WO2005033338A1 - Method of evaluating risk for viral infection - Google Patents

Abstract

One or more single nucleotide polymorphisms (for example, single nucleotide polymorphisms at the -8921, -7, 1638, 1656, 3519, 4792, 4960, 5252, 6301 and 6444 positions) in human Toll-like receptor 3 gene are detected and the risk for viral infection is evaluated based on the genotypes of the single nucleotide polymorphisms thus detected.

Description

 Specification

 Risk assessment method for virus infection

 Technical field

 [0001] The present invention relates to polymorphisms associated with mutations in genes related to human allergic diseases, in particular, polymorphisms in human toll-like receptor 3 (hereinafter referred to as human TLR3 or simply TLR3), which is a genetic predisposition marker for allergic diseases. And its use. More specifically, the present invention relates to a method for evaluating the risk of viral infection using a TLR3 gene polymorphism, a primer or probe for detecting the same, a method for reducing the risk thereof, and the like.

 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 they are frequently present in the genome, they are important as markers in gene analysis. It is being used in search.

[0004] Genome analysis using gene polymorphisms including single nucleotide polymorphisms has revealed that Studies are underway to elucidate nucleotide polymorphisms and genetic polymorphisms that cause differences in susceptibility to diseases. 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 evaluate 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.

 [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).

[0008] On the other hand, viruses are infectious substances having DNA or RNA as genes and growing in host cells. Invades the cells of animals and plants and proliferates using the metabolic system in host cells. The propagated virus can damage and kill the host cell. Viruses causing such infectious diseases have a large number of powers. Generally, they are roughly classified into groups according to the mechanism of producing virus products (for example, retroviruses represented by HIV, etc.). Viruses that cause infectious diseases on a daily basis. Adenowinores (adenovirus) etc. Human rhinovirus (hereinafter referred to as rhinovirus) belongs to the genus Rhinovirus belonging to the Picornavirus family and is a small virus having a genome of + -strand RNA, and has a nasal cold, Rhinovirus inflammation often occurs in the upper respiratory tract (upper respiratory tract inflammation), and secondary infection with bacteria may further progress to bronchitis, sinusitis, otitis media, etc. The cold caused by the rhinovirus is different from that caused by the influenza virus, and is seen throughout the year. It is said that there are many treatments for this infection in the spring and fall, but chemotherapy and vaccine therapy have been studied, but no definitive effective treatment has yet been established. Precautionary measures against infection become important.

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

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

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

 Disclosure of the invention

 Problems to be solved by the invention

 [0010] As described above, a treatment method has been established, and a preventive measure for a virus infection is desired! /

 In particular, there is a strong demand for prevention of rhinovirus infection in children as soon as they become ill, and elucidation of the etiology and causes. A correlation has been suspected between viral infections and allergic diseases, and a genetic predisposition may be involved in the infection. Therefore, if it becomes possible to predict the risk of infection of a subject (also referred to as a subject or an individual) and the severity at the time of infection, it will be possible to provide effective lifestyle guidance and preventive measures taking into account the individual constitution. Become. In addition, preventive measures can be taken for co-occurrence with allergic diseases, which will guide the development of more effective treatment methods and therapeutic drugs.

[0011] Therefore, the present invention provides identification of a predisposition gene for an allergic disease, and The main object of the present invention is to identify existing gene polymorphisms associated with a disease and to provide a method for evaluating the risk of virus infection using the gene polymorphism.

 [0012] Further, the present invention aims to provide a specific primer or probe for the allele constituting the gene polymorphism, and to use it for evaluating the risk of virus infection.

 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, which are thought to have a direct effect on susceptibility (disease susceptibility) by selecting candidate genes to be compared, between the allergic disease group and the control group, both in association analysis using the candidate gene approach We searched for genes related to allergic diseases. 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. Furthermore, they found that those polymorphisms showed a statistically significant relationship with susceptibility to viral infections (particularly human rhinovirus infection).

 [0014] Therefore, the above-mentioned object is achieved by the present invention described below. That is, the present invention detects a TLR3 gene polymorphism as a genetic predisposition marker (risk of development) of an allergic disease, and evaluates whether a subject having the polymorphism is susceptible to viral infection (infection risk). In particular, the present invention provides the following (1)-(19).

