KR102193658B1 - SNP markers for diagnosing Soeumin of sasang constitution and use thereof - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphism (SNP) marker for diagnosing soeumin (a type of sasang constitutional medicine) among sasang constitution. More specifically, the present invention relates to a composition for diagnosis of soeumin comprising a SNP marker specific to soeumin, a kit or a microarray containing the composition, a method for diagnosing soeumin among sasang constitution using the marker, and a method for selecting the SNP for diagnosis of soeumin.

Description

SNP markers for diagnosing Soeumin of sasang constitution and use thereof

The present invention relates to a SNP (single nucleotide polymorphism) marker for diagnosing noise in sasang constitution, and more specifically, a composition for diagnosis in noise including a specific SNP marker for noise, a kit or microarray containing the composition, and sasang constitution using the marker It relates to a method of providing information for predicting or diagnosing a noise person, and a method of selecting a SNP for diagnosis of a noise person.

Sasang Medicine is known as Korea's unique constitutional medicine, founded by Ijema about 100 years ago. Sasang Medicine classifies human constitution into four categories: sun, soyang, taeum, and noise, which synthesizes the proportional functional physiological phenomenon of a person's constitution, which is somewhat strong and weak, and sexuality, behavior, attitude, and appearance. And classified. At this time, the meaning of some (多少) means a functional physiological heosil (虛實), that is, strength and weakness (强弱).

The above four constitutions are known to show differences not only in reactivity to drugs, but also in appearance, body type, personality, book function, and diet. Ijema argued that through Donguisusebowon, which he wrote in 1894, that humans naturally have books and demeanor, and that the resulting emotions of happiness and sorrows act to create physiological phenomena, the physiology, pathology, diagnosis, discrimination, treatment and From drug to food intake, a new method was suggested that could be applied to clinical settings in connection with the constitution.

According to Donguisusebowon, the constitution is considered to remain unchanged for a lifetime if it is determined by nature, and it can be said that it is a biologically genetic trait. In relation to this, the constitution ratio in the population is Taeeumin: Soyangin: Soeumin = 5:3:2, which has remained almost unchanged for more than 100 years after the theory of Sasang constitution was asserted, and even in a study on the inheritance of constitution in twins and families It has been found that the genetic literacy by constitution reaches around 50%. Therefore, it is believed that the constitution determined in the mother's womb does not change throughout life, so constitution formation and genetic differences are considered to be highly related to each other.

On the other hand, the discrimination of the Sasang constitution has traditionally been performed by comprehensively using an individual's voice, appearance, body shape, personality, and various external biological indicators, and diagnosis is made based on subjective judgment by skilled experts. In modern times, various methods such as pulsing method, body shape index, questionnaire test method, facial shape classification method, drug use method, infrared imaging method and O-ring test method have been developed.

In addition, a constitutional questionnaire was devised for objectification of constitution classification, systematically synthesizing various constitution determination indicators to enable scientific constitution diagnosis, and an effort to find differences and associations by constitution by examining constitution in terms of genetics. This has been done.

However, although the genetic background of constitution has been established by the existing studies, it is difficult to classify it by the conventional genome analysis method due to the characteristics of the trait in which the genetic/environmental predisposition is complex. In addition, constitution classification using statistical data collected by oriental medicine hospitals and oriental clinics or patient information provided by the Korea Centers for Disease Control and Prevention and Health Insurance Corporation has a disadvantage in that the scale is very small and performance evaluation is difficult.

In addition, there are disclosed and registered patents related to the diagnosis of Sasang constitution, and exemplary patents are as follows. Registration No. 10-0455996 relates to a device for determining sasang constitution by measuring a body reaction after a finger is brought into contact with a golden ring and a silver ring; Registration No. 10-0427243 relates to a method for discriminating sasang constitution by analyzing the pitch of the voice; Registration No. 10-0563127 relates to a device that determines the sasang constitution through the pressure of the wrist when in contact with food or medicine suitable for the constitution. However, despite these studies, a scientific and highly accurate Sasang constitution diagnosis method has not yet been developed. Therefore, it is urgent to develop a method for diagnosing Sasang constitution with high accuracy, reproducibility and reliability.

Under this background, the present inventors conducted a study on SNPs related to the Sasang constitution based on the heritability of the Sasang constitution, and tried to find a marker capable of scientifically and objectively diagnosing the Sasang constitution. As a result, it was confirmed that the noise group among SNPs has a specific genotype in specific SNPs, and the present invention was completed.

One object of the present invention is to provide a diagnostic SNP (single nucleotide polymorphism) marker, which is noise in sasang constitution.

