore, when the nucleic acid derived from the subject is a double-stranded nucleic acid such as genomic DNA, it is preferable to detect both opposing alleles.

[0045] The method of detecting the SNP for determining sensitivity to cold in vitro is not particularly limited. As described above, the SNP for determining sensitivity to cold is a known SNP, so existing methods such as a method for detecting known SNPs and / or a method for determining the type of SNP (SNP typing method; genotyping method) may be used. For example, but not limited to, a nucleic acid amplification method, a DNA microarray method, or an AOPL (Amplified Product Length Polymorphism) method may be used.

[0046] Examples of nucleic acid amplification methods include a method in which a specific region on a template nucleic acid is amplified by a PCR method or the like using SNP-specific or SNP allele-specific primers (and a probe, if necessary), and the target SNP is detected by detecting the allele in the amplified product through fluorescence or luminescence. Examples of SNP detection based on nucleic acid amplification include PCR-RFLP (restriction fragment length polymorphism), PCR-SSCP (single-strand conformation polymorphism), PCR-SSO (sequence specific oligonucleotide), direct sequencing, ASO (allele specific oligonucleotide) hybridization, ASP-PCR (allele specific primer-PCR), Snapshot, ARMS (amplification refracting mutation system), real-time (RT-) PCR, invader, MALDI-TOF / MS, RNase A cleavage, DOL (dye-labeled oligonucleotide ligation), TDI (template-directed dye-terminator incorporation), etc. These are well known in the art and can be performed by known procedures.

[0047] In addition, SNP detection kits and SNP typing kits using nucleic acid amplification methods are commercially available from life science manufacturers, and the target SNP can be detected using these. For example, TaqMan SNP Genotyping Assays (Thermo Fisher Scientific) using real-time PCR or real-time RT-PCR is exemplified. In TaqMan SNP Genotyping Assays, a set of primer pairs capable of amplifying a region containing a target SNP (here, a SNP for determining poor circulation) on a template nucleic acid such as a genome and multiple TaqMan (registered trademark) probes corresponding to the target SNP on the template nucleic acid are prepared. Each TaqMan (registered trademark) probe has a sequence complementary to each allele. These primer pairs and probes are mixed with a template nucleic acid such as genomic DNA to perform real-time PCR or real-time RT-PCR, and each allele can be detected by detecting fluorescence from each probe.

[0048] The reaction conditions in the nucleic acid amplification method may be determined appropriately based on the known general conditions in each nucleic acid amplification method, taking into consideration the base length of the nucleic acid fragment to be amplified, the amount of template nucleic acid, the base length and Tm value of the primer used, the optimal reaction temperature and optimal pH of the nucleic acid polymerase used, etc. As an example, in the case of the PCR method, the denaturation reaction is usually performed at 94 to 95 ° C for 5 seconds to 5 minutes, the annealing reaction is performed at 50 to 70 ° C for 10 seconds to 1 minute, and the extension reaction is performed at 68 to 72 ° C for 30 seconds to 3 minutes, and this is considered as one cycle, which is repeated for about 15 to 40 cycles, and finally, an extension reaction is performed at 68 to 72 ° C for 30 seconds to 10 minutes. When using a commercially available kit, in principle, the protocol attached to the kit may be followed.

[0049] 1-3-3. Cold sensitivity assessment process The "step of assessing sensitivity to cold" is a step of assessing whether or not the subject has a genetic predisposition to sensitivity to cold based on the detected SNPs that assess sensitivity to cold.

[0050] In this step, based on the bases of the cold sensitivity determining SNP detected in the cold sensitivity determining SNP determination step, when at least one of the bases in the allele is a risk allele or an allele of an SNP that is in linkage disequilibrium with the risk allele, it is determined that the subject has a genetic predisposition to cold sensitivity.

[0051] Specifically, for example, when the base in rs750707 is a risk allele G, when the base in rs1869201 is a risk allele A, when the base in rs62295776 is a risk allele T, when the base in rs10016576 is a risk allele G, when the base in rs74513903 is a risk allele C, when the base in rs2656514 is a risk allele A, when the base in rs12761019 is a risk allele G, when the base in rs17098278 is a risk allele G, When the base is a squalene T, when the base in rs10220484 is a risk allele C, when the base in rs2876278 is a risk allele G, and when the base in rs4818919 is a risk allele G, and when it is an allele of an SNP that is in linkage disequilibrium with the risk allele of each of the above SNPs, subjects who have these alleles are judged to have a relatively high possibility of having poor circulation compared to individuals who do not have the risk allele, and to have a genetic predisposition to poor circulation, that is, to be more likely to be aware of poor circulation and to be more likely to develop poor circulation.

[0052] 1-4. Biomarkers for determining sensitivity to cold In the present invention, the SNPs for determining sensitivity to cold, which are the subject of detection from nucleic acid derived from a subject, and the SNPs in linkage disequilibrium with the SNPs can be biomarkers for determining genetic predisposition to sensitivity to cold. For example, the SNPs for determining sensitivity to cold are selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278, and rs4818919, which are SNPs for determining sensitivity to cold, or the SNPs in linkage disequilibrium with any SNP selected from the group.

[0053] These biomarkers make it possible to determine whether or not a subject has a genetic predisposition to poor circulation, based on whether or not the allele contained in the nucleic acid derived from the subject is the base of the risk allele described above.