 (1) A method for assessing the risk of viral infection, comprising detecting at least one single nucleotide polymorphism in the human Toll-like receptor 3 gene and detecting the virus infection based on the detected single nucleotide polymorphism genotype. An evaluation method characterized by evaluating risk.

 (2) The evaluation method according to (1), wherein the virus is a rhinovirus.

(3) The at least one single nucleotide polymorphism force The 8921th single nucleotide polymorphism, the 7th single nucleotide polymorphism, the 1638th single nucleotide polymorphism, the 1656th single nucleotide polymorphism, the 3519th single nucleotide polymorphism Polymorphism, single nucleotide polymorphism at position 4792, single nucleotide polymorphism at position 4960, single nucleotide polymorphism at position 5252 Wherein the polymorphism is selected from the group consisting of a single nucleotide polymorphism at position 6301, a single nucleotide polymorphism at position 6444, and a single nucleotide polymorphism in linkage disequilibrium with the single base polymorphism at position 6444. Evaluation method described in 2).

 (4) The method according to (3), wherein the single nucleotide polymorphism is the 6444th single nucleotide polymorphism.

 (5) The evaluation method according to (4), wherein the risk of viral infection is evaluated as high if the genotype of the detected single nucleotide polymorphism is CC.

 (6) The evaluation method according to the above (4), wherein the risk of virus infection is evaluated as high when the genotype of the detected single nucleotide polymorphism is CT.

 (7) The evaluation method according to (4), wherein the risk of virus infection is evaluated as low when the genotype of the detected single nucleotide polymorphism is TT.

 (8) The method according to the above (3), wherein the single nucleotide polymorphism is the 3519th single nucleotide polymorphism.

 (9) The evaluation method according to (8), wherein the risk of virus infection is evaluated as high when the detected single nucleotide polymorphism genotype is AA.

 (10) The evaluation method according to (8), wherein the risk of virus infection is evaluated as high if the detected single nucleotide polymorphism genotype is AT.

 (11) The evaluation method according to (8), wherein the risk of virus infection is evaluated as low if the genotype of the detected single nucleotide polymorphism is TT.

 (12) The method according to (3), wherein the single nucleotide polymorphism is the 4792th single nucleotide polymorphism.

 (13) The evaluation method according to (12), wherein the risk of virus infection is evaluated to be high when the detected single nucleotide polymorphism genotype is GG.

 (14) The evaluation method according to (12), wherein when the detected single nucleotide polymorphism genotype is GC, the risk of virus infection is evaluated to be high.

 (15) The evaluation method according to (12), wherein the risk of virus infection is evaluated to be low when the genotype of the detected single nucleotide polymorphism is CC.

(16) To evaluate the risk of viral infection, the human toll-like receptor 3 gene 8921 single nucleotide polymorphism, 7th single nucleotide polymorphism, 1638th single nucleotide polymorphism, 1656th single nucleotide polymorphism, 3519th single nucleotide polymorphism, 4792th single nucleotide polymorphism Use of a specific primer or a specific probe for the 4960th single nucleotide polymorphism, the 5252nd single nucleotide polymorphism, the 6301th single nucleotide polymorphism or the 644th single nucleotide polymorphism.

(17) A method for reducing the risk of viral infection in a human or mammal, comprising administering to the human or mammal an effective amount of a compound having an activity of regulating the expression of the human toll-like receptor 3 gene. Method.

 (18) A method for assessing the risk of viral infection and susceptibility to allergic diseases, comprising detecting at least one single nucleotide polymorphism in the human toll-like receptor 3 gene and determining the genotype of the detected single nucleotide polymorphism. The susceptibility of a virus infection or allergic disease to a subject.

 (19) The evaluation method according to (18), wherein the virus is rhinovirus and the allergic disease is pediatric asthma.