Another object of the present invention is to provide a diagnostic composition for noise-in comprising a probe capable of detecting the SNP marker for diagnostic use in noise-in or a primer capable of amplifying it.

Another object of the present invention is to provide a method of providing information for predicting or diagnosing a noise person comprising the step of confirming whether or not a SNP specifically expressed as a noise person is detected using a nucleotide sequence obtained from a sample collected from a subject. To provide.

Another object of the present invention is to provide a kit for diagnosis of noise, comprising a composition for diagnosis of the subject's noise.

Another object of the present invention is to provide a microarray for diagnosis of noise comprising a composition for diagnosis of the noise of a subject.

Another object of the present invention is to provide a method for selecting the SNP marker for diagnosis of the noise.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

In addition, those of ordinary skill in the art can recognize or ascertain using only routine experimentation a number of equivalents to the specific aspects of the invention described herein. Also, such equivalents are intended to be included in the present invention.

In order to achieve the above object, one aspect of the present invention provides a diagnostic SNP (single nucleotide polymorphism) marker for noise in sasang constitution.

The marker is specifically selected from polynucleotides consisting of 5 to 100 consecutive bases, including the 16th base of each sequence, which is the SNP position, in one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 15 It may be a diagnostic marker for noise, including one or more polynucleotides, or complementary polynucleotides thereof.

The term "polymorphism" used in the present invention refers to a case in which two or more alleles exist in one locus. Among polymorphic sites, only a single base differs from one person to another. It is called single nucleotide polymorphism (SNP). Specific polymorphic markers have two or more alleles that exhibit a frequency of occurrence of at least 1%, more specifically at least 5% or 10% in the selected population.

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

The term "rs_id" used in the present invention refers to rs-ID, which is an independent marker assigned to all SNPs initially registered by NCBI, which started accumulating SNP information from 1998. In the present invention, it is described in the same form as rs12667492. The rs_id described in this table means the SNP marker, which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily identify the location and sequence of the SNP using the rs_id. The specific sequence corresponding to the NCBI's dbSNP (The Single Nucleotide Polymorphism Database) number, rs_id, 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 the altered sequence.

The “SEQ ID NOs: 1 to 15” are polymorphic sequences including polymorphic sites. The polymorphic sequence refers to a sequence including a polymorphic site including 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 3, and when the polymorphic site of the SNP marker of the individual is an allele indicated as an effect allele, the sasang constitution of the individual can be determined as noise.

The term "sasang constitution" as used in the present invention means that a person's constitution is classified into four categories: Taeyangin, Soyangin, Taeeumin, and Soeuminin by Sasang Medicine. The constitution is congenital and does not change over a lifetime, so it is biologically genetic. It can be seen as having characteristics. Since there is a genetic difference for each constitution, if it is possible to provide a specific marker for the sasang constitution, it is expected that a more accurate diagnosis of the sasang constitution can be made. Was provided. For the purposes of the present invention, it provides a SNP marker specific to Taeeumin, Soyangin and Soeumin, specifically Soeumin, except for Taeyangin, whose frequency is very low in Koreans.

The term "diagnosis" as used in the present invention includes all types of analysis used to determine or discriminate sasang constitution.

Specifically, the SNP marker is a diagnostic SNP marker for noise in, among the polynucleotides consisting of SEQ ID NOs: 1 to 15, in rs12667492 described in SEQ ID NO: 1, the 16th base is A or G, including the 16th base 5 A polynucleotide consisting of from to 100 consecutive bases, or a complementary polynucleotide thereof;

In rs74028907 shown in SEQ ID NO: 2, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is T or G, or a complementary polynucleotide thereof;

In rs4909803 shown in SEQ ID NO: 3, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is G or T, or a complementary polynucleotide thereof;

A polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is A or G, or a complementary polynucleotide thereof according to rs11646443 shown in SEQ ID NO: 4;

In rs75575584 shown in SEQ ID NO: 5, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is A or G, or a complementary polynucleotide thereof;

In rs17669146 shown in SEQ ID NO: 6, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is C or T, or a complementary polynucleotide thereof;

A polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is G or A, or a complementary polynucleotide thereof according to rs4688636 shown in SEQ ID NO: 7;

In rs1017409 shown in SEQ ID NO: 8, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is C or T, or a complementary polynucleotide thereof;

In rs981760 shown in SEQ ID NO: 9, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is T or A, or a complementary polynucleotide thereof;

In rs1671413 shown in SEQ ID NO: 10, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is T or C, or a complementary polynucleotide thereof;

In rs7013390 shown in SEQ ID NO: 11, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is A or G, or a complementary polynucleotide thereof;

In rs10955446 shown in SEQ ID NO: 12, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is C or T, or a complementary polynucleotide thereof;

In rs7015202 shown in SEQ ID NO: 13, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is A or C, or a complementary polynucleotide thereof;

In rs1115971 shown in SEQ ID NO: 14, a polynucleotide consisting of 5 to 100 consecutive bases including a 16th base in which the 16th base is C or T, or a complementary polynucleotide thereof;

In rs7138393 described in SEQ ID NO: 15, at least one selected from the group consisting of 5 to 100 consecutive bases including the 16th base in which the 16th base is G or C, or complementary polynucleotides thereof It can be a polynucleotide.

