KR102348688B1 - SNP markers for diagnosing Cold Hands/Feet Syndrome and use thereof - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphism (SNP) marker for diagnosing cold syndrome, and more particularly, a composition for diagnosing cold syndrome including a cold syndrome-specific SNP marker, a kit or microarray comprising the composition, a method for diagnosing cold syndrome using the marker, and poor circulation It relates to a method for screening a diagnostic SNP.

Description

SNP markers for diagnosing Cold Hands/Feet Syndrome and use thereof

The present invention relates to a single nucleotide polymorphism (SNP) marker for diagnosing cold syndrome, and more particularly, a composition for diagnosing cold syndrome including a cold syndrome-specific SNP marker, a kit or microarray comprising the composition, a method for diagnosing cold syndrome using the marker, and poor circulation It relates to a method for screening a diagnostic SNP.

Coldness refers to a state of feeling cold at a temperature that other people do not feel cold. The cold symptoms generally appear as cold hands and feet, which means that the hands or feet become cold at a temperature that other people do not feel cold, and the discomfort in daily life is great.

Causes of poor circulation include decreased physical strength due to gastrointestinal disorders, anemia, hypotension, constriction of capillaries due to autonomic nerve abnormalities, congestion in the pelvis, water metabolism disorder, postpartum wind, vasculitis, scleroderma, hypertension, diabetes, hyperlipidemia, etc. Blood circulation disorders due to arteriosclerosis, etc. caused by diseases may be mentioned, and may appear accompanying the above-mentioned cause diseases. In particular, in women, changes in female hormones such as menstruation, childbirth, and menopause affect the autonomic nervous system, and when an external stimulus such as cold is received, the sympathetic nerve becomes sensitive and blood vessels constrict, which leads to a decrease in blood supply to peripheral parts such as hands and feet. You may feel cold.

Although the poor circulation is a disease that causes inconvenience in daily life, a scientific and high-precision diagnostic method for this has not yet been developed. Therefore, there is an urgent need to develop a method for diagnosing poor circulation with high accuracy, reproducibility and reliability.

In addition, the genetic factors involved in temperature are highly correlated with metabolic-related diseases such as obesity, cholesterol levels, heart disease, and diabetes. It is considered that other prescriptions for metabolic diseases caused by this are possible.

Under this background, the present inventors conducted research on SNPs related to poor circulation based on genetic factors related to temperature, and as a result of efforts to find markers that can scientifically and objectively diagnose poor circulation, specific SNPs It was confirmed that it has a specific genotype, and the present invention was completed.

One object of the present invention is to provide a composition for diagnosing cold syndrome comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing cold syndrome or a primer capable of amplifying it.

Another object of the present invention is to provide a kit for diagnosing cold syndrome comprising the composition for diagnosing cold syndrome.

Another object of the present invention is to provide a microarray for diagnosis of cold syndrome, which includes a probe capable of detecting the SNP marker for diagnosis of cold syndrome or an agent capable of amplifying it.

Another object of the present invention is to provide a method of providing information for predicting or diagnosing poor circulation.

Another object of the present invention is to provide a method for selecting a SNP marker for diagnosing poor circulation.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

In order to achieve the above object, one aspect of the present invention provides a composition for diagnosis of cold syndrome comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosis of cold syndrome or a primer capable of amplifying it.

Specifically, the SNP marker for diagnosis of cold syndrome may be one or more polynucleotides selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 18 and combinations thereof, or a polynucleotide complementary thereto. More specifically, the SNP marker for diagnosing cold syndrome includes one or more polynucleotides selected from polynucleotides comprising the 20th base in each base sequence as SNP and consisting of 5 to 100 consecutive bases, or a complementary polynucleotide thereof. may include.

As used herein, the term “coldness” refers to a state in which a person feels cold at a temperature that other people do not feel cold. In the present invention, the cold syndrome may be a condition in which hands or feet feel cold and cold at a temperature that other people do not feel cold, that is, cold hands and feet, but is not limited thereto.

If it is possible to provide a cold syndrome-specific marker, it is expected that a more accurate diagnosis of poor circulation can be made. Therefore, the present inventors first provided a cold syndrome-specific SNP marker for diagnosing poor circulation.

As used herein, the term “diagnosis” includes any type of analysis used to determine or discriminate cold syndrome.

As used herein, the term "polymorphism" refers to a case in which two or more alleles exist at one locus, and among polymorphic sites, only a single base differs from one person to another. It is called single nucleotide polymorphism (SNP). A specific polymorphic marker has two or more alleles that exhibit an incidence of at least 1%, more specifically at least 5% or 10%, in a selected population.

