WO2005033339A1

WO2005033339A1 - Human toll-like receptor 3 gene polymorphism as marker for inherited factor of allergic disease and utilization of the same

Info

Publication number: WO2005033339A1
Application number: PCT/JP2004/014608
Authority: WO
Inventors: Kazuhiko Obara; Yusuke Nakamura; Mayumi Tamari
Original assignee: Hitachi Chemical Co., Ltd.; Riken
Priority date: 2003-10-03
Filing date: 2004-10-04
Publication date: 2005-04-14
Also published as: JP4533979B2; JPWO2005033339A1; TW200516153A

Abstract

An inherited factor for an allergic disease is examined by detecting the gene polymorphism at least one of single nucleotide polymorphism sites located at the -8921, -7, 1638, 1656, 3519, 4792, 4960, 5252, 6301 and 6444 positions in human Toll-like receptor 3 (TLR3) gene.

Description

Specification

Human toll-like receptor as a genetic predisposition marker for allergic diseases: 3 gene polymorphisms and their use

Technical field

[0001] The present invention relates to polymorphisms involving mutations in genes associated with human allergic diseases, in particular, polymorphisms in human toll-like receptor 3 (hereinafter referred to as human TLR3 or simply TLR3) as a genetic predisposition marker for allergic diseases. And its use. More specifically, a TLR3 gene polymorphism, a primer or probe for detecting the same, a method for testing a genetic predisposition to an allergic disease using the same, a method for determining susceptibility to a viral disease, and a method for screening a therapeutic agent for an allergic disease And therapeutic agents for allergic diseases.

Background art

[0002] Studies of twins and families have shown that a genetic predisposition is strongly involved in allergic diseases. Unlike hereditary diseases, in which changes in the nucleotide sequence of a gene region directly cause a disease, diabetes and hypertension, including allergic diseases, are called “common diseases”. A common disease is thought to be caused by the interaction between genetic factors and environmental factors.A large number of genes are involved in the disease as genetic factors, and the amount attributable to the polymorphism of the disease-related gene is considered. It is said that qualitative and qualitative (functional) differences affect the diversity of the disease.

[0003] In the nucleotide sequence in the human genome, it has been found that there are sequence differences at many sites between individuals, and this difference is a gene polymorphism. This sequence difference is considered to be one of the causes of diversity such as individual phenotype and susceptibility to disease (susceptibility). A single nucleotide polymorphism (SNP: Single Nucleotide Polymorphism) is a gene defined as being present at a frequency of 1% or more in the population at a site where a single nucleotide difference occurs in the DNA base sequence between individuals. It is a polymorphism and is said to have 300 to 1,000,000 places in the human genome. SNPs can cause qualitative and quantitative changes in gene expression, and because of their high frequency in the genome, they can be used as markers in gene analysis. It is being used in studies of association with gene function and in searching for disease-related genes.

[0004] Through genomic analysis using genetic polymorphisms including single nucleotide polymorphisms, research has been conducted to clarify single nucleotide polymorphisms that are strongly associated with diseases and gene polymorphisms that cause differences in susceptibility to diseases. Has been done. By clarifying the single nucleotide polymorphism site that causes the disease and the causative substance by gene identification, the difference in the nucleotide sequence of the gene polymorphism is determined to determine the risk of disease, elucidate the mechanism of disease onset, Research on the search and identification of disease-related genes by genetic polymorphisms has attracted attention because there are many possibilities, such as the presentation of preventive guidelines for the disease, the mechanism of disease development, and drug development through target clarification.

[0005] Asthma, which is also a representative of allergic diseases, is a disease characterized by paroxysmal and reversible bronchial obstruction caused by excessive hyperresponsiveness of the bronchial system to various stimuli. It causes symptoms such as smooth muscle thickening and airway hyperreactivity. The existence of a genetic predisposition in asthma has been studied by twins and pedigrees before the establishment of molecular biological techniques, and the identity of traits is higher in identical twins than in dizygotic twins. Numerous reports, including high levels, have implicated a genetic predisposition. Asthma is on the rise worldwide, with more than 150 million asthma patients worldwide, according to the National Institutes of Health (NIH). The number of asthma patients in Japan is more than 3 million, and the prevalence rate has increased remarkably, more than double 30 years ago, and about 6,000 people die in one year due to difficulty breathing due to seizures.

[0006] Human TLR is a molecule that recognizes pathogens in innate immunity, and 10 types of ligands are recognized at present, and form a TLR family (Patent Documents 1 and 2). TLR is a receptor type I membrane protein.It has a part containing a motif called leucine-rich repeat (LRR) involved in binding between proteins as an extracellular part, and a signal similar to the IL-1 receptor as an intracellular part. There is a portion that includes a transduction domain. TLRs recognize foreign molecules that are components of pathogens such as double-stranded RNA, lipopolysaccharide, peptide darican, flagellin, and bacterial DNA, and eliminate the pathogens through innate immunity (macrophage neutrophils or It is thought that it is involved in the activation of 榭 (dendritic cells) and also involved in the activation of acquired immunity. [0007] TLR3 is a molecule that recognizes double-stranded RNA as a ligand in the TLR family, and is considered to be involved in protection against virus infection. This has also been reported by Medzhitov et al. (Non-Patent Document 1).

