KR101346476B1 - Single Nucleotide Polymorphismic Marker Associated with Asthma Risk and Use Thereof - Google Patents

Abstract

The present invention relates to kits for predicting the risk of developing asthma and methods of providing useful information for predicting the risk of developing asthma. According to the present invention, by detecting genotypes of four known SNPs present in the promoter region of the β-catenin gene, it is possible to provide very useful information for predicting the risk of asthma in a subject, and based on this information Necessary and appropriate treatment can be provided to prospective subjects or asthmatic patients. In addition, according to the present invention, it is possible to construct a screening system (HTS, High-throughput screening) capable of searching for a high-speed, large-capacity asthma candidate substance that is expected to be effective in the human body.

Description

Single Nucleotide Polymorphismic Marker Associated with Asthma Risk and Use Thereof}

The present invention relates to monobasic polymorphic markers associated with asthma risks and their use, more particularly kits for predicting the risk of developing asthma, methods of providing information useful for predicting the risk of developing asthma, candidates for treating asthma A method for screening a material and a kit used in the method.

Β-catenin encoded by the CTNNB1 gene is a member of the cadherin cell-adhesion complex and also acts as a transcription promoter [1]. β-catenin is known to play a very important role in the regulation of differentiation, proliferation, development and tissue regeneration [2, 3]. β-catenin is also known to act as a transcriptional co-regulator in the WNT / β-catenin pathway. When the WNT / β-catenin pathway is activated, β-catenin migrates into the nucleus, followed by transcription of target genes such as cyclin D1, PPARδ, and the like [4]. Recently, studies have reported that β-catenin plays an opposite role in immune responses induced by bacteria and lipopolysaccharides. Duan et al. Reported that β-catenin negatively regulates the inflammatory response induced by Salmonella typhimurium , while Kim et al. Reported that β-catenin is liposomes via the toll-like receptor 4 pathway. It has been reported to positively regulate NADPH oxidase expression and production of reactive oxygen species in polysaccharide-stimulated macrophages [5, 6].

In a previous study, we compared the gene expression patterns of peripheral blood nuclear cells in asthma patients and normal subjects via microarrays, so that expression levels of certain genes in the WNT / β-catenin pathway were increased in asthmatic patients and normal patients. A difference was found between the subjects. It was also found that β-catenin negatively regulates cytokine production in lipopolysaccharide stimulated macrophages [7]. Studying the genetic relevance of human subjects is very important in identifying what role β-catenin plays in the immune response. However, little is known about how β-catenin plays a role in the immune disease of human subjects.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Lee et al., Immunopharmacol Immunotoxicol. 2012 Feb; 34 (1): 56-65

The present inventors have tried to find molecular biological markers that can predict the risk of asthma. As a result, the present invention was completed by discovering that a specific haplotype of four single nucleotide polymorphisms (SNPs) of the promoter region of the β-catenin gene is closely related to the risk of asthma.

It is therefore an object of the present invention to provide a kit for use in predicting the risk of developing asthma, including oligonucleotides capable of detecting four SNPs at the promoter region of the β-catenin gene.

Another object of the present invention is to provide a method for providing useful information for predicting the risk of developing asthma, including detecting four SNPs at a promoter region of a β-catenin gene.

Another object of the present invention is to provide a method for screening a candidate for asthma treatment and a kit for use in the method.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention (i) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 1, comprising 8-100 consecutive nucleotide sequence comprising the 501st nucleotide or its complementary base sequence Polynucleotides; (Ii) a polynucleotide comprising a DNA nucleotide sequence of SEQ ID NO: 2, comprising 8-100 contiguous nucleotide sequences comprising the 301th nucleotide or a complementary nucleotide sequence thereof; (Iii) a polynucleotide comprising the DNA nucleotide sequence of SEQ ID NO: 3, comprising 8-100 contiguous nucleotide sequences comprising the 301th nucleotide or a complementary nucleotide sequence thereof; And (iii) a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the risk of developing asthma comprising a polynucleotide comprising 8-100 contiguous sequences comprising the 406th nucleotide or complementary sequences thereof. Provided are kits for predictive use.