[0054] Effects According to the method for determining poor circulation of the present invention, the presence or absence of a previously unknown genetic predisposition to poor circulation can be determined in vitro, and the method can be applied to the diagnosis of a constitutional predisposition to poor circulation.

[0055] 2. How to determine your cold sensitivity type 2-1. Overview The second aspect of the present invention is a method for determining a type of sensitivity to cold. The method of the present invention determines the genetic predisposition of a subject to a type of sensitivity to cold in vitro based on a biomarker, a SNP determining type of sensitivity to cold. According to the method of the present invention, it is possible to easily determine in vitro whether a subject has a genetic predisposition to a type of sensitivity to cold from nucleic acid derived from the subject.

[0056] 2-2. Method The method for determining a type of sensitivity to cold of the present invention includes a step of detecting SNPs for determining a type of sensitivity to cold and a step of determining a type of sensitivity to cold as essential steps. In addition, prior to the above steps, the method for determining a type of sensitivity to cold of the present invention may include a step of extracting nucleic acid, a step of detecting SNPs for determining sensitivity to cold, and a step of determining sensitivity to cold as optional steps. In other words, the method for determining a type of sensitivity to cold of the present invention can be carried out independently on a subject, and after carrying out the method for determining sensitivity to cold of the first embodiment, the method can also be carried out on a subject who has been determined to have a genetic predisposition to sensitivity to cold in order to determine the type of sensitivity to cold. The nucleic acid extraction step can be selected when it is necessary to extract nucleic acid from a biological sample derived from a subject, regardless of whether or not the method for determining sensitivity to cold of the first embodiment has been carried out.

[0057] Each step will be described below, but since the nucleic acid extraction step, the SNP detection step for determining poor circulation, and the step for determining poor circulation are similar to the steps described in the first embodiment, their explanations will be omitted here.

[0058] 2-2-1. SNP detection process for determining sensitivity to cold The "chilliness type determining SNP detection process" is a process of detecting chilliness type determining SNP in vitro from nucleic acid derived from a subject. This process is basically similar to the "chilliness type determining SNP detection process" of the first embodiment, except that the detection target is a chilliness type determining SNP. Therefore, the description of the common procedures is omitted, and the following description focuses on the characteristics of this process.

[0059] The SNPs for determining the type of sensitivity to cold to be detected in this step differ depending on the site where sensitivity to cold occurs. The types of sensitivity to cold to be detected in this specification are, but are not limited to, the three types of sensitivity to cold to the extremities, the lower body type, and the whole body type, as described above.

[0060] The SNP for determining the lower body type of cold sensitivity is, but is not limited to, an SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006, and rs13376933, or an SNP that is in linkage disequilibrium with the base of the risk allele in any SNP selected from the above group.

[0061] The SNP for determining the type of cold extremities is an SNP selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186, and rs4818919, or an SNP that is in linkage disequilibrium with the risk allele base in any SNP selected from the above group.

[0062] The SNP for determining the generalized type of cold sensitivity is an SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343, and rs6506395, or an SNP that is in linkage disequilibrium with the risk allele base in any SNP selected from the above group.

[0063] The linkage disequilibrium relationship between each of the SNPs for determining the cold sensitivity type may be 0.8 or more, 0.85 or more, 0.9 or more, 0.95 or more, or 1.0 in terms of linkage disequilibrium coefficient.

[0064] "Detecting a SNP for determining a type of sensitivity to cold" refers to identifying the base of an allele in a SNP for determining a type of sensitivity to cold. The detection may be one allele of the opposing allele of the SNP for determining a type of sensitivity to cold, or both alleles. In the step of determining a type of sensitivity to cold described below, sensitivity to cold is determined based on whether or not at least one allele in the SNP for determining a type of sensitivity to cold is a risk allele. Therefore, when the nucleic acid derived from the subject is a double-stranded nucleic acid such as genomic DNA, it is preferable to detect both opposing alleles.

[0065] 2-2-2. Process for determining type of sensitivity to cold The "step of determining the type of sensitivity to cold" is a step of determining that sensitivity to cold in the subject may occur in a site of that sensitivity to cold type when the allele of the detected SNP for determining the type of sensitivity to cold is a risk allele.

[0066] Except for the fact that the detection target in this step is a SNP for determining sensitivity to cold type, the basic operation procedure is similar to that of the "step for determining sensitivity to cold" in the first embodiment. Based on the bases of the SNP for determining sensitivity to cold type detected in the step for determining SNP for determining sensitivity to cold type, when at least one of the bases in the allele is a risk allele or is an allele of an SNP in a linkage disequilibrium relationship with the risk allele, the subject is determined to have a genetic predisposition to sensitivity to cold type.

[0067] Specifically, for example, in the case of SNPs for determining the type of cold sensitivity to the lower body, when the base in rs750707 is the risk allele G, when the base in rs76388277 is the risk allele C, when the base in rs6819504 is the risk allele C, when the base in rs9994962 is the risk allele C, when the base in rs72719006 is the risk allele C, and when the base in rs13376933 is the risk allele C, and when the allele is an SNP that is in linkage disequilibrium with the risk allele of each of the above SNPs, subjects having these alleles are relatively more likely to have cold sensitivity to the lower body compared to individuals not having the risk allele, and are judged to have a genetic predisposition to cold sensitivity to the lower body, that is, to be prone to developing cold sensitivity to the legs and waist.