As used herein, the term “allele” refers to several types of one gene present at the same locus on homologous chromosomes. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.

The term "rs_id" used in the present invention means rs-ID, which is an independent marker given to all SNPs initially registered by the NCBI, which has been accumulating SNP information since 1998. In the present invention, it was described in the same form as rs150186451. rs_id described in this table means a SNP marker, which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily identify the position and sequence of the SNP using the rs_id. The specific sequence corresponding to rs_id, which is the NCBI's dbSNP (The Single Nucleotide Polymorphism Database) number, may change slightly over time. It will be apparent to those skilled in the art that the scope of the present invention also extends to such altered sequences.

The "SEQ ID NOs: 1 to 18" is a polymorphic sequence comprising a polymorphic site. The polymorphic sequence refers to a sequence including a polymorphic site including a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

The SNP marker of the present invention is a marker shown in Table 1, and when the polymorphic site of the SNP marker of the individual is the allele indicated by the effect allele, it is possible to diagnose the coldness of the individual.

As used herein, the term "probe" refers to an allele-specific probe, in which a polymorphic site exists in nucleic acid fragments derived from two members of the same species, so that it hybridizes to a DNA fragment derived from one member, but is derived from another member. There is no hybridization in one fragment. In this case, the hybridization conditions must be sufficiently stringent to hybridize to only one of the alleles, showing a significant difference in the intensity of hybridization between alleles. This can lead to good hybridization differences between different allelic forms.

As used herein, the term "probe capable of detecting a SNP marker for diagnosis of poor circulation" refers to a composition capable of diagnosing poor circulation by specifically hybridizing with the polymorphic site of the SNP of the present invention. A specific method of gene analysis is not particularly limited, and any gene detection method known in the art to which the present invention pertains may be used.

The probe can be used in a method for diagnosing cold syndrome by detecting an allele. The prediction or diagnosis method includes detection methods based on hybridization of nucleic acids, such as Southern blotting, and may be provided in a form previously bound to a substrate of a DNA chip in a method using a DNA chip. The hybridization may be usually performed under stringent conditions, for example, a salt concentration of 1M or less and a temperature of 25° C. or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 °C may be suitable for allele-specific probe hybridization.

As used herein, the term "primer" refers to a nucleotide sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and prevent template strand copying. A short sequence that serves as a starting point for Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. By performing PCR amplification, it is possible to predict cold syndrome-specific metabolic syndrome through the generation of a desired product. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.

As used herein, the term "primer capable of amplifying the SNP marker for diagnosis of cold syndrome" refers to a composition capable of diagnosing cold syndrome by confirming the polymorphic site of the SNP of the present invention through amplification, specifically, the marker for diagnosis of cold syndrome. It refers to a primer capable of specifically amplifying a polynucleotide of

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

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

In the present invention, the SNP marker for diagnosing cold syndrome is rs150186451 described in SEQ ID NO: 1 among the polynucleotides consisting of SEQ ID NOs: 1 to 18, wherein the 20th base is G or A, 5 to 100 including the 20th base a polynucleotide consisting of consecutive bases, or a complementary polynucleotide thereof;

The polynucleotide of rs75132839 set forth in SEQ ID NO: 2, wherein the 20th base is T or C, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs1436897 set forth in SEQ ID NO: 3, wherein the 20th base is C or T, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs60473221 set forth in SEQ ID NO: 4, wherein the 20th base is C or G, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs79924644 set forth in SEQ ID NO: 5, wherein the 20th base is T or C, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs1317437 set forth in SEQ ID NO: 6, wherein the 20th base is T or C, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs28558876 set forth in SEQ ID NO: 7, wherein the 20th base is G or T, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs2560299 set forth in SEQ ID NO: 8, wherein the 20th base is G or A, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide according to rs114675604 set forth in SEQ ID NO: 9, wherein the 20th base is C or T, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide according to rs7782355 set forth in SEQ ID NO: 10, wherein the 20th base is T or C, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

The polynucleotide of rs2961149 set forth in SEQ ID NO: 11, wherein the 20th base is G or T, the polynucleotide consisting of 5 to 100 consecutive bases including the 20th base, or a complementary polynucleotide thereof;

In rs2961161 set forth in SEQ ID NO: 12, at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base in which the 20th base is G or C, or a complementary polynucleotide thereof polynucleotides;

In rs76371309 set forth in SEQ ID NO: 13, at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base in which the 20th base is C or A, or a complementary polynucleotide thereof polynucleotides;

rs189591476 set forth in SEQ ID NO: 14, wherein at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base in which the 20th base is G or T, or a complementary polynucleotide thereof polynucleotides;

