JP2007135489A - METHOD FOR EXAMINING SENSITIVITY TO Th2 CYTOKINE INHIBITOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a method for examining the sensitivity to a Th2 cytokine inhibitor. <P>SOLUTION: The examination of the sensitivity to the Th2 cytokine inhibitor is carried out as follows. A polymorphic genotype which is a polymorphism in a leukotriene C4 synthase gene and/or an interleukin 13 gene promoting or suppressing the expression of the gene possessed by a subject is determined. The examination is carried out by predicting the sensitivity of the subject to the Th2 cytokine inhibitor on the basis of the determined genotype. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a test method for sensitivity to a Th2 cytokine inhibitor, a nucleic acid molecule used in the test method, a test kit, and the like.

  In recent years, allergic diseases such as bronchial asthma, allergic rhinitis, atopic dermatitis, food allergies have increased remarkably. There are currently reports that about 30% have some allergic disease in Japan, and more than 70% have some predisposition to allergic disease. There are environmental predisposing factors and genetic predisposing factors for such allergic diseases, but these predisposing factors are thought to be intertwined.

  Currently, there are mediator release inhibitors, histamine H1 receptor antagonists, thromboxane inhibitors, leukotriene antagonists, Th2 cytokine inhibitors as antiallergic agents, but the sensitivity to drugs may vary greatly in individual patients . For this reason, in the prevention and treatment of allergic diseases, trial and error prescriptions are not uncommon, which is not preferable in terms of treatment and medical economics.

Here, Th2 cytokine inhibitors such as plast tosylate suppress the production of IL-4 and IL-5, which are closely related to the development of allergic diseases, from lymphocytes, and suppress IgE antibody production and chemical mediator release. (Non-patent Documents 1 and 2). Many other effects have been reported. For example, effects such as eosinophil production inhibitory action have been reported (Non-patent Document 3).
ALLERGY VOL.4, NO.2 PAGE.206-214,1997 Jpn J Pharmacol VOL.61, NO.1PAGE.31-39 1993 Allergy VOL.44, NO.3-2 PAGE.375 1995

  However, as described above, allergic diseases are multifactorial diseases. Even if these various mechanisms of action are apparent for Th2 cytokine inhibitors, genetic polymorphisms that can be clearly associated with their clinical effects There are currently no reports.

  Accordingly, an object of the present invention is to provide a test method for sensitivity to a Th2 cytokine inhibitor, a nucleic acid molecule for the test method, and a test kit containing the nucleic acid molecule.

  In order to clarify the relationship between the effect of the Th2 cytokine inhibitor and the gene polymorphism, the inventors of the present invention have not changed the cases and the pathology of bronchial asthma patients who received the Th2 cytokine inhibitor. Alternatively, genetic analysis of the leukotriene C4 synthesis gene (LTC4S gene) and the interleukin 13 gene (IL13 gene) was performed on exacerbated cases. As a result, it was found that the sensitivity of a Th2 cytokine inhibitor can be predicted using polymorphisms related to these genes as markers, and the present invention has been completed.

  Conventionally, there is no known method for predicting the sensitivity to a Th2 cytokine inhibitor based on the polymorphism of the gene. For this reason, it is possible to determine the sensitivity to a Th2 cytokine inhibitor unless actually administered. could not. Therefore, there is a possibility that wasteful medication may occur. According to the present invention, since sensitivity to a Th2 cytokine inhibitor can be predicted based on the polymorphism of a gene, effective medication for treatment and prevention of allergic diseases is possible, and accurate drug selection is possible. became. That is, according to the present invention, the following means are provided.

According to one aspect of the present invention, there is provided a method for testing sensitivity to a Th2 cytokine inhibitor, which is a polymorphism in a leukotriene C4 synthase gene and / or interleukin 13 gene possessed by a subject, A genotype determination step for determining a genotype of a polymorphism that promotes or suppresses expression;
Predicting a subject's sensitivity to a Th2 cytokine inhibitor based on the genotype determined in the genotype determination step.

  In this embodiment, the polymorphism is preferably a single nucleotide polymorphism. In this embodiment, the polymorphism of the leukotriene C4 synthase gene is preferably the polymorphism at position −444 of the promoter region of the leukotriene C4 synthase gene, and the polymorphism at position −444 of the promoter region of the leukotriene C4 synthase gene. When the types are C and A and the genotype is AA, it can be predicted to have the sensitivity. Further, when the polymorphism at position −444 in the promoter region of the leukotriene C4 synthetic gene is C and A, and the genotype has a C allele, it can be predicted that the sensitivity is not present.

  In this embodiment, the polymorphism of the interleukin 13 gene is preferably a polymorphism at the site encoding the amino acid at position 110 of the mature interleukin 13 protein, more preferably the amino acid is arginine. The polymorphism in which the arginine is substituted with glutamine, specifically, a polymorphism in which the base at position 389 of the interleukin 13 gene is G and A, and when the genotype is GG, the sensitivity is It can be predicted to have. Moreover, when the 389th base of the interleukin 13 gene is a polymorphism of G and A, and it has an A allele in the genotype, it can be predicted that it does not have the sensitivity.

  Furthermore, in this form, the polymorphism A-C at position −444 in the promoter region of the leukotriene C4 synthetic gene and the polymorphism at the position encoding arginine at position 110 in the mature interleukin 13 protein are used. The genotype of the polymorphism G / A of the base at position 389 may be determined. In this aspect, when the polymorphic genotype at position −444 of the promoter region of the leukotriene C4 synthase gene is AA, and the polymorphic genotype at position 389 of the interleukin 13 gene is GG, the sensitivity Can be predicted. When the -44 polymorphic genotype of the promoter region of the leukotriene C4 synthase gene has a C allele and the polymorphic genotype of the 389th base of the interleukin 13 gene has an A allele, It can be predicted that there is no sensitivity.

  Furthermore, in this embodiment, the subject can be a patient with bronchial asthma.

