JP2011004619A - Method for examining immunological disease, and method for screening medicine for preventing or treating immunological disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an examination method for predicting an immunological disease, and to provide a method for screening a medicine for preventing or treating immunological disease.SOLUTION: The examination method comprises analyzing the single nucleotide polymorphism of a base existing on IBTK gene or a base in linkage disequilibrium to the base, and then examining an immunological disease on the basis of the analysis result. Or, the examination method comprises measuring the expression amount of the mRNA or protein of IBTK gene long form and then examining the immunological disease on the basis of the measurement result. The method for screening a medicine for preventing or treating the immunological disease includes a process for selecting a substance for changing the expression amount of the IBTK gene long form.

Description

  The present invention relates to a method for examining immune diseases, probes and primers used therefor, and a method for screening drugs for preventing or treating immune diseases.

Currently, about 3% of Japanese adults and about 6% of children are said to have bronchial asthma. This bronchial asthma is clinically characterized by chronic reversible airway obstruction with wheezing, coughing, shortness of breath and the like. The bronchial asthma is one of the diseases which has been increasing in recent years, and elucidation of the pathological condition is awaited. Adult bronchial asthma is considered to be a multifactorial disease that develops due to a comprehensive contribution of genetic and environmental factors. Regarding genetic factors, polymorphism studies on candidate genes that are predicted to be functionally involved in the development of asthma have been conducted.
T-bet gene (patent document 1), Iκβ-related protein gene (patent document 2), IL-12B gene promoter (patent document 3), TRAIL receptor gene (patent document 4), TSLP gene A method for examining the risk of bronchial asthma based on a polymorphism such as (Patent Document 5) is known.

  IBTK (Inhibitor of Bruton's Tyrosine Kinase) is a factor that binds to the PH domain of Bruton's tyrosine kinase (Btk), which plays an important role in B cell differentiation, and suppresses Btk activity (Non-patent Document 1). It is known that there are multiple transcripts of the IBTK gene. And it is shown that the short form which is a minor transcription product suppresses the information transmission of B cell receptor stimulation (nonpatent literature 2). On the other hand, regarding long form, the function, the relationship with the disease, etc. remained unclear. In addition, no correlation between IBTK gene polymorphism and immune disease has been reported so far.

JP 2006-149276 JP JP 2002-218997 A Japanese Unexamined Patent Publication No. 2006-101847 JP 2005-027595 JP 2008-109915 A

Liu, W., I. Quinto, X. Chen, et al. 2001. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat Immunol 2: 939-946. Spatuzza, C., M. Schiavone, E. Di Salle, et al. 2008. Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk. Nucleic Acids Res 36: 4402-4406.

  An object of the present invention is to provide a test method for accurately predicting the onset and progression of an immune disease. Another object of the present invention is to provide a screening method for preventive or therapeutic agents for immune diseases.

  The present inventors conducted analysis to identify single nucleotide polymorphism (SNP) related to immune diseases such as bronchial asthma. As a result, it was found that SNPs of bases present on the IBTK gene or SNPs in linkage disequilibrium with the bases are related to adult bronchial asthma. Furthermore, it was found that long form expression of the IBTK gene was induced by pseudo-stimulation of infection, thereby enhancing production of cytokines involved in exacerbation of inflammation such as IL-6, TNF-α, IL-23, etc. The present inventors have found that substances that suppress the expression of are useful as preventive or therapeutic agents for immune diseases, and have completed the present invention.

That is, the present invention is as follows.
(1) A method of analyzing an immune disease based on the analysis result by analyzing a base present on the IBTK gene or a single nucleotide polymorphism of a base in linkage disequilibrium with the base.
(2) The single nucleotide polymorphism is a polymorphism in a base corresponding to the base number 61 in the base sequence of any one of SEQ ID NOs: 1 to 6 or a base in linkage disequilibrium with the base (1 )the method of.
(2) The method according to (2), wherein the base in the linkage disequilibrium is a base corresponding to the 61st base in the base sequence of any one of SEQ ID NOs: 7 to 37.
(4) The method according to any one of (1) to (2), wherein the immune disease is an allergic disease.
(5) The method according to (4), wherein the allergic disease is bronchial asthma.
(6) A probe for testing immunological diseases having a sequence of 10 bases or more including the 61st base in the base sequence of SEQ ID NOs: 1 to 37 or a complementary sequence thereof.
(7) A primer for an immune disease test capable of amplifying a region containing the base at the 61st base number in any one of the base sequences of SEQ ID NOs: 1 to 37.
(8) adding a drug candidate substance to a cell expressing a reporter gene linked to a promoter of IBTK gene long form or IBTK gene long form, measuring the expression level of IBTK gene long form or reporter gene, and A method for screening a prophylactic or therapeutic agent for immune diseases, comprising a step of selecting a substance that reduces the expression level.
(9) A method for testing an immune disease, comprising a step of measuring the expression level of mRNA or protein of IBTK gene long form.