[0068] In addition, in the case of SNPs for determining the cold extremity type, when the base in rs556161 is the risk allele G, when the base in rs10911744 is the risk allele C, when the base in rs1452609 is the risk allele A, when the base in rs61998228 is the risk allele T, when the base in rs849057 is the risk allele T, when the base in rs6476958 is the risk allele T, when the base in rs6518186 is the risk allele C, and when the base in rs4818919 is the risk allele G, and when the allele is an SNP that is in a linkage disequilibrium relationship with the risk allele of each of the above SNPs, subjects having these alleles are relatively more likely to have cold extremity type compared to individuals not having the risk allele, and are judged to have a genetic predisposition to cold extremity type, that is, to be prone to developing cold hands and feet.

[0069] Furthermore, in the case of SNPs for determining the type of cold sensitivity to the whole body, when the base in rs12615626 is the risk allele A, when the base in rs1513696 is the risk allele C, when the base in rs79196079 is the risk allele G, when the base in rs9777637 is the risk allele T, when the base in rs78458106 is the risk allele T, when the base in rs150099343 is the risk allele A, and when the base in rs6506395 is the risk allele G, and when they are alleles of SNPs that are in a linkage disequilibrium relationship with the risk alleles of each of the above SNPs, subjects having these alleles are relatively more likely to have cold sensitivity to the whole body compared to individuals not having the risk allele, and are judged to have a genetic predisposition to cold sensitivity to the whole body, that is, to be prone to developing cold sensitivity to the whole body.

[0070] 2-3. Biomarkers for determining the type of cold sensitivity In the present invention, the SNP determining the type of sensitivity to cold, which is the subject of detection from nucleic acid derived from a subject, and SNPs in a linkage disequilibrium relationship with the SNP, can be biomarkers for determining the presence or absence of a genetic predisposition to sensitivity to cold.

[0071] For example, an SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006, and rs13376933, or an SNP that is in linkage disequilibrium with any SNP selected from the above group, can be a biomarker for determining the presence or absence of a genetic predisposition to lower body cold sensitivity.

[0072] In addition, an SNP selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186, and rs4818919, or an SNP that is in linkage disequilibrium with any SNP selected from the above group, can be a biomarker for determining the presence or absence of a genetic predisposition to cold extremity type.

[0073] Furthermore, an SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343, and rs6506395, or an SNP that is in linkage disequilibrium with any SNP selected from the above group, can be a biomarker for determining the presence or absence of a genetic predisposition to systemic cold sensitivity.

[0074] 2-4.Effects According to the method for determining the type of sensitivity to cold of the present invention, the presence or absence of a genetic predisposition to sensitivity to cold can be determined in vitro, thereby making it possible to predict and determine the site where sensitivity to cold will occur.

[0075] Furthermore, by combining this with the first aspect of the method for assessing poor circulation, it becomes possible to stratify subjects who have been assessed as having poor circulation, enabling the prescription of appropriate medicines or herbal medicines according to each type of poor circulation.

[0076] 3. Cold sensitivity detection kit and device 3-1. Overview The third aspect of the present invention is a kit and device for detecting poor circulation. The kit and device for detecting poor circulation of the present invention are configured to specifically detect SNPs for determining poor circulation and / or SNPs in a linkage disequilibrium relationship therewith, and to identify the bases of the alleles in the SNPs. The kit and device for detecting poor circulation of the present invention can easily and simply detect SNPs for determining poor circulation of a subject, and determine the genetic predisposition to poor circulation.

[0077] 3-2.Configuration 3-2-1. Components The kit and device for detecting poor circulation in the present specification include, as essential components, a probe and / or a primer for detecting at least one SNP for determining poor circulation, which is a biomarker for determining poor circulation, and / or a SNP in a linkage disequilibrium relationship with the SNP. Each component will be specifically described below.

[0078] (1) Probe: SNP detection probe for determining sensitivity to cold The probe contained in the cold sensitivity detection kit and device of the present invention (often referred to in this specification as the "cold sensitivity determining SNP detection probe") is configured to be capable of specifically binding to the cold sensitivity determining SNP and / or an SNP that is in linkage disequilibrium with that SNP, as well as nucleotides in the vicinity thereof.

[0079] For example, the probe is composed of a polynucleotide containing a base sequence capable of hybridizing to one or more SNP sites selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278, and rs4818919, which are SNPs for determining sensitivity to cold, or SNP sites in a linkage disequilibrium relationship with the SNPs, and the base sequence of the nucleotides in the vicinity thereof. The term "nucleotides in the vicinity" refers to nucleotides located consecutively upstream and / or downstream of the SNP site to be detected on the template nucleic acid. In other words, the SNP detection probe for determining sensitivity to cold is configured to contain an antisense strand of the base sequence surrounding the SNP site for determining sensitivity to cold, or the SNP site in a linkage disequilibrium relationship with the SNP.

[0080] This polynucleotide constituting the cold sensitivity determining SNP detection probe can contain a deletion, substitution, or addition of one or several bases in the base sequence of the antisense strand so that it can hybridize to any allele of the cold sensitivity determining SNP site or an SNP that is in linkage disequilibrium with that SNP.

[0081] The SNP detection probe for determining poor circulation is composed of natural nucleotides and / or non-natural nucleotides. It is usually composed of natural nucleotides of DNA or RNA. If necessary, it may be a probe composed of a hybrid of natural nucleotides of DNA and RNA, or may be a probe partially containing non-natural nucleotides such as LNA or PNA.

