Cold constitution determination method and cold intolerance type determination method

Abstract

The invention aims at finding out the genetic correlation between the cold constitution and the cold intolerance and developing and providing a method for judging the disease site of the cold constitution and the cold intolerance according to the obtained genetic information. Questionnaire survey is carried out on coldness, comprehensive SNP analysis of a whole genome is carried out, and SNPs only related to cold constitution and various cold intolerance types are found out. Also provided is a method for determining the site of onset of cold constitution and cold intolerance, in which the SNP is used as a biomarker for cold constitution and the like.

Description

Technical Field

[0001] The present invention relates to a method for determining a cold constitution, a method for determining a site where chills occur, and a suit or device used in the method. Background Art

[0002] Chills refer to the phenomenon that the whole body or part of the body feels cold without any organic abnormality. In addition, a cold constitution refers to a constitution that causes chills, and chills sensitivity refers to a symptom that causes physical pain due to chills and causes interference with daily life.

[0003] Generally speaking, the main complaint rate of feeling cold is higher in women than in men. Therefore, feeling cold has become one of the clinical main complaints of female menopausal syndrome at present. However, studies have shown that the cases of feeling cold in young women have increased in recent years (non-patent literature 1). In addition, it is known that women with fear of cold will not only have the subjective symptoms of feeling cold, but also will have accompanying symptoms such as insomnia, fatigue, edema and pain (non-patent literature 2). Therefore, it is currently believed that feeling cold will not only reduce women's QOL through the feeling of cold, but will also become the inducement of other diseases, and mastering its actual situation and establishing therapy become important issues.

[0004] The physiological causes of chills are believed to be vascular motor nerve disorders caused by autonomic dysfunction, abnormal female hormone balance caused by estrogen decline, and decreased body temperature regulation caused by muscle loss. On the other hand, it is reported that 60% of mothers of women with cold constitutions have cold constitutions, which also indicates that chills are caused by genetic background (Non-patent Documents 3, 4). However, there are almost no reports on genetic studies related to chills, and the influence of genetic factors on chills is still unclear.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Kouhei Fujita et al., 2017, Sports Health Science Research 39: 19-27 (Kouhei Fujita et al., 2017, Sports Health Science Research 39: 19-27)

[0008] Non-patent literature 2: Yoshino T., et al., 2019, Complementary Therapies in Medicine, 45: 7-13

[0009] Non-patent document 3: Kondo Masahiko, Okamura Yasushi, 1987, Journal of the Japanese Society of Obstetrics and Gynecology, 39(11): 2000-2004. (Kondo Masahiko, Okamura Yasushi, 1987, Journal of the Japanese Society of Obstetrics and Gynecology, 39(11): 2000-2004.)

[0010] Non-patent document 4: Arakawa K., et al., 2015, Jpn. J. Biometeor., 52(4): 199-211 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The present invention identifies the genetic correlation between cold constitution and fear of cold, and develops a method for determining the onset site of cold constitution and fear of cold based on the obtained genetic information, and also develops a kit or equipment for the determination method.

[0013] Technical solutions to solve problems

[0014] In order to solve the above problems, the inventors conducted questionnaire surveys and SNP analysis on a number of adult female volunteers aged 20 to 60, and used a genome-wide association study (GWAS) to conduct a comprehensive analysis of the genetic association between chills and SNPs. As a result, in the comparison of the two groups of people related to the presence or absence of chills, multiple SNPs closely related to cold constitution were found. In addition, in the comparison of the "no chills" group and the "cold waist and legs", "cold hands and feet", and "cold whole body" groups, multiple SNPs that are different for each type of chills were also found. The present invention is a related invention of cold constitution and chills based on the genetic information of the subjects, which was developed based on the above analysis results, and provides the following content.

[0015] (1) A method for determining the genetic cause of a subject's cold constitution in vitro, comprising: a cold constitution determination SNP detection step, detecting a single nucleotide polymorphism (cold constitution determination SNP) associated with a cold constitution based on nucleic acid from the subject; and a cold constitution determination step, determining that the subject has the genetic cause of a cold constitution when at least one of the bases in the detected alleles of the cold constitution determination SNP is a risk allele.

[0016] (2) The method according to (1), wherein the cold constitution determination SNP is any one of 1 to 11 in the following Table 1, 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 1 to 11 cold constitution determination SNPs.

[0017] [Table 1]

[0018] serial number SNP ID Base 1 rs750707 G 2 rs1 869201 A 3 rs62295776 T 4 rs1 0016576 G 5 rs74513903 C 6 rs2656514 A 7 rs12761019 G 8 rs1 7098278 T 9 rs1 0220484 C 10 rs2876278 G 11 rs4818919 G .

[0019] (3) The method according to (2), wherein the linkage disequilibrium coefficient between the SNPs is greater than or equal to 0.8.

[0020] (4) A method for determining the genetic cause of a subject's chills type in vitro, comprising: a chills type determination SNP detection step, detecting a single nucleotide polymorphism (colds type determination SNP) based on a site where the chills occur based on nucleic acid from the subject; and a colds type determination step, determining that the subject's chills will occur at the site of the colds type when at least one of the bases in the detected alleles of the colds type determination SNP is a risk allele.

[0021] (5) The method according to (4), wherein the type of cold sensitivity is any one of a lower body type, an extremity type, or a whole body type.

[0022] (6) The method according to (5), wherein the SNP for determining the type of chills is any one of a to u in Table 2 below, and the risk allele is a base shown in Table 2 below, or any one of the SNPs for determining the type of chills among a to u is a SNP that is in linkage disequilibrium with the risk allele shown in Table 2.

[0023] [Table 2]

[0024] serial number Types of cold intolerance SNP ID Base a Lower body type rs750707 G b Lower body type rs76388277 C C Lower body type rs6819504 C d Lower body type rs9994962 C e Lower body type rs72719006 C f Lower body type rs1 3376933 C g Extremity type rs556161 G h Extremity type rs10911744 C i Extremity type rs1452609 A j End-of-limb type rs61998228 T k Extremity type rs849057 T l Extremity type rs6476958 T m Extremity type rs6518186 C n Extremity type rs4818919 G o Whole body type rs12615626 A p Whole body type rs1513696 C q Whole body type rs79196079 G r Whole body type rs9777637 T S Whole body type rs78458106 T t Whole body type rs150099343 A U Whole body type rs6506395 G .

[0025] (7) The method according to (6), wherein the linkage disequilibrium coefficient between the SNPs is greater than or equal to 0.8.

[0026] (8) The method according to any one of (4) to (7), wherein the subject is a subject determined to have a genetic cause of cold constitution by the determination method according to any one of (1) to (3).

[0027] (9) A kit for detecting the genetic cause of cold constitution, comprising: a probe that can specifically bind to a cold constitution determining SNP associated with cold constitution and / or a SNP that is in linkage disequilibrium with the cold constitution determining SNP and the nucleotides in the vicinity thereof; and / or a primer set that specifically recognizes and amplifies the cold constitution determining SNP and / or a SNP that is in linkage disequilibrium with the cold constitution determining SNP and the nucleotides in the vicinity thereof.

[0028] (10) The set according to (9), wherein the cold constitution determination SNP is any one of 1 to 11 in Table 1 of (2).

[0029] (11) A kit for determining the type of chills, comprising: a probe that can specifically bind to a SNP for determining the type of chills based on the site of occurrence of the chills and / or a SNP that is in linkage disequilibrium with the SNP for determining the type of chills and its nearby nucleotides; and / or a primer set that specifically recognizes and amplifies the SNP for determining the type of chills and / or a SNP that is in linkage disequilibrium with the SNP for determining the type of chills and its nearby nucleotides.

[0030] (12) The kit according to (11), wherein the chills type determination SNP is any one of a to u in Table 2 of (6).

[0031] (13) The kit according to (11) or (12), further comprising the probe and / or primer set according to (9) or (19).