ADVANTAGE OF THE INVENTION According to this invention, the onset risk of immune diseases, such as adult bronchial asthma, can be estimated correctly and simply. Thereby, the dangerous state which concerns on a patient's life can be avoided.
Moreover, according to the screening method of the present invention, a therapeutic or prophylactic agent for immune diseases such as bronchial asthma can be obtained.

The figure which shows the procedure of the SNPs search relevant to adult bronchial asthma. The figure which shows LD (linkage disequilibrium) map around the IBTK gene. The figure which shows the position on the IBTK gene of 8 types of selected SNPs. The figure which shows the structure of the transcription product and translation product of IBTK long form and short form. The figure which shows the expression level of IBTK long form and short form in each tissue and each cell. The figure which shows the stimulus-responsive expression pattern of IBTK long form and short form in NHBE (normal human bronchial epithelium) cell, NHLF (normal human lung fibroblast) cell, and BSMC (normal human bronchial smooth muscle) cell. The figure which shows the expression of NF (kappa) B-Luc (what connected the luciferase gene to the NF (kappa) B promoter) in the stable expression cell of IBTK long form. The figure which shows the expression of various cytokines in the stable expression cell of a parent strain and IBTK long form. The figure which shows the expression of various cytokines in a parent strain and the knockdown cell of IBTK long form. Non target indicates that non-specific sequence siRNA is introduced, and IBTK indicates that IBTK long form siRNA is introduced. The figure which shows the stimulus responsive expression of various cytokines in a parent strain and the knockdown cell of IBTK long form. Non target indicates that non-specific sequence siRNA is introduced, and IBTK indicates that IBTK long form siRNA is introduced. The figure which shows stimulation-responsive NF (kappa) B-Luc activation in various TLR expression cells which do not introduce | transduce IBTK long form or introduce | transduced. Stimulation (MALP-) in TLR2 / 6 expressing cells without IBTK or with IBTK long form (49393C> T (rs9449444) major or minor allele) or IBTK short form (49393C> T (rs9449444) major or minor allele) 2: Macrophage-activated lipopeptides) responsive NFκB-Luc activation. The figure which shows the luciferase activity expressed from the construct which has 3'-UTR containing the major or minor allele of 69371A> G (rs991781).

<1> Test Method Based on SNP The test method of the present invention analyzes a base SNP present on the IBTK gene or a base SNP in linkage disequilibrium with the base, and tests an immune disease based on the analysis. Is the method. Although it does not specifically limit as an immune disease, For example, allergic diseases, such as bronchial asthma and atopic dermatitis, are preferable, and bronchial asthma is more preferable. Bronchial asthma includes adult bronchial asthma, childhood bronchial asthma, atopic bronchial asthma, etc., but adult bronchial asthma is preferred. In the present invention, “test” includes a test for the risk of developing an immune disease and a test for the presence or absence of the disease.

The IBTK gene is preferably a human IBTK gene, and examples thereof include a gene having a sequence registered in GenBank Accession No. NC_000006.10, 82936675..83014167 (complement). However, the gene is not limited to the gene of the above sequence because substitution or deletion may exist in one or a plurality of bases due to differences in race.
The SNP of the IBTK gene related to immune diseases is not particularly limited, and examples thereof include SNP-1 to SNP-6 shown in Table 1 below.