[0082] The base length and base sequence of the SNP detection probe for determining poor circulation are not particularly limited as long as the probe is designed to have a Tm value in the range of 55°C to 80°C, preferably 60°C to 75°C, so that it can specifically hybridize to each SNP and its neighboring region. In general, the base length of the probe is preferably a base length and base sequence that can hybridize to consecutive bases on the template nucleic acid chain. For example, the length of the primer may be 15 to 100 consecutive bases, 15 to 80 consecutive bases, 15 to 60 consecutive bases, 16 to 50 consecutive bases, 17 to 40 consecutive bases, 18 to 30 consecutive bases, or 20 to 25 consecutive bases, which are commonly used in the art as primer lengths.

[0083] The SNP detection probe for determining poor circulation of the present invention may have some or all of the phosphate groups, sugars and / or bases of the nucleotides constituting the probe labeled with a labeling substance. The labeling position of the labeling substance in the probe may be appropriately determined according to the properties of the labeling substance and the purpose of use, and is not limited, but is preferably the 5' end, the 3' end, and / or the SNP site to be detected. Any substance known in the art can be used as the labeling substance. Examples of the labeling substance include radioisotopes, DIG, fluorescent substances, quenchers, chemiluminescent substances, biotin, magnetic beads, etc. Labeling of each labeling substance to a nucleotide can be performed by a known method.

[0084] The term "radioisotope" refers to an element that emits radiation among isotopes with different mass numbers. For example, 32 P, 33 P, or 35 Examples include S.

[0085] The "fluorescent substance" refers to a substance that has the property of becoming excited by absorbing excitation light of a specific wavelength and emitting fluorescence when returning to the original ground state. Examples of the fluorescent substance include FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar (registered trademark) 670, Quasar (registered trademark) 705, CAL Fluor (registered trademark) Red 610, fluorescamine or a derivative thereof, or rhodamine or a derivative thereof. However, the fluorescent substance is not limited to these.

[0086] The "quencher" refers to a substance that absorbs the excitation energy of the fluorescent substance and has the property of suppressing fluorescence. Examples include AMRA, DABCYL, BHQ-1, BHQ-2, and BHQ-3. In general, a quencher is used in combination with a fluorescent substance. Since the quencher has different suppression wavelength ranges for fluorescence depending on the type, a combination of a fluorescent substance and a quencher is made by combining the fluorescent substance with a quencher that can suppress the fluorescence of the fluorescent substance used for labeling. For example, when the fluorescent substance is FAM, HEX, TET, etc., it is sufficient to combine BHQ1, which has a suppression wavelength range of 480 nm to 580 nm, and when the fluorescent substance is Cy3, Cy5, ROX, TAMRA, Texas, etc., it is sufficient to combine it with BHQ2, which has a suppression wavelength range of 550 nm to 650 nm. The arrangement of the fluorescent substance and the quencher in the probe is not particularly limited as long as both substances are arranged so that the quencher can suppress the fluorescence of the fluorescent substance. For example, one end of the probe (the 5' end or the 3' end) may be labeled with a fluorescent substance, and the other end (the 3' end or the 5' end, respectively) may be labeled with a quencher.

[0087] The term "chemiluminescent substance" refers to a substance that has the property of emitting the difference in energy as light when it returns to the ground state after being excited by a chemical reaction. Examples of such substances include acridinium esters.

[0088] The kit or device for detecting poor circulation of the present invention may contain two or more types of detection probes for SNPs for determining poor circulation, such as probes that specifically hybridize to different alleles of the SNPs for determining poor circulation to be detected.

[0089] (2) Primer: SNP detection primer for determining sensitivity to cold The primers contained in the cold sensitivity detection kit and device of the present invention (often referred to in this specification as "chill sensitivity determining SNP detection primers") are configured to specifically recognize and amplify cold sensitivity determining SNPs and / or SNPs that are in linkage disequilibrium with those SNPs, as well as nucleotides in the vicinity thereof.

[0090] The SNP detection primer for determining poor circulation of the present invention is, in principle, configured as a primer set consisting of a forward (Fw) primer and a reverse (Rv) primer, but the kit and device for detecting poor circulation of the present invention may be configured to include only one of the primers. This primer is configured as a polynucleotide containing a base sequence that hybridizes to a base sequence such as a nucleotide near a SNP for determining poor circulation or a SNP in linkage disequilibrium with the SNP in a template nucleic acid such as the genomic DNA of a subject, and amplifies a region surrounding the SNP for determining poor circulation or a SNP in linkage disequilibrium with the SNP.

[0091] For example, the primer is composed of a Fw primer and an Rv primer, which are polynucleotides that hybridize to the base sequence of nucleotides adjacent to any one or more SNP sites selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278, and rs4818919, which are SNPs for determining sensitivity to cold, or SNP sites that are in a linkage disequilibrium relationship with the SNPs, and that can amplify the nucleic acid region surrounding the SNP site. The "neighboring nucleotides" refer to nucleotides that are consecutively located upstream and / or downstream of the SNP site to be detected on the template nucleic acid.

[0092] Each of the primers is composed of natural nucleotides and / or non-natural nucleotides. Usually, it is composed of natural nucleotides of DNA or RNA. Among them, DNA is particularly preferable because it is highly stable, easy to synthesize, and inexpensive. If necessary, natural nucleotides of DNA and RNA can be combined, or non-natural nucleotides such as chemically modified nucleic acids and pseudo nucleic acids can be partially combined. Examples of chemically modified nucleic acids and pseudo nucleic acids include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA.