According to another aspect of the present invention, a primer for testing sensitivity to a Th2 cytokine inhibitor is a polymorphism in a leukotriene C4 synthase gene and / or a mature interleukin 13 gene possessed by a subject. Thus, a primer comprising an oligonucleotide that synthesizes a region containing a polymorphic site that promotes or suppresses the expression of these genes is provided. In this embodiment, the polymorphic site is preferably any of the following.
(A) polymorphism at position -444 of the promoter region of leukotriene C4 synthase gene (b) polymorphism at the site encoding arginine at position 110 of mature interleukin 13 protein

According to another aspect of the present invention, a probe for testing sensitivity to a Th2 cytokine inhibitor, which is a polymorphism in a leukotriene C4 synthase gene and / or interleukin 13 gene possessed by a subject, Probes comprising oligonucleotides that hybridize to regions containing polymorphic sites that promote or suppress expression of these genes are provided. In this embodiment, the polymorphic site is preferably any of the sites described below.
(A) polymorphism at position -444 of the promoter region of leukotriene C4 synthase gene (b) polymorphism at the site encoding arginine at position 110 of mature interleukin 13 protein

  According to another aspect of the present invention, there is provided a kit for testing sensitivity to a Th2 cytokine inhibitor, comprising any oligonucleotide selected from the primers and the probes.

  In the present specification, the term interleukin 13 gene used for specifying a polymorphism means cDNA encoding interleukin 13 protein (including ATG encoding methionine). Therefore, when the polymorphism in the interleukin 13 gene is referred to, the sequence notation in the gene sequence for specifying the polymorphism is the base sequence of cDNA. These identification methods use the base sequence of cDNA to identify the polymorphic site. In addition to mutations detected on cDNA, it corresponds to polymorphic sites on cDNA on genomic DNA and mRNA. All the polymorphisms in the site to be included are included in the polymorphism in the present specification.

  The present invention relates to a method for testing sensitivity to a Th2 cytokine inhibitor, and a subject has a leukotriene C4 synthase gene (hereinafter simply referred to as LTC4S gene) and / or an interleukin 13 gene (hereinafter simply referred to as IL13 gene). And genotype determination step for determining the genotype of the polymorphism that promotes or suppresses the expression of these genes, and Th2 of the subject based on the genotype determined in the genotype determination step Predicting sensitivity to cytokine inhibitors.

  In the present invention, the gene to be examined is selected from the LTC4S gene and the IL13 gene. Either of these genes may be sufficient, and both may be sufficient. In addition, by combining these genes, it is possible to accurately predict the sensitivity of the subject to the Th2 cytokine agent.

  Polymorphisms in the LTC4S gene and the IL13 gene include those that promote or suppress the expression of these genes, but preferably those that promote the expression of these genes. Here, to promote gene expression means to promote gene expression in a hetero or mutant homo polymorphism rather than in a normal homo polymorphism. Further, to suppress gene expression means to suppress gene expression in a hetero or mutant homo polymorphism rather than a normal homo polymorphism. Although the present invention is not theoretically restricted, it is predicted that the polymorphism that can promote the expression of the LTC4S gene does not have sensitivity to a Th2 cytokine inhibitor. Moreover, when it has the polymorphism which can promote the expression of IL13 gene, it is estimated that it does not have the sensitivity of a Th2 cytokine inhibitor.

  Note that the strength of the statistical relationship between the gene polymorphism and the sensitivity of the Th2 cytokine inhibitor is evaluated by the P value. That is, it can be said that the thing with small P value shown in the Example has a stronger relationship with the sensitivity of a Th2 cytokine inhibitor.

  In the present invention, allergy is a state in which strong reactivity is shown when the same antigen is re-entered into a living body sensitized with a certain antigen. Allergies are generally classified into types I to IV. In the present invention, the allergic disease means a state or disease in which allergic inflammation is caused by such allergy. Examples of allergic diseases include bronchial asthma, allergic rhinitis, atopic dermatitis, food allergy, and urticaria. In the present invention, it can be preferably used for prediction of sensitivity to a sensitive Th2 cytokine inhibitor such as bronchial asthma.

  The gene to be tested in the present invention has been found by the present inventors to be associated with sensitivity to a Th2 cytokine inhibitor on the gene. In the present invention, the “gene” includes an expression control region necessary for transcription of a structural gene in addition to a structural gene encoding an amino acid sequence. Therefore, the “gene” is not limited to a structural gene and includes a region having a control function such as a promoter or an operator. Furthermore, the structural gene is composed of exons and introns. A polymorphism found to be associated with sensitivity to a Th2 cytokine inhibitor is in the LTC4S gene promoter region and is present in the coding region of the mature IL13 protein of the IL13 gene.

  In addition, polymorphism is generally defined genetically as a change in a certain base in one gene that exists at a frequency of 1% or more in the population. However, the “polymorphism” of the present invention is not limited to this definition, and even a base change of less than 1% is included in the “polymorphism” of the present invention. Examples of the polymorphism in the present invention include, but are not limited to, polymorphisms in which several tens of bases are deleted, substituted, or inserted from single nucleotide polymorphisms (SNPs).

  As described above, according to the present invention, a polymorphism having a relationship with sensitivity to a Th2 cytokine inhibitor was found on a gene to be examined (including a promoter region). Therefore, those skilled in the art, for example, by searching for various polymorphisms on these genes in patients with allergic diseases, and analyzing the genotypes of these polymorphisms, etc., showed sensitivity to Th2 cytokine inhibitors. A polymorphism preferable for prediction can be selected and the polymorphism can be used as a marker. In addition, the prediction of the sensitivity to the Th2 cytokine inhibitor due to the polymorphism in the test target gene (promoter region) in the present invention can be applied regardless of the race, but preferably applied to the Orientals, Preferably applied to Japanese.

  In the present invention, the gene to be examined associated with sensitivity to a Th2 cytokine inhibitor is preferably a single nucleotide polymorphism (SNPs). SNPs are considered to exist at a rate of 1 in 1000 bases on the human genome. Therefore, theoretically, about 20 SNPs are considered to exist in a control locus consisting of 20 kb, for example. From these SNPs, SNPs having an association with Th2 cytokine inhibitor sensitivity can be found by linkage analysis.