According to another aspect of the present invention, the present invention provides a method of providing information useful for predicting the risk of developing asthma, comprising the steps of: (a) removing a nucleic acid molecule from a biological sample isolated from a subject; Extracting; And (b) a polynucleotide consisting of (iii) a DNA nucleotide sequence of SEQ ID NO: 1 from the nucleic acid molecule, the base type of the 501st nucleotide; (Ii) the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 2, the base type of the 301 th nucleotide; (Iii) the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 3, the base type of the 301 th nucleotide; And (iii) detecting the base type of the 406th nucleotide in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 4.

The present invention finds that certain haplotypes of four single nucleotide polymorphisms (SNPs) in the promoter region of the β-catenin gene are very closely associated with an increase or decrease in the risk of asthma. Based.

As used herein, the term "risk of asthma" refers to the degree of potential (sensitivity) to asthma.

As used herein, the term “nucleotide” is a deoxyribonucleotide or ribonucleotide present in single- or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New). York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term “monopolymorphism” refers to a variation in the DNA sequence in which a single nucleotide A, T, C or G in the genome is different within the same species or between chromosomal pairs of each individual.

In the present invention, in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 1, the single nucleotide polymorphism represented by the 501th nucleotide is also described as "-10,288C> T" or "rs7630377".

In addition, in the present specification, in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 2, the single nucleotide polymorphism represented by the 301th nucleotide is also indicated and expressed as "-6,426C> G" or "rs9859392".

In addition, in the present specification, in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 3, the single nucleotide polymorphism represented by the 301th nucleotide is also indicated as "-4,361G> C" or "rs9870255".

In addition, in the present specification, in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 4, the single nucleotide polymorphism represented by the 406th nucleotide is also described as "-765G> A" or "rs3864004".

According to a preferred embodiment of the invention, (iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 1, the 501st nucleotide is a T base; (Ii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 2, the 301th nucleotide is a G base; (Iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 3, the 301th nucleotide is a C base; (Iii) In the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 4, the 406th nucleotide is A base.

According to another preferred embodiment of the invention, (iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 1, the 501st nucleotide is a C base; (Ii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 2, the 301th nucleotide is a C base; (Iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 3, the 301th nucleotide is a G base; (Iii) In the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 4, the 406th nucleotide is a G base.

In the present invention, a haploid genotype consisting of specific combinations of alleles of four SNPs, -10,288C> T, -6,426C> G, -4,361G> C, and -765G> A has a strong asthma risk and strong asthma risk. Correlation

In the present invention, a haploid genotype ht2 [TGCA] consisting of -10,288C> T base, -6,426C> G base, -4,361G> C base, and -765G> A base, is β-catenin. It is associated with increased expression levels of mRNA and increased risk of developing asthma.

In the present invention, the haploid genotype ht1 [CCGG] consisting of -10,288C> T C base, -6,426C> G C base, -4,361G> C G base, and -765G> A G base is β-catenin It is associated with decreased expression levels of mRNA and reduced risk of developing asthma.

The present invention relates to a nucleotide variant of each SNP position in the DNA base sequence of SEQ ID NO: 1 to 4, but when such a SNP nucleotide variation is found in double stranded gDNA (genomic DNA), it is complementary to the nucleotide sequence described above It is also interpreted to include polynucleotide sequences. Thus, the base at the SNP position in the complementary polynucleotide sequence is a complementary base.

The present invention provides a method of providing information useful for predicting the risk of developing asthma, comprising analyzing the base types of the four SNPs described above from biological samples isolated from a subject.

The term "nucleic acid molecule" as used herein has the meaning encompassing DNA (gDNA and cDNA) and RNA molecules inclusively.

In the method of the present invention, the nucleic acid molecule can be obtained from the various types of “biological samples” such as supernatants of tissues, whole blood, serum, plasma, body fluids, urine, cells, cell debris or cell cultures. Including but not limited to, preferably whole blood, serum or plasma.

In the method of the present invention, when the nucleic acid molecule is gDNA, isolation of the gDNA can be carried out according to a conventional method known in the art (see Rogers & Bendich (1994)), for example, when separating from blood, GDNA can be isolated using the QIAamp DNA Blood Maxi Kit. If the starting material is mRNA, total RNA is isolated by conventional methods known in the art. See Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Ausubel, F.M. et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); And Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987). The isolated total RNA is synthesized by cDNA using reverse transcriptase. Since the total RNA is isolated from animal cells, it has a poly-A tail at the end of the mRNA, and cDNA can be easily synthesized using oligo dT primer and reverse transcriptase using this sequence characteristic (see PNAS). USA, 85: 8998 (1988); Libert F, et al., Science, 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001) )).