[0093] In the SNP detection primer for determining poor circulation, the Fw primer and the Rv primer are designed so that their Tm values ​​are in the range of 55° C. to 80° C., preferably 60° C. to 75° C., though not limited thereto. The base length and base sequence of each primer are not particularly limited, but for example, the base length may be a base sequence complementary to 15 or more, 18 or more, 20 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 35 or more, or 40 or more consecutive bases on the template nucleic acid strand.

[0094] The base length of the amplified fragment by the primer set of SNP detection primers for determining cold sensitivity is not limited, but may be within the range of 100 to 1500 bases, 150 to 1200 bases, 200 to 1000 bases, 250 to 800 bases, 300 to 600 bases, or 400 to 500 bases.

[0095] The SNP detection primer for determining poor circulation of the present invention can include an additional sequence different from the base sequence of the template nucleic acid at its 5'-end. The additional sequence can be any base sequence. For example, a base sequence including a restriction enzyme site or a new primer binding site can be included.

[0096] Furthermore, the SNP detection primer for determining poor circulation of the present invention may be labeled with a labeling substance at the phosphate group, sugar and / or base. The labeling position of the labeling substance in the primer may be appropriately determined according to the properties of the labeling substance and the purpose of use, and is not limited, but is preferably the 5' end, which does not usually contribute to the extension reaction. Any substance known in the art can be used as the nucleic acid labeling substance. For example, the above-mentioned radioisotopes, DIG, fluorescent dyes, chemiluminescent substances, etc. can be mentioned.

[0097] The primer set of the SNP detection primer for determining poor circulation may be one set, but may also be a set of multiple sets of primers such as nested primers. Furthermore, it may also include a control primer set capable of amplifying a control nucleic acid other than the SNP to be detected, which will be described later.

[0098] (3) Internal control primer (IC primer) The kit or device for detecting poor circulation of the present invention may contain an internal control (IC) primer as an optional component.

[0099] The IC primer is configured to be capable of amplifying a non-target template nucleic acid molecule present in a sample as an internal control. The IC primer is added as necessary to monitor the progress or state of the amplification reaction when amplifying the SNP for determining poor circulation and its surrounding base sequence using the kit or device for detecting poor circulation of the present invention.

[0100] The template nucleic acid molecule serving as an internal control may be a template nucleic acid molecule obtained by adding an appropriate nucleic acid molecule from the outside to the sample, or an endogenous nucleic acid molecule (such as a gene transcription product) present in the sample may be used as the template nucleic acid molecule. It is preferable to select a gene that is constitutively expressed, such as a housekeeping gene, as the endogenous nucleic acid molecule. In the case of the cold sensitivity detection kit or device of the present invention, suitable internal controls include, but are not limited to, transcription products (mRNA) of 18S rRNA, β-actin, GAPDH, EF1α, tubulin, and the like contained in human blood.

[0101] The base length of the amplified fragment of the IC primer, the basic structural characteristics, components, base length, and base sequence of the Fw primer and Rv primer that constitute the IC primer are not limited, but should basically conform to the structure of the SNP detection primer for determining cold sensitivity.

[0102] (4) Internal control probe (IC probe) The kit or device for detecting poor circulation of the present invention may also contain an internal control probe as an optional component.

[0103] The IC probe is configured to be capable of hybridizing to an amplified fragment of an internal control amplified by the IC primers (often referred to as an "IC amplified fragment" in this specification) and to be capable of detecting the IC amplified fragment. Therefore, the probe is used in combination with the IC primer set in the cold sensitivity detection kit or device of the present invention.

[0104] The structural characteristics, constituent components, base length, and base sequence of the IC probe are not limited, but may basically be similar to those of the SNP detection probe for determining poor circulation.

[0105] 3-2-2. Configuration of the cold sensitivity detection kit and device The configuration of the cold sensitivity detection kit of the present invention may be a configuration conforming to that of a general detection kit. For example, in addition to the aforementioned essential components, the SNP detection probe for determining cold sensitivity and / or primer, the kit may be configured to include a protocol describing appropriate reaction conditions and a method for detecting SNPs. In addition, the SNP detection primer for determining cold sensitivity may further include, for example, reagents required for a nucleic acid amplification reaction (dNTPs, DNA polymerase, etc.) and reagents for detecting the amplified nucleic acid fragments (fluorescence / luminescence reagents, etc.).

[0106] On the other hand, the device for detecting poor circulation of the present invention may have a configuration of a nucleic acid array (including a DNA chip) to which a nucleic acid array technique can be applied, but is not limited thereto. For example, a nucleic acid array in which a plurality of SNP detection probes for determining poor circulation are immobilized on a substrate (chip) can be mentioned.

[0107] 4. Chilliness type determination kit and device 4-1. Overview The fourth aspect of the present invention is a kit and device for determining a type of sensitivity to cold. The kit and device for determining a type of sensitivity to cold of the present invention are configured to specifically detect a SNP for determining a type of sensitivity to cold and / or an SNP in a linkage disequilibrium relationship therewith, and to identify the base of an allele in the SNP. The kit and device for determining a type of sensitivity to cold of the present invention can easily and simply determine the type of sensitivity to cold of a subject. In addition, by combining with the kit and / or device for detecting sensitivity to cold of the third aspect, more specific stratification of subjects determined to have sensitivity to cold can be easily performed.