[0032] (14) A device for detecting the genetic cause of cold constitution, comprising: a probe that can specifically bind to a cold constitution determination SNP associated with cold constitution and / or a SNP that is in linkage disequilibrium with the cold constitution determination SNP and the nucleotides in its vicinity; and / or a primer set that specifically recognizes and amplifies the cold constitution determination SNP and / or the SNP that is in linkage disequilibrium with the cold constitution determination SNP and the nucleotides in its vicinity.

[0033] (15) The device according to (14), wherein the cold constitution determination SNP is any one of 1 to 11 in Table 1 of (2).

[0034] (16) A device for determining the type of chills, comprising: a probe that can specifically bind to a SNP for determining the type of chills based on the site of occurrence of the chills and / or a SNP that is in linkage disequilibrium with the SNP for determining the type of chills and its nearby nucleotides; and / or a primer set that specifically recognizes and amplifies the SNP for determining the type of chills and / or a SNP that is in linkage disequilibrium with the SNP for determining the type of chills and its nearby nucleotides.

[0035] (17) The device according to (16), wherein the chills type determination SNP is any one of a to u in Table 2 of (6).

[0036] (18) The device according to (16) or (17), further comprising the probe and / or primer set according to (14) or (15).

[0037] (19) The device according to any one of (14) to (18), which is a device for performing measurement using a hybridization technique.

[0038] (20) The device according to (19), wherein the hybridization technology is nucleic acid array technology.

[0039] (21) A biomarker consisting of a cold constitution determining SNP for determining the genetic cause of a cold constitution.

[0040] (22) The biomarker according to (21), wherein the cold constitution determining SNP 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.

[0041] (23) A biomarker consisting of a chills type determining SNP for determining the chills type.

[0042] (24) A biomarker according to (23), wherein the type of chills is a lower body type, and the chills type determination SNP is a SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006 and rs13376933, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group.

[0043] (25) A biomarker according to (23), wherein the type of chills is an extremity type, and the chills type determination SNP 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 one of the SNPs selected from the group.

[0044] (26) A biomarker according to (23), wherein the type of chills is systemic, and the chills type determination SNP is a SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343 and rs6506395, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group.

[0045] Effects of the Invention

[0046] According to the method for determining a cold constitution of the present invention, it is possible to determine in vitro whether a subject has a genetic cause for a cold constitution.

[0047] According to the method for determining the site of onset of chills of the present invention, it is possible to determine in vitro where in the body of a subject the chills will occur and its genetic cause.

[0048] According to the cold constitution determination kit or device of the present invention, it is possible to simply and easily detect and determine in vitro whether a subject has a cold constitution.

[0049] According to the cold constitution onset site determination kit or device of the present invention, the onset site of the chills in the subject's body can be simply and easily detected and determined in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A represents the Manhattan plot of GWAS results related to the presence or absence of chills; Figure 1 B represents the QQ (quantile-quantile) graph;

[0051] Figure 2 A represents the Manhattan plot of GWAS results related to the chill sites (waist and legs); Figure 2 B represents the QQ (quantile-quantile) graph;

[0052] Figure 3 A represents the Manhattan plot of GWAS results related to the chill sites (hands and feet); Figure 3 B represents the QQ (quantile-quantile) graph;

[0053] Figure 4 A represents the Manhattan plot of GWAS results related to the chill site (whole body); Figure 4 B represents a QQ (quantile-quantile) plot. DETAILED DESCRIPTION

[0054] 1. Methods for determining cold constitution

[0055] 1-1. Overview

[0056] The first aspect of the present invention is a method for determining a cold constitution. The method of the present invention determines the genetic cause of a subject's cold constitution in vitro based on a biomarker, namely a cold constitution determination SNP. According to the method of the present invention, it is possible to easily determine in vitro whether the subject has a genetic cause of a cold constitution based on nucleic acid derived from the subject.

[0057] 1-2. Definition of terms

[0058] The following terms used in this specification are defined below.

[0059] In this specification, "chills" refers to the phenomenon that the whole body or part of the body feels cold without organic abnormalities as mentioned above. It should be noted that in this specification, "chills" is sometimes recorded as a term including constitution (cold constitution) and symptoms (chills fear) related to chills in addition to the above meanings.

[0060] In this specification, "cold constitution" refers to a constitution that causes chills.

[0061] In this specification, "cold intolerance" refers to a symptom that causes difficulty in daily life, such as a feeling of physical burden caused by one's own feeling of cold, a persistent feeling, and a slow recovery of the skin surface temperature after cold stress.

[0062] In this specification, "type of chills" refers to the type (category) distinguished according to the body part and state where chills occur. There are three known types: extremity type, lower body type and whole body type. "Extremity type" is a type in which chills occur in the hands and feet. "Lower body type" is a type in which chills occur in the lower body, such as the legs and waist. When accompanied by upper body heat or dryness, it is sometimes described as upper heat and lower cold type. "Whole body type" is a type in which chills occur throughout the body. There is no limitation in this specification, and it mainly targets three types based on the body part where chills occur, namely, extremity type, lower body type and whole body type.

[0063] In this specification, "genetic cause" refers to the possibility of suffering from a specific disease caused by genetic factors including mutations such as base substitutions.

[0064] "Gene polymorphism" refers to the mutation of the base sequence of the allele of the homologous locus on the genome between individuals in a population composed of the same species. In particular, it refers to a mutation that exists at a frequency of 1% or more in a population. Gene polymorphism includes SNP (single-nucleotide polymorphism), indel (insertion / deletion), STRP (short tandem repeat polymorphism), VNTR (variable number of tandem repeat) and CNV (copy number polymorphism) and the like.

[0065] "Alleles" are also called alleles, which refer to the individual genes that make up a genetic polymorphism. If they are identical genes, they do not have to be present in homologous loci, and can also be present in other loci through transposition or duplication. In this specification, alleles, in principle, refer to genes that are congenitally present in the genome, but can also include genes acquired due to mutations. Moreover, each allele is, in principle, a gene that exists in all cells that make up an individual, but can also be a gene that exists only in some cells, tissues or organs.

[0066] In this specification, "risk allele" refers to an allele that is closely associated with the site of occurrence of cold constitution or fear of cold in the cold constitution determination SNP or fear of cold type determination SNP described later. Therefore, for an individual who has a base of the risk allele in at least one of the genotypes, it can be determined that the cold constitution or fear of cold will occur at the presumed site.

[0067] "Single nucleotide polymorphism" (single nucleotide polymorphism, often referred to as "SNP" in this specification) refers to a gene polymorphism in which a base in the base sequence of an allele is replaced by a different base. The SNP in this specification is a SNP registered in the SNP database (dbSNP; http: / / www.ncbi.nlm.nih.gov / SNP / ) of the National Center for Biotechnology Information (NCBI), a public database, and can be searched by its reference number, i.e., rs number.

[0068] In the present specification, the "cold constitution determining SNP" refers to a SNP that is closely related to the cold constitution and can be used to determine the cold constitution.

[0069] In the present specification, the "cold sensitivity type determination SNP" refers to an SNP that is associated with the site where cold sensitivity occurs and can be used to determine the type of cold sensitivity.

[0070] In this specification, "genotype" refers to a combination of alleles at a SNP site.

[0071] In the present specification, "linkage disequilibrium" refers to a phenomenon in which alleles or gene polymorphisms at multiple loci are not inherited independently but in a correlated manner.

[0072] In this specification, "subject" refers to a human individual (living individual) provided as a subject in the methods, kits and devices of the present invention. The age and gender of the subject are not limited. For example, preferably, the subject is an East Asian, including Japanese, or a Southeast Asian woman aged 15 to 70 or 20 to 60.

[0073] In the present specification, "nucleic acid derived from a subject" refers to a nucleic acid derived from the above-mentioned subject and contained in a biological sample of the above-mentioned subject.

