KR20180118279A - Novel SNP marker for pattern identification of asthma patients and uses thereof - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphism (SNP) marker for pattern identification of an asthma patient and a use thereof. More specifically, the present invention relates to an SNP marker composition, a kit, a microarray for pattern identification of an asthma patient and a method for providing information for pattern identification of an asthma patient. The SNP marker of the present invention can objectively identify a pattern of the asthma patient, thereby being usefully used for customized treatment of the asthma patient. The SNP marker composition for pattern identification of an asthma patient comprises a polynucleotide consisting of 5 to 300 continuous bases including a 141^st base, wherein the 141^st base of SERPINA3 gene represented by a nucleotide sequence of sequence number 1 is T or G, or a polynucleotide in complementary thereto.

Description

SNP markers for the diagnosis of asthma in patients with asthma and their use [0002]

The present invention relates to SNP (single nucleotide polymorphism) markers for asthma patients and their use, and more particularly, to SNP marker compositions, kits, microarrays, .

Asthma is a common allergic disease caused by combined genetic and environmental factors, such as dyspnea, coughing, and rough breathing. Factors that aggravate asthma include cold, tobacco smoke, indoor pollution, air pollution, food additives, climate change, dustiness, and stress. Especially, industrialization causes a decrease in air quality, which is a major cause of increasing the incidence of asthma .

Despite the growth of medical technology based on Western medicine, due to the nature of allergic diseases, it is difficult to cure and the cost of treatment increases. Therefore, interest and preference for CAM is increasing. Among them, oriental medicine treatment is highly satisfied with improvement of quality of life, and it is recognized as an effective treatment for alleviating side effects of bilateral treatment.

On the other hand, asthma in bronchial asthma is divided into aphasia and demonstration according to the dialectical tool. Because the treatment method and medication are very different from each other, accurate diagnosis is needed. The traditional diagnosis is based on the experience of the expert, the opinions, or the content of the old documents, thus including reliability and validity (J Physiol & Pathol Korean Med 28 6): 585-592). In addition, a variety of errors are likely to occur depending on the psychological situation of the patient responding to the questionnaire, and a standardized diagnosis method is required.

In this regard, there has been developed a method for discriminating a paralytic dialect (Korean Patent Laid-Open No. 10-2010-0052284), a method for discriminating an obesity dialect (Korean Patent Laid-Open No. 10-2011-0060142) Have not been developed.

Under these circumstances, the present inventors have made intensive efforts to develop an objective discrimination method for asthmatic patients so as to apply the medical treatment to the genetic and constitutional characteristics of the patient. As a result, when using the SNP contained in the SERPINA3 gene, It is possible to classify the patient's dialectically with high accuracy, thus completing the present invention.

One object of the present invention is to provide a polynucleotide consisting of 5 to 300 consecutive bases, wherein the 141st base of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 is T or G, Which comprises the polynucleotide of the present invention.

It is another object of the present invention to provide a composition for distinguishing between asthmatic patients, which comprises an agent capable of detecting or amplifying the SNP marker composition.

It is still another object of the present invention to provide a kit for the classification of asthma patients.

It is another object of the present invention to provide a polynucleotide consisting of 5 to 300 consecutive bases comprising the 141st base, wherein the 141st base of SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 is T or G, The present invention provides a microarray for distinguishing a polymorphism in an asthmatic patient, which comprises a complementary polynucleotide.

Another object of the present invention is to amplify a polymorphic site comprising a SNP of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide thereof from the DNA of a sample isolated from an individual step; And (b) determining the base of the amplified polymorphic site. The present invention also provides a method for providing information for distinguishing a dialect of a patient suffering from asthma.

In one aspect of the present invention, the 141th base of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 is T or G, and the 5'to 300 consecutive bases including the 141st base Wherein the polynucleotide comprises a polynucleotide or a polynucleotide complementary thereto.

The term " pattern identification (syndrome differentiation) " of the present invention refers to the diagnosis of a patient by analyzing the cause, characteristic, type, and symptom of the disease, (WHO international standard terminologies on traditional medicine in the Western Pacific Region, 2007). In the present invention, the above-mentioned dialectic may be classified as hypochromic or empirical.