 The invention's effect

 [0015] According to the present invention, a specific allele of the genetic polymorphism is detected from a sample such as a genomic DNA of a subject (individual) using a genetic polymorphism associated with a genetic predisposition to an allergic disease. This assesses the risk of viral infection in the subject. By implementing the evaluation method of the present invention, the susceptibility to infection with a specific virus (susceptibility to infection) can be predicted, and life guidance for infection prevention can be provided. Furthermore, simultaneous evaluation with susceptibility to allergic diseases is possible. In addition, the findings obtained in the present invention can also be applied to the development of prophylactic and therapeutic agents for viral infection.

 Brief Description of Drawings

FIG. 1 is a schematic diagram showing the action of the immune system during virus infection.

 [Fig. 2] Fig. 2 is a graph showing the results of measuring the rhinovirus infection rate of a subject by RT-PCR throughout the year.

[Figure 3] Figure 3 shows that the expression level of TLR3 expressed when CD14 + cells of samples from different genotypes were stimulated with poly (I: C) was plotted against poly (I: C) concentration. It is the figure which plotted. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

 [0018] As used herein, the term "gene polymorphism" means a portion where the nucleotide sequence on the human genome differs between individuals. "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.

[0019] As used herein, the term "specifically hybridizes" is synonymous with the term "hybridizes under stringent conditions" as recognized by those skilled in the art. Or fragments) are described in Sambrook, J., "Expression of cloned genes in E. coli", Morecuff. Cloning: A. Laboratory. _{Yuanore. (M oi e cular Cloning:} A laboratory manual), the United States, cold 'spring' Nono over server ^ ~ laboratory ^ ~ Press (cold Sprin g Harbor laboratory Press) , 1989 years, pp 9. 47-9. 62, pp. 11. 45-11. It means to hybridize with each other under the hybridization conditions described in 61.

 [0020] More specifically, the above-mentioned "stringent conditions" means that hybridization is performed at 6.6 X SSC at about 45 ° C, and then 2.0 X SSC at 50 ° C. 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.

[0021] The origin of the nucleic acid test sample (also referred to as a specimen) 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. The sample contains not only the extracted genomic DNA but also a part of the genomic DNA base sequence. May be. That is, before analysis of the allele, a sample obtained by amplifying the entire or partial base sequence (DNA fragment) in the sample nucleic acid by a suitable method, for example, PCR, etc., as a nucleic acid test sample is used. Is also good. The DNA can be extracted using a known method, for example, a commercially available extraction kit such as “QIAamp mono kit” (manufactured by QIAGEN).

 [0022] The primer used in the evaluation method of 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 that does not contain a single nucleotide polymorphism site but obtains a nucleic acid fragment containing a single nucleotide polymorphism site by PCR or the like. Is included. As the latter primer, for example, a probe designed to correspond to the base sequence immediately before the predetermined single nucleotide polymorphism to be detected or a base sequence several bases before, that is, the 3 ′ end or the 5 ′ end of the probe is an SNP. Primers designed to be upstream or downstream of or near one base 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, more preferably 15 to 30 nucleotides. Specifically, such a primer can be synthesized as a forward primer and a reverse primer, for example, using an automatic synthesizer, based on allele and base sequence information on or near Z. 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 preferable that the primer has a sequence complementary to the nucleic acid to be type III, but if necessary, one or more base pairs that are not complementary to each other unless desired and have no effect are included in the primer. Mismatch).

[0023] The polynucleotide probe used in the evaluation method of the present invention can be prepared by any suitable synthesis method. The polynucleotide probe may have any suitable length that matches the detection method and the like, but is continuous 10 or more 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 locations may be used unless undesirable effects occur on the polynucleotide probe. The above non-complementary base pairs may be introduced. Probes may also carry one or more labels, similar to primers!

 [0024] The present invention also includes a nucleic acid fragment containing one or more single nucleotide polymorphism sites. For example, nucleic acids 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 amplified to 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.

[0025] Numerous methods are known 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) 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, Judgment of polymorphism), Restriction Fragment Length Polymorphisms (RFLP) Allele detection by hybridization such as DNA microarray, SPR (Surface Plasmon Resonance), SSCP (Single Strand Conformation Polymorphism), mouth double-strand analysis, WAVE system (base difference detection by chromatography), A molecular beacon (Sniper method or the like) is exemplified. In any of such known detection methods, it is possible to detect a predetermined single nucleotide polymorphism according to the evaluation method of the present invention using the probe of the present invention, Z or a primer, Z or a polynucleotide, or the like. it can.