In the present invention, a method for analyzing the base type of SNP, that is, SNP genotyping may be performed by applying various methods known in the art. For example, a method using an Affymetrix SNP chip, PCR-RFLP (Restriction Fragment Length Polymorphism) method, PCR-SSCP (Single Strand Conformation Polymorphism) method, PCR-SSO (Specification Sequence Oligonucleotide) method, dot Hybridization method, Allele Specific Oligonucleotide (ASO) hybridization method combining PCR-SSO and dot hybridization method, TaqMan method, Pyrosequencing method, Invader method, SNaPShot method (allele specific gene amplification method), MassArray method Including, but not limited to, Dynamic Allele-Specific Hybridization (DASH) method, Microplate Array Diagonal Gel Electrophoresis (MADG) method, GoldenGate method, SNPlex method and BeadArray method It is not.

In the method of the present invention, a nucleic acid molecule amplification primer or a nucleic acid molecule detection probe may be used for SNP genotyping.

As used herein, the term "primer" refers to a single-stranded oligonucleotide, wherein the template- under suitable conditions (the presence of four different nucleoside triphosphates and polymerases such as DNA or RNA polymerases), suitable temperature and suitable buffer- It is meant to act as a starting point for initiating directed DNA synthesis. The suitable length of the primer is determined by the nature of the primer to be used, but is usually used as a length of 15 to 30 bp. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.

Where amplification of nucleic acid molecules is applied in the methods of the invention, it is important to design suitable primers to identify the SNP bases of the invention.

Nucleic acid molecule amplification techniques that can be used in the methods of the present invention include PCR amplification (Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822)), ligase chain reaction (LCR, Wu) , DY et al., Genomics 4: 560 (1989)), polymerase ligase chain reaction (Barany, PCR Methods and Applic., 1: 5-16 (1991)), Gap-LCR (WO 90/01069), Repair chain reaction (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86: 1173 (1989)) and NASBA (US Pat. No. 5,130,238).

As described in a specific embodiment of the present invention, TaqMan method may be used for SNP genotyping in the present invention, and this method uses the 5'-nuclease property of Tap polymerase. A pair of TaqMan probes and a pair of PCR primers are used. A TaqMan probe with a reporter dye at the 5'-end and a covalently bonded absorber dye at the 3'-end is used, and a match between the polymorphic site and the TaqMan probe The difference between mismatches is determined by using fluorescent materials. When both the reporter dye and the absorbing dye are combined in a TaqMan probe, the reporter dye is not illuminated by the absorbing dye. However, TaqMan probes, which are complementary to the site where the SNP is located, are bound at the annealing stage of PCR and at the 5'-terminal end by the 5'-nuclease activity of the Taq polymerase at the extension stage. The reporter dye (FAM or VIC) is separated from the absorbing dye to give inherent fluorescence. If the probe mismatches with the target SNP, no further reaction proceeds because the probe is pulled away from the template.

In the present invention, SNP genotyping may also be performed through an analysis method using a hybridization signal. In this case, a probe complementary to the sequence comprising the SNP of the present invention is used. In this technique, hybridization signals of probes and target sequences are detected to directly determine whether SNP variants are present.

As used herein, the term “probe” refers to a linear oligomer having naturally occurring or modified monomers or bonds, including deoxyribonucleotides and ribonucleotides that can hybridize to a particular nucleotide sequence. Preferably, the probe is single stranded for maximum efficiency in hybridization. The probe is preferably deoxyribonucleotide. As the probe used in the present invention, a sequence that is completely complementary to the sequence including the SNP may be used, but a sequence that is substantially complementary within a range that does not prevent specific hybridization may be used. Suitable conditions for hybridization include, but are not limited to, Nucleic Acid Hybridization, A Practical Approach, Hayes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IRL Press, Washington, DC (1985). ≪ / RTI >

The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

On the other hand, when the kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases, heat stable DNA polymerases, DNA polymerase cofactors and dNTPs. It may include.