[0074] In this specification, "biological sample" refers to nucleic acid derived from a subject, preferably a sample containing genomic DNA, such as body fluids, cells, tissues (including nails, hair, epidermis, oral epithelium, etc.), and cell cultures.

[0075] In this specification, "body fluid" refers to a liquid biological sample containing nucleic acid or cells containing nucleic acid. Examples include, but are not limited to: blood, cerebrospinal fluid (cerebrospinal fluid), lymph, secretions, mucus, ascites, pleural effusion, sputum, nasal mucus, saliva, lacrimal gland secretions, semen, vaginal fluid, urine, pharyngeal swab fluid, nasal swab fluid, nerve root periarthritis fluid, sweat, and extracts of various tissues or cells.

[0076] In the present specification, "natural nucleotide" refers to a nucleotide that exists naturally, that is, in nature. Examples thereof include ribonucleotides known as RNA constituent units and deoxyribonucleotides known as DNA constituent units.

[0077] In this specification, "non-natural nucleotides" refer to nucleotides that have properties and / or structures similar to the above-mentioned natural nucleotides and do not exist in nature. Specifically, it refers to artificially constructed or artificially chemically modified nucleotides, or nucleotides containing non-natural nucleosides or non-natural bases. The above-mentioned artificially constructed nucleotides include, for example, peptide nucleic acids (PNA: Peptide Nucleic Acid), peptide nucleic acids with phosphate groups (PHONA), cross-linked nucleic acids [BNA / LNA: Bridged Nucleic Acid / Locked Nucleic Acid], morpholino nucleic acids, etc. Artificially chemically modified nucleotides include, for example, chemically modified nucleic acids and nucleic acid analogs such as methylphosphonate DNA / RNA, phosphorothioate DNA / RNA, phosphoramidite DNA / RNA, and 2'-O-methyl DNA / RNA.

[0078] In this specification, "nucleic acid array" is also called "microarray" or "nucleic acid chip", which refers to a high-density integrated array of different nucleic acids arranged and fixed on a substrate. By using microfabrication technology in semiconductor manufacturing such as photolithography or inkjet method, a large number of nucleic acids are integrated on the chip at a high density, thereby enabling parallel analysis of a large number of types and quantities of nucleic acids. A nucleic acid array with DNA fragments arranged on a substrate is called a DNA microarray or DNA chip.

[0079] 1-3. Methods

[0080] In the cold constitution determination method of the present invention, the necessary steps include the cold constitution determination SNP detection step and the cold constitution determination step, and the optional steps include the nucleic acid extraction step. Each step is described below.

[0081] 1-3-1. Nucleic acid extraction steps

[0082] The "nucleic acid extraction step" is a step of extracting nucleic acid from a biological sample derived from a subject. This step is an optional step of the method of the present invention and can be implemented as needed.

[0083] The type of biological sample used in this step is not limited. Considering that there is no need to select the collection time, it is easy to obtain, and it is low in invasiveness to the subject, it is preferably body fluid or oral epithelium. Body fluid is particularly preferably saliva, throat swab fluid, and nasal swab fluid. If a biological sample of more than 1 mL is required, blood (whole blood) or lymph fluid is preferably used.

[0084] Biological samples can be collected by known methods without limitation. For example, saliva can be collected directly. Throat swab fluid, nasal swab fluid, or oral epithelium can be sampled at specified locations using cotton swabs or the like. For blood, for example, in addition to blood collected directly from a vein or the like by injection, the following blood can also be used as samples: blood anticoagulated by adding heparin or the like to the collected whole blood, blood separated into plasma or serum, blood temporarily stored in a refrigerated or frozen manner, etc. Collection can be performed using a commercially available kit. The collected biological sample can be purified using known methods as needed, and can also be stored at low temperatures, such as -20°C or other temperatures below freezing, until this step is performed.

[0085] In this step, nucleic acid is extracted from the above-mentioned biological sample. The nucleic acid extracted in this step is not limited, and is preferably DNA, particularly genomic DNA. For example, if the SNP to be detected is present in mature mRNA, it can be RNA, particularly mRNA and total RNA. The method for extracting nucleic acid from a biological sample can be prepared according to the common methods in this field according to the type of nucleic acid. For example, the nucleic acid extraction method recorded in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York can be used for extraction. In addition, life science manufacturers also sell kits for extracting nucleic acids from biological samples on the market, and these kits can also be used. When using the kit, the specific extraction method can be operated according to or in accordance with the attached instructions.

[0086] 1-3-2. SNP detection steps for cold constitution determination

[0087] The "cold constitution determining SNP detection step" is a step of detecting the cold constitution determining SNP in vitro based on the nucleic acid derived from the subject.

[0088] The cold constitution determination SNP to be detected in this step is not limited, and 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 SNPs in linkage disequilibrium with the bases of the risk alleles shown in Table 1 among these SNPs and any one of the cold constitution determination SNPs in 1 to 11 above. In the above linkage disequilibrium relationship, the linkage disequilibrium coefficient may be 0.8 or more, 0.85 or more, 0.9 or more, 0.95 or more, or 1.0.

[0089] "Detecting the cold constitution determination SNP" refers to identifying the base of the allele in the cold constitution determination SNP. The detection can be either one allele or both alleles of the paired allele of the cold constitution determination SNP. In the cold constitution determination step described later, the cold constitution is determined based on whether at least one allele in the cold constitution determination SNP 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 preferred to detect both pairs of paired alleles.

[0090] There is no particular limitation on the method for detecting SNPs for cold constitution determination in vitro. The cold constitution determination SNPs are the known SNPs mentioned above, so existing methods such as known SNP detection methods and / or SNP type determination methods (SNP typing methods, genotyping methods) can be used. For example, but not limited to: nucleic acid amplification method, DNA microarray method, or AOPL (Amplified Product Length Polymorphism) method, etc. can be used.

[0091] Examples of nucleic acid amplification methods include the following: using SNP-specific or SNP allele-specific primers (and probes if necessary) to amplify a specified region on a template nucleic acid by PCR or the like, and detecting the target SNP by fluorescence or luminescence of the allele in the amplified product. Examples of SNP detection based on nucleic acid amplification methods include: PCR-RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-strand conformation polymorphism) method, PCR-SSO (sequence specific oligonucleotide) method, direct sequencing method, ASO (Allele Specific Oligonucleotide) hybridization method, ASP-PCR (Allele Specific Primer-PCR) method, Snapshot method, ARMS (Amplification Refracting Mutation System) method, real-time (RT-) PCR method, Invader method, MALDI-TOF / MS method, RNase A cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TDI (Template-directed Dye-terminator Incorporation, template-directed dye terminator incorporation reaction) etc. These are well-known methods in the art and can be operated by well-known steps.

[0092] In addition, life science manufacturers sell SNP detection kits and SNP typing kits using nucleic acid amplification methods on the market, which can also be used to detect target SNPs. For example, TaqMan SNP genotyping detection kits (Thermo Fisher Scientific) using real-time PCR or real-time RT-PCR methods can be cited. In the TaqMan SNP genotyping detection kit, a set of primer pairs that can amplify the region containing the target SNP (here, the cold constitution determination SNP) on the template nucleic acid of the genome, etc., 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 allyl group. The primer pair and the probe are mixed with the template nucleic acid of genomic DNA, etc., and real-time PCR or real-time RT-PCR is performed to detect the fluorescence of each probe, so that each allyl group can be detected.