Patent Document 1: Japanese Patent Publication No. 2002-514083 (P2002-514083A)

Patent Document 2: JP 2000-128900 (P2000-128900A)

Non-Patent Document 1: Medzhitov et al., Nature, 413, 732-738, 2001

Disclosure of the invention

Problems to be solved by the invention

[0009] As described above, in recent years, the number of patients suffering from allergic diseases is rapidly increasing, and prevention and elucidation of the onset mechanism are desired. In particular, asthma presents serious symptoms as an allergic disease, and there is a strong demand for prevention and elucidation of the mechanism of its occurrence. Genetic predisposition is involved in allergic diseases, but if it becomes possible to predict the onset risk and severity of patients, it will be possible to provide effective lifestyle guidance and preventive measures that take individual constitution into account. In addition, identification of allergic disease-related genes will be an important guideline for developing more effective treatment methods and therapeutic agents.

[0010] Accordingly, the present invention provides identification of a predisposition gene for an allergic disease, clarification of a disease-related gene polymorphism existing in the gene region, and a genetic predisposition for an allergic disease utilizing the gene polymorphism. The main purpose is to provide an inspection method.

[0011] Further, the present invention aims to provide a primer or a probe for detecting a single nucleotide polymorphism constituting the gene polymorphism, and to use them for diagnosis of an allergic disease.

[0012] In addition, the present invention provides a method for determining the susceptibility of a viral disease, a method for screening a therapeutic agent for an allergic disease, a therapeutic agent for an allergic disease, and the like, utilizing the gene polymorphism. Also aim.

Means for solving the problem

[0013] The present inventors have proposed linkage disequilibrium mapping that narrows down chromosomal regions that are associated with allergic diseases by linkage analysis and the like, and predict from known information that they are related to the pathology of allergic diseases such as asthma and atopy The frequency of SNPs that are thought to have a direct effect on susceptibility (disease susceptibility) by selecting candidate genes to be Genes associated with allergic diseases were searched for, both in association analysis using the candidate gene approach to be compared. Furthermore, as a result of repeated studies using polymorphism analysis of candidate gene regions and correlation analysis based on patient-control studies, allele frequencies in the TLR3 gene region were significantly different between the allergic disease group and the control group (that is, (Correlated with) was successfully identified. The present invention has been completed based on strong knowledge.

[0014] Therefore, the above-mentioned object is achieved by the present invention described below. That is, the present invention provides a TLR3 gene polymorphism as a genetic predisposition marker (risk of development) for allergic diseases and its use in diagnosis and treatment of allergic diseases. (1) One (17).

(1) A genetic predisposition marker for an allergic disease consisting of any one of the polynucleotides selected from the group consisting of the polynucleotides described in (a) to (j) and (V) below:

(a) a polynucleotide specified in SEQ ID NO: 1 or SEQ ID NO: 2, comprising a single nucleotide polymorphism site present at the seventh position in the untranslated region of the human Toll-like receptor 3 gene;

(b) a polynucleotide comprising the single nucleotide polymorphism site located at position 1638 of the untranslated region of the human Toll-like receptor 3 gene identified by SEQ ID NO: 3 or SEQ ID NO: 4;

(c) a polynucleotide comprising the single nucleotide polymorphism site located at position 1656 of the untranslated region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 5 or SEQ ID NO: 6;

(d) a polynucleotide comprising the single nucleotide polymorphism site located at position 3519 of the untranslated region of the human toll-like receptor 3 gene specified by SEQ ID NO: 7 or SEQ ID NO: 8;

(e) a polynucleotide comprising the single nucleotide polymorphism site identified at SEQ ID NO: 9 or SEQ ID NO: 10 and located at position 4792 of the untranslated region of the human Toll-like receptor 3 gene;

(f) a polynucleotide specified in SEQ ID NO: 11 or 12 and containing a single nucleotide polymorphism site at position 4960 of the untranslated region of the human Toll-like receptor 3 gene;

(g) a polynucleotide comprising the single nucleotide polymorphism site located at position 5252 of the untranslated region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 13 or 14;

(h) a polynucleotide comprising the single nucleotide polymorphism site located at position 6301 of the translated region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 15 or 16; (i) a polynucleotide comprising the single nucleotide polymorphism site located at position 6444 of the translation region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 17 or 18;

(V) a polynucleotide comprising the single nucleotide polymorphism site at position 8921 of the untranslated region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 19 or 20, and (j) the polynucleotide described above, A polynucleotide having a sequence complementary to a nucleotide (2) any one or more polynucleotides selected from the group consisting of the polynucleotides described in (a) to (j) and (V) below: A polynucleotide that hybridizes specifically with a genetic predisposition marker for a powerful allergic disease and has a base length of 10 or more to detect the marker:

(h) a polynucleotide comprising the single nucleotide polymorphism site located at position 6301 of the translated region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 15 or 16;

(i) a polynucleotide comprising the single nucleotide polymorphism site located at position 6444 of the translation region of the human Toll-like receptor 3 gene specified by SEQ ID NO: 17 or 18; (v) a polynucleotide comprising the single nucleotide polymorphism site at position 8921 of the untranslated region of the human toll-like receptor 3 gene specified by SEQ ID NO: 19 or SEQ ID NO: 20; and A polynucleotide having a sequence complementary to a nucleotide

(3) A primer for detecting a genetic predisposition marker for allergic disease, comprising at least one kind of the polynucleotide according to (2).