In Table 1, for example, SNP-1 means a polymorphism of guanine (G) / adenine (A) at the -5507th base counted from the base corresponding to the translation start point on the genome. In some cases, the possibility of an immune disease is high. Moreover, when analyzing in consideration of alleles, SNP-1 has a high possibility of an immune disease in the order of AA>GA> GG. dbSNP indicates the registration number of the dbSNP database (//www.ncbi.nlm.nih.gov/projects/SNP/) of National Center for Biotechnology Information.
The same applies to other SNPs, but in the case of SNP-2, T is more likely to be an immune disease if A> T, and in the case of SNP-3, G is an immune disease if T is> G In the case of SNP-4, if T>G> T, the possibility of an immune disease is high. In the case of SNP-5, if A>G> A, the possibility of an immune disease In the case of SNP-6, if A> C, C is more likely to be an immune disease.

Regarding SNP-1 to SNP-6, a sequence having a total length of 121 bp including the SNP base and the region of 60 bp before and after that is shown in SEQ ID NOs: 1 to 6 (the 61st base has a polymorphism) ( The sequence of the antisense strand is shown except for SNP-2).
The polymorphism corresponding to this base is analyzed by the method of the present invention. Here, “corresponding” means a corresponding base in the region having the sequence on the human IBTK gene, and even if the sequence is slightly changed at a position other than SNP due to a difference in race, It also includes analyzing the corresponding base in it.

An immune disease can be examined by examining the type of base of the SNP. The sequence of the IBTK gene may be analyzed for the sense strand or the antisense strand. For example, in the case of SNP-1 (rs2132287), the polymorphism when analyzing the sense strand is G / A, but the polymorphism when analyzing the antisense strand is C / T.
In addition, the base to be analyzed in the present invention is not limited to the above SNP, and a polymorphism that is in linkage disequilibrium with the above base may be analyzed. Here, “polymorphism in linkage disequilibrium with the above-mentioned base” refers to a base that satisfies the relationship of r ² > 0.5 with the above-described polymorphism.

For example, since the above SNP-2 (rs1495558) is in a linkage disequilibrium (r ² = 1) relationship with the following plurality of SNPs, polymorphisms of any of the following bases may be analyzed. “3585C> T” means a polymorphism of cytosine (C) / thymine (T) at the 3585th base counted from the base corresponding to the translation start point on the genome, and the same applies to other SNPs. is there. A sequence having a total length of 121 bp including each SNP base and a region of 60 bp before and after the SNP base is indicated by each SEQ ID NO.
3585C> T (rs6939841) (SEQ ID NO: 7),
8548G> C (rs9449451) (SEQ ID NO: 8),
9046A> G (rs7766372) (SEQ ID NO: 9),
9187A> C (rs7766046) (SEQ ID NO: 10),
10627A> G (rs9353058) (SEQ ID NO: 11),
16895G> A (rs9361904) (SEQ ID NO: 12),
19558T> G (rs9344259) (SEQ ID NO: 13),
22713G> A (rs2273194) (SEQ ID NO: 14),
23426A> G (rs7743511) (SEQ ID NO: 15),
27816G> A (rs9353057) (SEQ ID NO: 16),
28865C> T (rs9341913) (SEQ ID NO: 17),
30357T> C (rs9361901) (SEQ ID NO: 18),
31317G> A (rs6454260) (SEQ ID NO: 19),
32300T> G (rs4706942) (SEQ ID NO: 20),
32513T> C (rs7381409) (SEQ ID NO: 21),
33115T> G (rs9443969) (SEQ ID NO: 22),
34944G> C (rs9294251) (SEQ ID NO: 23),
35744C> T (rs9361899) (SEQ ID NO: 24),
36233C> A (rs6454258) (SEQ ID NO: 25),
40656G> C (rs6929155) (SEQ ID NO: 26),
41801G> A (rs9294250) (SEQ ID NO: 27),
42170G> A (rs4706937) (SEQ ID NO: 28),
49126G> C (rs9350944) (SEQ ID NO: 29),
49393C> T (rs9449444) (SEQ ID NO: 30),
50507G> A (rs9359515) (SEQ ID NO: 31),
51766T> C (rs4706936) (SEQ ID NO: 32),
54332T> C (rs9361896) (SEQ ID NO: 33),
64340A> G (rs9344252) (SEQ ID NO: 34),
64340T> C (rs9449442) (SEQ ID NO: 35),
69371A> G (rs991781) (SEQ ID NO: 36),
72754A> G (rs9344251) (SEQ ID NO: 37)
SEQ ID NOs: 8, 23, 26, and 29 show the sequence of the sense strand, and the others are the sequences of the antisense strand.