More specifically, it may be a set of 2 or more, more specifically 3 to 14 or more, and most specifically all 15 SNP markers. When the 16th base of each SNP marker is an effect allele base of Table 3, the marker can be diagnosed as Soeumin, and the accuracy may increase as the number of SNP markers of an individual increases.

The present inventors confirmed through the following verification that the SNP is a diagnostic marker for noise. Specifically, SNPs related to constitution were obtained for those who confirmed constitution based on a questionnaire (Sasang Constitut Med, 2015, 27(2): 211-221) through the shortened Sasang Constitution Diagnosis Tool (KS-15). .

The shortened Sasang Constitutional Diagnosis Questionnaire (KS-15) was developed to classify the constitution by selecting important items of body type, personality, and condition from the data of the Constitutional Information Bank, and the score given the weight of constitution characteristics obtained through the KS-15 questionnaire Based on this, genome-related analysis was conducted. The KS-15 questionnaire includes body shape factor, personality, and symptom factor. That is, the Body Mass Index (BMI), which is generated through height and weight, was selected as the body shape factor question, and the personality and symptom factor questions from the personality questions and subtest questions collected by the Constitutional Information Bank. Afterwards, 14 questions were finally selected through a discussion of Sasang constitutional experts.

Constitution weights are given for each question, and the sum of the weights is derived as three points for each constitution, and Multinomial Logistic Regression was conducted with one BMI as an independent variable and constitution as a dependent variable. Using the regression coefficient obtained through the analysis, the prediction probability for each constitution was calculated. The predicted probability for each constitution refers to the sum of weights corresponding to the score of the questionnaire for each constitution (Sasang Constitut Med, 2015, 27(2): 211-221).

The predicted probability for each constitution was used as basic data to select a genetic candidate by analyzing the association with the genotype.

In other words, the genetic candidate material was selected by deriving the linear relationship between the numerical value of the score of KS-15 and the genotype, and the biological function score of SNP was assigned using the location information of the candidate materials and used for final selection.

Each of the genetic candidate marker SNPs is located in a specific region of the genome. In this case, in addition to the gene, various proteins that control the gene or epigenetic material are attached. Accordingly, a procedure for assigning a function score by collecting all of the function information that controls genes was added. The assignment of the function score is to extract the SNP function information pattern within a selected block, while learning using the learning case/control data sets, the higher the score is given as the correct answer set is similar. Summed up. The summation results of all scores were converted into scores of 0 to 1 through distribution transformation. It was determined that the pattern was similar to those of the correct answer sets as it approached 1, and the markers selected in the present invention were selected by ranking from those with a score of 1.

Since each SNP has one location information, the pattern was analyzed by collecting SNPs that can be involved together by expanding to the surrounding 1M area to give a function score. Since the block of the SNP to be trained is already constitution-related candidate SNPs, it can be seen that their patterns are functional information related to constitution. The process of deriving the pattern is as follows.

Based on the p-value, the SNP with the strongest correlation is selected as the lead (clue) SNP, and among the SNPs with a p-value of 5 x 10 ^-4 or less among candidate SNPs within 1 Mb of one lead SNP and its surroundings Up to 30 SNPs were selected in the highest order and set as one block. It was divided into training, validation, and testing sets, and the functional domains of SNPs were mapped. By applying a deep learning algorithm, the function of SNP was scored to select candidate SNPs for each constitution. After a verification procedure through a person diagnosed with constitution, a SNP for diagnosis of specific Sasang constitution was finally selected.

Another aspect of the present invention provides a diagnostic composition for noise-in, comprising a probe capable of detecting a SNP marker for diagnosis of noise-in or an agent capable of amplifying it.

The term used in the present invention, "a probe capable of detecting a noise-in diagnostic SNP marker" refers to a composition capable of diagnosing so-eum-in among sasang constitutions by specifically identifying the polymorphic site of the above-described gene through a hybridization reaction, and , There is no particular limitation on a specific method of such gene analysis, and may be performed by any method for detecting genes known in the art to which this invention belongs.