Preferred polymorphisms of each gene to be examined in the present invention are as follows. According to the analysis of the present inventors, in allergic disease patients having the following genotypes among these polymorphisms of the gene to be examined, the sensitivity to a Th2 cytokine inhibitor is significantly high. It was. Therefore, it can be predicted that patients with allergic diseases having the following genotypes have sensitivity to a Th2 cytokine inhibitor.
[Polymorphism A / C at position −444 of the promoter region of LTC4S gene]
AA genotype patient [Polymorphism at amino acid 110 (arginine) of mature IL13 protein: polymorphism G / A at position 389 of IL13 gene]
GG genotype patients

On the other hand, it can be predicted that patients with allergic diseases having the following genotype markers or alleles in these genes have no sensitivity to Th2 cytokine inhibitors.
[Polymorphism A / C at position −444 of the promoter region of LTC4S gene]
Patients with genotypes of AA and CC or patients with C allele [polymorphism at amino acid 110 (arginine) of mature IL13 protein: polymorphism G / A at position 389 of IL13 gene]
Patients with genotypes GA and AA or patients with A allele

  The nucleotide sequence containing the polymorphism at position −444 of the promoter region of the LTC4S gene is shown in SEQ ID NO: 1. This base sequence has already been registered in the data bank (GeneBank accession # U50136). In the base sequence shown in SEQ ID NO: 1, it corresponds to the translation start point 1447 position. Therefore, -444 shown as the polymorphic site corresponds to position 1003 in the base sequence of SEQ ID NO: 1, the wild type is A, and the mutant type is C.

  The base sequence containing the IL13 gene region is shown in SEQ ID NO: 2. This base sequence has already been registered in the data bank (GeneBank accession # L06801). In the base sequence shown in SEQ ID NO: 2, the translation start point of the IL13 protein precursor corresponds to position 45, and the coding region of the mature IL13 protein is from position 105 to position 443 (including the stop codon). As for the polymorphism in the IL13 gene in this specification, the 110th amino acid of the mature IL13 protein is arginine in the wild type and glutamine in the mutant type. Further, position 389 shown as the polymorphic site on the IL13 gene corresponds to position 433 in SEQ ID NO: 2, the wild type is G, and the mutant type is A.

  Depending on the subject, there is a possibility that the position of the polymorphism in the base sequence described in each SEQ ID NO. May be shifted due to deletion or insertion of bases at other sites. Even if the number of bases from the transcription start point is deviated, those skilled in the art can specify the position of the base to be typed by alignment with the base sequences before and after the polymorphism shown in each SEQ ID NO. A base position that can be collated in this way is called a homologous position. The method of the present invention can also be carried out by genotyping a base at a position homologous to each polymorphism.

  The subject's “determination of genotype” can be carried out by clarifying the base sequence of the subject locus to be examined, as an example. To perform genotyping, a DNA sample is prepared from a subject. The DNA sample can be prepared based on, for example, chromosomal DNA extracted from a biological sample of a subject. As biological samples, tissues or cells such as peripheral blood leukocytes, skin, oral mucosa, tears, saliva, urine, feces, hair, and the like can be used. The DNA sequence prepared here is used to determine the nucleotide sequence of a site having a polymorphism associated with sensitivity to a Th2 cytokine inhibitor in the gene to be examined.

  More specifically, polymorphism genotyping associated with Th2 cytokine inhibitor sensitivity is performed by determining the base sequence of the region and confirming which base it is. When each polymorphism has an allele as described above, the subject is predicted to have Th2 cytokine inhibitor sensitivity.

  Furthermore, these genotypes can be determined indirectly based on polymorphisms that are tags having a linkage disequilibrium relationship with these polymorphisms such as haplotype blocks. Haplotype block is a term that refers to a group of polymorphisms in a linkage disequilibrium relationship. The polymorphisms that make up the haplotype are inherited by linkage with a high probability. Therefore, the genotype of each polymorphism can also be determined using the other polymorphisms constituting the haplotype block together with the polymorphism associated with Th2 cytokine inhibitor sensitivity found by the present inventors. Based on the results of genome analysis, haplotype maps are being developed. Based on the result of this analysis, a haplotype block containing the polymorphism shown by the present inventors can be identified, and genotyping in the present invention can be performed based on the other polymorphism in the block.

  In this way, polymorphic genotypes related to the promotion or suppression of the expression of these genes are determined in the gene to be examined, and the presence or absence of Th2 cytokine inhibitor sensitivity is predicted. Here, a Th2 cytokine inhibitor is administered to an allergic disease patient who is predicted to have Th2 cytokine inhibitor sensitivity, and an allergic disease patient who is predicted to have no Th2 cytokine inhibitor sensitivity By selecting a drug to administer an antiallergic agent other than those, it is possible to reliably and promptly treat and prevent allergic diseases.

  As described above, the method for directly determining the base sequence of the polymorphic site related to the Th2 cytokine inhibitor patient has been exemplified as a method for determining the genotype, but in addition to determining the base sequence, the genotype can be determined more quickly. Various methods are known that can be determined. As an analysis method using the PCR method, TaqMan PCR method, AcycroPrimer method, fluorescent magnetic bead method, use of single molecule fluorescence analysis system, MALDI-TOF / MS method and the like can be used. In addition, the Invader method and the RCA method are known as methods for genotyping independent of PCR. In addition, genotypes can be determined using DNA arrays. These methods are briefly described below. Any of the methods described herein can be applied to genotyping in the present invention.

[TaqMan PCR method]
The principle of TaqMan PCR method is as follows. The TaqMan PCR method is an analysis method using a primer set that can amplify a region containing an allele and a TaqMan probe. The TaqMan probe is designed to hybridize to the region containing the allele amplified by this primer set.

If the target base sequence is hybridized under conditions close to the Tm of the TaqMan probe, the hybridization efficiency of the TaqMan probe is significantly reduced due to the difference of one base. When PCR is performed in the presence of the TaqMan probe, the extension reaction from the primer eventually reaches the hybridized TaqMan probe. At this time, the TaqMan probe is degraded from its 5 ′ end by the 5′-3 ′ exonuclease activity of DNA polymerase. If the TaqMan probe is labeled with a reporter dye and a quencher, the degradation of the TaqMan probe can be followed as a change in fluorescence signal. In other words, when the TaqMan probe is decomposed, the reporter dye is released and the distance from the quencher is increased to generate a fluorescent signal. If the hybridization of the TaqMan probe decreases due to a difference in one base, the TaqMan probe does not decompose and a fluorescent signal is not generated.