In rs141241568 set forth in SEQ ID NO: 15, at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base in which the 20th base is T or C, or a complementary polynucleotide thereof polynucleotides;

In rs2286425 set forth in SEQ ID NO: 16, at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base in which the 20th base is T or C, or a complementary polynucleotide thereof polynucleotides;

In rs68140471 set forth in SEQ ID NO: 17, at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base in which the 20th base is G or A, or a complementary polynucleotide thereof polynucleotides;

In rs73161005 set forth in SEQ ID NO: 18, at least one selected from the group consisting of a polynucleotide consisting of 5 to 100 consecutive bases including the 20th base where the 20th base is C or T, or a complementary polynucleotide thereof polynucleotides; Or it may include one or more selected from the group consisting of combinations thereof.

In addition, the SNP marker of the present invention may be a set of SNP markers including 2 or more of the above SNP markers, more specifically 3 to 11 or more, and most specifically all 18 of the SNP markers. The marker can be diagnosed as poor circulation when the 20th base of each SNP marker is the effect allele base of Table 1, and the higher the number of SNP markers in the subject, the higher the accuracy can be.

Specifically, the SNP marker is A in the case of the 20th base of SEQ ID NO: 1; C for the 20th base of SEQ ID NO: 2; T for the 20th base of SEQ ID NO: 3; G for the 20th base of SEQ ID NO: 4; C for the 20th base of SEQ ID NO: 5; C for the 20th base of SEQ ID NO:6; T for the 20th base of SEQ ID NO: 7; A for the 20th base of SEQ ID NO: 8; T for the 20th base of SEQ ID NO: 9; C for the 20th base of SEQ ID NO: 10; T for the 20th base of SEQ ID NO: 11; C for the 20th base of SEQ ID NO: 12; A for the 20th base of SEQ ID NO: 13; T for the 20th base of SEQ ID NO: 14; C for the 20th base of SEQ ID NO: 15; C for the 20th base of SEQ ID NO: 16; A for the 20th base of SEQ ID NO: 17; In the case of the 20th base of SEQ ID NO: 18, T; may be included as an effect allele base, but is not limited thereto.

The present inventors have demonstrated that the SNP is a marker for cold diagnosis as follows.

Specifically, coldness was defined as a subject who responded that both hands and feet were cold in the questionnaire. Thereafter, genomic data were produced using SNP chips, specifically PMRA (Precision Medicine Research Array) chips, for a total of 2000 subjects, including 700 people in the group showing cold symptoms and 1300 people in the group not showing cold symptoms. The above data were collected from 19 oriental hospitals from 2007 to 2012, and all of them had improved poor circulation due to constitutional prescription. In the case of genotypes with deletions in the PMRA chip, LD (linkage disequilibrium) blocks are calculated through haplotype phasing for the 2,000 population, and 400 Asians provided by the 1000 Genome Project An imputation was performed by creating a panel for the Imputated SNPs were selected as markers when the results of association were derived for full-length SNPs through genome-wide association analysis, and when P<0.00005 and a missing rate were 0. The selected markers are shown in Table 1.

Another aspect of the present invention provides a kit for diagnosing cold syndrome comprising the composition for diagnosing cold syndrome.

The terms used herein are the same as described above.

The kit may be an RT-PCR kit or a kit for DNA analysis (eg, a DNA chip).

The kit of the present invention can diagnose cold syndrome by amplifying the SNP marker, which is a marker for diagnosis of poor circulation, or by checking the expression level of the mRNA by checking the expression level of the SNP marker.

In a specific embodiment, the kit of the present invention may be a kit for diagnosing cold syndrome including essential elements necessary for performing a DNA chip. A DNA chip kit is a method in which nucleic acid species are attached in a gridd array to a generally flat solid support plate, typically a glass surface no larger than a microscope slide, and the nucleic acids are uniformly arranged on the chip surface, and the DNA chip It is a tool that allows multiple hybridization reactions to occur between the nucleic acids on the chip surface and the complementary nucleic acids contained in the solution treated on the chip surface, enabling massively parallel analysis.

Another aspect of the present invention provides a microarray for diagnosis of cold syndrome, comprising a probe capable of detecting the SNP marker for diagnosis of cold syndrome or an agent capable of amplifying it.

The terms used herein are the same as described above.

The microarray may include DNA or RNA polynucleotides. The microarray consists of a conventional microarray, except that it contains a probe capable of detecting the SNP marker for cold diagnosis of the present invention or an agent capable of amplifying it.