According to another aspect of the invention, the invention provides a method for screening a candidate for asthma treatment comprising the following steps:

Step (a): (iii) the 501th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 1 is the T base, (ii) the 301th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 2 is the G base, and (iii) A promoter of β-catenin gene comprising a ht2 genotype wherein the 301th nucleotide is a C base in the DNA sequence and the 406th nucleotide is an A base in the DNA nucleotide sequence of SEQ ID NO: 4; And processing the candidate in a cell transformed with the vector comprising a reporter coding nucleic acid molecule operatively linked to the promoter.

Step (b): measuring reporter expression levels in the candidate treated cells and reporter expression levels in the candidate untreated cells; And

Step (c): determining that the reporter expression level in the candidate treated cells is asthmatic drug candidate when the reporter expression level in the candidate untreated cells is reduced.

According to another aspect of the present invention, (iii) the 501th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 1 is a T base, (ii) the 301th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 2 is a G base, A promoter of β-catenin gene comprising the ht2 genotype, in which the 301th nucleotide is a C base in the DNA nucleotide sequence of SEQ ID NO: 3, and the 406th nucleotide is the A base in the DNA base sequence of SEQ ID NO: 4; And a vector comprising a reporter coding nucleic acid molecule operatively linked to the promoter, or a kit for screening a candidate for asthma treatment, comprising a cell transformed with the vector as an active ingredient.

In the present specification, the expression “promoter of β-catenin gene comprising ht2 genotype” refers to a T base of -10,288C> T SNP, G base of -6,426C> G SNP, in a DNA sequence of β-catenin gene promoter. Β-catenin gene promoter comprising the ht2 [TGCA] genotype consisting of the C base of 4,361G> C SNP, and the A base of -765G> A SNP.

In the present invention, a method for screening a candidate for asthma treatment and a kit used therein include four SNPs, -10,288C> T, -6,426C> G, -4,361G> C, and -765G present in the promoter of the β-catenin gene. In> A, haploid genotype ht2 [TGCA] is β-catenin consisting of -10,288C> T T bases, -6,426C> G bases, -4,361G> C bases, and -765G> A bases It is based on the fact that it increases the level of expression of the gene and increases the risk of developing asthma in the human body.

Candidates exhibiting activity that inhibits the transcriptional activity of β-catenin gene promoters, including haploid genotype ht2 [TGCA], which is associated with risk of asthma in humans, are candidates for asthma treatment, and asthma in humans is higher than conventional methods. Therapeutic efficacy can be expected.

As used herein, the term “promoter” refers to a DNA sequence that regulates the expression of a coding sequence or functional RNA.

As used herein, the term “operatively linked” refers to a functional bond between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, The regulatory sequence thereby regulates the transcription and / or translation of the other nucleic acid sequence.

As used herein, the term “reporter” or “reporter gene” is a gene that is operably linked to a specific promoter to enable analysis of the transcriptional activity of the promoter by measuring the expression pattern or expression level of the gene. Preferably, the gene of luciferase, the gene of chloramphenicol acetyltransferase, the gene of β-galactosidase, the gene of human growth hormone, the green fluorescent protein, the secretory placental egg Secreted placental alkaline phosphatase and the like, but is not limited thereto.

The amount of protein expressed by the reporter gene was determined in de Wet J. et al, Mol. Cell Biol., 7: 725-7 37 (1987), Gorman C. et al, Mol. For chloramphenicol acetyltransferase. Cell Biol., 2: 104 4-1051 (1982), in the case of β-galactosidase, Hall C.V. et al, J. Mol. Appl. Genet., 2: 101-109 (1983), for Selden R. et al., Mol. Cell Biol., 6: 3173-3179 (1986), in the case of green fluorescent protein, Chalfie M. et al, Science, 263: 802-805 (1994), and secretory placental alkaline phosphate In the case of iz, it is measured according to the method disclosed in Berger, J. et al, Gene, 66: 1-10 (1988).

Since the vector of the present invention is generally transformed and used in eukaryotic cells, preferably animal cells, more preferably mammalian cells, it requires a replication origin that operates in eukaryotic cells, and the origin of replication of eukaryotic cells is f1. Replication origin, SV 40 replication origin, pMB1, and the like, but is not limited thereto. Vectors of the present invention require a polyadenylation signal sequence as a transcription termination signal. In addition, in order to select transformed eukaryotic cells, resistance genes for antibiotics that act on eukaryotic cells must be included, and preferably, there are resistant meteors to neomycin, geneticin (G418) and kanamycin.

