WO2005033339A1 - Polymorphisme du gene du recepteur de type toll-3 humain utilise en tant que marqueur du facteur herite d'une maladie allergique et utilisation de ce marqueur - Google Patents

Polymorphisme du gene du recepteur de type toll-3 humain utilise en tant que marqueur du facteur herite d'une maladie allergique et utilisation de ce marqueur Download PDF

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WO2005033339A1
WO2005033339A1 PCT/JP2004/014608 JP2004014608W WO2005033339A1 WO 2005033339 A1 WO2005033339 A1 WO 2005033339A1 JP 2004014608 W JP2004014608 W JP 2004014608W WO 2005033339 A1 WO2005033339 A1 WO 2005033339A1
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seq
gene
receptor
single nucleotide
nucleotide polymorphism
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PCT/JP2004/014608
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English (en)
Japanese (ja)
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Kazuhiko Obara
Yusuke Nakamura
Mayumi Tamari
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Hitachi Chemical Co., Ltd.
Riken
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Priority to JP2005514485A priority Critical patent/JP4533979B2/ja
Publication of WO2005033339A1 publication Critical patent/WO2005033339A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Human toll-like receptor as a genetic predisposition marker for allergic diseases 3 gene polymorphisms and their use
  • 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.
  • human TLR3 human toll-like receptor 3
  • 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.
  • 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.
  • NASH National Institutes of Health
  • TLR 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.
  • LRR leucine-rich repeat
  • 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 ⁇ (dendritic cells) and also involved in the activation of acquired immunity.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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;
  • (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:
  • 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;
  • 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;
  • 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 primer for detecting a genetic predisposition marker for allergic disease comprising at least one kind of the polynucleotide according to (2).
  • a probe for detecting a genetic predisposition marker for an allergic disease comprising the polynucleotide according to (2).
  • a kit for detecting a genetic predisposition marker for an allergic disease comprising the detection primer according to (3).
  • a kit for detecting a genetic predisposition marker for an allergic disease comprising the detection probe according to (4).
  • 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.
  • 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:
  • 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;
  • 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.
  • a method for determining the susceptibility of a viral disease 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • polynucleotide refers to a substance in which two or more nucleotides are linked by a phosphodiester bond.
  • Polynucleotide includes “ribonucleotide (RNA)” and “deoxyribonucleotide (DNA)”.
  • 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.
  • nucleic acid is used synonymously or interchangeably with the above “polynucleotide” and means DNA or RNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention also includes a nucleic acid fragment containing one or more single nucleotide polymorphism sites.
  • 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.
  • 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.
  • nucleotide sequence analysis sequence
  • detection reaction with invader assay Cleavase, allele-specific probe and FRET probe
  • 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
  • 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.
  • a gene polymorphism associated with the development of an allergic disease can be detected, for example, as follows.
  • 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.
  • SNPs associated with allergic diseases 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.
  • 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.
  • SEQ ID NO: 2 corresponds to the above (k)
  • SEQ ID NO: 4 corresponds to the above (k)
  • SEQ ID NO: 6 corresponds to the above (k)
  • SEQ ID NO: 8 corresponds to the above (k)
  • SEQ ID NO: 10 corresponds to the above (k)
  • SEQ ID NO: 12 corresponds to the above (k)
  • SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20 corresponds to the above (k)
  • the other allele shown in (t) is a continuous base sequence of 10 bases each on the 5 'side and 3' side from the single nucleotide polymorphism site.
  • 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.
  • the inspection can be performed as follows.
  • 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)).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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: 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.
  • the probe and the primer of the present invention are preferably provided as a test kit containing each of them as an effective component.
  • 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.
  • dNTP deoxynucleotide triphosphate
  • the size marker e.g., a size marker, and the like.
  • it may contain an appropriate buffer, detergent, reaction stop solution and the like.
  • an antibody specific to a polymorphism in the TLR3 gene can be used for detection.
  • 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.
  • 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 ⁇ g / mL of TLR3 ligand (poly (l: C), manufactured by Amersham Fanolemasia).
  • 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.
  • poly (l: C) was supplemented with 10 ⁇ g 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.
  • 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.
  • 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.
  • Whether or not the candidate drug regulates the expression of TLR3 gene may be determined directly as in Example 2, as in Example 2.
  • a gene fusion of the ORF of the TLR3 gene and a known reporter gene eg, a firefly luciferase gene
  • a known reporter gene eg, a firefly luciferase gene
  • the present invention also includes a method for screening a therapeutic agent for an allergic disease, which is performed as described above.
  • 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).
  • 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.
  • the present invention also includes a method for determining susceptibility to a viral disease (pre-onset diagnosis) using a polymorphism in the TLR3 gene.
  • 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.

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  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

Un facteur hérité associé à une maladie allergique est étudié au moyen de la détection du polymorphisme d'un gène au niveau d'au moins un site de polymorphisme nucléotidique simple situé sur les positions 8921, -7, 1638, 1656, 3519, 4792, 4960, 5252, 6301 et 6444 dans le gène du récepteur de type Toll-3 (TLR3) humain.
PCT/JP2004/014608 2003-10-03 2004-10-04 Polymorphisme du gene du recepteur de type toll-3 humain utilise en tant que marqueur du facteur herite d'une maladie allergique et utilisation de ce marqueur WO2005033339A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077204A1 (fr) * 1999-06-10 2000-12-21 University Of Iowa Research Foundation Acide nucleique tlr4 variant et ses utilisations
JP2002514083A (ja) * 1997-05-07 2002-05-14 シェーリング コーポレイション ヒトＴｏｌｌ様レセプタータンパク質、関連する試薬および方法
WO2003050137A2 (fr) * 2001-12-11 2003-06-19 David Schwartz Mutations de recepteur de type toll 4

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002514083A (ja) * 1997-05-07 2002-05-14 シェーリング コーポレイション ヒトＴｏｌｌ様レセプタータンパク質、関連する試薬および方法
WO2000077204A1 (fr) * 1999-06-10 2000-12-21 University Of Iowa Research Foundation Acide nucleique tlr4 variant et ses utilisations
WO2003050137A2 (fr) * 2001-12-11 2003-06-19 David Schwartz Mutations de recepteur de type toll 4

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALEXOPOULOU L. ET AL.: "Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3", NATURE, vol. 413, no. 6857, 2001, pages 732 - 738, XP002968529 *
EINSELE H. ET AL: "Gene-polymorphisms in toll like receptor genes are associated with the risk of CMV infection after HLA-identical stem cell transplantation (688)", BLOOD, vol. 102, no. 11, November 2003 (2003-11-01), pages 197A, XP002984473 *
MATSUSHIMA H. ET AL.: "Hifu mast saibo wa TLR3 oyobi TLR9 o kaishite byogentai no kakusan o ninshiki shi, tokui na saibo oto o shimesu", ALLERGY, vol. 51, no. 9/10, 2002, pages 265, XP002989354 *
NOGUCHI E. ET AL.: "An association study of asthma and total serum immunoglobin E levels for Toll-like receptor polymorphisms in a Japanese population", CLIN. EXP. ALLERGY, vol. 34, no. 2, February 2004 (2004-02-01), pages 177 - 183, XP002073840 *
ROCK F.L. ET AL.: "A family of human receptors structurally related to Drosophila toll", PROC. NATL. ACAD. SCI. USA, vol. 95, no. 2, 1998, pages 588 - 593, XP002073840 *

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JP4533979B2 (ja) 2010-09-01
TW200516153A (en) 2005-05-16

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