  The sample used for the analysis of the SNP of the IBTK gene is not particularly limited as long as it is a sample containing chromosomal DNA. Examples thereof include body fluid samples such as blood and urine, body hair such as cells and hair, and nails. Although these samples can be used directly for the analysis of SNP, it is preferable to isolate chromosomal DNA from these samples by a conventional method and analyze them.

  Analysis of SNP of the IBTK gene can be performed by a usual SNP analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, and the like.

  The sequence can be performed by a usual method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5 ′ side of a base showing polymorphism, and the type of base at the corresponding position is determined from the analysis result. can do. When sequencing is performed, it is preferable to amplify a fragment containing a polymorphism in advance by PCR or the like.

  It can also be analyzed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base showing a polymorphism and corresponding to each polymorphism is prepared. PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of the amplification product.

  It is also possible to amplify a DNA fragment containing a polymorphism and determine which type of polymorphism is based on the difference in mobility in electrophoresis of the amplified product. Examples of such a method include a PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1; 12 (1): 139-146.). Specifically, first, DNA containing a polymorphic site of the IBTK gene is amplified, and the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel, and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.

  Furthermore, when a base showing polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method). In this case, first, a DNA sample is amplified by PCR and cut with a restriction enzyme. The DNA fragments can then be separated and the type of polymorphism determined by the size of the detected DNA fragment.

It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization. That is, it is possible to determine which base an SNP is by preparing probes corresponding to each base and examining which probe hybridizes.
Thus, by determining which base an SNP is, data for examining an immune disease can be obtained.

<2> Reagent for immune disease test | inspection This invention also provides test reagents, such as a primer and a probe for test | inspecting an immune disease. Examples of such a probe include a probe that includes the polymorphic site in the IBTK gene and can determine the type of base at the polymorphic site based on the presence or absence of hybridization. Specifically, a probe having a length of 10 bases or more having a sequence containing the 61st base of the base sequence in SEQ ID NOs: 1 to 37 or a complementary sequence thereof can be mentioned. The length of the probe is more preferably 15 to 35 bases, and further preferably 20 to 35 bases.

Examples of the primer include a primer that can be used for PCR for amplifying the polymorphic site in the IBTK gene, or a primer that can be used for sequence analysis (sequencing) of the polymorphic site. Specifically, a primer that can amplify or sequence the region containing the 61st base of the base sequence of SEQ ID NOs: 1 to 37 can be mentioned. The length of such a primer is preferably 10 to 50 bases, more preferably 15 to 35 bases, and even more preferably 20 to 35 bases.
As a primer for sequencing the polymorphic site, a primer having a sequence 5′-side of the base, preferably 30 to 100 bases upstream, or a 3′-side region of the base, preferably 30 to 100 bases. A primer having a sequence complementary to the downstream region is exemplified. As a primer used for judging polymorphism by the presence or absence of amplification by PCR, it has a sequence containing the above base, has a primer containing the above base on the 3 ′ side, and a complementary sequence of the sequence containing the above base, Examples include a primer containing a base complementary to the above base on the 3 ′ side.
The test reagent of the present invention may contain PCR polymerases, buffers, hybridization reagents, etc. in addition to these primers and probes.

  In the test method of the present invention, in addition to polymorphisms of the IBTK gene, polymorphisms of other genes may be analyzed, and an immune disease test may be performed based on the combination of the polymorphisms of those genes. Other genes include T-bet gene (JP 2006-149276), Iκβ-related protein gene (JP 2002-218997), IL-12B gene promoter (JP 2006-101847), Examples include TRAIL receptor gene (Japanese Patent Laid-Open No. 2005-027595), TSLP gene (Japanese Patent Laid-Open No. 2008-109915), and the like.