In the present invention, the method of transforming the vector into cells may employ a method of transforming a vector into a eukaryotic cell known in the art, for example, a micro-injection method (Capecchi, MR, Cell, 22: 479 (1980) )), Calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposomes -Mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), gene bambard Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990), Lithium acetate-DMSO method (Hill et al., Nucleic Acid Res., 19, 5791 (1991)), etc. Can be used.

The screening method of the present invention is suitable for high-throughput screening (HTS).

Candidates in the present invention may include a library of compounds produced by natural products or chemical synthesis methods and combinations. For example, non-peptidic organic molecules, peptides, polypeptides, sugars, hormones, nucleic acid molecules, small molecule libraries are included.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention is based on the finding that the specific haploid genotypes of the four SNPs present in the promoter region of the β-catenin gene are closely associated with an increase or decrease in the risk of developing asthma in the human body.

(Ii) By using the kit or method of the present invention, by identifying the surrogate genotypes of the four SNPs described above, it is possible to provide useful information in predicting the risk of asthma, thereby providing a prospective subject or asthmatic patient with It can provide the optimal treatment needed.

(Iii) Asthma therapeutic candidates that are expected to have good effects in the human body based on the correlation between the haploid genotype ht2 [TGCA] present in the β-catenin gene promoter region identified in the present invention and the increased risk of asthma in humans. It is possible to build a screening system that can search in a high speed and large capacity.

The present invention relates to kits for predicting the risk of developing asthma and methods of providing useful information for predicting the risk of developing asthma. According to the present invention, by detecting genotypes of four known SNPs present in the promoter region of the β-catenin gene, it is possible to provide very useful information for predicting the risk of asthma in a subject, and based on this information Necessary and appropriate treatment can be provided to prospective subjects or asthmatic patients. In addition, according to the present invention, it is possible to construct a screening system capable of searching for a high-speed, high-capacity method for asthma candidates that are expected to have good effects in the human body.

Figure 1 shows the structure of the SNP and haploid genotype of the promoter region of the β-catenin gene in Korean subjects. Panel A shows the location and gene map of the SNPs in the promoter of β-catenin gene present on staining 3p21. Black bars are coding exons and 5 'and 3' UTRs are marked with white bars. The initial base of the transcriptional position is indicated by nucleotide +1. Panel B shows the LD coefficients among the four SNPs of 684 Korean subjects. Panel C shows promoter haploid genotypes of β-catenin and their frequency in 684 Korean subjects.
2 shows the effect of the promoter haploid genotype of β-catenin on mRNA expression in peripheral blood nuclear cells of human subjects. Β-catenin mRNA levels in peripheral blood nuclei obtained from a subgroup of 185 subjects were compared according to the haploid genotype of the promoter using ANCOVA with age and gender adjustment (n: number of subjects).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

1. Subject

The subjects were recruited from Respiratory Allergy Medicine, Chung-Ang University Hospital. Currently, patients with asthma have been identified as having one or more asthma symptoms by physical examination according to the American Thoracic Society Asthma Diagnosis Regulation [8]. Normal subjects were recruited from the general public who responded negatively to a screening questionnaire about respiratory symptoms. The subjects were a total of 684, including 400 controls and 284 asthma patients. General characteristics of the test subjects are summarized in Table 1 below.

2. Genotyping and Haploid  Genotype composition

The genome DNA of each subject was extracted from whole blood using the aQIAampDNA Blood Mini Kit (Qiagen. Hilden, Germany). SNPs in the promoter region of the β-catenin gene in the Asian population (CHB + JPT, China and Japan) from HapMap data release 22 were analyzed using the Haploview program (http://www.broadinstitute.org/scientific-community/software). It was confirmed [9]. Genotyping of identified SNPs was done according to Taq man methods known in the art [10] and PCR primer / probe sets for genotyping were purchased from Applied Biosystems (Foster City, CA, USA) (Table 2). .

Calculation of linkage disequilibrium (LD) coefficients, D ', and r2, and construction of haploid genotypes for each subject were performed using the SNPAnalyzer 2.0 program (http://snp.istech21.com). .