(4) A probe for detecting a genetic predisposition marker for an allergic disease, comprising the polynucleotide according to (2).

(5) A kit for detecting a genetic predisposition marker for an allergic disease, comprising the detection primer according to (3).

(6) A kit for detecting a genetic predisposition marker for an allergic disease, comprising the detection probe according to (4).

(7) An amplification product obtained by PCR-amplifying the human toll-like receptor 3 gene with the primers described in (3) above.

(8) A method for testing a genetic predisposition for an allergic disease, comprising the steps of:

An inspection method characterized by detecting gene polymorphisms in three genes.

(9) The gene polymorphism is at least one single nucleotide polymorphism selected from the group consisting of single nucleotide polymorphism site forces described in (k) and (u) below: Inspection method described in (8) above:

(k) a single nucleotide polymorphism site located at position -7 of the untranslated region of the human toll-like receptor 3 gene specified in SEQ ID NO: 1 or SEQ ID NO: 2;

(1) a single nucleotide polymorphism site located at position 1638 of the untranslated region of the human toll-like receptor 3 gene specified by SEQ ID NO: 3 or SEQ ID NO: 4;

(m) a single nucleotide polymorphism site identified at SEQ ID NO: 5 or SEQ ID NO: 6 at position 1656 in the untranslated region of the human Toll-like receptor 3 gene;

(n) a single nucleotide polymorphism site located at position 3519 of the untranslated region of the human toll-like receptor 3 gene specified by SEQ ID NO: 7 or SEQ ID NO: 8;

(o) non-inverted human Toll-like receptor 3 gene identified by SEQ ID NO: 9 or SEQ ID NO: 10 A single nucleotide polymorphism site at position 4792 of the translation region;

(P) a single nucleotide polymorphism site identified at SEQ ID NO: 11 or SEQ ID NO: 12 and located at position 4960 of the untranslated region of the human Toll-like receptor 3 gene;

(q) a single nucleotide polymorphism site identified at SEQ ID NO: 13 or SEQ ID NO: 14 and located at position 5252 of the untranslated region of the human Toll-like receptor 3 gene;

(r) a single nucleotide polymorphism site identified at SEQ ID NO: 15 or SEQ ID NO: 16 and located at position 6301 of the translation region of the human Toll-like receptor 3 gene;

(s) a single nucleotide polymorphism site identified at SEQ ID NO: 17 or SEQ ID NO: 18 and located at position 6444 of the translation region of the human Toll-like receptor 3 gene;

(t) a single nucleotide polymorphism site identified at SEQ ID NO: 19 or SEQ ID NO: 20, located at position-8921 of the untranslated region of the human Toll-like receptor 3 gene; and

(u) a single nucleotide polymorphism site in the human toll-like receptor 3 gene that is in linkage disequilibrium with the single nucleotide polymorphism site at position 6444 of the translation region of the human toll-like receptor 3 gene;

(10) The test method according to the above (8) or (9), wherein the allergic disease is asthma.

(11) The test method according to (10), wherein the asthma is child asthma.

(12) A method for determining an individual's susceptibility to an allergic disease, which comprises detecting a polymorphism in the human toll-like receptor 3 gene.

(13) A method for determining the susceptibility of a viral disease, the method comprising a step of performing an analysis in which a genetic polymorphism in the human Toll-like receptor 3 gene is an indicator of the morbidity.

(14) The above-mentioned (13), wherein the gene polymorphism is at least one single-nucleotide polymorphism selected from the group consisting of the single-nucleotide polymorphism sites described in (9) above. Judgment method described in.

(15) A method for screening a therapeutic agent for an allergic disease, comprising contacting a cell expressing the human toll-like receptor 13 gene with a candidate drug and attenuating the expression of the human toll-like receptor 3 gene. .

(16) Contains an agent that regulates the expression of the human toll-like receptor 3 gene as an active ingredient A therapeutic agent for an allergic disease, which is characterized in that:

(17) Use of the polynucleotide according to (2) for a genetic predisposition test for an allergic disease.

The invention's effect

According to the present invention, it is possible to examine a genetic predisposition to an allergic disease, using the polynucleotide of the present invention to prepare a sample of a subject (individual) such as genomic DNA or the like. By detecting a genetic predisposition marker. When the test method of the present invention is used, the susceptibility (susceptibility) of a subject to an allergic disease can be predicted, so that life guidance for the prevention of an allergic disease becomes possible. In addition, it will enable the onset of allergic diseases to be delayed and detected earlier. The findings obtained in the present invention can also be applied to elucidation of the pathogenesis of allergic diseases and development of therapeutic drugs.