[0093] The reaction conditions in the nucleic acid amplification method are based on the known general conditions in each nucleic acid amplification method, and vary according to the base length of the amplified nucleic acid fragment and the amount of template nucleic acid, the base length and Tm value of the primer used, the optimal reaction temperature and the optimal pH of the nucleic acid polymerase used, etc., so these conditions can be appropriately determined. As an example, in the case of the PCR method, a denaturation reaction can generally be performed at 94-95°C for 5 seconds to 5 minutes, an annealing reaction can be performed at 50-70°C for 10 seconds to 1 minute, and an extension reaction can be performed at 68-72°C for 30 seconds to 3 minutes. The above reaction is repeated for about 15 to 40 cycles as one cycle, and finally an extension reaction can be performed at 68-72°C for 30 seconds to 10 minutes. When using a commercially available kit, in principle, the operation can be performed according to the instructions attached to the kit.

[0094] 1-3-3. Steps to determine cold constitution

[0095] The "cold constitution determination step" is a step of determining whether the subject has the genetic cause of cold constitution based on the detected cold constitution determination SNP.

[0096] In this step, when at least one of the bases in the allele of the cold constitution determination SNP detected in the cold constitution determination SNP determination step is a risk allele, or is an allele of the SNP that is in linkage disequilibrium with the risk allele, the subject is determined to have the genetic cause of cold constitution.

[0097] Specifically, when the base in rs750707 is the risk allele G, when the base in rs1869201 is the risk allele A, when the base in rs62295776 is the risk allele T, when the base in rs10016576 is the risk allele G, when the base in rs74513903 is the risk allele C, when the base in rs2656514 is the risk allele A, when the base in rs12761019 is the risk allele G, when the base in rs17098278 is the risk allele T When the base in rs10220484 is the risk allele C, when the base in rs2876278 is the risk allele G, when the base in rs4818919 is the risk allele G, and when the allele is an allele of a SNP that is in linkage disequilibrium with the risk alleles of the above-mentioned SNPs, it is determined that the subjects with these alleles are relatively more likely to have a cold constitution than individuals without the risk allele, and have the genetic cause of a cold constitution, that is, they are prone to feel cold themselves, and people with a cold constitution are prone to develop cold constitution diseases.

[0098] 1-4. Biomarkers for cold constitution

[0099] In the present invention, the detection object of the nucleic acid from the subject source, i.e., the cold constitution determination SNP, and the SNP in linkage disequilibrium with the SNP can be referred to as a biomarker for determining the genetic cause of the cold constitution. For example, it is a SNP selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278 and rs4818919 as the cold constitution determination SNP, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group.

[0100] Through these biomarkers, it is possible to determine whether the subject has a genetic cause for a cold constitution based on whether the allele contained in the nucleic acid derived from the subject is the base of the above-mentioned risk allele.

[0101] 1-5. Effect

[0102] According to the method for determining cold constitution of the present invention, it is possible to determine in vitro whether there is a previously unknown genetic cause of cold constitution, thereby being applied to the physical diagnosis of cold constitution.

[0103] 2. Method for determining the type of chills

[0104] 2-1. Overview

[0105] The second aspect of the present invention is a method for determining the type of chills. The method of the present invention determines the genetic cause of the type of chills of a subject in vitro based on a biomarker, i.e., a chills type determination SNP. According to the method of the present invention, it is possible to easily determine in vitro whether the subject has any type of genetic cause of chills based on nucleic acid derived from the subject.

[0106] 2-2. Methods

[0107] In the method for determining the type of chills of the present invention, the necessary steps include a chills type determination SNP detection step and a chills type determination step. In addition, before the above steps, the optional steps include the nucleic acid extraction step of the first aspect, the cold constitution determination SNP detection step, and the cold constitution determination step. That is to say, the method for determining the type of chills of the present invention can be implemented independently on the subject, or after implementing the first aspect of the cold constitution determination method, the method for determining the type of chills of the present invention can be further implemented on the subject determined to have the genetic cause of the cold constitution to determine the type of chills. It should be noted that regardless of whether the first aspect of the cold constitution determination method is implemented, the nucleic acid extraction step can be selected when it is necessary to extract nucleic acid from a biological sample derived from the subject.

[0108] Each step is described below. The nucleic acid extraction step, the SNP detection step for determining a cold constitution, and the cold constitution determination step are based on the steps described in the first aspect, so they will not be repeated here.

[0109] 2-2-1. SNP detection steps for determining the type of chills

[0110] The "SNP detection step for determining the type of cold syndrome" is a step for detecting the SNP for determining the type of cold syndrome in vitro based on the nucleic acid from the subject. In this step, except that the detection object is the SNP for determining the type of cold syndrome, the basic operation steps are based on the "SNP detection step for determining the cold constitution" of the first aspect. Therefore, the common steps will not be repeated here, and the description here will focus on the characteristic points in this step.

[0111] The chills type determination SNP to be detected in this step varies depending on the site of occurrence of chills. The chills type to be detected in this specification is not limited, and as mentioned above, there are three types: extremity type, lower body type, and whole body type.

[0112] The SNP for determining the type of cold sensitivity in the lower body is not limited and is an SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006 and rs13376933, or an SNP in linkage disequilibrium with the base of the risk allele of any one of the SNPs selected from the above group.

[0113] The SNP for determining the type of cold fear in the extremities 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 the base of the risk allele of any one of the SNPs selected from the above group.

[0114] The systemic cold sensitivity type determining SNP is a SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343 and rs6506395, or a SNP in linkage disequilibrium with the base of the risk allele of any one of the SNPs selected from the above group.

[0115] In the linkage disequilibrium relationship with each of the above-mentioned chills type determining SNPs, the linkage disequilibrium coefficient may be 0.8 or more, 0.85 or more, 0.9 or more, 0.95 or more, or 1.0.

[0116] "Detecting the chills type determination SNP" refers to identifying the base of the allele in the chills type determination SNP. The detection can be either one allele or both alleles of the parallel allele of the chills type determination SNP. In the chills type determination step described later, the chills type is determined based on whether at least one allele in the chills type determination SNP 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 preferred to detect both parallel alleles.

[0117] 2-2-2. Steps for determining the type of chills

[0118] The "chill fear type determination step" is a step of determining that the subject's chill fear will occur at the site of the chill fear type when the detected allele of the chill fear type determination SNP is a risk allele.

[0119] In this step, except that the detection object is the cold fear type determination SNP, the basic operation steps are based on the "cold constitution determination step" of the first aspect. When at least one of the bases in the allele of the cold fear type determination SNP detected in the cold fear type determination SNP determination step is a risk allele, or is an allele of the SNP in a linkage disequilibrium relationship with the risk allele, the subject is determined to have a genetic cause of cold fear type.

[0120] Specifically, for example, in the SNPs for determining the type of cold fear in 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, when the base in rs13376933 is the risk allele C, and when the allele is an allele of a SNP that is in linkage disequilibrium with the risk alleles of the above-mentioned SNPs, it is determined that the subjects with these alleles are relatively more likely to have cold fear in the lower body than individuals without the risk allele, and have the genetic cause of cold fear in the lower body, that is, cold fear is likely to occur in the waist and legs.

[0121] In addition, among the extremity type cold fear type determination SNPs, 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, when the base in rs4818919 is the risk allele G, and when the allele is an allele of a SNP that is in linkage disequilibrium with the risk allele of the above-mentioned SNPs, it is determined that the subjects having these alleles are relatively more likely to have extremity cold fear than individuals without the risk allele, and have a genetic cause of extremity cold fear, that is, cold fear is likely to occur in the hands and feet.

[0122] Furthermore, among the SNPs for determining the type of systemic chills, 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, when the base in rs6506395 is the risk allele G, and when the allele is an allele of a SNP that is in linkage disequilibrium with the risk allele of the above-mentioned SNPs, it is determined that the subjects having these alleles are relatively more likely to have systemic chills than individuals without the risk allele, and have the genetic cause of systemic chills, that is, chills are likely to occur throughout the body.

[0123] 2-3. Biomarkers for determining the type of chills

[0124] In the present invention, the cold sensitivity type determination SNP that is the detection target of nucleic acid derived from a subject, and the SNP in linkage disequilibrium with the SNP can be referred to as a biomarker for determining the presence or absence of a genetic cause of the cold sensitivity type.