The term "hypoglycemia" of the present invention refers to a condition in which the function is weakened due to insufficient blood or blood loss as a result of palpitation, This means that the body's resistance and physiological functions are weak. It is difficult to say, there is little sound coughing, a low sound is heard, a lot of sweating during the day, short and difficult to breathe, and the symptoms of shortness of breath when moving, sweating after sleeping, It is characterized by the symptom of breathlessness when it moves, the symptom of the pain of the back, the back of the syringe, the heart and the loss of the heart, and the symptom of the loss of the heart.

The term " demonstration " of the present invention refers to a state in which fragility is vigorous as one of the eight riverside states, and the blood of the body is depressed due to a dysfunction of the body do. It is cold, there is a headache, the body is sick everywhere, there is a lot of phlegm, the sputum does not refresh even if it spits, the inside is gourmet, the sputum is white, the face is dark and blue is blue, Is characterized by showing signs of symptoms such as breathing fast and rapid, yellow sputum yellow, thirsty and wanting to drink a lot of water, .

The term " SERPINA3 gene " of the present invention means a gene encoding a protein called Serpin Family A Member 3 or Alpha-1-Antichymotrypsin. It has been known that mutations of the gene have been found in patients with liver disease or Alzheimer's disease, but the association of asthma with the gene is unknown. The specific base sequence and protein information of the gene are known from NCBI (Accession: AAH70265, etc.).

The term " SNP marker " of the present invention means a single nucleotide polymorphism (SNP) allele base pair on the DNA sequence. The SNP refers to a polymorphic site in which two or more alleles exist in one locus, and only a single base is different, which is relatively frequent and stable and distributed throughout the genome , Resulting in genetic diversity of the individual. In addition, SNPs may generally involve a change in phenotype associated with single nucleotide polymorphism. The SNPs of the present invention may indicate a difference in symptoms, such as hypersomnia or demonstration of asthmatic patients. Specifically, the SNP may be rs1884082, and in the present specification, the 'SNP marker' may be named in combination with 'SNP'.

In addition, the SNP marker may be a polynucleotide of all or a part including the SNP region of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1, more specifically, the 141nd nucleotide of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: And most specifically, the 141th base of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 is T or G, and the 5'to 300 consecutive nucleotides including the 141 < th > base may be all or some of the polynucleotides But are not limited to, a polynucleotide consisting of a base, or a polynucleotide complementary thereto.

In a specific embodiment of the present invention, patients diagnosed with asthma disease were divided into apathy and hypometabolism by questionnaire, and genotypes of the SERPINA3 gene SNP (rs1884082) were analyzed As a result, patients confirmed to be hypoglycemia had G / T or G / G genotypes of the SNPs, and patients with T / T proved to be positive (Table 2). This suggests that the SNP marker composition according to the present invention can be usefully used for the classification of apnea in asthmatic patients.

Another aspect of the present invention provides a composition for distinguishing hyperparathyroidism from asthmatic patients, which comprises an agent capable of detecting or amplifying a SNP marker composition for discriminating a subject's asthma.

The term " agent capable of detecting or amplifying a marker composition " of the present invention means a preparation capable of specifically recognizing SNPs contained in the SNP marker composition and capable of amplifying or amplifying the SNPs. As a specific example, a probe capable of specifically binding to a polymorphic site containing a SNP; Or a primer capable of specifically amplifying a polynucleotide comprising the SNP or a complementary polynucleotide thereof, but is not limited thereto.

The term " probe " of the present invention means a nucleic acid fragment which is labeled with RNA or DNA corresponding to a few bases or a few hundred bases, which can achieve specific binding with mRNA. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.

In the present invention, a probe that binds to and recognizes a SNP marker includes a sequence complementary to a polynucleotide sequence including a SNP, but may be in the form of DNA, RNA or DNA-RNA hybrid. Further, fluorescent markers, radiation markers, and the like can be additionally attached to the 5 'or 3' ends of the probe so as to be visually recognizable.