Brief Description of Drawings

[FIG. 1] FIG. 1 shows the expression level of TLR3 expressed when CD14 + cells of samples from different genotypes were stimulated with poly (I: C), and the poly (I: C) concentration FIG. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

[0035] As used herein, the term "polynucleotide" refers to a substance in which two or more nucleotides are linked by a phosphodiester bond. “Polynucleotide” includes “ribonucleotide (RNA)” and “deoxyribonucleotide (DNA)”. Here, DNA includes cDNA and genomic DNA. The “polynucleotide” is not limited to these, but also includes artificially synthesized nucleic acids such as peptide nucleic acids, morpholino nucleic acids, methylphosphonate nucleic acids, and S-oligonucleic acids. As used herein, the term "nucleic acid" is used synonymously or interchangeably with the above "polynucleotide" and means DNA or RNA. Further, the terms "nucleic acid" and "polynucleotide" refer to isolated nucleic acids and polynucleotides that, when prepared by recombinant DNA techniques or the like, contain substantially no cellular material or culture media. Refers to nucleic acids or polynucleotides that are substantially free of precursor chemicals or other chemicals when chemically synthesized.

[0036] As used herein, the term "gene polymorphism" refers to a nucleotide sequence on the human genome of each individual. It means different parts between them. "Single nucleotide polymorphism (SNP)" refers to a gene polymorphism that appears as a mutation in a single nucleotide nucleic acid. Further, “nucleic acid fragment” refers to a fragment having a partial sequence and a Z or full-length sequence of a nucleic acid. The “gene region” refers to a non-translated region such as a translation region encoding a protein, a promoter other than Z or the protein coding region, an intron region, and an unidentified region near the gene.

[0037] As used herein, "specifically hybridizes" is synonymous with the term "hybridizes under stringent conditions" as recognized by those skilled in the art, and includes two nucleic acids ( Or fragments) are described in Sambrook, J., “Expression of cloned genes in E. coli”, More-Chufu 'Cloning: A' Laboratory. Yuanore (Molecular Cloning: A laboratory manual), Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, 1989, pp. 9.47—9.62, pp. 11. 45—11.61 means to hybridize to each other under the hybridization conditions described in 1.

[0038] More specifically, the above-mentioned "stringent conditions" means that hybridization is performed at about 45 ° C at 6.6 X SSC, and then at 50 ° C at 2.0 X SSC. Refers to washing. For selection of stringency, the salt concentration in the washing step may be, for example, about 2.0 × SSC at low stringency, at 50 ° C., about 0.2 × SSC at high stringency, 50 × SSC. Up to ° C. In addition, the temperature of the washing step can be increased from low stringency conditions at room temperature, about 22 ° C, to high stringency conditions at about 65 ° C.

[0039] The source of the nucleic acid test sample (also referred to as a sample) used in the present invention is not limited as long as it is an individual somatic cell. For example, blood, skin, tissue, oral mucosal cells, and the like are preferably used from the viewpoint of ease of sample collection, content, and type of extraction reagent. In addition, the sample may contain a partial base sequence of the genomic DNA in addition to the extracted genomic DNA. That is, before analysis of the single nucleotide polymorphism site, the whole or partial nucleotide sequence (DNA fragment) in the sample nucleic acid that matches the purpose is amplified by any suitable method such as PCR. The widened sample may be used as a nucleic acid test sample. The DNA can be extracted by a known method, for example, using a commercially available extraction kit such as “QIAamp mono kit” (manufactured by QIAGEN).

[0040] The primer used in the present invention can be prepared by any suitable preparation method such as a chemical synthesis method. Primers include both a primer containing a polymorphic site on the TLR3 gene region and a primer for obtaining a nucleic acid fragment containing a single nucleotide polymorphic site by PCR, etc. Is included. As the latter primer, for example, a probe designed to correspond to the base sequence immediately before or several bases before the target SNP to be detected, that is, the 3 'end or 5' end of the probe is up or down one base of the SNP Are preferred. The primer may have any suitable length according to the detection method to be used and the like, but has 10 or more consecutive nucleotides, preferably 15 to 50 nucleotides, and more preferably 15 to 30 nucleotides. Specifically, such a primer can be synthesized as a forward 'primer and a reverse' primer based on single nucleotide polymorphism site and base sequence information at or near Z, for example, using an automatic synthesizer. A primer may carry one or more labels if necessary. Preferred labels include enzymes, biotin, fluorescent materials, haptens, antigens, antibodies, radioactive materials, luminophores, and the like. It is desirable that the primer have a sequence complementary to the nucleic acid to be type III, but if necessary, one or more base pairs (mismatches) in the primer that are not complementary unless undesirable effects occur. You can introduce it.

[0041] The polynucleotide probe used in the present invention can be prepared by any suitable synthesis method. The polynucleotide probe may have any suitable length according to the detection method and the like, but has a length of 10 or more consecutive nucleotides, preferably 15 to 50 nucleotides, and more preferably 15 to 30 nucleotides. The probe contains a nucleotide sequence complementary to any one allele of the corresponding single nucleotide polymorphism site in the TLR3 gene region. However, if necessary, one or more non-complementary base pairs may be introduced as long as the polynucleotide probe has no undesirable effect. Further, the probe may carry one or more labels, similarly to the primer.