Specifically, the microarray includes one or more polynucleotides selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 18 and combinations thereof or a complementary polynucleotide thereof, and the nucleotide sequence includes the 20th nucleotide as a SNP. can More specifically, the 20th base of SEQ ID NO: 1 is A; The 20th base of SEQ ID NO: 2 is C; The 20th base of SEQ ID NO: 3 is T; The 20th base of SEQ ID NO: 4 is G; The 20th base of SEQ ID NO: 5 is C; The 20th base of SEQ ID NO: 6 is C; The 20th base of SEQ ID NO: 7 is T; The 20th base of SEQ ID NO: 8 is A; The 20th base of SEQ ID NO: 9 is T; The 20th base of SEQ ID NO: 10 is C; The 20th base of SEQ ID NO: 11 is T; The 20th base of SEQ ID NO: 12 is C; The 20th base of SEQ ID NO: 13 is A; The 20th base of SEQ ID NO: 14 is T; The 20th base of SEQ ID NO: 15 is C; The 20th base of SEQ ID NO: 16 is C; The 20th base of SEQ ID NO: 17 is A; The 20th base of SEQ ID NO: 18 may be T;, but is not limited thereto.

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

In another aspect of the present invention, (a) polymorphism comprising one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 18 and combinations thereof from DNA of a sample isolated from an individual, or a SNP of a complementary polynucleotide thereof amplifying the site or hybridizing with the probe; (b) determining the base of the amplified or hybridized polymorphic site of step (a); And (c) when the SNP of the base sequence determined in step (b) includes an effect allele base, diagnosing the subject as cold syndrome. Provides a method for predicting or diagnosing poor circulation. .

As used herein, the term “subject” may be a subject for predicting or diagnosing poor circulation. DNA may be obtained from samples such as hair, urine, blood, various body fluids, tissues, cells, or saliva isolated from the subject, but is not limited thereto.

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

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

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

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

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

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

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

Then, as in step (c), among the nucleotide sequences determined in step (b), in rs150186451 described in SEQ ID NO: 1, when the 20th base is A; In rs75132839 set forth in SEQ ID NO: 2, when the 20th base is C; in rs1436897 set forth in SEQ ID NO: 3, when the 20th base is T; In rs60473221 set forth in SEQ ID NO: 4, when the 20th base is G; In rs79924644 set forth in SEQ ID NO: 5, when the 20th base is C; for rs1317437 set forth in SEQ ID NO: 6, when the 20th base is C; In rs28558876 set forth in SEQ ID NO: 7, when the 20th base is T; In rs2560299 set forth in SEQ ID NO: 8, when the 20th base is A; for rs114675604 set forth in SEQ ID NO: 9, wherein the 20th base is T; In rs7782355 set forth in SEQ ID NO: 10, when the 20th base is C; for rs2961149 set forth in SEQ ID NO: 11, wherein the 20th base is T; In rs2961161 set forth in SEQ ID NO: 12, when the 20th base is C; In rs76371309 set forth in SEQ ID NO: 13, when the 20th base is A; rs189591476 set forth in SEQ ID NO: 14, wherein the 20th base is T; In rs141241568 set forth in SEQ ID NO: 15, when the 20th base is C; for rs2286425 set forth in SEQ ID NO: 16, wherein the 20th base is C; In rs68140471 set forth in SEQ ID NO: 17, when the 20th base is A; In rs73161005 set forth in SEQ ID NO: 18, when the 20th base is T; Or a combination thereof; that is, when the SNP contains an effector allele base, the subject can be diagnosed as poor circulation.

Another aspect of the present invention provides a method for selecting a SNP marker for diagnosis of cold syndrome.

Specifically, the method comprises the steps of: (a) producing genomic data with a SNP chip for a cold group including individuals who responded that both hands and feet are cold and a group not including the individual; (b) performing an imputation in the case of a genotype in which a deletion exists in the SNP chip; (c) analyzing the imputed SNPs for genome-wide association; and (d) selecting the SNP for diagnosing cold syndrome when P<0.00005 and the Missing Rate are 0 as a result of the analysis.

In step (b), in the case of a genotype in which a deletion exists in the SNP chip, the haplotype including both the cold syndrome group including the individuals who responded that both the hands and feet of step (a) are cold and the group not including the individual It may be to calculate the linkage disequilibrium (LD) block through haplotype phasing, generate a panel for about 400 Asians provided by the 1000 Genome Project, and execute the imputation, but this may be limited doesn't happen

The step (c) may be to derive a result of association of the imputed SNPs to the full-length SNP target through full-genome association analysis, but is not limited thereto.