The term " primer " of the present invention means a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, It means a short sequence functioning as a point. In the present invention, the primers used for the detection and amplification of SNP markers can be obtained by hybridization under appropriate conditions in suitable buffers (for example, four different nucleoside triphosphates and DNA, RNA polymerase or reverse transcriptase) Stranded oligonucleotides that can serve as a starting point for template-directed DNA synthesis under the control of the primers. The appropriate length of the primers may vary depending on the intended use. In addition, the primer sequence need not be completely complementary to the polynucleotide comprising the SNP marker or its complementary polynucleotide, and can be used if it is sufficiently complementary to hybridize.

In addition, primers can be modified as appropriate, including but not limited to methylation, capping, substitution of nucleotides, or modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate , Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, the primer may be a polynucleotide consisting of the nucleotide sequences of SEQ ID NOS: 2 and 3, but is not limited thereto.

The nucleotide sequences used in the present invention are interpreted to include sequences showing substantial identity with the sequences listed in the sequence listing, considering mutations having biologically equivalent activities. The term " substantial identity " means aligning the sequence of the present invention to any other sequence as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, Means a sequence showing 60% homology, more specifically 70% homology, even more specifically 80% homology, most specifically 90% homology.

Therefore, the nucleotide sequence having high homology with the nucleotide sequence shown in SEQ ID NO: 1 to 3, for example, the homology thereof is 70% or more, specifically 80% or more, more specifically 90% or more Nucleotide sequences are also to be construed as falling within the scope of the present invention.

In one specific embodiment of the present invention, primers represented by SEQ ID NOS: 2 and 3 were prepared to amplify a DNA fragment containing the SNP (rs1884082) of the SERPINA3 gene (Example 2) The genotypes of the SNPs were identified to identify the apnea patients (Table 2). This suggests that the composition according to the present invention, which comprises a preparation capable of detecting or amplifying the SNP, can be usefully used for the classification of apnea in asthmatic patients.

Another aspect of the present invention provides a kit for separating dialysis of a patient suffering from asthma, comprising the composition for distinguishing a dialyzer from a patient suffering from asthma.

The kit of the present invention can discriminate apnea patients by detecting and detecting bases of SNP markers provided in the present invention using the above-described composition for discriminating hypercholesterolemia. Specifically, the kit may be an RT-PCR kit or a DNA chip kit, but is not limited thereto.

In one example, the kit may be a kit containing the necessary elements necessary to perform RT-PCR. For example, RT-PCR kits can be used in conjunction with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), ddNTPs ), Enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

As another example, the kit of the present invention may be a DNA chip kit for discrimination of asthma patients, which includes essential elements necessary for carrying out a DNA chip.

The term " DNA chip " of the present invention means one of DNA microarrays capable of identifying each base of hundreds of thousands of DNAs at a time. The DNA chip kit is formed by attaching nucleic acid species to a glass surface, which is generally not larger than a flat solid support plate, typically a slide for a microscope, in a gridded array. The nucleic acid is uniformly arranged on the chip surface, It is a tool that enables multiple parallel hybridization reactions between the nucleic acid on the chip and the complementary nucleic acid contained in the treated solution on the chip surface.

Another aspect of the present invention provides a microarray for discriminating asthmatic patients, comprising the SNP marker composition for discriminating the asthmatic patients.

Specifically, the microarray comprises a polynucleotide consisting of 5 to 300 consecutive bases, wherein the 141st base of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 is T or G and the 141st nucleotide, Gt; polynucleotides < / RTI >

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe in which 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 the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can detect an allele and can be used for the classification of the apnea of asthmatic patients. The division includes detection methods based on hybridization of nucleic acids such as Southern blots, etc., and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization may be carried out under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher.

The process of immobilizing the probe polynucleotide associated with the apical differentiation of asthmatic patients of the present invention on a substrate can also be easily performed using conventional techniques. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. As a specific example, the detection may be performed by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal including a fluorescent substance, then hybridizing the label on a microarray, and detecting a hybridization result by detecting a signal generated from the labeling substance .