[0042] The present invention also includes a nucleic acid fragment containing one or more single nucleotide polymorphism sites. For example, Nucleic acids and the like amplified by PCR or the like using a primer having a sequence in the vicinity thereof that does not include the single nucleotide polymorphism site and include the single nucleotide polymorphism site. Nucleic acids containing one or more monobasic polymorphism sites can be used for multiple gene polymorphisms on separate nucleic acid fragments, even if each gene polymorphism is on a separate nucleic acid fragment, such as multi-primer PCR amplification products. May be present. The length of the nucleic acid fragment is preferably 15b-1kb because of conditions such as the limit of the length of the sample that can be amplified in PCR, the accuracy of base complementation in the extension reaction, and the ease of analysis. Such a nucleic acid fragment can be used, for example, as a sample for base sequence analysis (sequence) or SSCP, for a sample for determining a single nucleotide polymorphism site, a probe for a chip 'microarray, or the like. A nucleic acid fragment may carry one or more labels, if necessary. There are many known methods for detecting single nucleotide polymorphisms. For example, using nucleotide sequence analysis (sequence) and detection reaction with invader assay (Cleavase, allele-specific probe and FRET probe), if the allele-specific probe is complementary to the polymorphic site and a triplex is formed, , The reaction by Cleavase proceeds to generate fluorescence), TaqmanPCR (PCR using a Taqman probe having a sequence complementary to each allele of the gene polymorphism part in the probe and a PCR primer, the Taqman probe Fluorescence is generated when the base sequence at the base polymorphism site is complementary), base determination by MS (single base extension reaction using primers up to immediately before the gene polymorphism portion, and the extended base Mass spectrometry with MS), neurosequencing (pyrophosphate generated by base extension reaction is converted to ATP and measured by bioluminescence, and the presence or absence of complementary strand synthesis is determined by luminescence) , SnaPshot (performs a single-base extension reaction with labeled ddNTPs (labels differing for each of the four bases) using the primers immediately before the single nucleotide polymorphism site, and identifies the extended bases by the type of label), allele-specific Primer method (The ability to extend by PCR when the sequence at the single nucleotide polymorphism site is complementary Designs the primers of the sequence that do not extend in the case of mismatch, Polymorphisms), restriction fragment length polymorphisms (RFLP), gene polymorphism detection by hybridization such as DNA microarray, SPR (Surface Plasmon Resonance), SSCP (Single Strand Conformation) Polymorphism), Hetero-duplex analysis, WAVE system (Detection of base differences by chromatography), Molecular biology -Cone (Sniper method, etc.). In any of such known detection methods, the desired single nucleotide polymorphism site can be detected according to the test method of the present invention using the probe of the present invention, Z or a primer, Z or a polynucleotide, or the like. It comes out.

[0044] In one embodiment of the present invention, a gene polymorphism associated with the development of an allergic disease can be detected, for example, as follows.

[0045] SNPs are determined using samples from a group of patients with allergic diseases (eg, asthma patients), and primary candidate SNPs are selected from among them (primary screening). Furthermore, the number of samples is increased to narrow down candidate SNPs (secondary screening). On the other hand, SNPs are determined using samples from the control group of healthy subjects, and primary candidate SNPs are selected from among them. From these SNPs and the SNPs for which the secondary screening ability was also obtained, a high-density SNP map was created and target association analysis was performed to identify genetic polymorphisms, and to determine whether or not the ability to establish linkage disequilibrium between SNPs. Upon validation, those that are confirmed to be in linkage disequilibrium with each other are identified as SNPs associated with allergic diseases. That is, there is a significant difference in the allele frequency between the allergic disease patient group and the control group for each of these polymorphisms.

As described above, in the present invention, 10 SNPs in the translation region or the non-translation region of the TLR3 gene shown in (k) and (t) below were identified.

(o) a single nucleotide polymorphism site identified at SEQ ID NO: 9 or SEQ ID NO: 10 and located at position 4792 of the untranslated region of the human Toll-like receptor 3 gene;

(P) the non-human Toll-like receptor 3 gene identified by SEQ ID NO: 11 or 12 A single nucleotide polymorphism site at position 4960 of the translation region;

(s) a single nucleotide polymorphism site identified at SEQ ID NO: 17 or SEQ ID NO: 18 and located at position 6444 of the translation region of the human Toll-like receptor 3 gene; and

(t) a single nucleotide polymorphism site identified at SEQ ID NO: 19 or SEQ ID NO: 20, which is located at position-8921 in the untranslated region of the human Toll-like receptor 3 gene;

[0047] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 correspond to the above (k) One allele shown in (t), which is a continuous base sequence of 10 bases each on the 5 ′ side and 3 ′ side from the single nucleotide polymorphism site.

[0048] SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20 correspond to the above (k) The other allele shown in (t), which is different from the above, is a continuous base sequence of 10 bases each on the 5 'side and 3' side from the single nucleotide polymorphism site.

[0049] A polynucleotide consisting of or containing the above-listed sequences is a genetic predisposition marker for allergic disease, and the genetic method of allergic disease is detected by detecting each marker in the test method of the present invention. Can be checked.