The SNP chip may specifically be a PMRA (Precision Medicine Research Array) chip, but is not limited thereto.

The SNPs selected through the above method are rs150186451, rs75132839, rs1436897, rs60473221, rs79924644, rs1317437, rs28558876, rs2560299. It may be one or more selected from the group consisting of rs114675604, rs7782355, rs2961149, rs2961161, rs76371309, rs189591476, rs141241568, rs2286425, rs68140471, rs73161005, and combinations thereof, but is not limited thereto.

The SNP marker of the present invention is a cold syndrome-specific SNP marker, and can accurately and objectively diagnose cold syndrome.

1 is a schematic flowchart of a method for screening SNPs of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Example 1. Selection of markers related to poor circulation

Cold syndrome-related markers were selected in the same order as in FIG. 1 .

Specifically, in the questionnaire about poor circulation, the subjects who responded that both hands and feet were cold was defined as poor circulation. Thereafter, genomic data was produced using a PMRA (Precision Medicine Research Array) chip for a total of 2000 subjects, including 700 people in the group showing cold symptoms and 1300 people in the group not showing cold symptoms. The above data were collected from 19 oriental hospitals from 2007 to 2012, and all of them had improved poor circulation due to constitutional prescription. In the case of genotypes with deletions in the PMRA chip, LD (linkage disequilibrium) blocks are calculated through haplotype phasing for the 2,000 population, and 400 Asians provided by the 1000 Genome Project An imputation was performed by creating a panel for the Imputated SNPs were selected as markers when the results of association were derived for full-length SNPs through genome-wide association analysis, and when P<0.00005 and a missing rate were 0.

The selected markers are shown in Table 1 below.

SEQ ID NO: Chromosome Position SNPid Ref ALT Odd Ratio P value Gene One One 109388739 rs150186451 G A 2.37966 3.859E-06 AKNAD1 2 One 120076422 rs75132839 T C 2.14555 3.624E-05 GAPDHP32 3 One 217005202 rs1436897 C T 1.39171 9.614E-06 ESRRG 4 2 46013166 rs60473221 C G 1.65979 1.18E-05 PRKCE 5 3 106655578 rs79924644 T C 1.57234 1.914E-05 LINC00882 6 4 180878476 rs1317437 T C 0.647211 1.312E-05 7 4 180879888 rs28558876 G T 0.649254 1.596E-05 8 5 5448263 rs2560299 G A 1.34967 3.518E-05 ICE1 9 7 101131018 rs114675604 C T 2.07366 4.088E-06 COL26A1 10 7 105665364 rs7782355 T C 2.73356 4.325E-05 CDHR3 11 7 143807515 rs2961149 G T 0.678533 3.46E-05 OR2A2 12 7 143826697 rs2961161 G C 0.67585 3.171E-05 OR2A14 13 10 43982069 rs76371309 C A 2.52918 1.191E-05 ZNF487 14 11 35349822 rs189591476 G T 1.94031 1.326E-05 SLC1A2 15 12 108847287 rs141241568 T C 2.84962 4.083E-05 LINC01498 16 14 74815239 rs2286425 T C 1.37022 1.395E-05 VRTN 17 18 42801202 rs68140471 G A 1.59295 8.532E-06 SLC14A2 18 22 30143240 rs73161005 C T 1.5776 1.406E-05 ZMAT5

Example 2. Functional Region Mapping of SNPs

Functional scores were assigned to SNPs by learning specific patterns of epigenetic information present at the positions of each SNP. The epigenetic information is a DNase Hypersensitive site (DHS) provided by the ENCODE project (The ENCODE Project Consortium, 2012) and the Roadmap Epigenomics Consortium, 2015), histone modifications, target gene functions, transcriptional regulators are combined It is a gene region contained within the The epigenetic information relates to a site to which a protein responsible for regulating the expression of nearby genes is attached or a metabolic pathway in which the gene acts. Based on this, a function score is assigned to the SNP. That is, binary data was constructed in which 1 is the case where the SNP corresponds to the functional region and 0 is 0 when the SNP does not correspond to the functional region.

Example 3. SNP function scoring to which a deep learning algorithm is applied

A deep learning framework based on convolutional neural networks was used to extract biological function patterns existing in genetic regions that are statistically related to constitutional values.

Specifically, after the whole genomes were sorted in the order of strongest association based on the p-value, the SNP with the strongest association was selected as the lead SNP, and the lead SNPs were selected so as not to overlap within 1 Mb of each other.