As used herein, the term "primer capable of amplifying the SNP marker for noise-in diagnosis" refers to a composition capable of diagnosing so-eum-in by confirming the polymorphic site of the above gene through amplification, and specifically, the diagnostic marker for noise-in It refers to a primer that can specifically amplify the polynucleotide of.

The primers used for the amplification of the polymorphic markers are template-directed DNA under appropriate conditions (e.g., 4 different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer. It 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 a lower temperature to form a stable hybrid 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 term "primer" used in the present invention is a base sequence having a short free 3'hydroxyl group, and can form a base pair with a complementary template, and template strand copying is performed. It refers to a short sequence that functions as a starting point for. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffers and temperatures. By performing PCR amplification, it is possible to predict the specific metabolic syndrome of sasang constitution through whether or not the desired product is produced. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

The probe or primer of the present invention can be chemically synthesized using a phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, e.g., uncharged linkers (e.g., methyl phosphonate, phosphorester, phosphoro Amidates, carbamates, etc.) or to charged linkers (eg phosphorothioate, phosphorodithioate, etc.).

In another aspect of the present invention, (a) a polymorphic site comprising a SNP of one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 12, or a complementary polynucleotide thereof from DNA of a sample isolated from an individual Amplifying or hybridizing with the probe; (b) It provides a method of providing information for prediction or diagnosis of noise comprising the step of determining the base of the amplified or hybridized polymorphic site in step (a).

The term "individual" in the present invention may be a subject for predicting or diagnosing sasang constitution. DNA may be obtained from samples such as hair, urine, blood, various body fluids, separated tissues, separated cells, or saliva from the individual, but is not limited thereto.

The step of amplifying the polymorphic site of the SNP marker from the DNA of step (a) or hybridizing with a probe may be performed by any method known to those skilled in the art. For example, it can be obtained by amplifying a target nucleic acid through PCR and purifying it. Other 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.

Of the above methods, determining the base of the polymorphic site in step (b) is sequence analysis, hybridization by microarray, allele specific PCR, and dynamic allele-specific hybridization. , DASH), PCR extension analysis, PCR-SSCP, 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 (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays). It doesn't work. One or more alleles in polymorphic markers, including microsatellites, SNPs or other types of polymorphic markers, can be identified by the above methods or other methods available to those skilled in the art to which the present invention pertains. Determining the base of such a polymorphic site can be specifically performed through an SNP chip.

In the present invention, the term "SNP chip" means one of the DNA microarrays capable of identifying each base of hundreds of thousands of SNPs at once.

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

In the above, the sequencing analysis may be performed using a conventional method for determining the base sequence, and may be performed using an automated genetic analyzer. In addition, allele-specific PCR refers to a PCR method for amplifying a DNA fragment in which the SNP is located with a primer set including a primer designed with the base at the 3'end of the SNP. The principle of the method is, for example, when a specific base is substituted from A to G, a primer containing the 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 performing the reaction, when the base at the SNP position is A, the amplification reaction is normally performed and the 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 is due to the fact that the amplification reaction is not performed properly because the 3'end cannot perform complementary bonding. DASH may be performed by a conventional method, and specifically, may be performed by a method by a prince or the like.

On the other hand, in the PCR extension analysis, a DNA fragment containing a base on which a single base polymorphism is located is first amplified with a primer pair, and then all nucleotides added to the reaction are dephosphorylated to inactivate, and the SNP-specific extension primer, A dNTP mixture, didioxynucleotide, reaction buffer, and DNA polymerase are added to perform a primer extension reaction. At this time, the extension primer uses the base immediately adjacent to the 5'direction of the base where the SNP is located as the 3'end, and the nucleic acid having the same base as the didioxynucleotide is excluded from the dNTP mixture, and the didioxynucleotide represents the SNP. It is selected from one of the base types. For example, when there is a substitution from A to G, a mixture of dGTP, dCTP, and TTP and ddATP are added to the reaction, the primer at the base where the substitution occurs is extended by DNA polymerase, and after several bases, A 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 length of the extended primers.

At this time, as a detection method, in the case of fluorescently labeled extension primers or dodioxynucleotides, the SNP can be detected by detecting fluorescence using a genetic analyzer (eg, ABI's Model 3700, etc.) used for general nucleotide sequence determination. The SNP can be detected by measuring the molecular weight using a matrix assisted laser desorption ionization-time of flight (MALDI-TOF) technique in the case of using a non-labeled extension primer and a didioxynucleotide.