[0125] For example, a SNP selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006 and rs13376933, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group can be a biomarker for determining the presence or absence of a genetic cause for the lower body type of cold intolerance.

[0126] In addition, 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 one of the SNPs selected from the group can be a biomarker for determining the presence or absence of a genetic cause for the extremity type of cold fear.

[0127] Furthermore, a SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343 and rs6506395, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group can serve as a biomarker for determining the presence or absence of a genetic cause for the systemic type of cold sensitivity.

[0128] 2-4. Effect

[0129] According to the method for determining the type of chills, it is possible to determine in vitro whether there is a genetic cause of the type of chills, thereby predicting and determining the site of occurrence of chills.

[0130] In addition, in combination with the cold constitution determination method of the first aspect, subjects determined to have a cold constitution can be stratified, so that appropriate drugs or Kampo medicines can be prescribed according to each type of cold sensitivity.

[0131] 3. Cold constitution test kit and equipment

[0132] 3-1. Overview

[0133] The third aspect of the present invention is a cold constitution detection kit and device. The cold constitution detection kit and device of the present invention are configured to specifically detect a cold constitution determination SNP and / or a SNP in linkage disequilibrium with the SNP, and can identify the base of the allele in the SNP. According to the cold constitution detection kit and device of the present invention, the cold constitution determination SNP of the subject can be easily and simply detected to determine the genetic cause of the cold constitution.

[0134] 3-2. Composition

[0135] 3-2-1. Components

[0136] In the cold constitution detection kit and device of the present invention, the essential components include probes and / or primers for detecting at least one of the cold constitution determination biomarkers, i.e., cold constitution determination SNPs and / or SNPs in linkage disequilibrium with the SNPs. Each component is described in detail below.

[0137] (1) Probe: Cold constitution determination SNP detection probe

[0138] The probes included in the cold constitution detection kit and equipment of the present invention (often referred to as "cold constitution determination SNP detection probes" in this specification) are configured to specifically bind to the cold constitution determination SNP and / or a SNP in linkage disequilibrium with the SNP and the nucleotides nearby.

[0139] For example, the probe is composed of a polynucleotide comprising a base sequence that can hybridize with the following sites and base sequences: any one or more SNP sites selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278 and rs4818919 as SNPs for cold constitution determination; or, a SNP site in a linkage disequilibrium relationship with the SNP; and, the base sequence of nucleotides nearby. "Nucleotides nearby" refers to nucleotides adjacent to the SNP site as the detection object in the upstream and / or downstream on the template nucleic acid. That is, the cold constitution determining SNP detection probe is configured to include the antisense strand of the base sequence surrounding it, and the base sequence surrounding it includes the cold constitution determining SNP site, or a SNP site in linkage disequilibrium with the SNP site.

[0140] The polynucleotide constituting the cold constitution determination SNP detection probe may contain one or more base deletions, substitutions or additions in the base sequence of the above-mentioned antisense chain, so that hybridization can be performed regardless of the allele of the cold constitution determination SNP site or the SNP in linkage disequilibrium with the SNP.

[0141] The SNP detection probe for cold constitution determination is composed of natural nucleotides and / or non-natural nucleotides. It is generally composed of natural nucleotides of DNA or RNA. In addition, as needed, a probe composed of a mixture of natural nucleotides of DNA or RNA can be used, or a probe partially containing non-natural nucleotides such as LNA and PNA can be used.

[0142] In order to be able to specifically hybridize with each of the above-mentioned SNPs and their vicinity regions, the base length and Tm value of the base sequence of the cold constitution determination SNP detection probe are not particularly limited as long as they are designed in the range of 55°C to 80°C, preferably 60°C to 75°C. The base length of the probe is generally preferably a base length and base sequence that can hybridize with continuous bases on the template nucleic acid chain. For example, it can be 15 to 100 continuous bases, 15 to 80 continuous bases, 15 to 60 continuous bases, 16 to 50 continuous bases, 17 to 40 continuous bases, or 18 to 30 continuous bases, or 20 to 25 continuous bases, which are commonly used as primer lengths in this field.

[0143] The phosphate groups, sugars and / or bases of part or all of the nucleotides constituting the cold constitution determination SNP detection probe of the present invention can be labeled with a labeling substance. The labeling position of the labeling substance in the probe can be appropriately determined according to the characteristics and purpose of use of the labeling substance, without limitation, and is preferably the 5' end, the 3' end, and / or the SNP site of the detection object. The labeling substance can utilize all substances known in the art. For example, radioactive isotopes, DIG, fluorescent substances, quenchers, chemiluminescent substances, biotin or magnetic beads can be cited. The labeling of nucleotides by each labeling substance can be carried out by a known method.

[0144] The above-mentioned "radioactive isotope" refers to an element that emits radiation among isotopes with different mass numbers. Examples include 32 P. 33 P or 35 S.

[0145] The above-mentioned "fluorescent substance" refers to a substance that has the property of emitting fluorescence when it returns to the original ground state after absorbing excitation light of a specific wavelength and becomes excited. Examples thereof include: FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar (registered trademark) 670, Quasar (registered trademark) 705, CALFluor (registered trademark) Red 610, fluorescamine or its derivatives, or rhodamine or its derivatives, etc. However, the present invention is not limited to the above-mentioned contents.

[0146] The above-mentioned "quencher" refers to a substance that has the property of absorbing the excitation energy of the above-mentioned fluorescent substance and inhibiting fluorescence. For example, AMRA, DABCYL, BHQ-1, BHQ-2 or BHQ-3 can be cited. Quenchers are generally used in combination with fluorescent substances. The quencher has different fluorescence inhibition wavelength ranges depending on its type. Therefore, in the combination of fluorescent substances and quenchers, it is necessary to combine with a quencher that can inhibit the fluorescence of the fluorescent substance used for labeling. For example, when the fluorescent substance is FAM, HEX, TET, etc., it can be combined with BHQ1 with an inhibition wavelength range of 480nm to 580nm; when the fluorescent substance is Cy3, Cy5, ROX, TAMRA, Texas, etc., it can be combined with BHQ2 with an inhibition wavelength range of 550nm to 650nm. There is no particular limitation on the configuration of the fluorescent substance and the quencher in the probe, as long as the two substances are configured so that the quencher can inhibit the fluorescence emitted by the fluorescent substance. For example, one end (5' end or 3' end) of the probe may be labeled with a fluorescent substance, and the other end (3' end or 5' end, respectively) may be labeled with a quencher.

[0147] The above-mentioned "chemiluminescent substance" refers to a substance having the property of releasing differential energy as light when returning to a ground state after being excited by a chemical reaction, and examples thereof include acridinium esters and the like.

[0148] The cold constitution detection kit or device of the present invention may include two or more cold constitution determination SNP detection probes, for example, probes that specifically hybridize with different alleles of the cold constitution determination SNP of the detection object.

[0149] (2) Primers: Cold constitution determination SNP detection primers

[0150] The primers included in the cold constitution detection kit and equipment of the present invention (often referred to as "cold constitution determination SNP detection primers" in this specification) are configured to specifically recognize and amplify the cold constitution determination SNP and / or a SNP in linkage disequilibrium with the SNP and the nucleotides nearby.

[0151] The cold constitution determination SNP detection primers of the present invention are in principle composed of a set of primers as a forward (Fw) primer and a reverse (Rv) primer, but the cold constitution detection kit and device of the present invention may also include only primers of either side. The primers are composed of a polynucleotide that hybridizes with a base sequence of a nucleotide near a cold constitution determination SNP or a SNP in a linkage disequilibrium relationship with the SNP in a template nucleic acid such as the genomic DNA of the subject and amplifies the base sequence of the cold constitution determination SNP or a SNP surrounding region in a linkage disequilibrium relationship with the SNP.