The correlation of the genome was calculated as a value of a phenotype according to a genotype allele type. The degree of association was interpreted as a statistical value of odds ratio or beta value, and significance was determined as a p-value. Candidate SNPs were determined by setting the p-value to cut off, and leadSNPs were sequentially selected by sorting them.

A block was defined by selecting up to 30 SNPs in the order of highest relevance among ^{SNPs with a p-value of 5 x 10 -4} or less among one lead SNP and candidate SNPs within 1 Mb of its surrounding.

One generated SNP section becomes one training image data, and based on the fact that there is at least one causal variation within this section, the convolutional neural network framework was used to learn the characteristics of the cause variation.

The model used for learning can be divided into a pattern search part and a part scoring function scores for each SNP, and the following process was performed.

3-1. Construction of input images from whole genome association analysis results

After sorting in the order of strong association based on the p-value for the whole genome, the lead SNPs were selected so that they did not overlap within 1 Mb, starting with the strongest SNP. Among candidate SNPs including one lead SNP and a SNP selected within 1 Mb of its surrounding, in the order of highest relevance, up to 30 SNPs were defined as a block. At this time, training image data defined as one image is generated from one section, and the causal variation is used using a CNN framework that can extract partial features of the image by using the fact that there is at least one causal variation within this section. characteristics were learned.

3-2. model design

The CNN framework consists of pattern detectors that can be tuned. The pattern searchers are called a kernel or a filter, which detects a partial pattern with local connectivity and repeatedly performs an operation on the entire input field.

3-3. Denoising- Pre-training using Auto Encoder (DA)

The auto-encoder can learn more general features of the training data before fine-tuning the kernels in deep learning, and start learning from a more optimized point so as not to fall into a local minimum. It is an unsupervised learning technique. Pre-learning was performed using denoising-autoencoder (DA), which learns by mixing random noise with the input value so that the model can learn more robust features.

3-4. hyper parameter

A deep learning model depends on a number of hyperparameters, each of which significantly affects the performance of the model. Hyperparameters are used in the noise level (corruption level) applied to the input value in the DA pre-learning process, the learning rate in the fine-tuning process, momentum (momemtum), and the elastic net regularization process parameters are included.

Optimized conditions were found based on a set of randomly selected hyperparameters.

3-5. Functional scoring of SNPs

In the first layer (convolution layer) C1, an image matrix defining one first layer input image is input. A convolution process was performed using the first layer input image and the first layer kernel, which is the convolution kernel, to generate a matrix for whether each candidate SNP matches each type of pattern. Each element of the array representing the first layer kernel has a value of 0 or 1.

In the second layer (convolution layer) (C2), it was possible to analyze nonlinear complex patterns based on the extracted pattern results. Each element of the array in the second layer kernel has a value of 0 or 1.

The closer the prediction score to 1, the more common control patterns in risk genetic sections were detected.

Finally, the score finally derived by performing max-pooling was derived as the score of the SNP that best matched the risk pattern within one association section.

Example 4. Cold syndrome prediction method

A total of 2000 people, including 700 people in the group showing poor circulation and 1300 people in the group not showing poor circulation of Example 1, were divided into training data (70%) and test data (30%) and classified, In order to overcome the numerical difference between the groups, which is not hypersensitivity, up-sampling (in all subjects, there are two groups A and B, and if the difference in the number of groups A and B is large, the number of the large group is Sampling method in which a small group is sampled to match the number of large groups), down (the number of small groups when there are two groups A and B in all subjects and the difference between the numbers of groups A and B is large) (a sampling method in which the large group is sampled to fit the same sampling method as the number of small groups) was applied and analyzed. The training and test data were randomly generated to create a classification model, and the one with the best classification performance was adopted by repeating the entire process 100 times. In this case, data mining methods such as a random forest, a support vector machine, and a neural network were used.

Table 2 shows the average AUC value of the classification model developed using the data mining method and the up and down sampling method on the coldness data of the 2,000 people.

Model cold classification RF_UP 0.723191 RF_DOWN 0.723852 RF 0.726737 SVM_UP 0.736364 SVM_DOWN 0.736864 SVM 0.736524 NN_UP 0.705419 NN_DOWN 0.699588 NN 0.711891

* RF: Random Forest, RF_UP: RF up sampling, RF_DOWN: RF down sampling, SVM: Support Vector Machine,SVM_UP: SVM up sampling,