  If a TaqMan probe corresponding to a polymorphism is designed and a different signal is generated by the degradation of each probe, the genotype can be determined at the same time. For example, as a reporter dye, 6-carboxy-fluorescein (FAM) is used for the TaqMan probe of allele A of an allele, and VIC is used for the probe of allele B. In a state where the probe is not decomposed, generation of a fluorescent signal of the reporter dye is suppressed by the quencher. If each probe hybridizes to the corresponding allele, a fluorescence signal corresponding to the hybridization is observed. That is, when either FAM or VIC signal is stronger than the other, it becomes clear that allele A or allele B is homozygous. On the other hand, when the allele is hetero, both signals are detected at substantially the same level. By using the TaqMan PCR method, PCR and genotyping can be performed simultaneously on a genome as an analysis target without a time-consuming step such as separation on a gel. Therefore, the TaqMan PCR method is useful as a method for genotyping a large number of subjects.

[Acyclo Prime method]
As a genotyping method using the PCR method, the Acyclo Prime method has also been put into practical use. In the Acyclo Prime method, one set of primers for genome amplification and one set of primers for SNPs detection are used. First, a region containing genomic SNPs is amplified by PCR. This process is the same as normal genomic PCR. Next, the obtained PCR product is annealed with a primer for detecting SNPs, and an extension reaction is performed. Primers for detecting SNPs are designed to anneal to a region adjacent to the SNPs to be detected.

  At this time, a nucleotide derivative (terminator) labeled with a fluorescent polarizing dye and blocked with 3′-OH is used as a nucleotide substrate for the extension reaction. As a result, only one base complementary to the base at the position corresponding to SNPs is incorporated, and the extension reaction stops. Incorporation of a nucleotide derivative into a primer can be detected by an increase in fluorescence polarization (FP) due to an increase in molecular weight. If two types of labels having different wavelengths are used for the fluorescent polarizing dye, it is possible to specify which of the two types of bases the specific SNPs are. Since the level of fluorescence polarization can be quantified, it is also possible to determine whether an allele is homo or hetero in one analysis.

[MALDI-TOF / MS method]
Genotypes can also be analyzed by analyzing PCR products with MALDI-TOF / MS. Since MALDI-TOF / MS can know the molecular weight very accurately, MALDI-TOF / MS is used as an analysis method that can clearly identify slight differences in protein amino acid sequences and DNA base sequences. For genotyping by MALDI-TOF / MS, first, the region containing the allele to be analyzed is amplified by PCR. The amplification product is then isolated and its molecular weight is measured by MALDI-TOF / MS. Since the base sequence of the allele is known in advance, the base sequence of the amplification product is uniquely determined based on the molecular weight.

Genotyping using MALDI-TOF / MS requires a PCR product separation step, but accurate genotyping can be expected without using labeled primers or labeled probes. It can also be applied to simultaneous detection of polymorphisms at multiple locations.

[SNPs-specific labeling method using type IIs restriction enzyme]
A method capable of genotyping at higher speed using the PCR method has also been reported. For example, SNPs are genotyped using type IIs restriction enzymes. In this method, a primer having a recognition sequence for type IIs restriction enzyme is used for PCR. A general restriction enzyme (type II) used for gene recombination recognizes a specific base sequence and cleaves a specific site in the base sequence. In contrast, type IIs restriction enzymes recognize specific base sequences and cleave sites away from the recognized base sequences. The number of bases between the recognition sequence and the cleavage site is determined by the enzyme. Therefore, if the primer containing the recognition sequence of the type IIs restriction enzyme is annealed at a position separated by the number of bases, the amplified product can be cleaved at the site of the SNPs by the type IIs restriction enzyme.

  At the end of the amplification product cleaved with the type IIs restriction enzyme, a cohesive end containing the base of SNPs is formed. Here, an adapter having a base sequence corresponding to the sticky end of the amplification product is ligated. The adapter has different base sequences including bases corresponding to SNPs, and can be labeled with different fluorescent dyes. Finally, the amplification product is labeled with a fluorescent dye corresponding to the base of the SNPs site.

  When PCR is performed by combining a primer containing the IIs type restriction enzyme recognition sequence with a capture primer, the amplification product is fluorescently labeled and can be immobilized using the capture primer. For example, if a biotin-labeled primer is used as a capture primer, the amplification product can be captured on avidin-bound beads. The genotype can be determined by tracking the fluorescent dye of the amplification product thus captured.

[Multiple SNPs typing using magnetic fluorescent beads]
A technique capable of analyzing a plurality of alleles in parallel in a single reaction system is also known. Analyzing a plurality of alleles in parallel is called multiplexing. In general, a typing method using a fluorescent signal requires fluorescent components having different fluorescent wavelengths for multiplexing. However, there are not so many fluorescent components that can be used for actual analysis. On the other hand, when a plurality of types of fluorescent components are mixed in a resin or the like, a variety of fluorescent signals that can be distinguished from each other can be obtained even with limited types of fluorescent components. Furthermore, if a component that is magnetically adsorbed in the resin is added, it is possible to obtain beads that emit fluorescence and can be separated magnetically.

  In multiplexed SNPs typing using magnetic fluorescent beads, a probe having a base complementary to the polymorphic site of each allele at its end is immobilized on the magnetic fluorescent beads. Both are combined so that each allele corresponds to a magnetic fluorescent bead having a unique fluorescent signal. On the other hand, when a probe fixed to a magnetic fluorescent bead is hybridized to a complementary sequence, a fluorescently labeled oligo DNA having a base sequence complementary to an adjacent region on the allele is prepared.

  The region containing the allele is amplified by asymmetric PCR, the above-mentioned magnetic fluorescent bead-immobilized probe and the fluorescently labeled oligo DNA are hybridized, and both are further ligated. When the end of the magnetic fluorescent bead-immobilized probe has a base sequence complementary to SNPs, it is efficiently ligated. On the other hand, if the terminal bases are different due to polymorphism, the ligation efficiency of the two decreases. As a result, the fluorescent labeled oligo DNA is bound to each magnetic fluorescent bead only when the sample is a genotype complementary to the magnetic fluorescent bead.