Specifically, among the nucleotide sequences determined in step (b), in rs12667492 represented by SEQ ID NO: 1, when the 16th base is A; In rs74028907 shown in SEQ ID NO: 2, when the 16th base is T; In rs4909803 shown in SEQ ID NO: 3, when the 16th base is G; In rs11646443 shown in SEQ ID NO: 4, when the 16th base is A; In rs75575584 shown in SEQ ID NO: 5, when the 16th base is A; In rs17669146 shown in SEQ ID NO: 6, when the 16th base is C; In rs4688636 shown in SEQ ID NO: 7, when the 16th base is G; In rs1017409 shown in SEQ ID NO: 8, when the 16th base is C; In rs981760 shown in SEQ ID NO: 9, when the 16th base is T; In rs1671413 shown in SEQ ID NO: 10, when the 16th base is T; In rs7013390 shown in SEQ ID NO: 11, when the 16th base is A; In rs10955446 shown in SEQ ID NO: 12, when the 16th base is C; In rs7015202 shown in SEQ ID NO: 13, when the 16th base is A; In rs1115971 shown in SEQ ID NO: 14, when the 16th base is C; In rs7138393 described in SEQ ID NO: 15, when the 16th base is G, it can be determined as Soeumin among sasang constitutions.

Another aspect of the present invention provides a kit for diagnosis of noise, comprising a composition for diagnosis of noise.

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 sasang constitution by confirming the SNP marker, which is a diagnostic marker for soumin, through amplification, or by checking the expression level of the SNP marker and the mRNA expression level.

In a specific embodiment, the kit of the present invention may be a diagnostic kit, which is a noise including essential elements necessary to perform a DNA chip. A DNA chip kit is a gridded array of nucleic acid species attached to a generally flat solid support plate, typically a glass surface that is not larger than a microscope slide, in which nucleic acids are uniformly arranged on the chip surface. It is a tool that enables mass-parallel analysis by causing multiple hybridization reactions between the nucleic acid on the image and the complementary nucleic acid contained in the solution treated on the chip surface.

Another aspect of the present invention provides a microarray for diagnosis of noise comprising a composition for diagnosis of noise.

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

A method of preparing a microarray by immobilizing a probe polynucleotide on a substrate is well known in the art. The probe polynucleotide refers to a polynucleotide capable of hybridizing, and refers to an oligonucleotide capable of sequence-specific binding to a complementary strand of a nucleic acid.

The probe of the present invention is an allele-specific probe, and a polymorphic site exists in a nucleic acid fragment derived from two members of the same species, and hybridizes to a DNA fragment derived from one member, but does not hybridize to a fragment derived from another member. . In this case, the hybridization conditions show a significant difference in hybridization strength between alleles, and must be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allele types.

The probe of the present invention can be used for diagnosing noise in sasang constitution by detecting alleles. The prediction or diagnosis method includes detection methods based on hybridization of nucleic acids such as Southern blot, and may be provided in the form of being previously bound to a substrate of a DNA chip in a method using a DNA chip. The hybridization may be usually carried out under stringent conditions, for example, a salt concentration of 1M or less and a temperature of 25°C or more. For example, 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and conditions of 2530° C. may be suitable for allele-specific probe hybridization.

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

Another aspect of the present invention provides a method of selecting a diagnostic SNP marker.

Specifically, the method comprises the steps of: (a) selecting SNPs associated with constitution through a full-length genome analysis (Genome-Wide Association Study, GWAS); (b) selecting an SNP set and mapping a functional region based on the selected SNP in step (a); (c) scoring a SNP function score by applying a deep learning algorithm; And (d) selecting SNPs having a high functional score in step (c).

Step (a) is the introduction of a convolution layer, a Restricted Boltzmann machine (RBM), or an auto-encoder in order to effectively extract the SNPs that are related to constitution from complex data. SNP can be selected through, but is not limited thereto. Specifically, SNPs having a correlation of P <0.0001 for each constitution can be selected.

In the step (b), a SNP set may be selected based on the constitution-related SNP selected in step (a) and a functional region may be mapped.

Since there may be SNPs that play a major role in the actual trait in the LD region in which linkage disequilibrium exists around SNPs with high statistical significance, the functional pattern was investigated by expanding to the region surrounding the SNP instead of one SNP.

In the step (c), the SNP function score may be scored by applying a deep learning algorithm.

In the step (d), the SNP having a high functional score in step (c) may be selected.

The SNP marker of the present invention is a specific SNP marker for diagnosing Soeumin among Sasang constitutions, and it will be possible to accurately and objectively diagnose Sasang constitution.

1 is a simplified flowchart of a method for selecting 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 more specifically, and the scope of the present invention is not limited by these examples.