<3> Screening method for preventive or therapeutic agent for immune disease
There are at least two types of expression products in IBTK: long form (also called α form; SEQ ID NO: 38) and short form (SEQ ID NO: 40) (FIG. 4). In the present invention, expression of the long form of IBTK is induced in response to stimulation of an infection mimetic substance, thereby enhancing production of cytokines involved in exacerbation of inflammation such as IL-6, TNF-α, and IL-23. It was also shown that suppression of IBTK long form expression suppressed the production of these cytokines. Therefore, a substance that can be used as a prophylactic or therapeutic drug for immune diseases can be obtained by screening a drug using the activity of reducing the expression of IBTK long form as an index.
That is, the screening method of the present invention includes a step of adding a drug candidate substance to a cell expressing a reporter gene linked to a promoter of IBTK gene long form or IBTK gene long form, an expression level of IBTK gene long form or reporter gene And a screening method for a prophylactic or therapeutic agent for immune diseases, which comprises a step of measuring the above and a step of selecting a substance that decreases the expression level.
For example, when the expression level of the IBTK gene long form or reporter gene is suppressed in the presence of a drug candidate substance compared to the absence of the drug candidate substance, the drug candidate substance is used as a prophylactic or therapeutic drug for immune diseases. Can be selected as a candidate. In addition, when measuring the expression level, a stimulating substance such as LPS (lipopolysaccharide) is added together with the drug candidate substance, and the indication is whether the induction of long form upon stimulation is suppressed compared to when no drug candidate substance is added. It is also possible to screen for preventive or therapeutic agents for immune diseases.
As cells expressing the IBTK gene long form, monocytic cells THP-1 cells and the like can be used.

When a reporter gene linked to the IBTK gene long form promoter is used, the IBTK gene long form promoter is preferably a region containing about 2 kbp upstream of the long form transcription start site, more preferably a region containing about 5 kbp upstream. . The sequence information of the promoter can be obtained from Nucleic Acids Res. 2008 August; 36 (13): 4402-4416.
Examples of reporter genes include luciferase gene, GFP gene, chloramphenicol acetyltransferase gene and the like. These reporter genes are linked to a promoter of the IBTK gene long form, incorporated into a plasmid used for introducing the gene into mammalian cells, and transfected into cells by a usual method such as lipofection.
A drug candidate substance is added to a cell expressing the IBTK gene long form as described above or a cell into which the reporter gene has been introduced, and the expression level of the IBTK gene long form or the reporter gene is measured.

  There is no restriction | limiting in particular as a pharmaceutical candidate substance, For example, a low molecular synthetic compound may be sufficient and the compound contained in a natural product may be sufficient. Further, it may be a peptide or a nucleic acid. Although individual test substances may be used for screening, a compound library containing these substances may be used. By selecting a candidate substance that lowers the expression level of the IBTK gene long form or reporter gene (compared to the non-added case), a substance that can be an immunological disease drug can be obtained.

The expression level of the IBTK gene can be measured by methods such as RT-PCR, quantitative PCR, Northern blot, ELISA, Western blotting, In situ hybridization, and immunohistochemical staining.
Although the expression level of the reporter gene depends on the type of reporter gene, it can be measured by fluorescence intensity, luminescence intensity, radioactivity intensity, and the like.

<5> Test Method Based on Expression Level of IBTK Gene Long Form The present invention also provides a test method for immune disease (a method for obtaining test data) including a step of measuring the expression level of mRNA or protein of IBTK gene long form. provide. When the expression level of mRNA or protein of IBTK gene long form is increased compared to a control such as a healthy person, it can be determined that the patient is suffering from an immune disease or has a high risk of immune disease.
The expression level of the IBTK gene can be examined by RT-PCR, Northern blot, microarray method and the like. In addition, the expression level of the IBTK gene long form product can be examined by ELISA, Western blot, or the like. As the antibody, a commercially available antibody can be used, and an antibody prepared by using a part of the amino acid sequence of IBTK long form protein (for example, SEQ ID NO: 39) as an antigen can also be used. Examples of the base sequence of the coding region of IBTK long form include (SEQ ID NO: 38), and primers or probes for expression analysis can be designed or obtained using this sequence or the like. Examples of RT-PCR primers include the primer sets of SEQ ID NOs: 42 and 43.
Samples used for the examination include blood samples, bronchial epithelial cells or oral cells.

  In the present invention, the expression level of the long form of the IBTK gene can be compared in the presence and absence of the immunological disease therapeutic agent to determine the effect of the immunological disease therapeutic agent. For example, it can be determined by using as an index whether long form induction is suppressed when an immune disease treatment is added to bronchial epithelial cells, etc. .