[0152] For example, the Fw primer and the Rv primer are composed of the following polynucleotides: the polynucleotides contain a base sequence that hybridizes with any one or more SNP sites selected from the group consisting of rs750707, rs1869201, rs62295776, rs10016576, rs74513903, rs2656514, rs12761019, rs17098278, rs10220484, rs2876278 and rs4818919 as cold constitution determination SNPs, or a base sequence of nucleotides near the SNP site in a linkage disequilibrium relationship with the SNP and can amplify the base sequence of the nucleic acid region around the SNP site. The above-mentioned "nearby nucleotides" refer to nucleotides that are adjacent to the SNP site as the detection object in the upstream and / or downstream on the template nucleic acid.

[0153] Each of the above primers is composed of natural nucleotides and / or non-natural nucleotides. Generally, it is composed of natural nucleotides of DNA or RNA. Among them, DNA with high stability, easy synthesis and low cost is particularly preferred. In addition, natural nucleotides of DNA and RNA can be combined as needed, or non-natural nucleotides such as chemically modified nucleic acids and pseudo nucleic acids can be partially combined. Chemically modified nucleic acids and pseudo nucleic acids can include, for example, PNA (Peptide Nucleic Acid, peptide nucleic acid), LNA (Locked Nucleic Acid, locked nucleic acid; registered trademark), methylphosphonate DNA, phosphorothioate DNA, 2'-O-methyl RNA, etc.

[0154] Among the SNP detection primers for cold constitution determination, the Fw primer and the Rv primer are not limited, and are designed to have Tm values ​​of 55° C. to 80° C., preferably 60° C. to 75° C., respectively. In addition, the base length and base sequence of each primer are not particularly limited. For example, the base length can 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 chain.

[0155] There is no limit to the base length of the amplified fragment of the primer set of the cold constitution determination SNP detection primers, and it can be in the range of 100 bases to 1500 bases, 150 bases to 1200 bases, 200 bases to 1000 bases, 250 bases to 800 bases, 300 bases to 600 bases or 400 bases to 500 bases.

[0156] The cold constitution determination SNP detection primer of the present invention may contain an additional sequence different from the base sequence of the template nucleic acid at its 5' end. The additional sequence may be any base sequence. For example, a base sequence including a restriction enzyme site and a new primer binding site may be cited.

[0157] In addition, the phosphate group, sugar and / or base of the cold constitution determination SNP detection probe of the present invention can be labeled with a labeling substance. The labeling position of the labeling substance in the primer can be appropriately determined according to the characteristics of the labeling substance and the purpose of use, without limitation, and is generally preferably at the 5' end portion that does not contribute to the extension reaction. The nucleic acid labeling substance can utilize all substances known in the art. For example, it can include: the above-mentioned radioactive isotopes, DIG, fluorescent dyes, chemiluminescent substances, etc.

[0158] The primer set of cold constitution determination SNP detection primers may be one set or multiple sets such as nested primers. In addition, a control primer set capable of amplifying a control nucleic acid other than the SNP to be detected described later may also be included.

[0159] (3) Internal control primer (IC primer)

[0160] Optional components of the cold constitution detection kit or device of the present invention may include internal control (Internal Control: IC) primers.

[0161] IC primers are configured to amplify non-target template nucleic acid molecules present in the sample as internal controls. When the cold constitution detection kit or device of the present invention is used to amplify the cold constitution determination SNP and the base sequence around it, if it is necessary to monitor whether the amplification reaction can proceed and the state of the reaction, IC primers are added.

[0162] Regarding the template nucleic acid molecule used as an internal control, an appropriate nucleic acid molecule can be added to the sample from the outside and used as a template nucleic acid molecule, or an endogenous nucleic acid molecule (gene transcription product, etc.) present in the sample can be used as a template nucleic acid molecule. Endogenous nucleic acid molecules are preferably selected from genes that are constantly expressed, such as housekeeping genes. The cold constitution detection kit or device of the present invention has no restrictions on suitable internal controls, for example, transcription products (mRNA) such as 18S rRNA, β-actin, GAPDH, EF1α, and tubulin contained in human blood can be cited.

[0163] There are no restrictions on 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, as long as the composition of the SNP detection primer for determining the cold constitution mentioned above is basically based on it.

[0164] (4) Internal control probe (IC probe)

[0165] Optional components of the cold constitution detection kit or device of the present invention may also include an internal control probe.

[0166] The IC probe is configured to hybridize with the amplified fragment of the internal control amplified by the IC primer (often referred to as "IC amplified fragment" in this specification) and detect the IC amplified fragment. Therefore, the probe is used in conjunction with the IC primer set in the cold constitution detection kit or device of the present invention.

[0167] There are no restrictions on the structural characteristics, composition, base length and base sequence of the IC probe, as long as the SNP detection probe is basically based on the cold constitution determination.

[0168] 3-2-2. Composition of cold constitution test kit and equipment

[0169] The composition of the cold constitution detection kit of the present invention only needs to have the composition based on the ordinary detection kit. For example, in addition to the cold constitution determination SNP detection probe and / or primer as the above-mentioned essential components, the composition including the instructions for recording the appropriate reaction conditions and the SNP detection method can also be cited. In addition, if it is a cold constitution determination SNP detection primer, it can also include, for example, reagents required for nucleic acid amplification reaction (dNTP, DNA polymerase, etc.), and reagents for detecting amplified nucleic acid fragments (fluorescent reagents, luminescent reagents, etc.).

[0170] On the other hand, the cold constitution detection device of the present invention is not limited and can have a composition of a nucleic acid array (including a DNA chip) that can be applied to nucleic acid array technology. For example, a nucleic acid array having a plurality of SNP detection probes for cold constitution determination fixed on a substrate (chip) can be cited.

[0171] 4. Kits and equipment for determining the type of chills

[0172] 4-1. Overview

[0173] The fourth aspect of the present invention is a cold fear type determination kit and device. The cold fear type determination kit and device of the present invention are configured to specifically detect the cold fear type determination SNP and / or the SNP in linkage disequilibrium with the SNP, and can identify the base of the allele in the SNP. According to the cold fear type determination kit and device of the present invention, the cold fear type of the subject can be easily and simply determined. In addition, it is easy to carry out more specific stratification of subjects determined to have a cold constitution by combining with the cold constitution detection kit and / or device of the third aspect.

[0174] 4-2. Composition

[0175] 4-2-1. Components

[0176] In the cold fear type determination kit and device of the present invention, the essential components include probes and / or primers for detecting at least one of the cold fear type determination biomarkers, i.e., cold fear type determination SNPs and / or SNPs in linkage disequilibrium with the SNPs. Each component is described in detail below.

[0177] The basic composition of the kit and device for determining the type of chills shall be based on the composition of the "kit and device for determining the type of chills" in the third aspect, except that the detection object is the SNP for determining the type of chills and / or the SNP in linkage disequilibrium with the SNP. Therefore, the common composition will not be repeated here, and the description here will focus on the characteristic points of the kit and device of this aspect.

[0178] (1) Probe: SNP detection probe for determining the type of chills

[0179] The probes included in the chills type determination kit and equipment of the present invention (often referred to as "chills type determination SNP detection probes" in this specification) are configured to specifically bind to the chills type determination SNP and / or a SNP in linkage disequilibrium with the SNP and the nucleotides nearby.

[0180] The cold sensitivity type determining SNP detection probe is provided so as to be capable of detecting each cold sensitivity type determining SNP detection biomarker described in the "Cold sensitivity type determining method" of the second aspect.

[0181] For example, when the type of chills to be detected is the lower body type, the probe is composed of a polynucleotide comprising a base sequence that can hybridize with the following sites and base sequences: a SNP site selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006 and rs13376933, which are SNPs for determining the lower body type chills; or, a SNP site that is in linkage disequilibrium with any one of the SNPs selected from the above group; and, the base sequence of the nucleotides nearby.