 [0026] 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.

 [0027] SNPs are determined using a sample 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.

 [0028] As described above, in the present invention, the following (k) and (10) untranslated region or 10 SNPs in the untranslated region of the TLR3 gene were identified.

 (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) the untranslated region of the human toll-like receptor 3 gene identified by SEQ ID NO: 9 or SEQ ID NO: 10

 4792 single nucleotide polymorphism site;

 (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; 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;

[0029] 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.

 [0030] 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.

 [0031] A polynucleotide consisting of or containing the above-listed sequences is a genetic predisposition marker for allergic disease. By detecting each marker in the evaluation method of the present invention, the risk of viral infection is increased. Can be evaluated.

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

[0033] 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 (region containing a genetic predisposition marker), and the TLR3 gene was detected using various single nucleotide polymorphism detection methods. Genetic polymorphism in one or more of each polymorphic site (-8921, -7, 1638, 1656, 3519, 4792, 4960, 5252, 6301, 6444) in the child region Determine the type of base for the type. As described above, from the correlation between the SNP obtained from the association analysis and the pathology (including severity) of the allergic disease, the risk of viral infection of the subject is determined by referring to the determined TLR3 gene polymorphism. To evaluate.

 [0034] It has been confirmed in the examples below that there is a strong correlation between the SNP at position 6444 of the TLR3 gene and asthmatic patients. Therefore, the risk of virus infection can be evaluated by detecting the SNP at position 6444 of the TLR3 gene. Also, instead of detecting the SNP at position 6444 of the TLR3 gene, the risk of viral infection can be assessed by detecting the SNP site at the TLR3 gene in linkage disequilibrium with the SNP site at position 6444 of the TLR3 gene. It is possible to do. SNP sites in the TLR3 gene that are in linkage disequilibrium with the SNP site 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.

 [0035] More specifically, PCR is performed in a region containing the single nucleotide polymorphism in the DNA sample, and 1) the type of base is determined by the SSCP method; or 2) the PCR amplification product is directly sequenced (Sanger Law or Maxam-Gilbert Law). Alternatively, a probe specifically hybridizing to a region containing a single nucleotide polymorphism is used to directly detect a single nucleotide polymorphism from the DNA sample or its PCR amplification product (such as Invader Atsushi). Furthermore, a single base extension reaction is performed using a primer having a sequence up to (before) the predetermined single nucleotide polymorphism, and the extended base is analyzed by MS (mass spectrometer).

 [0036] For detection and analysis of the polymorphic site, the polynucleotide of the present invention is preferably used. 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 at least one nucleotide sequence of the nucleotide sequences of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 and a nucleotide sequence complementary thereto.

In the case where 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, Sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 , Preferably containing one base sequence.

 [0038] In the present invention, the probe and primer of the present invention are preferably provided as a test kit containing each of them as an effective component. In particular, the evaluation kit of the present invention comprising a primer may include a DNA polymerase, four types of deoxynucleotide triphosphates (dNTPs), a size marker, and the like. In addition, a suitable buffer, detergent, reaction stop solution and the like may be included.

 [0039] In addition, an antibody specific to a polymorphism in the TLR3 gene can be used for detection.

 [0040] The test for determining whether or not the subject is actually infected with a virus is performed using a virus culture solution separated from a clinical specimen (for example, a nasal washing solution or the like). The assay can be performed by recovering the virus from the culture solution and using an antiserum (specific for the virus). In the case of rhinovirus, virus growth is slow in cultured cells and virus isolation is difficult. Therefore, preferably, RNA is extracted from a sample such as a culture solution, and cDNA is obtained and DNA is amplified by reverse transcription PCR (RT-PCR). The amplification product is separated on an agarose gel and subjected to V ヽ sequencing according to a conventional method. By comparing the determined nucleotide sequence with the rhinovirus nucleotide sequence, the presence of the virus in the sample can be assayed.