(A) amplifying a polymorphic site comprising a SNP of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide thereof from the DNA of the sample isolated from the individual; And (b) determining a base of the amplified polymorphic site.

As described above, the SNP marker composition for discriminating asthmatic patients according to the present invention comprises a polynucleotide of SEQ ID NO: 1, specifically, a SNP marker contained in a polynucleotide of SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 Therefore,

When the 141 th nucleotide of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is G and the base of the complementary polynucleotide is T or G,

When the 141th nucleotide of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is T and the base of the complementary polynucleotide is T, it can be judged to be a demonstration.

The term " sample " of the present invention refers to a substance derived from a person who wants to distinguish dialysis, and may specifically be a tumor, blood, serum, urine, or hair. More specifically, The type thereof is not particularly limited as long as it can be ensured.

In the present invention, amplification of the polymorphic site containing the SNP from the DNA of step (a) may be performed by any method known to those skilled in the art. Specific examples include, but are not limited to, PCR, ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, nucleic acid based sequence amplification (NASBA), and the like.

Further, the step of determining the base of the amplified polymorphic site in step (b) may be any method known to a person skilled in the art. Specific examples include sequencing, mini sequencing, direct sequencing, allele specific PCR, dynamic allele-specifichybridization (DASH), PCR extension analysis (e.g., single base extension SBE), PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (eg MassenRAY system of Sequenom), Bio-Plex system (BioRad) But is not limited thereto.

Specifically, the sequencing, mini-sequencing, or direct sequencing analysis can be performed using conventional methods for sequencing, and can be performed using an automated gene analyzer.

In a specific embodiment of the present invention, DNA was isolated from the blood of patients with apical differentiation, and a portion of the DNA containing the SNP (rs1884082) of the SERPINA3 gene was amplified by PCR and then subjected to direct sequencing, (G / T) or G / G (G / G) genotypes of the SNP, and T / T of the patients who were found to be positive (Table 2). This suggests that the marker composition according to the present invention including the SNP marker can be usefully used for the classification of asthma patients.

The SNP markers of the present invention can be used for the personalized treatment of asthmatic patients because it can objectively discriminate the apnea patients.

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

Example  1. Setting criteria for screening and dialectic subjects

For the present invention, a total of 53 asthmatic patients diagnosed with asthma by pulmonary function tests were included. Of the 53 patients, 19 were male and 34 were female, and the mean age was 47.8 ± 13.8 years. Thereafter, they were classified as either hypoatric or empirical through a hypothetical demonstration questionnaire as shown in Table 1 below. The questionnaire was also used in a previous study (Journal of korean oriental internal medicine, v.37, no.1, 2016, pp.47-64).

Specifically, if the item corresponds to the item listed in the item (external appearance, intrinsic resistance, ruins, physical or mental handicap or loss), 1 point, 0 if not applicable, Two points were scored. After summing the scores for each item, empirical test when the total score of the items with external and internal resistance is high; If the sum of the scores of the ruins, mental and physical handicaps, and the lossless items is high, it is judged to be valid.

Example  2. Identification of genetic polymorphisms in asthmatic patients

In order to develop a genetic marker for discriminating the asthmatic patients, the polymorphism of the asthmatic patients having the apnea discriminated in Example 1 was confirmed. At this time, single nucleotide polymorphism (SNP) of SERPINA3 gene 'rs1884082' was used.

Specifically, DNA was extracted from the patient's blood using a kit (Roche, High Pure PCR Template Preparation Kit). The 260/280 value of the DNA was found to be 1.8 or more and the amount of DNA was found to be 10 ng / 쨉 l or more through a nanodrop device. Thereafter, PCR was performed to amplify the DNA fragment containing the SNP of the SERPINA3 gene, and the sense and reverse primers of SEQ ID NOS: 2 and 3 were used (SEQ ID NO: 2: 5'-GACCTTTGGAGGAAGAGATTAGAGC-3 And SEQ ID NO: 3: 5'-TCCAGGCTGCTGCCTAAAACTCTC-3 '). The PCR conditions were as follows: denaturation step (20 sec at 95 캜), binding step (20 sec at 59 캜), and extension step (30 sec at 72 캜) were repeated 39 times. After completion of the PCR, 1.8% agarose gel PCR products were identified using UV lamps. Then, SNP genotypes were confirmed by direct sequencing of the PCR products, and the results are shown in Table 2 below. In Table 2, OR denotes an odd ratio, and CI denotes a confidence interval.