[0182] In addition, when the type of chills to be detected is the extremity type, the probe is composed of a polynucleotide comprising a base sequence that can hybridize with the following sites and base sequences: a SNP site selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186 and rs4818919, which are SNPs for determining extremity chills; or a SNP site that is in linkage disequilibrium with any one of the SNPs selected from the above group; and the base sequence of the nucleotides nearby.

[0183] In addition, when the type of chills to be detected is generalized, the probe is composed of a polynucleotide comprising a base sequence that can hybridize with the following sites and base sequences: a SNP site selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343 and rs6506395, which are SNPs for determining generalized chills; or a SNP site that is in linkage disequilibrium with any one of the SNPs selected from the above group; and the base sequence of the nucleotides nearby.

[0184] The chills type determination kit or device of the present invention may include two or more chills type determination SNP detection probes, for example, probes that specifically hybridize with different alleles of the chills type determination SNP of the test object and probes for detecting SNPs of different chills types.

[0185] (2) Primers: SNP detection primers for determining the type of chills

[0186] The primers included in the chills type determination kit and device of the present invention (often referred to as "chills type determination SNP detection primers" in this specification) are configured to specifically recognize and amplify the chills type determination SNP and / or a SNP in linkage disequilibrium with the SNP and the nucleotides nearby.

[0187] The cold sensitivity type determining SNP detection primers are provided so as to be capable of detecting each cold sensitivity type determining SNP detection biomarker described in the "Cold sensitivity type determining method" of the second aspect.

[0188] For example, when the type of chills to be detected is the lower body type, it is composed of the following Fw primer and Rv primer, and the Fw primer and Rv primer are composed of the following polynucleotide: the polynucleotide contains a SNP site selected from the group consisting of rs750707, rs76388277, rs6819504, rs9994962, rs72719006 and rs13376933 which are SNPs for determining the type of chills in the lower body, or a base sequence of nucleotides near a SNP site that is in a linkage disequilibrium relationship with the SNP and can amplify the base sequence of the nucleic acid region surrounding the above-mentioned SNP site.

[0189] In addition, when the type of chills to be detected is the extremity type, it is composed of the following Fw primer and Rv primer, and the Fw primer and Rv primer are composed of the following polynucleotide: the polynucleotide contains a SNP site selected from the group consisting of rs556161, rs10911744, rs1452609, rs61998228, rs849057, rs6476958, rs6518186 and rs4818919 which are SNPs for determining the type of chills, or a base sequence of nucleotides near a SNP site that is in linkage disequilibrium with the SNP and can amplify the base sequence of the nucleic acid region surrounding the above-mentioned SNP site.

[0190] In addition, when the type of chills to be detected is generalized, it is composed of the following Fw primer and Rv primer, and the Fw primer and Rv primer are composed of the following polynucleotide: the polynucleotide contains a SNP site selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343 and rs6506395 which are SNPs for determining the type of generalized chills, or a base sequence of nucleotides near a SNP site that is in linkage disequilibrium with the SNP and is capable of amplifying a base sequence of a nucleic acid region surrounding the SNP site.

[0191] The SNP detection primer set for determining the type of chills may be one set, or may include multiple primer sets to determine multiple different types of chills. In addition, a control primer set that can amplify a control nucleic acid other than the SNP to be detected may also be included.

[0192] [Example]

[0193] <Exploration and selection of SNPs for determining cold constitution and cold sensitivity type>

[0194] (Purpose)

[0195] The SNPs with a genetic correlation that is closely related to the occurrence site of cold constitution or fear of cold syndrome are explored, and the SNPs for determining the cold constitution and the SNPs for determining the type of fear of cold syndrome are selected.

[0196] (method)

[0197] The subjects were 1,200 adult female volunteers aged 20 to 60 years old who had given their consent to this Example and used samples and information obtained through the genetic testing service "MYCODE" of DeNA Life Sciences, Inc. (hereinafter referred to as DLS).

[0198] Based on a questionnaire survey with attribute information such as age, height, weight, current medical history, subjective symptoms of chills, and the burden of physical symptoms, as well as genetic diversity information obtained in MYCODE, a genome-wide association study (GWAS) was conducted on traits related to chills in each volunteer.

[0199] First, based on the SNPs obtained through the SNP array, sample quality control (QC) was performed on the 1,200 samples that had answered the questionnaire, and finally 1,113 samples remained.

[0200] Next, using the 1113 samples after sample QC, before genotype imputation, SNP QC was performed on 684,428 biallelic SNPs on autosomes that were judged to be able to be analyzed by DLS and carried on the MYCODE array (Infinium OmniExpress-24+BeadChip, Human OmniExpress-24+BeadChip), and finally 492,418 SNPs were obtained. Genotype imputation was performed on these SNPs. In genotype imputation, SNPs in areas not carried on the SNP array were also analyzed, so the genotypes of areas not statistically typed were estimated based on the whole genome information used as a reference.

[0201] For all 43,835,851 SNPs after genotype filling, 36,208,933 SNPs with low precision whose Rsq value (the estimated value of the correlation coefficient between the true genotype and the estimated genotype output by Minimac3) was lower than 0.7, which is usually used as an indicator of filling accuracy. Afterwards, in order to reduce the possibility of false positives caused by population structuring, etc., and to confirm the uniformity of the genetic background of the sample, PCA (plink-indep-pairwise 50 5 0.5) was performed again on the SNPs after genotype filling. PCA was performed on the filled genomic data again, and samples that obviously deviated from the population were visually judged and removed through the graphs of the first and second principal components. PCA was performed again, and samples that deviated greatly from the population were visually judged and removed through the graphs. PCA was performed again on the remaining 1,111 samples, and it was visually confirmed that there were no samples that obviously deviated from the population.

[0202] The trait data obtained through the questionnaire were converted into two values: the study group and the control group.

[0203] GWAS analysis: For 7234812 SNPs that passed SNP QC after genotype filling, the GWAS analysis population of each trait was used as the object, and SNPs with Hardy-Weinberg equilibrium (HWE) <1e-06 and minor allele frequency (MAF) <0.05 were removed. Finally, 5077272 to 5077326 SNPs were used for GWAS. PLINK (version 1.9) was used to implement GWAS, and logistic regression analysis was performed on the study group / control group assuming an additive model. Age, BMI, amenorrhea, and the first to fifth principal components of the principal component analysis from 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 meet the suggestive significance level, the clump option of PLINK was used to group SNPs that showed the same correlation and define them as the same region, taking into account the linkage disequilibrium in SNPs that exist in the 10,000 kbp region (linkage disequilibrium coefficient R2=0.2 or more), and the SNPs with the lowest P value were used as SNPs (lead SNPs) representing the correlation in the field. For visualization of the analysis results, QQ plots (quantile-quantile) and Manhattan plots were made using the qqman package of R. The position information of SNPs is based on the reference genome version GRCh37.

[0204] (result)

[0205] As the correlation regions that satisfied the suggestive significance level for the chill-related trait, 32 regions were detected. The Manhattan plot and QQ plot of the GWAS results related to the presence or absence of chills are shown in Figure 1 ; Manhattan plot and QQ plot of GWAS results related to chilling sites (waist and legs) are shown in Figure 2 ; Manhattan plot and QQ plot of GWAS results related to cold sites (hands and feet) are shown in Figure 3 ; Manhattan plot and QQ plot of GWAS results related to chilling site (whole body) are shown in Figure 4 .

[0206] Among the 32 regions, the trait of "whether there is chills" was suggested to be associated with the 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; when the OR value is greater than 1.0, the minor allele (A1) is the risk allele. Therefore, for rs1869201, rs62295776, rs74513903, rs2656514, rs12761019 and rs17098278, the major allele is the risk allele for cold constitution; for rs750707, rs10016576, rs10220484, rs2876278 and rs4818919, the minor allele is the risk allele for cold constitution.