[0108] 4-2.Configuration 4-2-1. Components The kit and device for determining the type of sensitivity to cold in the present specification include, as essential components, a probe and / or a primer for detecting at least one SNP for determining the type of sensitivity to cold, which is a biomarker for determining the type of sensitivity to cold, and / or a SNP in a linkage disequilibrium relationship with the SNP. Each component will be specifically described below.

[0109] The basic configuration of the kit and device for determining the type of sensitivity to cold is similar to that of the "kit and device for determining sensitivity to cold" in the third embodiment, except that the detection target is a SNP for determining the type of sensitivity to cold and / or an SNP in a linkage disequilibrium relationship with the SNP. Therefore, the description of the common configuration is omitted, and the following description focuses on the features characteristic of the kit and device in this embodiment.

[0110] (1) Probe: SNP detection probe for determining cold sensitivity type The probe included in the cold sensitivity type determination kit and device of the present invention (often referred to in this specification as the "cold sensitivity type determination SNP detection probe") is configured to be capable of specifically binding to the cold sensitivity type determination SNP and / or an SNP that is in linkage disequilibrium with that SNP, as well as nucleotides in the vicinity thereof.

[0111] The SNP detection probe for determining sensitivity to cold type is configured to be capable of detecting each of the SNP detection biomarkers for determining sensitivity to cold type described in the "method for determining sensitivity to cold type" of the second aspect.

[0112] For example, when the type of cold sensitivity to be detected is lower body type, the polynucleotide is composed of a SNP site selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006, and rs13376933, which are SNPs for determining lower body type cold sensitivity, or a SNP site that is in linkage disequilibrium with any SNP selected from the above group, as well as a polynucleotide containing a base sequence that can hybridize to the base sequence of the nucleotides in the vicinity thereof.

[0113] In addition, when the type of cold sensitivity to the distal limbs is detected, the polynucleotide is composed of a SNP site selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186, and rs4818919, which are SNPs for determining cold sensitivity to the distal limbs type, or a SNP site that is in linkage disequilibrium with any SNP selected from the above group, as well as a polynucleotide containing a base sequence that can hybridize to the base sequence of the nucleotides in the vicinity thereof.

[0114] Furthermore, when the type of cold sensitivity to be detected is systemic, the polynucleotide is composed of a SNP site selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343, and rs6506395, which are SNPs for determining systemic cold sensitivity to type, or a SNP site that is in linkage disequilibrium with any SNP selected from the above group, as well as a polynucleotide containing a base sequence that can hybridize to the base sequence of the nucleotides in the vicinity thereof.

[0115] The kit or device for determining a type of sensitivity to cold of the present invention can include two or more types of detection probes for determining a type of sensitivity to cold SNP. For example, the probes can be specifically hybridized to different alleles of the SNP for determining a type of sensitivity to cold to be detected, or probes for each SNP for detecting a type of sensitivity to cold.

[0116] (2) Primer: SNP detection primer for determining cold sensitivity type The primers contained in the cold sensitivity type determination kit and device of the present invention (often referred to in this specification as "cold sensitivity type determination SNP detection primers") are configured to specifically recognize and amplify the cold sensitivity type determination SNP and / or SNPs that are in linkage disequilibrium with the SNP, as well as nucleotides in the vicinity thereof.

[0117] The SNP detection primer for determining sensitivity to cold type is configured to be capable of detecting each of the SNP detection biomarkers for determining sensitivity to cold type described in the "method for determining sensitivity to cold type" of the second aspect.

[0118] For example, when the type of cold sensitivity to be detected is lower body type, the primer is composed of an Fw primer and an Rv primer, which are polynucleotides containing a base sequence that hybridizes to a SNP site selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006, and rs13376933, which are SNPs for determining lower body type cold sensitivity, or to a nucleotide sequence of a nucleotide in the vicinity of a SNP site that is in a linkage disequilibrium relationship with the SNP, and amplifies the nucleic acid region including and surrounding the SNP site.

[0119] In addition, when the type of cold sensitivity to the distal limbs is detected, the primer is composed of an Fw primer and an Rv primer, which are polynucleotides containing a base sequence that hybridizes to a SNP site selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186, and rs4818919, which are SNPs for determining cold sensitivity to the distal limbs type, or to a nucleotide sequence of a nucleotide in the vicinity of a SNP site that is in a linkage disequilibrium relationship with the SNP, and amplifies the nucleic acid region including and surrounding the SNP site.

[0120] Furthermore, when the type of cold sensitivity to be detected is systemic, the primer is composed of an Fw primer and an Rv primer, which are polynucleotides containing a base sequence that hybridizes to a SNP site selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343, and rs6506395, which are SNPs for determining systemic cold sensitivity type, or to a nucleotide sequence of a nucleotide in the vicinity of a SNP site that is in a linkage disequilibrium relationship with the SNP, and amplifies the nucleic acid region including and surrounding the SNP site.

[0121] The primer set for detecting SNPs for determining the type of sensitivity to cold may be one set, but may contain multiple sets of primers to determine multiple different types of sensitivity to cold. Furthermore, the primer set may contain a control primer set capable of amplifying a control nucleic acid other than the SNP to be detected, which will be described later. EXAMPLES

[0122] <Searching and selection of SNPs for determining sensitivity to cold and SNPs for determining sensitivity to cold type> (the purpose) SNPs that have a close genetic association with poor circulation or the site of onset of poor circulation are searched for, and SNPs for determining poor circulation and SNPs for determining the type of poor circulation are selected.