3. Measurement of mRNA Expression Levels in Peripheral Blood Cells

MRNA levels in peripheral blood cells were measured for 185 subjects who could obtain a buffy coat fraction. The age and gender distribution of this subgroup did not differ significantly from that of the experimental group (p> 0.05). 10 ml of peripheral blood obtained from each subject was centrifuged at 4,000 rpm for 20 minutes to obtain 0.4 ml of each buffy coat fraction composed mainly of peripheral blood nuclear cells. The buffy coat fraction was mixed with 1.3 ml of RNAlater solution (Ambion, Austin, TX, USA) and stored at -70 ° C. Total RNA was extracted using the RiboPure-Blood Kit (Ambion). 1 μg of RNA was used as a template and synthesized into cDNA using a cDNA Reverse Transcription Kit (Applied Biosystems). The expression levels of β-catenin (Hs00170025_m1) and 18S rRNA (Hs99999901_s1) were evaluated using Assay-on-Demand Gene Expression products (Applied Biosystems). 18S rRNA was used as an endogenous control. For each sample, the levels of β-catenin mRNA were normalized similar to that of 18S rRNA and expressed according to comparative Ct method [12].

4. Statistical Analysis

All data are expressed as mean ± standard deviation (SE). A logistic regression analysis was performed to test the association between β-catenin promoter haplotype and the risk of asthma, using sex, age, and haplotype as covariates. Subjects' β-catenin mRNA expression levels were compared by ANCOVA and adjusted for age and sex. A p value of <0.05 was considered statistically significant. All analyzes were performed using SPSS version 19.0 (SPSS, Chicago, IL, USA).

Experiment result

1. Polymorphism Analysis of the β-catenin Promoter Site

In this experiment, genetic polymorphism in the promoter region of β-catenin gene was investigated to find genetic factors that could directly affect the expression level of human β-catenin. By examining the 41,205-41,216 kb site of chromosome 3 including the upstream 10 kb region of the human β-catenin gene, four single nucleotide polymorphisms (SNPs) were identified. These identified SNPs are -10,288C> T (rs7630377), -6,426C> G (rs9859392), -4,361G> C (rs9870255), and -765G> A, as shown in panel A and Table 2 of FIG. (rs3864004). Analysis of the genotypes of 684 Korean test subjects found that their minor allele frequency (MAF) was 0.220-0.291 in the Korean population, which is similar to the values of 0.221-0.320 in the Chinese and Japanese populations according to HapMap data. It was a level. Their genotype distribution was at the Hardy-Weinberg equilibrium (P> 0.05) (see Table 3).

Next, the association non-equilibrium (LD) coefficients and haplotype construction were analyzed for these SNPs [13]. LD coefficients between four SNPs are shown in panel B of FIG. 1. Three of these SNPs, -6,426C> G, -4,361G> C, and -765G> A, are | D '| = 1 and r2≥0.996, which was almost completely related, whereas -10,288C> T had a value of r2 <0.8 and was not associated with the other three SNPs. The constitution of the haploid genotype of the promoter region is shown in panel C of FIG. 1.

As the most common promoter haploid genotyping, ht1 and ht2 were found to be 0.708 and 0.219, respectively. A rare promoter haploid genotype construct, ht3, showed an allele frequency of 0.071. ht1 was composed of four alleles of four SNPs, -10,288C> T C, -6,426C> G C, -4,361G> C G, and -765G> A G. In contrast, the ht2 haploid genotype was composed of minor alleles in each SNP. The ht3 haploid genotype consists of a minor allele T of SNP -10,288C> T and a majority allele of three other SNPs, with ht1 and ht2 between alleles -10,288C> T and -6,426C> G recombining It is assumed to be one result (see panel C of FIG. 1). Since Ht4 and ht5 show very low frequencies of 0.0008 and 0.0007, respectively, and the effects on the genetic population are negligible, no further analysis was performed on this haploid genotype.