Example 1. Subject selection

The subjects were Taeumin (TE), Soeuminin (SE), and Soyangin (SY) whose constitutions were classified based on a questionnaire through the shortened Sasang Constitutional Diagnosis Tool (KS-15). Each constitution group consisted of 5797 people.

Example 2. Selection of genetic factors related to constitution

A full-length genome analysis (Genome-Wide Association Study, GWAS) was performed on 5797 people of the Soeumin group. In the experiment, the missing value was estimated using the ASN panel provided by the 1000 genome project.

Genetic factors related to constitution were selected through linear regression between genotype and constitution found through full-length genome analysis. At this time, statistical power was calculated and statistically significant SNPs were selected based on P-value <0.0001.

Example 3. SNP block generation and SNP set selection

After arranging the full-length genome in the order of strong correlation based on the p-value, the SNP with the strongest correlation was selected as the lead (clue) SNP, and the lead SNPs were selected so that they do not overlap within 1Mb of each other.

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

Among the SNPs with a p-value of 5 x 10 ^-4 or less among candidate SNPs within one lead SNP and its surrounding 1Mb, up to 30 SNPs were selected in the order of high correlation and defined as a block.

Statistical linkage variation is not statistically significant, but it is included in the genetic linkage disequilibrium section and may affect the trait, so the list of SNPs within the SNP block section is selected as shown in Table 1 below to include the surrounding linkage SNPs. I did.

In addition, data sets were classified based on chromosome information in order not to overlap between training, verification, and testing sets. Chromosomes 1 to 10 were used as training sets, and testing sets 11 to 14 were used to evaluate the final performance of the model. The remaining chromosomes from 15 to 22 were used as validation sets.

The numbers in Table 1 below represent the number of SNP blocks (sections) as the number of basic information of the SNP set used for learning.

Number of training, validation, and test sets GWAS SNP set (out off P value <5e ^-04 ) Noise (SE) Soyang (SY) Taeum (TE) Training Set (Chr. 1-10) 92 89 86 Validation Set (Chr. 15-22) 33 22 26 Test Set (Chr.11-14) 30 21 31

Example 4: Functional region mapping of SNP

Function points were assigned to SNPs by learning specific patterns of epigenetic information present at the positions of each SNP. The epigenetic information is the DNase Hypersensitive site (DHS) provided by the ENCODE Project (The ENCODE Project Consortium, 2012) and the Roadmap Epigenomics Consortium (2015), histone modification, target gene function, and transcriptional regulatory factors. It is a gene region contained within the region (Table 2). 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 through which the gene acts. Based on this, SNP is given a function score. That is, when the SNP corresponds to the functional site 1, the binary data was configured as 0 when it does not.

Number of feature mapping information used for training # feature Noise (SE) Soyang (SY) Taeum (TE) DHS 124 124 122 119 Histone modification 606 570 511 453 Target gene pathway 301 9 14 11 FMO 996 15 7 6

Example 5: Functional scoring of SNP applying deep learning algorithm

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

One SNP block generated in Example 2 becomes one training image data, and based on the fact that at least one cause mutation exists in this block, the features of the cause mutation were learned using the convolutional neural network framework.

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

5-1. Composition of input image from full-length genome correlation analysis results

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

5-2. Model design

The CNN framework consists of pattern detectors that can be tuned. The pattern searchers are called kernels or filters, which detect partial patterns with local connectivity and perform operations repeatedly on the entire input field.

5-3. Denoising-pre-training using auto encoder (DA)

The auto-encoder enables learning more general features of training data before fine-tuning the kernels in deep learning, and starts 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 a denoising-auto-encoder (DA) that mixes random noise into the input value so that the model can learn more robust features.

5-4. Hyper parameter

Deep learning models rely on a number of hyperparameters, and each hyperparameter has a significant impact on the model's performance. Hyper-parameters are used for the noise level (corruption level) applied to the input values in the DA pre-learning process, the learning rate in the fine tuning process, the momentum, and the elastic net regularization process. Parameters are included.

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

5-5. SNP function scoring

In the first layer (Convolution layer) C1, an image matrix defining one input image of the first layer is input. Using the first layer input image and the first layer kernel, which is a convolution kernel, a convolution process was performed to generate a matrix of 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), a nonlinear complex pattern could be analyzed based on the extracted pattern result. Each element of the array of the second layer kernel has a value of 0 or 1.

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

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

Example 6: Selection of SNP for diagnosing Sasang constitution through verification procedure

After the verification procedure through the constitution confirmed person, SNP for the diagnosis of specific Sasang constitution was finally selected.

The criteria for selecting a person with constitution are as follows.