SVM_DOWN: SVM down sampling,

NN: Neural Network,

NN_UP: NN up sampling,

NN_DOWN: NN down sampling

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

<110> Korea Institute of Oriental Medicine <120> SNP markers for diagnosing Cold Hands/Feet Syndrome and use it <130> KPA200387-KR <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 45 <212> DNA <213> Unknown <220> <223> rs150186451 <400> 1 tgtgtaacca ataagtagag gcacgacatc cgctccaaaa tccaa 45 <210> 2 <211> 45 <212> DNA <213> Unknown <220> <223> rs75132839 <400> 2 gtcgatcacc cttttgctat agccaaattc attgtcatac cagaa 45 <210> 3 <211> 45 <212> DNA <213> Unknown <220> <223> rs1436897 <400> 3 gaaagatgtt tgcaagtcac tgggcactta catgtaaata tatat 45 <210> 4 <211> 45 <212> DNA <213> Unknown <220> <223> rs60473221 <400> 4 ctcagcgctg ctgaggggtc tagagtctca cagccctgct gggtc 45 <210> 5 <211> 45 <212> DNA <213> Unknown <220> <223> rs79924644 <400> 5 gttcccattc tccttccttt ctttgggaca caacttgcct tctag 45 <210> 6 <211> 45 <212> DNA <213> Unknown <220> <223> rs1317437 <400> 6 ggcgctaata acataactct gatccaatac ttgaacatgg ggaga 45 <210> 7 <211> 45 <212> DNA <213> Unknown <220> <223> rs28558876 <400> 7 aaaattgata actacatagg cggcaaatta tgacttgtaa gccaa 45 <210> 8 <211> 45 <212> DNA <213> Unknown <220> <223> rs2560299 <400> 8 aactagttgg agatataaca tctgcttttt tcaattaatg aatac 45 <210> 9 <211> 45 <212> DNA <213> Unknown <220> <223> rs114675604 <400> 9 ctgtggttgg ccaccctgcc cgtaagataa gccccacgccc tgtca 45 <210> 10 <211> 45 <212> DNA <213> Unknown <220> <223> rs7782355 <400> 10 tatgccgtgc tcttatttgt agaatattta aaggatagta tggaa 45 <210> 11 <211> 45 <212> DNA <213> Unknown <220> <223> rs2961149 <400> 11 ccctgtttca cagtgtcttt aatccaatgc tgaaccccct gatct 45 <210> 12 <211> 45 <212> DNA <213> Unknown <220> <223> rs2961161 <400> 12 ctttagttct catcctgagc ctgcccttct gcgggcctca tgaaa 45 <210> 13 <211> 45 <212> DNA <213> Unknown <220> <223> rs76371309 <400> 13 taaaaggcaa ataagagcac ctttatttat atcaaagtgg aactt 45 <210> 14 <211> 45 <212> DNA <213> Unknown <220> <223> rs189591476 <400> 14 agggttcact ccaactctgg ttcttcaccg ggaaggactc cccca 45 <210> 15 <211> 45 <212> DNA <213> Unknown <220> <223> rs141241568 <400> 15 gtaatggagt cagagcctat gcactcctgc agcagccccg aaagc 45 <210> 16 <211> 45 <212> DNA <213> Unknown <220> <223> rs2286425 <400> 16 aactggttct ggagtgagac gagctcggct ggggacgcta cttga 45 <210> 17 <211> 45 <212> DNA <213> Unknown <220> <223> rs68140471 <400> 17 agattttctt cttctaaatg ttgtctttgc tcttgtcttt cagtc 45 <210> 18 <211> 45 <212> DNA <213> Unknown <220> <223> rs73161005 <400> 18 ccagcatgta gcaggcatgc gatccatgct cattccttgt ttgtg 45

Claims (8)