[0123] (method) The subjects were 1,200 adult female volunteers aged 20 to 60 years who had given their consent for this experiment and whose samples and information were obtained through DeNA Life Sciences Co., Ltd.'s (hereinafter referred to as DLS) genetic testing service "MYCODE."

[0124] Each volunteer was asked to complete a questionnaire to obtain demographic information such as age, height, and weight, as well as information on their current medical history, subjective symptoms of cold, and the burden of physical symptoms. Based on genetic diversity information, mainly SNPs, already obtained from MYCODE, a Genome Wide Association Study (GWAS) was conducted on traits related to cold.

[0125] First, sample quality control (QC) based on SNPs obtained by SNP array was performed on 1200 samples from whom questionnaire responses were obtained, and a final sample of 1113 people remained.

[0126] Next, using the samples from 1113 individuals after sample QC, SNP QC was performed on 684428 biallelic SNPs on autosomes that were included in the MYCODE array (Infinium OmniExpress-24+ BeadChip, Human OmniExpress-24+ BeadChip) and were determined to be analyzable by DLS before genotype imputation, ultimately obtaining 492418 SNPs. Genotype imputation was performed on these SNPs. In genotype imputation, SNPs in regions not included in the SNP array were also analyzed, so the genotypes of regions that were not statistically typed were estimated based on the reference whole genome information.

[0127] Of the total 43,835,851 SNPs after genotype imputation, 36,208,933 SNPs with low accuracy, whose Rsq value (estimated value of the correlation coefficient between the true genotype and the estimated genotype output by Minimac3), which is generally used as an index of imputation accuracy, was less than 0.7, were excluded. After that, in order to confirm the homogeneity of the genetic background of the samples in order to reduce the possibility of false positives due to population structuring, etc., PCA (plink-indep-pairwise 50 5 0.5) was performed again on the SNPs after genotype imputation. In addition, PCA was performed on the genome data after imputation, and samples that significantly deviated from the population were visually judged and removed in the plot of the first and second principal components obtained, and PCA was performed again, and samples that significantly deviated from the population were visually judged and removed in the plot. PCA was performed again on the remaining 1,111 samples, and it was confirmed that there were no samples that significantly deviated from the population visually.

[0128] The trait data obtained from the questionnaire was converted into two values: case group and control group.

[0129] GWAS analysis was performed on 7,234,812 SNPs that had passed SNP QC after genotype imputation. SNPs with Hardy-Weinberg equilibrium (HWE) < ​​1e-06 and SNPs with minor allele frequency (MAF) < 0.05 were removed from the GWAS analysis population for each trait, and 5,077,272 to 5,077,326 SNPs were finally used for GWAS. GWAS was performed using PLINK (version 1.9), assuming an additive model, and a logistic regression analysis was performed for the case group and the control group. Age, BMI, whether or not menopause occurred, and the first to fifth principal components from the principal component analysis of the SNP data were used as covariates. The genome-wide significance level was set to 5E-08, and the suggestive significance level was set to 1E-05. For SNPs that met the suggestive significance level, the clump option of PLINK was used to group SNPs that exist within a 10,000 kbp region of each other, taking into account linkage disequilibrium (linkage disequilibrium coefficient R2 = 0.2 or more), showing similar correlations, and define them as the same region. The SNP with the lowest P value among them was defined as the SNP that represents the correlation of that region (Lead SNP). To visualize the analysis results, the qqman package of R was used to create QQ plots (Quantile-quantile plots) and Manhattan plots. The location information of SNPs was based on the reference genome build GRCh37.

[0130] (result) Thirty-two regions were detected as associated with traits related to coldness that met the suggestive significance level. The Manhattan plot and QQ plot of the GWAS results for the presence or absence of coldness are shown in Figure 1, the Manhattan plot and QQ plot of the GWAS results for the location of coldness (legs and lower back) are shown in Figure 2, the Manhattan plot and QQ plot of the GWAS results for the location of coldness (hands and feet) are shown in Figure 3, and the Manhattan plot and QQ plot of the GWAS results for the location of coldness (whole body) are shown in Figure 4.

[0131] Among these 32 regions, the trait of "presence or absence of cold sensitivity" was found to have a suggestive association with SNPs in the 11 regions shown in Table 3. When the OR value is less than 1.0, the major allele (A2) is the risk allele, and when it is 1.0 or more, the minor allele (A1) is the risk allele. Therefore, the major alleles for rs1869201, rs62295776, rs74513903, rs2656514, rs12761019, and rs17098278, and the minor alleles for rs750707, rs10016576, rs10220484, rs2876278, and rs4818919 were risk alleles for cold sensitivity.

[0132] [Table 3]

[0133] A1: Minor allele A2: Major allele ·MAF: Minor allele frequency OR: Odds ratio of the A1 allele ·SE: Standard error of OR

[0134] In addition, for the three traits "Cold areas - legs and hips," "Cold areas - hands and feet," and "Cold areas - whole body," suggestive associations were observed with SNPs in 6, 8, and 7 regions, respectively, as shown in Table 4. When the OR value is less than 1.0, the major allele (A2) is the risk allele, and when it is 1.0 or more, the minor allele (A1) is the risk allele.