According to another embodiment of the present invention, the inspection can be performed as follows.

[0051] A nucleic acid sample (eg, tissue, cell, blood, etc.) is collected from a subject, and DNA or genomic DNA is extracted according to a conventional method. The obtained DNA sample was amplified by PCR (the region containing the genetic predisposition marker), and the various polymorphism sites in the TLR3 gene region (the 7th and 1638th , 1656th, 3519th, 4792th, 4960th, 5252th, 6301th, 6444th, —8921)). As described above, the TL determined from the correlation between the SNP obtained from the association analysis and the pathology (including severity) of the allergic disease The state of the individual is determined with reference to the R3 gene polymorphism.

[0052] The following examples confirm that there is a strong correlation between the SNP at position 6444 of the TLR3 gene and asthmatics. Therefore, by detecting the SNP at position 6444 of the TLR3 gene, it is possible to examine the genetic predisposition to allergic diseases. Also, instead of detecting the SNP at position 6444 of the TLR3 gene, detection of an SNP site in the TLR3 gene that is in linkage disequilibrium with the SNP position at position 644 of the TLR3 gene does not affect the genetic predisposition to allergic disease. It is possible to inspect. SNP sites in the TLR3 gene that are in linkage disequilibrium with the SNP site present at position 6444 of the TLR3 gene include, but are not limited to, SNPs at positions 3519 and 4792. Those skilled in the art can find SNP sites in linkage disequilibrium based on known methods.

[0053] More specifically, PCR is performed on the DNA sample in the region containing the genetic predisposition marker, and 1) the type of base is determined by the SSCP method, or 2) the PCR amplification product is directly sequenced ( Sanger method or Maxam-Gilbert method). Alternatively, a polymorphic site is directly detected from the DNA sample or its PCR amplification product using a probe that specifically hybridizes to a region containing a genetic predisposition marker (such as Invader Atssay). Furthermore, a single base extension reaction is performed using a primer having a sequence up to (before) the genetic predisposition marker, and the extended base is analyzed by MS (mass spectrometer).

The polynucleotide of the present invention is preferably used for the detection and analysis of the polymorphic site. Examples of the force probe include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, More preferably, it contains any one of the nucleotide sequences of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

When the polynucleotide of the present invention is used as a primer for the detection, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, Sequence It preferably contains any one of the nucleotide sequences of SEQ ID NO: 19 and SEQ ID NO: 20.

In the present invention, the probe and the primer of the present invention are preferably provided as a test kit containing each of them as an effective component. In particular, the test kit of the present invention containing primers may contain DNA polymerase, four types of deoxynucleotide triphosphate (dNTP), a size marker, and the like. In addition, it may contain an appropriate buffer, detergent, reaction stop solution and the like.

[0057] In addition, an antibody specific to a polymorphism in the TLR3 gene can be used for detection.

[0058] The test kit of the present invention is intended to detect a genetic predisposition marker for an allergic disease, and if necessary, in combination with a test kit for other disease-related factors (markers). The result of the test can determine the complex state of the subject.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

(Example 1)

Relevance studies

[0061] (1) Subjects

With sufficient informed consent, samples from patients with allergic diseases (pediatric and adult asthma) and controls (normal subjects) were collected along with clinical information. The subjects were all Japanese.

[0062] (2) SNP

The allele frequency in the TLR3 gene was calculated as described above. Testing for significant differences between the pediatric asthmatics and the control group for each polymorphism indicated that the base substitution at position 6444 was particularly common in pediatric asthmatics. That is, the T homozygotes in the healthy subjects were significantly higher than the homozygotes of force C and the heterozygotes of CT (ρ = 0.00000020). Table 1 shows the results. Therefore, if it is a homozygote of the polymorphism at position 6444 of the TLR3 gene region, This particular genetic polymorphism, which has a significantly higher probability of being a healthy individual than, has a strong association with the disease phenotype. In addition, for each polymorphism, a significant difference between the adult asthma patient group and the control group was tested.In this case, too, it was concluded that the base substitution at position 6444 was particularly abundant in adult asthmatic patients. . In other words, the homozygotes of Ding were significantly higher in the case of homozygotes in healthy subjects than in the homozygotes of C and heterozygotes of CT (p = 0.000097) .o The results are shown in Table 2. . Therefore, in the case of a homozygote having the polymorphism at position 6444 of the TLR3 gene region, the probability of being a healthy individual is significantly higher than that in the adult asthma disease group. The result is that they are related.

[0063] [Table 1]

[0064] [Table 2

[0065] In addition, when SNPs other than the 6444th SNP were examined! For CC, 3519 SNP homozygous CC or heterozygous CA, 4792 SNP homozygous GG or heterozygous GC, 630 1 SNP In the case of homozygous CC or heterozygous CT, and in the case of homozygous TT in the 8921st SNP, the proportion of asthmatic patients was higher than that of healthy subjects.