  The genotype is determined by collecting magnetic fluorescent beads by magnetism and detecting the presence of fluorescently labeled oligo DNA on each magnetic fluorescent bead. Since magnetic fluorescent beads can analyze the fluorescence signal for each bead using a flow cytometer, the signal can be easily separated even if various types of magnetic fluorescent beads are mixed. In other words, “multiplexing” in which many types of SNPs are analyzed in parallel in a single reaction vessel is achieved.

[Invader method]
A method for genotyping independent of the PCR method has also been put into practical use. For example, in the Invader method, genotyping is realized with only three types of oligonucleotides, an allele probe, an invader probe, and a FRET probe, and a special nuclease called cleavase. Of these probes, only the FRET probe requires labeling. Allele probes are designed to hybridize to regions adjacent to the allele to be detected. A flap consisting of a base sequence unrelated to hybridization is linked to the 5 ′ side of the allele probe. The allele probe has a structure that hybridizes to the 3 ′ side of the polymorphic site and is linked to a flap on the polymorphic site.

  On the other hand, the invader probe has a base sequence that hybridizes to the 5 ′ side of the polymorphic site. The base sequence of the invader probe is designed so that the 3 ′ end corresponds to the polymorphic site by hybridization. The base at the position corresponding to the polymorphic site in the invader probe may be arbitrary. That is, both base sequences are designed so that the invader probe and the allele probe hybridize adjacently across the polymorphic site.

  When the polymorphic site is a base complementary to the base sequence of the allele probe, when both the invader probe and the allele probe hybridize to the allele, the invader probe enters the base corresponding to the polymorphic site of the allele probe. An (invasion) structure is formed. The cleavase cleaves the strand on the invading side of the oligonucleotide having the invasion structure formed in this way. Since the cutting occurs on the intrusion structure, the result is that the allele probe flap is cut off. On the other hand, if the base at the polymorphic site is not complementary to the base of the allele probe, there is no competition between the invader probe and the allele probe at the polymorphic site, and no invasion structure is formed. Therefore, the flap is not cut by cleavase.

  The FRET probe is a probe for detecting the flap thus separated. The FRET probe constitutes a hairpin loop having a self-complementary sequence on the 5 ′ end side and a single-stranded portion arranged on the 3 ′ end side. The single-stranded portion arranged on the 3 ′ end side of the FRET probe has a base sequence complementary to the flap, and the flap can hybridize there. Both base sequences are designed so that when the flap hybridizes to the FRET probe, a structure is formed in which the 3 ′ end of the flap enters the 5 ′ end of the self-complementary sequence of the FRET probe. A cleavase recognizes and cleaves an invasion structure. If the FRET probe is cleaved by a cleavase and labeled with a reporter dye and quencher similar to TaqMan PCR, the FRET probe cleavage can be detected as a change in fluorescence signal.

  Theoretically, the flap should hybridize to the FRET probe even in the uncut state. However, in reality, there is a large difference in the binding efficiency to FRET between the cut flap and the flap present in the state of the allele probe. Therefore, it is possible to specifically detect the cleaved flap using the FRET probe.

  In order to determine the genotype based on the Invader method, two types of allele probes including base sequences complementary to each of allele A and allele B may be prepared. At this time, the base sequences of the flaps are different base sequences. If two types of FRET probes for detecting flaps are prepared and each reporter dye can be identified, the genotype can be analyzed in the same way as the TacMan PCR method.

  The advantage of the Invader method is that the FRET probe is the only oligonucleotide that needs to be labeled. The FRET probe can use the same oligonucleotide regardless of the base sequence to be detected. Therefore, mass production is possible. On the other hand, since the allele probe and the invader probe do not need to be labeled, a reagent for genotyping can be provided at low cost.

[RCA method]
An example of a method for genotyping that does not depend on the PCR method is the RCA method. A method for amplifying DNA based on a reaction in which a DNA polymerase having a strand displacement action synthesizes a long complementary strand using a circular single-stranded DNA as a template is the Rolling Circle Amplification (RCA) method (Lizardri PM et al., Nature Genetics 19, 225, 1998). In the RCA method, an amplification reaction is configured using a primer that anneals to a circular DNA and initiates complementary strand synthesis and a second primer that anneals to a long complementary strand generated by this primer.

  In the RCA method, a DNA polymerase having a strand displacement action is used. Therefore, the portion that has become double-stranded by complementary strand synthesis is replaced by a complementary strand synthesis reaction started from another primer annealed to the 5 ′ side. For example, the complementary strand synthesis reaction using circular DNA as a template does not end in one round. The complementary strand synthesis continues while replacing the previously synthesized complementary strand, and a long single-stranded DNA is produced. On the other hand, the second primer anneals to the long single-stranded DNA generated using the circular DNA as a template, and complementary strand synthesis starts. Since single-stranded DNA generated by the RCA method uses circular DNA as a template, its base sequence is a repetition of the same base sequence. Thus, continuous production of long single strands results in continuous annealing of the second primer. As a result, single-stranded portions that can be annealed by the primer are continuously generated without going through a denaturing step. Thus, DNA amplification is achieved.

  If the circular single-stranded DNA necessary for the RCA method is generated according to the SNPs base, the SNPs can be typed using the RCA method. For this purpose, a linear single-stranded padlock probe is used. The padlock probe has complementary base sequences on both sides of the SNPs to be detected at the 5 ′ end and 3 ′ end. These base sequences are linked at a portion consisting of a special base sequence called a backbone. If the SNPs part is a base sequence complementary to the end of the padlock probe, the end of the padlock probe hybridized to the allele can be ligated by DNA ligase. As a result, the linear padlock probe is circularized and the reaction of the RCA method is triggered. The reaction of DNA ligase is significantly reduced in efficiency when the terminal portion to be ligated is not completely complementary. Therefore, SNPs can be genotyped by confirming the presence or absence of ligation by the RCA method.