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) SNPs in the IBTK gene region
In the “Customized Medical Realization Project”, genome-wide screening was performed using an adult bronchial asthma patient group (diagnosed based on the diagnostic criteria of the American Thoracic Society) and a control group (FIG. 1). All races are Japanese, and in accordance with the rules of the Research Ethics Committee at each joint research institution such as The Institute of Physical and Chemical Research (RIKEN), SNP Research Center, Written informed consent was obtained from all to participate in the study.

  One of the landmark SNPs that showed a strong association with susceptibility to adult bronchial asthma in primary and secondary screening was confirmed as 17814A> T (rs1495558) present in the LD block containing the IBTK gene region (Figure 2). (Table 2).

This block can completely determine the genotype with a minor allele frequency of 10% or more for the IBTK gene region by genotyping the following 8 tag SNPs
(Figure 3).
-5507G> A (rs2132287),
17814A> T (rs1495558),
32300T> G (rs4706942),
56115T> A (rs2323642),
56627G> T (rs2132288),
63504G> A (rs9353054),
70943T> C (rs16893965),
77890A> C (rs2095671).
These SNPs were genotyped using a TaqMan ™ probe specific for the peripheral sequence of each allele.

(2) IBTK SNPs
As a third screening, genomic DNA was extracted from peripheral blood of 468 adult bronchial asthma patients and 740 non-patients, and IBTK was subjected to genotyping by the above method.In 6 SNPs (-5507G> A (rs2132287), 17814A> T (rs1495558), 32300T> G (rs4706942), 56627G> T (rs2132288), 63504G> A (rs9353054), 77890A> C (rs2095671)), significant susceptibility to disease in adult bronchial asthma The relationship was shown (Table 3). Statistical analysis was performed by chi-square test.

Among tag SNPs, in particular, -5507G> A (rs2132287), 17814A> T (rs1495558), and 32300T> G (rs4706942) showed a strong association with adult bronchial asthma patients (p = 0.0040, p = 0.0045, and p = 0.0090 with odds ratios of 2.28, 2.29, and 2.14). Based on the above, it was considered that genotyping of SNPs that showed significant correlation, or SNPs that had a relationship of linkage disequilibrium coefficient r ² = 1, was useful for predicting the onset of adult bronchial asthma. .

(3) IBTK expressing cells
The transcript of the IBTK gene has a long form and a short form (FIG. 4), and these expression patterns were examined in each tissue. The expression pattern was examined by RT-PCR and standardized with GAPDH. The primer used for long form detection is Long forward primer: 5'-TTACACTATGGGACTAAATGGTGGACAAC-3 '(SEQ ID NO: 42); Long reverse primer: 5'-GTCTTTATGGTGAAGGGCAGA-3' (SEQ ID NO: 43) The primers used in this were Short forward primer: 5′-TTTCTTATTATTTTTCTGCATTTGATG-3 ′ (SEQ ID NO: 44); Short reverse primer 5′-ACAAGAGGTGGATTATCAACTCAA-3 ′ (SEQ ID NO: 45).
As a result, long form and short form were expressed in various organs, and in both cases, it was confirmed that long form was about 50 times stronger (upper figure). In particular, long form expression was highly expressed in immune system tissues, lung tissues, and airway tissues.

In addition, the presence of pseudo-stimulation of infection in various immune cells (PBMC (peripheral blood mononuclear cells), CD4 ⁺ cells, CD8 ⁺ cells, B cells, NK (natural killer) cells, monocyte cells, DC (dendritic cells)) The expression of long form and short form was examined below and in the absence (FIG. 5 bottom). As a result, long forms of immune system cells tended to be induced when B cells or monocytes were stimulated with an infection mimic.
In addition, the long form tended to be induced when the epithelial cells (NHBE), fibroblasts (NHLF), and smooth muscle cells (BSMC) that compose the airway epithelial tissue were also stimulated with an infection mimetic substance ( (Fig. 6).
In other words, the long form of IBTK induced by innate immunity is highly expressed in immune system cells important for allergic inflammation and in the field of inflammation, suggesting the possibility of being involved in the development of bronchial asthma.