a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 15 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
a probe capable of detecting the twentieth base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 17 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying; and
A probe capable of detecting the 20th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 18 or the corresponding base of the complementary polynucleotide thereof or a primer capable of specifically amplifying;
The 20th base of SEQ ID NO: 1 is A; The 20th base of SEQ ID NO: 2 is C; The 20th base of SEQ ID NO: 3 is T; The 20th base of SEQ ID NO: 4 is G; The 20th base of SEQ ID NO: 5 is C; The 20th base of SEQ ID NO: 6 is C; The 20th base of SEQ ID NO: 7 is T; The 20th base of SEQ ID NO: 8 is A; The 20th base of SEQ ID NO: 9 is T; The 20th base of SEQ ID NO: 10 is C; The 20th base of SEQ ID NO: 11 is T; The 20th base of SEQ ID NO: 12 is C; The 20th base of SEQ ID NO: 13 is A; The 20th base of SEQ ID NO: 14 is T; The 20th base of SEQ ID NO: 15 is C; The 20th base of SEQ ID NO: 16 is C; The 20th base of SEQ ID NO: 17 is A; The 20th base of SEQ ID NO: 18 is T;
A kit for diagnosis of poor circulation comprising the composition of claim 1.
The kit according to claim 2, wherein the kit is an RT-PCR kit or a DNA chip kit.
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 15 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 17 or a complementary polynucleotide thereof; and
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 18 or a complementary polynucleotide thereof; includes, wherein the nucleotide sequence includes the 20th nucleotide as SNP (single nucleotide polymorphism),
The 20th base of SEQ ID NO: 1 is A; The 20th base of SEQ ID NO: 2 is C; The 20th base of SEQ ID NO: 3 is T; The 20th base of SEQ ID NO: 4 is G; The 20th base of SEQ ID NO: 5 is C; The 20th base of SEQ ID NO: 6 is C; The 20th base of SEQ ID NO: 7 is T; The 20th base of SEQ ID NO: 8 is A; The 20th base of SEQ ID NO: 9 is T; The 20th base of SEQ ID NO: 10 is C; The 20th base of SEQ ID NO: 11 is T; The 20th base of SEQ ID NO: 12 is C; The 20th base of SEQ ID NO: 13 is A; The 20th base of SEQ ID NO: 14 is T; The 20th base of SEQ ID NO: 15 is C; The 20th base of SEQ ID NO: 16 is C; The 20th base of SEQ ID NO: 17 is A; The 20th base of SEQ ID NO: 18 is T; a microarray for diagnosis of cold syndrome.
(a) from the DNA of the sample isolated from the subject,
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10, or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12, or a complementary polynucleotide thereof;
a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 15, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16, or a complementary polynucleotide thereof;
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 17, or a complementary polynucleotide thereof; and
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 18, or a polynucleotide complementary thereto; amplifying a polymorphic site including the SNP or hybridizing with a probe;
(b) determining the base of the amplified or hybridized polymorphic site of step (a); and
(c) of the nucleotide sequence determined in step (b),
In rs150186451 set forth in SEQ ID NO: 1, when the 20th base is A;
In rs75132839 set forth in SEQ ID NO: 2, when the 20th base is C;
in rs1436897 set forth in SEQ ID NO: 3, when the 20th base is T;
In rs60473221 set forth in SEQ ID NO: 4, when the 20th base is G;
In rs79924644 set forth in SEQ ID NO: 5, when the 20th base is C;
for rs1317437 set forth in SEQ ID NO: 6, when the 20th base is C;
In rs28558876 set forth in SEQ ID NO: 7, when the 20th base is T;
In rs2560299 set forth in SEQ ID NO: 8, when the 20th base is A;
for rs114675604 set forth in SEQ ID NO: 9, wherein the 20th base is T;
In rs7782355 set forth in SEQ ID NO: 10, when the 20th base is C;
for rs2961149 set forth in SEQ ID NO: 11, wherein the 20th base is T;
In rs2961161 set forth in SEQ ID NO: 12, when the 20th base is C;
In rs76371309 set forth in SEQ ID NO: 13, when the 20th base is A;
rs189591476 set forth in SEQ ID NO: 14, wherein the 20th base is T;
In rs141241568 set forth in SEQ ID NO: 15, when the 20th base is C;
for rs2286425 set forth in SEQ ID NO: 16, wherein the 20th base is C;
In rs68140471 set forth in SEQ ID NO: 17, when the 20th base is A;
In rs73161005 set forth in SEQ ID NO: 18, when the 20th base is T; or
Combinations thereof;
A method of providing information for predicting or diagnosing poor circulation, comprising judging the individual as having poor circulation.
The method of claim 5, wherein the nucleotide determination in step (b) comprises sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis, PCR-SSCP (PCR-single strand conformation polymorphism), PCR-RFLP (PCR-restriction fragment length polymorphism) and one or more methods selected from the group consisting of TagMan techniques, the method.
(a) producing genomic data with a SNP chip for a cold group including individuals who responded that both hands and feet are cold and a group not including the individual;
(b) performing an imputation in the case of a genotype in which a deletion exists in the SNP chip;
(c) analyzing the imputed SNPs for genome-wide association; and
(d) when the analysis result P < 0.00005 and the missing rate is 0, selecting the SNP marker for cold diagnosis;
The selected SNP markers are rs150186451, rs75132839, rs1436897, rs60473221, rs79924644, rs1317437, rs28558876, rs2560299. rs114675604, rs7782355, rs2961149, rs2961161, rs76371309, rs189591476, rs141241568, rs2286425, rs68140471 and rs73161005 A method for selecting a SNP marker for diagnosing poor circulation. delete

Images