  The RCA method can amplify DNA but does not generate a signal as it is. If only the presence or absence of amplification is used as an index, the genotype cannot be determined unless a reaction is performed for each allele. Methods are known in which these points are improved for genotyping. For example, genotyping can be performed with one tube based on the RCA method using a molecular beacon. A molecular beacon is a signal generation probe using a fluorescent dye and a quencher, as in the TaqMan method. The molecular beacons are composed of complementary base sequences at the 5 ′ end and 3 ′ end, and by themselves form a hairpin structure. If both ends are labeled with a fluorescent dye and a quencher, a fluorescent signal cannot be detected in a state where a hairpin structure is formed. If a part of the molecular beacon is set as a base sequence complementary to the amplification product of the RCA method, the molecular beacon hybridizes to the amplification product of the RCA method. Since the hairpin structure is eliminated by hybridization, a fluorescent signal is generated.

  The advantage of the molecular beacon is that the base sequence of the molecular beacon can be made common regardless of the detection target by using the base sequence of the backbone portion of the padlock probe. Genotyping is possible in one tube by changing the backbone base sequence for each allele and combining two types of molecular beacons with different fluorescence wavelengths. Since the cost of synthesis of fluorescently labeled probes is high, the ability to use a common probe regardless of the measurement target is an economic advantage.

[Single molecule fluorescence spectroscopy]
A system that enables fluorescence analysis of a minute region of 1 fL (femtoliter) has been put into practical use. With this system, the extension of the fluorescently labeled primer can be detected as an increase in translational diffusion time. Primers having base sequences complementary to the alleles to be typed are prepared. Each primer is bound with a distinguishable fluorescent label. PCR is performed using this primer, and the amplification product is measured for fluorescence by fluorescence correlation analysis (Fluorescence Correlation Spectroscopy). If the sample has a base sequence complementary to the primer, the primer is extended by PCR. The extended primer generates a fluctuation of fluorescence due to the large molecule. This fluorescence fluctuation is detected as an increase in translational diffusion time. If the base sequence complementary to the primer is not contained in the sample, no PCR amplification product is generated, and therefore no fluorescence change occurs.

  Specifically, PCR is performed in the same reaction solution using primers having different fluorescent labels for the two alleles A and B, respectively. In the fluorescence measurement of the amplified product, if a change in the fluorescence signal of either A or B is observed, it can be confirmed that either of the homologues is heterogeneous if both of the fluorescence signals change (PharmaGenomics, July / August 46-48, 2003). It is evaluated as an accurate and quick analysis method.

  All of these methods have been developed for genotyping a large amount of samples at high speed. Except for MALDI-TOF / MS, it is necessary to prepare a labeled probe in some way for each method. On the other hand, genotyping that does not rely on labeled probes has also been performed for a long time. As one of such methods, for example, a method using restriction fragment length polymorphism (RFLP), a PCR-RFLP method and the like can be mentioned.

  RFLP utilizes the fact that a restriction enzyme recognition site mutation or a base insertion or deletion in a DNA fragment caused by restriction enzyme treatment can be detected as a change in the size of the fragment produced after the restriction enzyme treatment. If there is a restriction enzyme that recognizes the base sequence containing the polymorphism to be detected, the base of the polymorphic site can be known by the principle of RFLP.

  As a method that does not require a labeled probe, a method for detecting a difference in base using a change in the secondary structure of DNA as an index is also known. PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its nucleotide sequence (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11. Genomics 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313- 1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). The PCR-SSCP method is performed by dissociating the PCR product into single-stranded DNA and separating it on a non-denaturing gel. Since the mobility on the gel varies depending on the secondary structure of the single-stranded DNA, if there is a difference in base at the polymorphic site, it can be detected as a difference in mobility.

  Other examples of methods that do not require a labeled probe include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated depending on the instability of each. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of a partially dissociated DNA fragment becomes very slow and can be separated from the mobility of a complete double-stranded DNA without a dissociated portion.

  Specifically, first, a region containing the SNPs site is amplified by PCR or the like. The amplification product is hybridized with a probe DNA whose base sequence is known to make a double strand. This is electrophoresed in a polyacrylamide gel that gradually increases as the concentration of denaturing agents such as urea moves, and is compared with a control. In a DNA fragment that has mismatched by hybridization with the probe DNA, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the moving speed becomes extremely slow. The presence or absence of mismatch can be detected by detecting the difference in mobility generated in this way.

  Furthermore, genotypes can also be determined using DNA arrays (Cell engineering separate volume “DNA microarray and the latest PCR method”, Shujunsha, published 2000.4 / 20, pp97-103 “SNP analysis using oligo DNA chip”, Sabae Shinichi). In the DNA array, sample DNA (or RNA) is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of SNPs simultaneously.

  In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous membrane such as a nitrocellulose membrane can also be used.

  In the present invention, examples of the nucleotide immobilization (array) method include an oligonucleotide-based array developed by Affymetrix. In an array of oligonucleotides, the oligonucleotides are usually synthesized in situ. For example, in-situ synthesis methods of oligonucleotides using photolithographic technology (Affymetrix) and inkjet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known. Can be used.

  The oligonucleotide is composed of a base sequence complementary to a region containing the SNPs to be detected. The length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bases, preferably 10 to 50 bases, more preferably 15 to 25 bases when the oligonucleotide is immobilized. Furthermore, in general, mismatch (MM) probes are used in the DNA array method in order to avoid errors due to cross hybridization (non-specific hybridization). The mismatch probe constitutes a pair with an oligonucleotide having a base sequence completely complementary to the target base sequence. An oligonucleotide consisting of a base sequence completely complementary to a mismatch probe is called a perfect match (PM) probe. In the process of data analysis, the influence of cross-hybridization can be reduced by eliminating the signal observed with the mismatch probe.

  A sample for genotyping by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a subject. The biological sample is not particularly limited. For example, a DNA sample can be prepared from chromosomal DNA extracted from tissues or cells of peripheral blood leukocytes, skin, oral mucosa, etc., tears, saliva, urine, feces or hair of the subject. A specific region of chromosomal DNA is amplified using a primer for amplifying a region containing SNPs to be determined. At this time, a plurality of regions can be simultaneously amplified by the multiplex PCR method. The multiplex PCR method is a PCR method using a plurality of primer sets in the same reaction solution. When analyzing a plurality of SNPs, a multiplex PCR method is useful.