(4) Functional analysis of IBTK long form A plurality of forced expression clone cells that stably express IBTK long form cDNA were established using the monocyte cell THP-1 and functional analysis was performed (Fig. 7). A pNF-κB-promoter vector (a construct in which a luciferase gene was linked downstream of the NF-κB promoter) was introduced into each established clone cell. After 24 hours, cells were stimulated with LPS, which is one of infection mimics, and chemiluminescence by luciferase depending on NF-κB activity was quantified. For standardization of gene introduction, a control vector expressing Renilla chemiluminescent protein was also introduced at the same time. When stimulated with LPS, the luciferase activity was observed stronger in all IBTK long form clonal cells than the parent cell line, confirming the enhancement of NF-κB activation.

  The influence of IBTK long form on the production ability of various cytokines induced depending on NF-κB was measured for the amount of mRNA induction by RT-PCR in the parent strain and IBTK long form stable expression strain 2B6 obtained in FIG. And ELISA (the following antibodies were used, respectively: IL-23: eBio473P19, C8.6; IL-12p70: B-T21, C8.6; IL-6: MQ2-13A5, MQ2-39C3; TNF-α: MABa, MAB11) were evaluated by measuring protein production (FIG. 8). As a result, when all the IBTK long form-expressing clone cells tested were compared with the parent cell line, it was confirmed that LPS stimulation enhanced the production of cytokines involved in exacerbation of inflammation such as IL-6 and IL-23.

  In THP-1 cells, the expression of IBTK long form is suppressed by knockdown method using siRNA (sequence siRNA A028643-13: UCUUGAUGUUUUAAGCGAU (SEQ ID NO: 46) and siRNA A028643-14: nucleic acid mixed with CAACCAUAUUUCAGAUUUA (SEQ ID NO: 47)). Then, cytokine production in response to LPS stimulation was examined (FIG. 9). As a result, compared to the negative control (non-inhibited group) during LPS stimulation, the IBTK expression-inhibited group can confirm the effect of reducing cytokine production related to exacerbation of inflammation such as IL-6, TNF-α, and IL-23. It was.

Inflammation-related cytokines in airway epithelial cells were evaluated by RT-PCR under the suppression of IBTK long form expression using siRNA (FIG. 10). Poly (I: C), which is highly reactive with airway epithelial cells, was used as an infection mimetic used for stimulation. As a result, compared to the negative control, in the IBTK expression suppression group, the production induction of physiologically active substances involved in the exacerbation of inflammation such as IL6, TNF, IL1B, and IL8 induced by stimulation decreased.
Based on the above, the long form of IBTK, a bronchial asthma-related molecule identified from correlation analysis, has the activity of enhancing the production of bioactive substances produced from monocyte cells and airway epithelial tissues during innate immune stimulation. Indicated. The results of these functional analyzes were thought to explain the mechanism of action of IBTK long form for the development of bronchial asthma.

(5) Types of TLRs receptor stimulation involving IBTK molecules
Since the long form of IBTK showed the activity of enhancing the production of physiologically active substances produced by stimulation of infection mimic substances, next, as part of clarifying the signal transduction pathways involved in long forms, infection mimic substances Comparison was made between the various TLRs receptors, which are considered to be receptors, and the activation ability by ligand stimulation (FIG. 11).
The pNF-κB-promoter vector and a vector construct expressing an IBTK long form protein were introduced into three types of HEK293 cells expressing TLR-2 / 6, TLR-4 and TLR-3 receptors, respectively. After 24 hours, stimulation with various infection mimetics was performed, and chemiluminescence emitted by luciferase protein depending on NF-κB activity was quantified. For the standardization of gene transfer, a control vector was also transferred at the same time. As a result, only when stimulated with specific ligands corresponding to the respective TLR receptors, NF-κB was activated, and the enhancement activity by IBTK long form was shown. On the other hand, in the short form, an enhancing effect on NF-κB activation could not be confirmed.
Based on the above, the long form of IBTK shows enhanced activation of NF-κB depending on TLR receptor stimulation, and by the stimulation of nucleic acids, proteins, and lipopolysaccharides derived from microorganisms, which is a typical ligand classification for infection stimulation. It was shown that all TLRs have a signaling effect on the signal transduction pathway. These functional analyzes suggested that IBTK long form is involved in exacerbation of bronchial asthma by increasing production of inflammation-related physiologically active substances through activation of NF-κB such as infection stimuli .