 The following results are obtained as a result of the analysis performed according to the above procedure.

 1.a) Detecting CC as the 6444 allele indicates that the subject has a high risk of viral infection.b) Detecting the 6444 allele with CT indicates an increased risk of viral infection. C) When TT is detected as the allele at position 6444, it indicates that the subject has a low risk of viral infection.

2.a) Detection of AA as the 3519 allele indicates that the subject is at high risk of viral infection.b) Low risk of viral infection when detecting the AT as the 3519 allele C) TT as 3519 allele When detected, indicates that the subject has a low risk of virus infection.

 3.a) When GG is detected as the 4792 allele, it indicates that the subject is at high risk of virus infection.b) When GC is detected as the 4792 allele, the risk of virus infection is low. C) Detection of CC as the allele at position 4792 indicates a low risk of viral infection, indicating that the subject is a subject.

 If the above analysis is further performed on other alleles (eg, 9 locations), haplotype analysis at the site of the allele can be performed.

 [0043] The detection kit according to the present invention is intended to detect an allele that is a marker for susceptibility to viral infection. If necessary, a kit for testing other disease-related factors (markers) may be used. In combination, the test result power can also determine the complex status of a subject.

 [0044] The correlation between the onset of an allergic disease and infection by a virus is not limited to a particular theory. Immune cells work against invasion of allergic antigens (allergens) to produce antibodies (IgE, etc.) and cytodynamics (IL4, IL6, IL8, γ-interferon (IFN-γ), etc.) I do. In the process, an inflammatory response is triggered. On the other hand, when a virus enters the body, a similar immune response is induced. Therefore, the size of an antigen of an allergic disease is similar to that of an allergic disease, and an individual is susceptible to the disease. Allergic diseases for which such a correlation is predicted include bronchial asthma (Rhinovirus Rsvirus), other atopic dermatitis (Herpes simplex virus), pollinosis (Influenza virus), Lugiic conjunctivitis (Adenovirus) and food allergy (Enterovirus). Thus, contracting the listed viral infections may trigger adaptive immunity and, at the same time, develop a corresponding allergic disease. Fig. 1 is a schematic diagram showing the function of the immune system due to virus infection.

 [0045] According to the present invention, the presence or absence of a predisposition to allergy can be determined by genetic diagnosis, and the susceptibility to viral infection can also be determined. Prevention of both diseases or sensitization with an allergen during virus infection can be performed. Specific preventive measures, such as avoidance, can be taken.

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

(Test Example 1)

 Association study between asthma and genetic polymorphism

[0048] (1) Subject

 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.

[0049] (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). The results are shown in Table 1. Therefore, when the homozygote of the polymorphism at position 6444 of the TLR3 gene region is homozygous, the probability of being a healthy individual is significantly higher than that of the childhood asthma disease group. The result showed that the data had a strong relationship with. 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.

[0050] [Table 1] Genotype control group (%) Pediatric asthma χ ² (Ρ value) Odds ratio

 Disease group (%) TT vs CC + CT (odds ratio)

 CC 95 (34.3%) 126 (38.3%)

 CT 1 19 (43.0%) 1 70 (51.7%) 18.2 (0.000020) 2.64

 (1 .67-4.1 7)

 TT 63 (22.7%) 33 (10.0%)

[0051] [Table: Genotype control group (%) Adult asthma χ ² (Ρ value) Odds ratio

 Disease group (¾) TT vs CC + CT (odds ratio)

 CC 95 (34.3%) 162 (35.1%)

 CT 1 19 (43.0%) 229 (49.7%) 6.69 1.64

 (0.000020) (1 .1 3-2.40)

 TT 63 (22.7%) 70 (15.0%)

[0052] In addition, when the SNPs other than the 6444th were examined, the homozygous CC for the -7th SNP and the homozygous CC or the heterozygous CA for the 3519th SNP were examined. In this case, homozygous GG or heterozygous GC at SNP 4792, homozygous CC or heterozygous CT at 630 1SNP, and homozygous at SNP 8921 at SNP 8921 In the case of body TT, the proportion of asthmatics was higher than in healthy individuals, respectively.