2. β- Catechin  Promoter Haploid  Genotype mRNA  Impact on expression level

To investigate the effect of haploid genotype structure of promoters on mRNA expression levels, β-catenin mRNA levels were compared in peripheral blood cells among subgroups of total RNA extracted from peripheral blood. As shown in panel A of FIG. 2, the Ht1 carrier heterozygous (-/ ht1) or isotype (ht1 / ht1) has significantly higher β-catenin mRNA levels compared to the non-carrier (-/-). It was reduced. On the other hand, as shown in panel B of FIG. 2, ht2 isotypes (ht2 / ht2) have significantly higher mRNA expression levels compared to non-carriers (-/-) and heterologous carriers (-/ ht2). Increased. The ht3 haploid genotype was found to have no effect on mRNA expression levels. Because of the very low frequency of the ht3 haploid genotype, the homozygous ht3 / ht3 was not found among the subject subgroups obtained by extracting total RNA. As shown in panel C of FIG. 2, the ht3 heterozygous (-/ ht3) showed similar levels of mRNA expression compared to the non-carrier (-/-).

3. β- Catechin  Promoter Haploid  Genotypes and Risk of Asthma Association degree

Logistic regression using covariate sex, age and haploid genotypes examined the relationship between the three types of haploid genotypes ht1, ht2, and ht3 present in the β-catenin promoter. The survey results are summarized in Table 4, and more detailed logistic regression results are shown in Table 5.

For the ht2 haploid genotype, homozygous carriers (ht2 / ht2) were found to be higher in asthma patients compared to control subjects (6.0% vs 2.8%), indicating that the ht2 haploid genotype increased the risk of asthma. It is strongly related to Logistic regression analysis showed that the ht2 homozygous carrier (ht2 / ht2) compared to the non-carrier (-/-) had an odds ratio of 2.340 at a 95% confidence interval (CI) of 1.067-5.132 (P = 0.034). ratio, OR), and a statistically significant increase in asthma risk. On the other hand, the heterologous carrier of ht2 (-/ ht2) showed no statistically significant change in asthma risk compared to the non-carrier (-/-).

On the other hand, for ht1 haploids, non-retainers (-/-) were found to be higher in asthmatic patients compared to control subjects (9.9% vs 5.8%), indicating that ht1 haploids reduce the risk of asthma. It is associated with. Logistic regression showed that the heterologous genotype carrier (-/ ht1) had an odds ratio of 0.532 at the 95% confidence interval of 0.292-0.970 (P = 0.039) and the homozygous carrier (ht1 / ht1) was 0.327- An 95% confidence interval (CI) of 1.070 (P = 0.083) showed an OR value of 0.591. The lack of significance in the homozygous genotype (ht1 / ht1) is due to the lack of the number of subjects in this experiment. All ht1 carriers, including heterozygous (-/ ht1) and homozygous (ht1 / ht1) had an OR value of 0.563 (P = 0.050), reducing the risk of asthma as compared to non-bearers. In contrast, the Ht3 haploid genotype was not associated with asthma risk.

4. Conclusion

Comparing the results of Figure 2 and Table 3, it was found that the haploid genotype of the β-catenin promoter region has the same effect on the mRNA expression level and their asthma risk. That is, ht1 decreased both β-catenin mRNA expression and asthma risk, while ht2 increased both β-catenin mRNA expression and asthma risk, while ht3 had no effect on β-catenin mRNA expression and asthma risk. It was not crazy. These results suggest that there is a constant association between β-catenin expression and asthma risk, suggesting that β-catenin plays a role in the development and progression of asthma.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

Willert et al., Curr Opin Genet Dev 1998; 8: 95-102.

2. Wodarz et al. Annu Rev Cell Dev Biol 1998; 14: 59-88.

3. Moon et al., Science 2002; 296: 1644-46.

4. Cadigan et al. J Cell Sci 2006; 119: 395-402.

5. Duan et al. Lab Invest 2007; 87: 613-24.

6. Kim et al., Febs J 2010; 277: 2830-7.

7. Lee et al. Immunopharmacol Immunotoxicol 2012; 34: 56-65.

Am Rev Respir Dis 1987; 136: 225-44.

9. Barrett et al., Bioinformatics 2005; 21: 263-5.

10. Lee et al., Clin Biochem; 43: 1195-200.

11. Yoo et al., Nucleic Acids Res 2005; 33: W483-8.

12. Livak et al., Method. Methods 2001; 25: 402-8.

13. Hedrick PW. Genetics 1987; 117: 331-41.

14.Pakis P. Curr Opin Genet Dev 2007; 17: 45-51.

15.Polakis P. Genes Dev 2000; 14: 1837-51.