Criterion 1: Subjects with a high probability of KS-15 for each constitution are given priority

Criterion 2: Constitutional expert's diagnosis and KS-15 probability value diagnosis constitution is the same

Criterion 3: BMI index 18.5 or higher

Criterion 4: In the case of Soeumin, divided into 30 normal weight groups with a BMI index of 18.5 to 25 and 30 overweight groups with a BMI index of 25 or more.

Criterion 5: No metabolic syndrome and no cancer or thyroid abnormalities in the history of disease

WGS (Whole Genome Sequencing) was performed on 30 Soumin, 30 Soyangin, 30 Fatty Groups, and 30 Normal Weight Groups for Taeumin, according to the selection criteria for constitutional diagnosis. Reproducibility was checked for the presence of candidate SNPs selected for each constitution in each constitutional confirmed group, and SNPs associated with other constitutional values were excluded.

Among the filtered SNPs, a SNP with a high Minor Allel Frequency and a high function score was selected in consideration of the frequency and function score of the SNP.

The constitution-specific SNPs finally selected through the candidate SNP selection procedure and verification procedure are shown in Table 3 below.

Soeumin specific SNP SNP ID Allel Type Minor Allel Frequency Chr. Position GWAS P-value Functional Score SE Allel Frequency rs12667492 A>G 0.1834 7 155965552 0.00002437 0.99 0.56 rs74028907 T>G 0.1791 15 87661906 0.00004146 0.99 0.6 rs4909803 G>T 0.1588 8 139509485 0.00002054 One 0.32 rs11646443 A>G 0.08159 16 84270476 0.00003038 0.99 0.5 rs75575584 A>G 0.05477 2 47393383 0.00002569 One 0.42 rs17669146 C>T 0.09453 3 61552092 0.0004771 One One rs4688636 G>A 0.08528 3 61555637 0.0001241 One One rs1017409 C>T 0.2349 5 59225621 0.0001596 0.833 One rs981760 T>A 0.2438 5 59234312 0.0001106 0.814 One rs1671413 T>C 0.001038 8 13226588 0.000307 One One rs7013390 A>G 0.1522 8 108288033 0.000234 0.95 One rs10955446 C>T 0.152 8 108288324 0.0001593 0.86 One rs7015202 A>C 0.02811 8 139501740 0.0000785 0.913 One rs1115971 C>T 0.1387 12 31836950 0.0003618 One One rs7138393 G>C 0.08139 12 33590787 0.0003389 One One

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later, and equivalent concepts, rather than the detailed description above, are included in the scope of the present invention.

<110> Korea Institute of Oriental Medicine <120> SNP markers for diagnosing Soeumin of sasang constitution and use thereof <130> KPA181218-KR <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> Y= A or G <400> 1 taggatgaga gttacygaat aaggtggtcc g 31 <210> 2 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> R= T or G <400> 2 ggtgaccttt ctctcrcctg ctctccagac t 31 <210> 3 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> Y= G or T <400> 3 ccgctgcgca ccgcgygggt agacgctgcg c 31 <210> 4 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> S= A or G <400> 4 ccatctcgcg cagccsgttc atgcacaggc c 31 <210> 5 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> K= A or G <400> 5 gagttcagac acattkcacc tttcacctaa t 31 <210> 6 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> R= C or T <400> 6 gattgaaagt aacttrcttg gcattcacgt g 31 <210> 7 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> R= G or A <400> 7 aaatgtgtct cataartctt ccaaatccct g 31 <210> 8 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> R= C or T <400> 8 cctaagacaa gcccarctta caaaagaggc t 31 <210> 9 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> R= T or A <400> 9 ccagccaaga tgaccratat gtctaaaacc a 31 <210> 10 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> K= T or C <400> 10 tgattgcctc agcctktgat gatgtttaga g 31 <210> 11 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> K= A or G <400> 11 caccttagat aatttkttgg aggcagtgct t 31 <210> 12 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> Y= C or T <400> 12 gtgcctctga agaccyagga agcctttttt g 31 <210> 13 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> Y= A or C <400> 13 ctccatgcct cctgtytaat ttacttttct c 31 <210> 14 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> Y= C or T <400> 14 aaaatcagtt gttaayataa taactgatgt c 31 <210> 15 <211> 31 <212> DNA <213> Homo sapiens <220> <221> variation <222> (16) <223> Y= G or C <400> 15 caacatgctc acacaytgcc ttccactgag t 31

Claims (11)