[0207] [Table 3]

[0208] serial number Lead SNP A1 A2 MAF P OR SE 1 rs750707 G A 0.42 1.24E-06 1.56 0.09 2 rs1869201 G A 0.42 2.04-E06 0.66 0.09 3 rs62295776 A T 0.27 8.91E-06 0.64 0.10 4 rs10016576 G C 0.48 5.07E-06 1.50 0.09 5 rs74513903 T C 0.20 9.98E-06 0.62 0.11 6 rs2656514 G A 0.30 8.35E-06 0.65 0.1O 7 rs12761019 A G 0.11 5.10E-06 0.52 0.14 8 rs17098278 C T 0.22 6.85E-06 0.61 0.11 9 rs10220484 C T 0.31 6.49E-06 1.55 0.10 10 rs2876278 G A 0.35 7.78E-06 1.51 0.09 11 rs4818919 G A 0.15 8.09E-06 1.73 0.12

[0209] A1: Minor allele

[0210] A2: Major allele

[0211] MAF: minor allele frequency

[0212] OR: odds ratio of A1 allele

[0213] SE: standard error of OR

[0214] In addition, the three traits of "Cold part_waist and legs", "Cold part_hands and feet", and "Cold part_whole body" showed suggestive associations with the SNPs in 6 regions, 8 regions, and 7 regions shown in Table 4, respectively. When the OR value is less than 1.0, the major allele (A2) is the risk allele; when the OR value is greater than 1.0, the minor allele (A1) is the risk allele.

[0215] Therefore, for the lead SNP of "Cold Location_Waist and Leg", the major allele in rs13376933 is the risk allele for the lower body type of cold intolerance, while the minor alleles in rs750707, rs76388277, rs6819504, rs9994962 and rs72719006 are the risk alleles for the lower body type of cold intolerance.

[0216] In addition, for the lead SNP of "Cold Location_Hands and Feet", the major alleles in rs1452609 and rs849057 are risk alleles for the extremity type cold fear type, while the minor alleles in rs556161, rs10911744, rs61998228, rs6476958, rs6518186 and rs4818919 are risk alleles for the extremity type cold fear type.

[0217] Furthermore, for "Chilling Location_Whole Body", the minor alleles in all the lead SNPs were risk alleles for the systemic chills type.

[0218] [Table 4]

[0219] serial number Types of cold intolerance Lead SNP A1 A2 MAF P OR SE a Lower body type rs750707 G A 0.42 3.42E-07 2.12 0.15 b Lower body type rs76388277 C A 0.09 1.25E-06 2.61 0.20 C Lower body type rs6819504 C T 0.33 2.86E-06 2.00 0.15 d Lower body type rs9994962 C A 0.31 5.08E-06 2.02 0.15 e Lower body type rs72719006 C G 0.09 7.22E-06 2.66 0.22 f Lower body type rs13376933 A C 0.36 4.96E-06 0.49 0.16 g Extremity type rs556161 G A 0.45 7.96E-06 1.55 0.10 h Extremity type rs10911744 C T 0.47 6.61E-06 1.54 0.10 i Extremity type rs1452609 G A 0.42 2.80E-06 0.63 0.10 j Extremity type rs61998228 T C 0.10 7.46E-06 2.12 0.17 k Extremity type rs849057 C T 0.36 8.66E-06 0.63 0.11 k Extremity type rs6476958 T G 0.47 8.92E-06 1.57 0.10 m End-of-limb type rs6518186 C T 0.29 6.49E-06 1.62 0.11 n Extremity type rs4818919 G A 0.15 1.76E-06 1.89 0.13 o Whole body type rs12615626 A G 0.15 6.14E-06 3.01 0.24 p Whole body type rs1513696 C T 0.08 9.76E-06 3.59 0.29 q Whole body type rs79196079 G A 0.11 3.59E-07 4.00 0.27 r Whole body type rs9777637 T A 0.17 5.03E-07 3.81 0.27 s Whole body type rs78458106 T C 0.07 2.17E-06 4.36 0.31 t Whole body type rs 150099343 A G 0.20 9.05E-06 2.73 0.23 U Whole body type rs6506395 G A 0.05 2.28E-06 4.54 0.32

[0220] The description of each item in the column of the table is the same as Table 3.

Claims

1. A method for determining the genetic cause of a subject's cold constitution in vitro, comprising: A cold constitution determination SNP detection step, detecting single nucleotide polymorphisms (cold constitution determination SNPs) associated with cold constitution based on nucleic acid from the subject; as well as, The cold constitution determination step is to determine that the subject has a genetic cause of cold constitution when at least one of the bases in the detected alleles of the cold constitution determination SNP is a risk allele.

2. The method according to claim 1, wherein: The cold constitution determination SNP 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 in linkage disequilibrium with the risk allele of any one of 1 to 11 cold constitution determination SNPs [Table 1] 。 3. The method according to claim 2, wherein: The linkage disequilibrium coefficient between the SNPs is greater than or equal to 0.

8.

4. A method for determining in vitro the genetic cause of a subject's type of chills, comprising: A chills type determination SNP detection step, detecting a single nucleotide polymorphism (chills type determination SNP) based on a chills occurrence site based on nucleic acid from a subject; as well as, The step of determining the type of cold sensitivity is to determine that the cold sensitivity of the subject will occur at a site of the type of cold sensitivity when at least one of the bases in the detected alleles of the cold sensitivity type determination SNP is a risk allele.

5. The method according to claim 4, wherein: The type of chills is any one of lower body type, extremity type or whole body type.

6. The method according to claim 5, wherein: The cold fear type determination SNP is any one of a to u in Table 2 below, and the risk allele is a base shown in Table 2 below, or any one of the cold fear type determination SNPs a to u is a SNP in linkage disequilibrium with the risk allele shown in Table 2 [Table 2] 。 7. The method according to claim 6, wherein: The linkage disequilibrium coefficient between the SNPs is greater than or equal to 0.

8.

8. The method according to claim 4, wherein: The subject is a subject determined to have a genetic cause of cold constitution by the determination method according to any one of claims 1 to 3.

9. A device or kit for detecting the genetic cause of a cold constitution, comprising: A probe that can specifically bind to a cold constitution determination SNP associated with a cold constitution and / or a SNP in linkage disequilibrium with the cold constitution determination SNP and nucleotides in the vicinity thereof; and / or, A primer set that specifically recognizes and amplifies the cold constitution determining SNP and / or a SNP in linkage disequilibrium with the cold constitution determining SNP and nucleotides in the vicinity thereof.

10. The device or kit according to claim 9, wherein: The cold constitution determination SNP is any one of 1 to 11 in Table 1 of claim 2.

11. A device or kit for determining the type of chills, comprising: A probe that can specifically bind to a SNP for determining the type of chills based on the site of occurrence of chills and / or a SNP in linkage disequilibrium with the SNP for determining the type of chills and nucleotides in the vicinity thereof; and / or, A primer set that specifically recognizes and amplifies the chills type determining SNP and / or a SNP in linkage disequilibrium with the chills type determining SNP and nearby nucleotides.

12. The device or kit according to claim 11, wherein: The chills type determination SNP 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 the probe and / or primer set according to claim 9 or 10.

14. A biomarker consisting of a cold constitution determination SNP for determining the genetic cause of a cold constitution.

15. The biomarker according to claim 14, wherein The cold constitution determination SNP 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 a chills type determining SNP for determining a chills type.

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

18. The biomarker according to claim 16, wherein The type of chills is extremity type, and the chills type determination SNP 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 one of the SNPs selected from the group.

19. The biomarker according to claim 16, wherein The type of chills is systemic, and the chills type determination SNP is a SNP selected from the group consisting of rs12615626, rs1513696, rs79196079, rs9777637, rs78458106, rs150099343 and rs6506395, or a SNP in linkage disequilibrium with any one of the SNPs selected from the group.

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