Attach an electronic file to a sequence list

Claims (9)

(Iii) a polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 1, a polynucleotide comprising 8-100 contiguous nucleotide sequences comprising the 501st nucleotide or complementary nucleotide sequences thereof;
(Ii) a polynucleotide comprising a DNA nucleotide sequence of SEQ ID NO: 2, comprising 8-100 contiguous nucleotide sequences comprising the 301th nucleotide or a complementary nucleotide sequence thereof;
(Iii) a polynucleotide comprising the DNA nucleotide sequence of SEQ ID NO: 3, comprising 8-100 contiguous nucleotide sequences comprising the 301th nucleotide or a complementary nucleotide sequence thereof; And
(Iii) predicting the risk of developing asthma in a polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 4, comprising a polynucleotide comprising 8-100 contiguous sequences comprising the 406th nucleotide or complementary sequences thereof Kit for use.
The method of claim 1, wherein (iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 1, the 501st nucleotide is a T base; (Ii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 2, the 301th nucleotide is a G base; (Iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 3, the 301th nucleotide is a C base; (Iii) A polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 4, wherein the 406th nucleotide is A kit.
The method of claim 1, wherein (iii) in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 1, the 501st nucleotide is a C base; (Ii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 2, the 301th nucleotide is a C base; (Iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 3, the 301th nucleotide is a G base; (Iii) A polynucleotide consisting of the DNA base sequence of SEQ ID NO: 4, wherein the 406th nucleotide is a G base.
The kit of claim 1, wherein the polynucleotide comprising 8-100 consecutive DNA sequences or complementary sequences thereof is a primer for nucleic acid amplification or a probe for detecting DNA sequences of nucleic acids. .
How to provide useful information to predict the risk of developing asthma, including the following steps:
(a) extracting a nucleic acid molecule from a biological sample isolated from the subject; And
(b) a base type of the 501st nucleotide in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 1 from the nucleic acid molecule; (Ii) the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 2, the base type of the 301 th nucleotide; (Iii) the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 3, the base type of the 301 th nucleotide; And (iii) detecting the base type of the 406th nucleotide in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 4.
The method of claim 5, wherein (iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 1, the 501st nucleotide is a T base; (Ii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 2, the 301th nucleotide is a G base; (Iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 3, the 301th nucleotide is a C base; (Iii) A polynucleotide consisting of the DNA base sequence of SEQ ID NO: 4, wherein the 406th nucleotide is A base.
The method of claim 5, wherein (iii) in the polynucleotide consisting of the DNA nucleotide sequence of SEQ ID NO: 1, the 501st nucleotide is a C base; (Ii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 2, the 301th nucleotide is a C base; (Iii) in the polynucleotide consisting of the DNA base sequence of SEQ ID NO: 3, the 301th nucleotide is a G base; (Iii) A polynucleotide consisting of the DNA base sequence of SEQ ID NO: 4, wherein the 406th nucleotide is a G base.
A method for screening a candidate for asthma treatment comprising the following steps:
Step (a): (iii) the 501th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 1 is the T base, (ii) the 301th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 2 is the G base, and (iii) A promoter of β-catenin gene comprising a ht2 genotype wherein the 301th nucleotide is a C base in the DNA sequence and the 406th nucleotide is an A base in the DNA nucleotide sequence of SEQ ID NO: 4; And processing the candidate in a cell transformed with the vector comprising a reporter coding nucleic acid molecule operatively linked to the promoter.
Step (b): measuring reporter expression levels in the candidate treated cells and reporter expression levels in the candidate untreated cells; And
Step (c): determining that the reporter expression level in the candidate treated cells is asthmatic drug candidate when the reporter expression level in the candidate untreated cells is reduced.
(Iii) the 501th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 1 is the T base, (ii) the 301th nucleotide in the DNA nucleotide sequence of SEQ ID NO: 2 is the G base, and (iii) the 301 in the DNA nucleotide sequence of SEQ ID NO: A promoter of β-catenin gene comprising an ht2 genotype wherein the nucleotide is the C base and the 406th nucleotide is the A base in the DNA sequence of SEQ ID NO: 4; And a vector comprising a reporter coding nucleic acid molecule operatively linked to this promoter or a kit for screening a candidate for an asthma therapeutic agent comprising a cell transformed with the vector as an active ingredient.

Images