 (Example 1)

 Association study between childhood asthma (rhinovirus infection) and genetic polymorphism

[0054] In the same manner as in Test Example 1, samples of pediatric asthmatic patients and control subjects (healthy subjects) were collected together with clinical information, and the presence or absence of rhinovirus infection was examined by the RT-PCR method. Here, we examined the homology of T in healthy subjects The union was significantly higher than the homozygotes with force A and the heterozygotes with AT (p = 0.00000018). Table 3 shows the results. Therefore, when the allele at position 3519 in the TLR3 gene region is a homozygote for T, this particular genetic polymorphism has a significantly higher probability of being healthy than the pediatric asthma disease group, and this particular polymorphism is a representation of the disease. The result is that it has a strong relationship with the mold. Inspection of each polymorphism for rhinovirus infection revealed that non-infected individuals were significantly more homozygous for T than homozygotes for A and heterozygotes for AT. I got high strength. Table 3 also shows the results. Therefore, when the 3519 allele of the TLR3 gene region is a homozygote of T, the probability of being non-infected with rhinovirus is significantly higher, in contrast to a homozygote of A and a heterozygote of AT. In the case of, the probability of being a virus infected person is significantly higher. The result showed that this genetic polymorphism had a strong association with viral infection. When RT-PCR was used to test for rhinovirus infection during asthma attack in patients with the adult asthma disease group, 71.6% were infected.

[Table 3]

(Example 2)

 Seasonality of rhinovirus infection

[0057] RT—The rate of rhinovirus infection in subjects measured by PCR throughout the year.

It can be seen that it rises in spring and autumn (May and September). The result is shown in FIG.

(Example 3)

 Association between genetic polymorphism and phenotype

[0059] (1) Experimental method

Allele of polymorphism at position 6444 of TLR3 gene region and TLR3 gene The correlation for expression 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.

[0060] (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 2 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.

 As described above, it was found from the results of Test Example 1 and Example 3 that the TLR3 gene polymorphism had a significant frequency difference between allergic patients and healthy subjects. In addition, we found a significant difference between the allele type and TLR3 expression level, and clarified that there is a relationship between allele type and function. Therefore, it has been clarified that the gene polymorphism is a polymorphism that has been clearly associated with an allergic disease, and is useful as a test index (including a marker) for the function of onset of allergic disease and genetic predisposition.

[0062] Further, the results shown in Example 3 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. Further, from the results shown in Example 3, it was confirmed that the T allele sample power was able to express a large amount of TLR3 by poly (I: C) stimulation as compared with 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 risk of virus infection and can be used as an index. This result is consistent with the result obtained in Example 1 (rhinovirus infection), and indicates that the risk of viral infection can be evaluated by detecting at least one allele of the TLR3 gene polymorphism. .

 [0064] Further, using such an index, it is also possible to analyze the correlation between the risk of virus infection of a specific subject and the susceptibility to an allergic disease.

 [0065] In addition, the agent that up-regulates or down-regulates the expression of TLR3, ie, a compound that has an activity of regulating the expression of the gene, may reduce the risk of viral infection in an individual.

 [0066] Whether or not a candidate drug regulates TLR3 gene expression may be determined directly as in Example 3, as in Example 3. 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 drug that reduces the risk of viral infection, which is performed in this manner.

[0067] Such agents are used, similar to known antiviral agents, but the preferred method of treatment is to prevent humans or mammals at risk of viral infection (such as horses, pigs, sheep, etc.) prophylactically. Administer. In practice, drugs are administered to animals in antiviral doses orally, rectally, transdermally, subcutaneously, intravenously, intramuscularly, intranasally, or by other routes of administration. The drug for administration is a pharmaceutical formulation in which an effective dose is formulated together with a pharmaceutically acceptable carrier, diluent, or excipient, and may be a tablet, powder, suspension, troche, syrup, or the like. Agents, aerosols and the like. Industrial applicability

 As described above, the polymorphism in the TLR3 gene region is a polymorphism that is significantly associated with allergic diseases (especially pediatric asthma). By detecting at least one allele, viral infection ( In particular, the risk of rhinovirus can be assessed. Thus, the alleles disclosed herein can be used as markers to determine whether a subject is predisposed to viral infection. The specific primers and probes according to the present invention for detecting the marker, and a detection kit containing them are useful for evaluating the risk of virus infection.