(Example 2)

Association between genetic polymorphism and phenotype

(1) Experimental method

The correlation between the allelic form of the polymorphism at position 6444 of the TLR3 gene region and the expression of the TLR3 gene was examined. A human peripheral blood monocyte fraction was obtained from peripheral blood of the T or C allele polymorphism at position 6444 in the TLR3 gene region using “Lymphoprep” (Axis shield). The isolated peripheral blood monocyte fraction was subjected to a magnetic separation method (MACS, Milteny i) to obtain a monocyte fraction having a differentiation antigen CD14. This fraction was cultured in an RPMI 1640 culture medium supplemented with 10 μg / mL of TLR3 ligand (poly (l: C), manufactured by Amersham Fanolemasia). Total RNA was extracted from the cultured cells using an RNA extract (Trizol, manufactured by Invitrogen). From the extracted total RNA, cDNA was synthesized using the cDNA synthesis reagent (Thirmos cript, RT-PCR System, manufactured by Invitrogen) with the mRNA in the total RNA as type III. The amount of TLR3 cDNA was determined by real-time PCR (Taqman PCR, manufactured by Applied Biosystems) to determine the expression level of TLR3.

[0068] (2) Experimental results

TLR3 expression levels were compared using 4 homozygotes of the single nucleotide polymorphism T allele at the 6444th base and 4 homozygotes of the C allele as samples. Figure 1 shows the average of the expression ratios for four samples of each allele. When poly (l: C) was supplemented with 10 μg ZmL and cultured, the TLR3 expression level in the T allele sample was about 8 times higher than that in the C allele sample. The same tendency was observed for all four alleles tested, indicating that there was a large difference in TLR3 expression levels due to the polymorphism of C6444T. That is, TLR3 expression was low in genotypic specimens susceptible to childhood asthma.

[0069] As described above, from the results of Examples 1-2, the gene polymorphism of TLR3 gene was It was found that there was a significant frequency difference with the normal person. In addition, we found a significant difference between the allele type and TLR3 expression level, and clarified the relationship between allele type and function. Therefore, it was found that the gene polymorphism was found to be associated with an allergic disease, and was useful as a test index (including markers) for the function of onset of allergic disease and genetic predisposition.

[0070] In addition, the results shown in Example 2 revealed that the expression level of TLR3 was associated with the phenotype of allergic disease. Regulation of this expression level may lead to treatment of allergic disease. . Therefore, an agent that up-regulates or down-regulates the expression of TLR3 can be a therapeutic agent for allergic diseases. The present invention also includes a therapeutic agent for an allergic disease having such a function.

[0071] Whether or not the candidate drug regulates the expression of TLR3 gene may be determined directly as in Example 2, as in Example 2. Alternatively, a gene fusion of the ORF of the TLR3 gene and a known reporter gene (eg, a firefly luciferase gene) is prepared, the cell line containing the same is exposed to a candidate drug, and the reporter gene between the control and The differential expression of the offspring may be monitored. The present invention also includes a method for screening a therapeutic agent for an allergic disease, which is performed as described above.

[0072] Furthermore, the results shown in Example 2 showed that TLR3 was expressed in a larger amount by poly (I: C) stimulation than the allele sample (described above). TLR3 is known to recognize viral double-stranded RNA (Medzhitov et al., Supra) and synthesize antiviral proteins (Immunity 17, 251, 2002). Thus, this result suggests that individuals with the T allele conjugate are more resistant to viral infection than individuals with the C allele conjugate. That is, the genetic polymorphism of the subject TLR3 gene is correlated with the susceptibility to a viral disease and can be used as an index. When analyzed using such indicators, the susceptibility of a particular subject to a viral disease can be determined. Furthermore, the correlation between the susceptibility of the subject to an allergic disease and the susceptibility to a viral disease can also be analyzed. The present invention also includes a method for determining susceptibility to a viral disease (pre-onset diagnosis) using a polymorphism in the TLR3 gene. Industrial applicability

As described above, the genetic polymorphism in the TLR3 gene region is a genetic polymorphism that is significantly associated with allergic diseases (especially childhood asthma), and can be used as a marker for testing the genetic predisposition of allergic disease development in individuals . Therefore, the detection kit of the present invention, which detects the above potential, is useful for testing allergic diseases.

Claims

The scope of the claims

[1] A genetic predisposition marker for an allergic disease, which is selected from the group consisting of the polynucleotides described in (a) to (j) and (v) below:

(a) a polynucleotide comprising the single nucleotide polymorphism site identified in SEQ ID NO: 1 or SEQ ID NO: 2 and present at the seventh position of the human Toll-like receptor 3 gene;

(b) a polynucleotide comprising the single nucleotide polymorphism site at position 1638 of the human Toll-like receptor 3 gene specified by SEQ ID NO: 3 or SEQ ID NO: 4;

(c) a polynucleotide specified in SEQ ID NO: 5 or SEQ ID NO: 6, comprising a single nucleotide polymorphism site present at position 1656 of the human Toll-like receptor 3 gene;

(d) a polynucleotide comprising the single nucleotide polymorphism site located at position 3519 of the human toll-like receptor 3 gene specified in SEQ ID NO: 7 or SEQ ID NO: 8,

(e) a polynucleotide comprising the single nucleotide polymorphism site located at position 4792 of the human toll-like receptor 3 gene specified by SEQ ID NO: 9 or SEQ ID NO: 10;