[0135] Therefore, for the lead SNP of "area of ​​coldness_legs and waist", the major allele at rs13376933 and the minor alleles at rs750707, rs76388277, rs6819504, rs9994962, and rs72719006 were risk alleles for lower body coldness type.

[0136] In addition, for the lead SNPs of "area of ​​coldness_hands and feet," major alleles in rs1452609 and rs849057, and minor alleles in rs556161, rs10911744, rs61998228, rs6476958, rs6518186, and rs4818919 were risk alleles for cold extremity type.

[0137] Furthermore, in the "area of ​​coldness - whole body" category, the minor alleles of all lead SNPs were risk alleles for the whole-body type of coldness.

[0138] [Table 4]

[0139] The explanation for each item in the upper column of the table is the same as in Table 3.

Claims

1. A method for determining a genetic predisposition to cold sensitivity in a subject in vitro, comprising: A step of detecting a gene polymorphism (a SNP that determines sensitivity to cold) associated with sensitivity to cold from nucleic acid derived from a subject; and a step of determining that the subject has a genetic predisposition to cold sensitivity when at least one of the bases in the allele of the detected cold sensitivity determining SNP is a risk allele; The method comprising:

2. The method according to claim 1, wherein the SNP for determining sensitivity to cold is any one of 1 to 11 in Table 1 below, and the risk allele is a base shown in Table 1, or a SNP that is in linkage disequilibrium with the risk allele of any one of the SNPs for determining sensitivity to cold, 1 to 11. 【Table 1】

3. The method according to claim 2 , wherein the linkage disequilibrium coefficient of the SNPs in the linkage disequilibrium relationship is 0.8 or more.

4. A method for determining a genetic predisposition to cold sensitivity in a subject in vitro, comprising: A step of detecting a SNP that determines the type of sensitivity to cold, which detects a single gene polymorphism (SNP that determines the type of sensitivity to cold) based on the site of occurrence of sensitivity to cold from nucleic acid derived from a subject; and a step of determining the type of sensitivity to cold in the subject when at least one of the bases in the allele of the detected sensitivity to cold type determining SNP is a risk allele, and determining that sensitivity to cold in the subject may occur in the area of ​​the sensitivity to cold type; The method comprising:

5. The method according to claim 4, wherein the cold sensitivity type is any one of lower body type, extremity peripheral type, and whole body type.

6. The method according to claim 5, wherein the SNP for determining the cold sensitivity type is any one of a to u in Table 2 below, and the risk allele is a base shown in Table 2 below, or the SNP for determining the cold sensitivity type is any one of a to u, and is in a linkage disequilibrium relationship with the risk allele shown in Table 2. 【Table 2】

7. The method according to claim 6 , wherein the linkage disequilibrium coefficient of the SNPs in the linkage disequilibrium relationship is 0.8 or more.

8. The method according to claim 4, wherein the subject is a subject who has been determined to have a genetic predisposition to cold sensitivity by the determination method according to any one of claims 1 to 3.

9. A device or kit for detecting a genetic predisposition to cold sensitivity, comprising: A probe capable of specifically binding to a cold sensitivity determining SNP associated with cold sensitivity and / or a SNP in a linkage disequilibrium relationship with the cold sensitivity determining SNP and a nucleotide adjacent thereto, and / or The present invention includes a primer set that specifically recognizes and amplifies the SNP for determining sensitivity to cold and / or an SNP that is in a linkage disequilibrium relationship with the SNP for determining sensitivity to cold, as well as nucleotides in the vicinity thereof. The device or kit.

10. The device or kit according to claim 9, wherein the SNP for determining cold sensitivity is any one of 1 to 11 in Table 1 of claim 2.

11. A device or kit for determining a cold sensitivity type, A probe capable of specifically binding to a SNP for determining a type of sensitivity to cold based on the site of occurrence of sensitivity to cold and / or a SNP in a linkage disequilibrium relationship with the SNP for determining a type of sensitivity to cold and a nucleotide adjacent thereto, and / or The present invention includes a primer set that specifically recognizes and amplifies the SNP for determining the cold sensitivity type and / or an SNP that is in a linkage disequilibrium relationship with the SNP for determining the cold sensitivity type, as well as nucleotides adjacent thereto. The device or kit.

12. The device or kit according to claim 11, wherein the SNP for determining the cold sensitivity type is any one of a to u in Table 2 of claim 6.

13. The device or kit according to claim 11 or 12, further comprising a probe and / or a primer set according to claim 9 or 10.

14. A biomarker consisting of SNPs that determine sensitivity to cold to determine genetic predisposition to cold.

15. The biomarker according to claim 14, wherein the SNP for determining poor circulation is a SNP selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278, and rs4818919, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group.

16. A biomarker consisting of SNPs that determine the type of sensitivity to cold to determine the type of sensitivity to cold.

17. The biomarker according to claim 16, wherein the cold sensitivity type is a lower body type, and the SNP for determining the cold sensitivity type is an SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006, and rs13376933, or an SNP in linkage disequilibrium with any one of the SNPs selected from the group.

18. The biomarker of claim 16, wherein the cold sensitivity type is a distal limb type, and the SNP for determining the cold sensitivity type is a SNP selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186, and rs4818919, or a SNP in linkage disequilibrium with any SNP selected from the group.

19. The biomarker of claim 16, wherein the type of cold sensitivity is systemic type, and the SNP for determining the type of cold sensitivity is an SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343, and rs6506395, or an SNP in linkage disequilibrium with any SNP selected from the group.

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