Claims

The scope of the claims  [I] A method for assessing the risk of viral infection, comprising detecting at least one single nucleotide polymorphism in the human toll-like receptor 3 gene and detecting the viral infection based on the detected single nucleotide polymorphism genotype. An evaluation method characterized by evaluating risk.  [2] The evaluation method according to claim 1, wherein the virus is a rhinovirus. [3] said at least one single nucleotide polymorphism force 8921th single nucleotide polymorphism, 7th single nucleotide polymorphism, 1638th single nucleotide polymorphism, 1656th single nucleotide polymorphism, 3519th single nucleotide polymorphism Type, 4792th single nucleotide polymorphism, 4960th single nucleotide polymorphism, 5252th single nucleotide polymorphism, 6301th single nucleotide polymorphism, 6444th single nucleotide polymorphism and 6444th single nucleotide polymorphism and 3. The evaluation method according to claim 2, wherein the method is selected from the group consisting of single nucleotide polymorphisms in linkage disequilibrium.  [4] The method according to claim 3, wherein the single nucleotide polymorphism is the 6444th single nucleotide polymorphism.  [5] The evaluation method according to claim 4, wherein, when the genotype of the detected single nucleotide polymorphism is CC, the risk of virus infection is evaluated to be high. [6] The evaluation method according to claim 4, wherein when the detected single nucleotide polymorphism genotype is CT, the risk of virus infection is evaluated to be high. [7] The evaluation method according to claim 4, wherein when the genotype of the detected single nucleotide polymorphism is TT, the risk of virus infection is evaluated to be low. [8] The method according to claim 3, wherein the single nucleotide polymorphism is the 3519th single nucleotide polymorphism.  [9] The evaluation method according to claim 8, wherein when the genotype of the detected single nucleotide polymorphism is AA, the risk of virus infection is evaluated to be high. [10] The evaluation method according to claim 8, wherein when the detected single nucleotide polymorphism genotype is AT, the risk of virus infection is evaluated to be high.  [II] The evaluation method according to claim 8, wherein when the genotype of the detected single nucleotide polymorphism is TT, the risk of virus infection is evaluated to be low. 12. The method according to claim 3, wherein the single nucleotide polymorphism is the 4792th single nucleotide polymorphism. The method described.  13. The evaluation method according to claim 12, wherein the risk of virus infection is evaluated to be high when the genotype of the detected single nucleotide polymorphism is GG. 14. The evaluation method according to claim 12, wherein the risk of virus infection is evaluated to be high when the genotype of the detected single nucleotide polymorphism is GC. 15. The evaluation method according to claim 12, wherein the risk of virus infection is evaluated to be low when the detected genotype of the single nucleotide polymorphism is CC. [16] To assess the risk of viral infection in the human toll-like receptor 3 gene Single nucleotide polymorphism 21st, Single nucleotide polymorphism 7th, Single nucleotide polymorphism 1638, Single nucleotide polymorphism 1656, Single nucleotide polymorphism 3519, Single nucleotide polymorphism 4792, 4960 Use of a specific primer or a probe for the 5th single nucleotide polymorphism, the 5252nd single nucleotide polymorphism, the 6301th single nucleotide polymorphism or the 6444th single nucleotide polymorphism. [17] A method for reducing the risk of viral infection in a human or mammal, comprising administering to the human or mammal an effective amount of a compound having an activity of regulating the expression of the human toll-like receptor 3 gene. . [18] A method for evaluating the risk of viral infection and susceptibility to allergic diseases, comprising detecting at least one single nucleotide polymorphism in the human toll-like receptor 3 gene and converting the detected single nucleotide polymorphism to a genotype. A method for evaluating the susceptibility of a virus infection and an allergic disease based on the evaluation method. [19] The evaluation method according to claim 18, wherein the virus is rhinovirus and the allergic disease is pediatric asthma.