Model
(Model) Genotype
(Genotype) In patients with hoarseness
Gene frequency In an empirical patient
Gene frequency OR (95% CI) P-value Co-dominance
(Codominant) T / T 25.0 51.7 One 0.049 G / T 45.8 41.4 0.40 (0.11 - 1.50) G / G 29.2 6.9 0.12 (0.02-0.77) dominant
(Dominant) T / T 25.0 51.7 One 0.047 G / T-G / G 75.0 48.3 0.30 (0.09-1.02) zeal
(Recessive) T / T-G / T 70.8 93.1 One 0.042 G / G 29.2 6.9 0.19 (0.03-1.07)

In Table 2, the result of the common dominant model comparing the genotypes of T / T, G / T or G / G, respectively, indicates that the genotype of T / T is a patient classified as an empirical patient ), And the genotype of G / T or G / G was found to be high in patients who were classified as blunted (hereinafter referred to as 'blind patients').

In addition, the genotype of T / T was found to be high in the empirical patients through the result of the dominant model comparing the T / T genotype with the genotype of G / T and G / G, T and G / G genotypes, the genotype of G / G was found to be higher in patients with hypoglycemia.

From the above results, it was confirmed that asthmatic patients can be distinguished from those with SERPINA3 gene SNP genotype of G / T or G / G, and those of T / T.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Novel SNP marker for pattern identification of asthma patients          and uses <130> KPA170237-KR <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 321 <212> DNA <213> Homo sapiens <400> 1 ctttggagga agagattaga gctagtccca taaccaggtt atttgagtag gtctaacaag 60 cccgtattac cagaaattat catctggtca tttccagtcc gagaacagaa cacttggttg 120 tcctggcatt tcccaagcag ngggaggagt tctctgcagg aataaataag cctcagcatt 180 catgaaaatc cactactcca gacagacggc tttggaatcc accagctaca tccagctccc 240 tgaggcaggt aatccatgat gttttacatc ctgggagcgg aggaatctgt ttttccagga 300 gagttttagg cagcagcctg g 321 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 2 gacctttgga ggaagagatt agagc 25 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 3 tccaggctgc tgcctaaaac tctc 24

Claims (11)

A polynucleotide consisting of 5 to 300 consecutive bases, wherein the 141st base of the SERPINA3 gene represented by the nucleotide sequence of SEQ ID NO: 1 is T or G, and the 141th base, or a polynucleotide complementary thereto; SNP marker composition for the differential diagnosis of asthma patients. 2. The marker composition of claim 1, wherein the apoptosis is categorized by aberrant or empirical. A composition for distinguishing between asthma patients and patients with asthma, comprising an agent capable of detecting or amplifying the SNP marker composition of claim 1 or 2. 4. The composition of claim 3, wherein the agent is a probe or a primer. 5. The composition according to claim 4, wherein the primer is a polynucleotide consisting of the nucleotide sequence of SEQ ID NOS: 2 and 3. A kit for the differential diagnosis of asthma patients comprising the composition of claim 3. 7. The kit of claim 6, wherein the kit is an RT-PCR kit or a DNA chip kit. 6. A microarray for the differential diagnosis of asthma patients comprising the SNP marker composition of claim 1 or 2. (a) amplifying a polymorphic site comprising a SNP of a polynucleotide consisting of the nucleotide sequence of SEQ. ID. NO. 1 or a complementary polynucleotide thereof from the DNA of the sample separated from the individual; And
(b) determining the base of the amplified polymorphic site. &lt; Desc / Clms Page number 19 &gt; 10. The method according to claim 9, wherein the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is G at position 141 and the nucleotide of complementary polynucleotide is T or G. 10. The method according to claim 9, wherein the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 is T at position 141 and the base of the polynucleotide complementary thereto is T.