  In general, in the DNA array method, a DNA sample is amplified by a PCR method and an amplification product is labeled. A labeled primer is used for labeling the amplification product. For example, genomic DNA is first amplified by PCR using a primer set specific to the region containing SNPs. Next, biotin-labeled DNA is synthesized by a labeling PCR method using a biotin-labeled primer. The biotin-labeled DNA synthesized in this way is hybridized to the oligonucleotide probe on the chip. The hybridization reaction solution and reaction conditions can be appropriately adjusted according to conditions such as the length of the nucleotide probe immobilized on the substrate and the reaction temperature. One skilled in the art can design appropriate hybridization conditions. In order to detect the hybridized DNA, avidin labeled with a fluorescent dye is added. The array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.

  In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allele-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where the SNPs to be detected are present. When ASO is hybridized to sample DNA, the efficiency of hybridization decreases if a mismatch occurs in the SNPs site due to polymorphism. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.

  The present invention also provides a probe or primer for the test method. In the above-described genotype analysis method, an oligonucleotide that can hybridize to the polymorphic sequence or a sequence in the vicinity thereof is often used as a primer or a probe. Therefore, the probe or primer for clarifying the base of the polymorphic site is an important element in the test method of the present invention and is useful.

  The primer is designed so that complementary strand synthesis can be initiated so as to include the polymorphic site, using DNA containing the polymorphic site as a template. The length of the primer is in the range of 15-100, generally 15-50, usually 15-30. The interval between the region where the primer hybridizes and the polymorphic site is arbitrary. For the interval between the two, a suitable number of bases can be selected according to the analysis method of the base at the polymorphic site. Further, the primer may be designed so that the primer hybridizes on the polymorphic site. In this case, only a polymorphic sequence determined to have a Th2 cytokine inhibitor sensitivity may be hybridized, and a sequence that does not hybridize on other polymorphisms (or is difficult to hybridize) may be designed. Moreover, the base sequence which comprises a primer can be selected based on the polymorphism and base sequence of a test object gene shown to sequence number: 1-sequence number: 2. However, the primer of the present invention is not limited to a sequence completely complementary to the template, and the complementary sequence may be appropriately changed. For example, when the sequence is changed, an arbitrary base sequence is added to a base sequence complementary to the base sequence of the genome. Examples of the sequence added here include a recognition sequence of a type IIs restriction enzyme. By adding a recognition sequence for type IIs restriction enzyme, it can be used as a primer for polymorphism analysis using type IIs restriction enzyme.

  In addition, the primer of the present invention includes those physically modified. Examples of physical modifications include labeling with isotopes, labeling with fluorescent substances, and labeling with binding affinity substances such as biotin or digoxin. Such labeled primers are useful in various genotyping methods.

  The probe of the present invention is designed so that it can hybridize with a polynucleotide having a base sequence in a region containing a polymorphic site. More specifically, a probe containing a polymorphic site in the base sequence of the probe is preferable as the probe of the present invention. Alternatively, depending on the base analysis method at the polymorphic site, the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, although the polymorphic site is not included in the base sequence of the probe itself, a probe including a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.

  In addition, a probe for determining genotype by haplotype block analysis can detect a tag polymorphism even if it does not hybridize to a polymorphic sequence related to Th2 cytokine inhibitor sensitivity or a sequence adjacent thereto. In addition, a probe capable of hybridizing to a tag polymorphism sequence or a sequence adjacent to the tag polymorphism is also included in the present invention.

  Like the primer, the probe of the present invention is not limited to a sequence that is completely complementary to the template, but can be altered in base sequence, added in base sequence, or modified. For example, a probe used in the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe of the present invention as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the gene locus to be examined described in SEQ ID NO: 1 to SEQ ID NO: 2. The length of the probe is substantially the same as the length described in the description of the DNA array method, and is in the range of 15 to 100, generally 15 to 50, and usually 15 to 30. Examples of probes used in the invader method include the probes described in SEQ ID NO: 3 to SEQ ID NO: 8.

  The primers and probes can be synthesized by any method used by those skilled in the art. In the synthesis of modified primers and modified probes, it is known to introduce arbitrary modifications to oligonucleotides using nucleotide derivatives modified with fluorescent dyes or biotin. A method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known. Modification of these oligonucleotides can also be performed using a commercially available kit or the like.

  According to the present invention, a Th2 cytokine inhibitor sensitivity test kit comprising at least one selected from such primers and probes is also provided. This kit may have only primers, may have only probes, or may have both of these. Primers and probes are appropriately selected and combined according to the genotyping technique employed.

[Identification of gene polymorphisms related to susceptibility to Th2 cytokine inhibitor by genetic analysis in patients with bronchial asthma who received Th2 cytokine inhibitor suplatast tosylate (trade name IPD, manufactured by Taiho Pharmaceutical Co., Ltd.]
Twenty patients with bronchial asthma (14 males, 6 females, 1 to 15 years old) who obtained informed consent for genetic analysis were targeted. Suplatast tosylate is administered for 8 weeks according to its dosage, and the overall improvement is divided into 5 stages: “significantly improved”, “improved”, “slightly improved”, “invariant” and “deteriorated”. “Slightly improved” or higher was considered effective, and “unchangeable” and “deteriorated” were determined to be invalid. Based on this determination, gene analysis was performed for the LTC4S gene promoter region and the mature IL13 protein coding region of the IL13 gene in 13 effective cases and 7 ineffective cases.

  For the gene analysis, the invader method (FRET probe and enzyme clevase for each allele under the trademark of Third Wave Japan Co., Ltd. was obtained from Third Wave Japan Co., Ltd. The promoter region of the LTC4S gene and the predetermined IL13 gene The probe base sequences (SEQ ID NOs: 3 to 8) for identifying alleles are shown below, where Fam or Vic was used as the reporter dye for each of the alleles, and MGB was used for the quencher. Were simultaneously analyzed to identify the genotype.