(6) Functional analysis of IBTK 49393C> T (rs9449444) Of the polymorphisms detected to be highly associated with bronchial asthma, 49393C> T (rs9449444) is present in the IBTK translational region and has an amino acid substitution. (A1185V), there was a possibility of functional differences.
A vector construct containing this SNP and expressing IBTK long form cDNA was prepared for each allele. Then, the vector construct was introduced into HEK293-TLR2 / 6 cells together with the pNF-κB-promoter vector, and stimulated with TLR ligand 24 hours later, and the chemiluminescence emitted by the luciferase protein depending on NF-κB activity was quantified. . For the standardization of gene transfer, a control vector was also transferred at the same time. As a result, when TLR receptor stimulation was added, IBTK long form was forced to enhance NF-κB activation, but rs944944 T allele (1185) A stronger enhancement of NF-κB activation was observed in the cells into which the construct having the amino acid of valine was introduced (FIG. 12).
From the above, it was shown that the group having rs9449444 T allele, which is frequently used in patients with bronchial asthma, has a stronger effect of activating IBTK long form NF-κB than the group of C allele. As a result, it was suggested that the production amount of physiologically active substances related to inflammation increases, which may increase the exacerbation of inflammation and lead to bronchial asthma. That is, the results of these functional analyzes reinforce the results of the association analysis between bronchial asthma and 49393C> T (rs9449444).

(7) Functional analysis of IBTK 69371A> G (rs991781) 69371A> G (rs991781) present in the 3 'untranslated region, which is associated with bronchial asthma, may contribute to the stability of IBTK mRNA. (Figure 13).
First, luciferase assay was performed in order to examine the difference in the expression level of IBTK by this SNP. For each allele, constructs were created by subcloning the DNA sequence of the IBTK 3 'untranslated region containing this SNP at the 3' end of the luciferase gene of pGL4.23-promoter vector (Promega). Genes were introduced into THP-1 cells, which are monocytic cell tumors, and chemiluminescence emitted by luciferase protein after 24 hours was quantified. For the standardization of gene transfer, a control vector was also transferred at the same time. As a result, stronger luciferase activity was observed in cells into which a construct having rs991781 G allele, which is frequently used in patients with bronchial asthma, was introduced.
In addition, a construct was created by subcloning the 3 times repeat sequence of the surrounding DNA sequence containing rs991781A allele and G allele, respectively, into the 3 'untranslated region of the luciferase gene of the pGL4.21-promoter vector, and contributing to stability as well. Evaluated. As a result, stronger luciferase activity was observed in cells into which a construct having a three-repeat sequence of rs9917811 G allele, which is frequently used in patients with bronchial asthma, was introduced.
Based on the above, in the population with rs991781 G allele, which has a high frequency in bronchial asthma, IBTK expression level increased due to strong IBTK stability, which promoted the production of physiologically active substances such as inflammatory cytokines. It was suggested that this could lead to illness. In other words, these functional analysis results reinforce the results of the association analysis between bronchial asthma and IBTK 69371A> G (rs991781).

Claims (9)

A method for analyzing an immune disease based on the analysis result by analyzing a base present on an IBTK gene or a single nucleotide polymorphism of a base in linkage disequilibrium with the base. The single nucleotide polymorphism is a polymorphism in a base corresponding to the base number 61 in the base sequence of any one of SEQ ID NOs: 1 to 6 or a base in linkage disequilibrium with the base. the method of. The method according to claim 2, wherein the base in linkage disequilibrium is a base corresponding to the 61st base of the base sequence of any one of SEQ ID NOs: 7 to 37. The method according to any one of claims 1 to 3, wherein the immune disease is an allergic disease. The method according to claim 4, wherein the allergic disease is bronchial asthma. A probe for immunological disease test, comprising a sequence of 10 bases or more including the 61st base in the base sequence of SEQ ID NOs: 1 to 37, or a complementary sequence thereof. A primer for immunological disease testing, which can amplify a region containing the base at position 61 in any one of SEQ ID NOs: 1 to 37. Adding a drug candidate substance to a cell expressing a reporter gene linked to a promoter of IBTK gene long form or IBTK gene long form, measuring an expression level of IBTK gene long form or reporter gene, and said expression level A method for screening for a prophylactic or therapeutic agent for an immune disease, comprising a step of selecting a substance to be reduced. A method for examining an immune disease, comprising measuring the expression level of mRNA or protein of IBTK gene long form.

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