Includes a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 or a complementary polynucleotide thereof, and the nucleotide sequence includes the 16th nucleotide as SNP (single nucleotide polymorphism),
The 16th base of SEQ ID NO: 6 is C, a diagnostic microarray of noise in sasang constitution.
The method of claim 1, wherein the polynucleotide further comprises one or more polynucleotides selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 5 and 7 to 15, wherein the nucleotide sequence is a 16th base as a single nucleotide polymorphism (SNP). ), and
The 16th base of SEQ ID NO: 1 is A, the 16th base of SEQ ID NO: 2 is T, the 16th base of SEQ ID NO: 3 is G, the 16th base of SEQ ID NO: 4 is A, and the 16th base of SEQ ID NO: 5 is A , The 16th base of SEQ ID NO: 7 is G, the 16th base of SEQ ID NO: 8 is C, the 16th base of SEQ ID NO: 9 is T, the 16th base of SEQ ID NO: 10 is T, and the 16th base of SEQ ID NO: 11 is A , The 16th base of SEQ ID NO: 12 is C, the 16th base of SEQ ID NO: 13 is A, the 16th base of SEQ ID NO: 14 is C, the 16th base of SEQ ID NO: 15 is G.
A probe capable of binding to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 or a complementary polynucleotide thereof, or a primer capable of specifically amplifying,
The 16th base of SEQ ID NO: 6 is C, a diagnostic composition for noise in sasang constitution.
The method of claim 3, wherein the polynucleotide further comprises one or more polynucleotides selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 5 and 7 to 15,
The 16th base of SEQ ID NO: 1 is A, the 16th base of SEQ ID NO: 2 is T, the 16th base of SEQ ID NO: 3 is G, the 16th base of SEQ ID NO: 4 is A, and the 16th base of SEQ ID NO: 5 is A , The 16th base of SEQ ID NO: 7 is G, the 16th base of SEQ ID NO: 8 is C, the 16th base of SEQ ID NO: 9 is T, the 16th base of SEQ ID NO: 10 is T, and the 16th base of SEQ ID NO: 11 is A , The 16th base of SEQ ID NO: 12 is C, the 16th base of SEQ ID NO: 13 is A, the 16th base of SEQ ID NO: 14 is C, the 16th base of SEQ ID NO: 15 is G.
A kit for diagnosis of noise in Sasang constitution comprising the composition of claim 3 or 4.
The kit of claim 5, wherein the kit is an RT-PCR kit or a DNA chip kit.
(a) amplifying a polymorphic site comprising a SNP of a nucleotide sequence of SEQ ID NO: 6 or a complementary polynucleotide thereof from DNA of a sample isolated from the individual, or hybridizing with a probe;
(b) determining the base of the amplified or hybridized polymorphic site of step (a); And
(c) Among the nucleotide sequences determined in step (b),
In rs17669146 of SEQ ID NO: 6, when the 16th base is C, the method of providing information for predicting or diagnosing noise in sasang constitution, comprising determining the individual as a soeumin.
The method of claim 7, wherein the polynucleotide further comprises one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 5 and 7 to 15,
Among the nucleotide sequences determined in step (b),
In rs17669146 described in SEQ ID NO: 6, the 16th base is C,
In rs12667492 shown in SEQ ID NO: 1, when the 16th base is A;
In rs74028907 shown in SEQ ID NO: 2, when the 16th base is T;
In rs4909803 shown in SEQ ID NO: 3, when the 16th base is G;
In rs11646443 shown in SEQ ID NO: 4, when the 16th base is A;
In rs75575584 shown in SEQ ID NO: 5, when the 16th base is A;
In rs4688636 shown in SEQ ID NO: 7, when the 16th base is G;
In rs1017409 shown in SEQ ID NO: 8, when the 16th base is C;
In rs981760 shown in SEQ ID NO: 9, when the 16th base is T;
In rs1671413 shown in SEQ ID NO: 10, when the 16th base is T;
In rs7013390 shown in SEQ ID NO: 11, when the 16th base is A;
In rs10955446 shown in SEQ ID NO: 12, when the 16th base is C;
In rs7015202 shown in SEQ ID NO: 13, when the 16th base is A;
In rs1115971 shown in SEQ ID NO: 14, when the 16th base is C; or
In rs7138393 described in SEQ ID NO: 15, when the 16th base is G,
Determining the subject to be a somber.
The method of claim 7 or 8, wherein the base determination in step (b) is sequence analysis, hybridization by microarray, allele specific PCR, and dynamic allele-specific hybridization. , DASH), PCR extension analysis, PCR-SSCP (PCR-single strand conformation polymorphism), PCR-RFLP (PCR-restriction fragment length polymorphism) and TaqMan method that is performed by one or more methods selected from the group consisting of .
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