(For polymorphism of the promoter region (position -444) of LTC4S gene)
Probe for wild-type allele: 5'-CGCGCCGAGGagggaacagataaggtgg-3 '(SEQ ID NO: 3)
Probe for mutant allele: 5'-ACGGACGCGGAGcgggaacagataaggtgg-3 '(SEQ ID NO: 4)
Invader probe: 5'-gccaggaacagcctggatggggact-3 '(SEQ ID NO: 5)

(For R110Q polymorphism of coding region of mature IL13 protein of IL13 gene)
Probe for wild type allele: 5'-CGCGCCGAGGcgtccctcgcgaa-3 '(SEQ ID NO: 6)
Probe for mutant allele: 5'-ACGGACGCGGAGtgtccctcgcgaaa-3 '(SEQ ID NO: 7)
Invader probe: 5'-tcctgtctctgcaaataatgatgctttcgaagtttcagttgaaca-3 '(SEQ ID NO: 8)

The invader method was performed according to a predetermined protocol. The results are shown in Tables 1 and 2.

  As shown in Table 1, among the 13 effective examples, there are 10 cases of the wild type (genotype is AA) of the polymorphism of the promoter region (position -444) of the LTC4S gene, and the mutant type (genotype is AC). There were 3 cases. On the other hand, among the 7 invalid cases, the wild type was 1 case and the mutant type (genotype was AC) was 6 cases. Furthermore, the X-square P value was 0.007. From the above, it was found that the wild type of the promoter region (position -444) of the LTC4S gene was significantly large in the effective examples.

  Moreover, as shown in Table 2, among the 13 effective examples, there are 8 polymorphic wild-types (genotype type GG) that cause a mutation in the amino acid at position 110 of the IL13 protein, whereas the A allele There were 5 mutants (GA or GG) having On the other hand, among the 7 ineffective cases, the wild type was 1 case and the above mutant type was 6 cases. Furthermore, the X-square P value was 0.043. From the above, it was found that the wild type of the polymorphism causing the amino acid mutation at position 110 of the mature IL13 protein of the IL13 gene in the effective example is significantly large.

  From the above, it has been found that both the polymorphism of the LTC4S gene and the polymorphism of the IL13 gene are useful for predicting the sensitivity of patients with allergic diseases such as asthma to a Th2 cytokine inhibitor. In particular, it was found that the analysis by the genotype of the polymorphism of the LTC4S gene and the biallelic analysis of the polymorphism of the IL13 gene can be used alone or in combination.

  SEQ ID NOs: 3 to 8: synthetic probe

Claims (17)

A method for testing sensitivity to a Th2 cytokine inhibitor,
A genotype determination step of determining a polymorphism in the leukotriene C4 synthase gene and / or interleukin 13 gene possessed by the subject, and determining the genotype of the polymorphism that promotes or suppresses the expression of these genes;
Predicting a subject's sensitivity to a Th2 cytokine inhibitor based on the genotype determined in the genotype determination step;
A method comprising:   The method according to claim 1, wherein the polymorphism is a single nucleotide polymorphism.   The method according to claim 2, wherein the polymorphism of the leukotriene C4 synthase gene is a polymorphism at position -444 of the promoter region of the leukotriene C4 synthase gene.   The method according to claim 3, wherein when the polymorphism at position -444 of the promoter region of the leukotriene C4 synthase gene is C and A and the genotype is AA, the sensitivity is predicted.   The method according to claim 3, wherein when the polymorphism at position −444 of the promoter region of the leukotriene C4 synthase gene is C and A and the genotype has a C allele, the method is predicted not to have the sensitivity.   The method according to claim 2, wherein the polymorphism of the interleukin 13 gene is a polymorphism of a site encoding arginine at position 110 of the mature interleukin 13 protein.   When the mature interleukin 13 protein is a polymorphism at the site encoding arginine at position 110, the polymorphism in which the base at position 389 of the interleukin 13 gene is G and A, and the genotype is GG, 7. The method of claim 6, wherein the method is predicted to be sensitive.   When it is a polymorphism of the site encoding arginine at position 110 of the mature interleukin 13 protein, the polymorphism where the base at position 389 of the interleukin 13 gene is G and A, and the genotype has an A allele, The method of claim 6, wherein the method is predicted to have no sensitivity.   A polymorphism at position −444 of the promoter region of the leukotriene C4 synthase gene and a polymorphism at the position encoding arginine at position 110 of the mature interleukin 13 protein, and a polymorphism at position 389 of the interleukin 13 gene The method of claim 1, wherein the genotype of type G / A is determined.   When the genotype of the polymorphism at position −444 of the promoter region of the leukotriene C4 synthase gene is AA and the genotype of the base polymorphism at position 389 of the interleukin 13 gene is GG, it is predicted to have the sensitivity. The method according to claim 9.   When the genotype at position −444 of the promoter region of the leukotriene C4 synthase gene has a C allele and the genotype at position 389 of the interleukin 13 gene has an A allele, the sensitivity The method of claim 9, wherein the method is predicted not to have.   The method according to claim 1, wherein the subject is a patient with bronchial asthma. A primer for testing sensitivity to a Th2 cytokine inhibitor,
A primer comprising an oligonucleotide that synthesizes a region containing a polymorphic site in a subject that is a polymorphism in the leukotriene C4 synthase gene and / or mature interleukin 13 gene, and that promotes or suppresses the expression of these genes. The primer according to claim 13, wherein the polymorphic site is any of the following sites.
(A) polymorphism at position -444 of the promoter region of leukotriene C4 synthase gene (b) polymorphism at the site encoding arginine at position 110 of mature interleukin 13 protein A probe for testing sensitivity to a Th2 cytokine inhibitor,
A probe comprising an oligonucleotide that hybridizes to a polymorphism in a leukotriene C4 synthase gene and / or interleukin 13 gene possessed by a subject and that includes a polymorphic site that promotes or suppresses the expression of these genes. The probe according to claim 15, wherein the polymorphic site is any of the following sites.
(A) polymorphism at position -444 of the promoter region of leukotriene C4 synthase gene (b) polymorphism at the site encoding arginine at position 110 of mature interleukin 13 protein   A kit for testing sensitivity to a Th2 cytokine inhibitor, comprising any one of the oligonucleotides selected from the primers according to claim 13 and 14 and the probes according to claims 15 and 16.