(f) a polynucleotide specified in SEQ ID NO: 11 or SEQ ID NO: 12, comprising a single nucleotide polymorphism site present at position 4960 of the human Toll-like receptor 3 gene;

(g) a polynucleotide comprising the single nucleotide polymorphism site located at position 525 of the human Toll-like receptor 3 gene specified in SEQ ID NO: 13 or 14;

(h) a polynucleotide specified in SEQ ID NO: 15 or SEQ ID NO: 16, comprising a single nucleotide polymorphism site present at position 630 of the human Toll-like receptor 3 gene;

(i) a polynucleotide comprising the single nucleotide polymorphism site located at position 644 of the human toll-like receptor 3 gene specified in SEQ ID NO: 17 or SEQ ID NO: 18;

(V) 89 of the human toll-like receptor 3 gene identified by SEQ ID NO: 19 or SEQ ID NO: 20

A polynucleotide comprising a single nucleotide polymorphism site present at position 21; and

(j) the above, a polynucleotide having a sequence complementary to any of the polynucleotides

[2] A genetic predisposition for an allergic disease, which is selected from the group consisting of the polynucleotides described in the following (a)-(j) and (V), and has one or more polynucleotide powers: Continuous base length for hybridizing specifically with Rikaichi and detecting the marker 1 Zero or more polynucleotides:

[3] A primer for detecting a genetic predisposition marker for an allergic disease, comprising at least one kind of the polynucleotide according to claim 2.

[4] A probe for detecting a genetic predisposition to an allergic disease, comprising the polynucleotide according to claim 2.

[5] A kit for detecting a genetic predisposition marker for an allergic disease, wherein the kit according to claim 3 is used. A detection kit comprising a detection primer.

[6] A kit for detecting a genetic predisposition marker for an allergic disease, comprising the detection probe according to claim 4.

[7] An amplification product obtained by PCR-amplifying the human toll-like receptor 3 gene with the primer according to claim 3.

[8] A method for testing a genetic predisposition to an allergic disease, which comprises detecting a gene polymorphism in the human Toll-like receptor 3 gene.

[9] The method according to [8], wherein the gene polymorphism is at least one single nucleotide polymorphism selected from the group consisting of the following single nucleotide polymorphisms (k) and (u). Inspection method described in:

(k) a single nucleotide polymorphism site identified by SEQ ID NO: 1 or SEQ ID NO: 2 and present at the seventh position of the human Toll-like receptor 3 gene;

(1) a single nucleotide polymorphism site identified at SEQ ID NO: 3 or SEQ ID NO: 4 and present at position 1638 of the human toll-like receptor 3 gene;

(m) a single nucleotide polymorphism site identified at SEQ ID NO: 5 or SEQ ID NO: 6 and present at position 1656 of the human toll-like receptor 3 gene;

(n) a single nucleotide polymorphism site located at position 3519 of the human toll-like receptor 3 gene specified by SEQ ID NO: 7 or SEQ ID NO: 8;

(o) a single nucleotide polymorphism site identified at SEQ ID NO: 9 or SEQ ID NO: 10 and present at position 4792 of the human Toll-like receptor 3 gene;

(P) a single nucleotide polymorphism site identified at SEQ ID NO: 11 or SEQ ID NO: 12 and present at position 4960 of the human Toll-like receptor 3 gene;

(q) a single nucleotide polymorphism site identified by SEQ ID NO: 13 or SEQ ID NO: 14 and located at position 525 of the human Toll-like receptor 3 gene;

(r) a single nucleotide polymorphism site identified as SEQ ID NO: 15 or SEQ ID NO: 16 and present at position 630 of the human Toll-like receptor 3 gene;

(s) a single nucleotide polymorphism site identified by SEQ ID NO: 17 or SEQ ID NO: 18 and present at position 644 of the human Toll-like receptor 3 gene; (t) a single nucleotide polymorphism site identified at SEQ ID NO: 19 or SEQ ID NO: 20, located at position 89 to 21 of the human Toll-like receptor 3 gene; and

(u) A single nucleotide polymorphism site in the human toll-like receptor 3 gene that is in linkage disequilibrium with the single nucleotide polymorphism site at position 6444 of the human toll-like receptor 3 gene.

[10] The test method according to claim 8 or 9, wherein the allergic disease is asthma.

11. The test method according to claim 10, wherein the asthma is child asthma.

[12] A method for determining the susceptibility of a viral disease, the method comprising a step of analyzing the polymorphism in the human Toll-like receptor 3 gene as an indicator of the susceptibility.

[13] The determination according to claim 12, wherein the gene polymorphism is at least one single nucleotide polymorphism selected from the group consisting of single nucleotide polymorphism sites according to claim 9. Method.

[14] A screening method for a therapeutic agent for an allergic disease, which comprises contacting a candidate agent with cells expressing the human toll-like receptor 3 gene, and attenuating the expression of the human toll-like receptor 3 gene.

[15] A therapeutic agent for allergic diseases, which comprises as an active ingredient a drug that regulates the expression of the human toll-like receptor 3 gene.