JP2013017398A - Method for examining immunologic disease or obstructive pulmonary disease based on single nucleotide polymorphism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for examining immunologic disease such as bronchial asthma, or obstructive pulmonary disease.SOLUTION: By analyzing single nucleotide polymorphism present in the fourth chromosome long arm 31 region (4q31 region), the fifth chromosome long arm 22 region (5q22 region), the sixth chromosome short arm 21 region (6p21 region), the tenth chromosome short arm 14 region (10p14 region) or the twelfth chromosome long arm 13 region (12q13 region), the presence or absence of the development risk and/or the development of immunologic disease or obstructive pulmonary disease are examined based on the result of the analysis.

Description

  The present invention relates to a test method for determining the onset risk and presence of an immune disease such as bronchial asthma or obstructive pulmonary disease, and a reagent used in the test method.

  Bronchial asthma is an inflammatory disease characterized by chronic reversible airway obstruction accompanied by wheezing, coughing, shortness of breath, and the like, and is considered to be one of immune diseases. Bronchial asthma has been increasing in recent years, and it is said that about 3% of Japanese adults and about 6% of children suffer from bronchial asthma. Bronchial asthma is considered to be a multifactorial disease that develops due to a comprehensive contribution of genetic and environmental factors, but there are still many unclear points regarding the mechanism of its onset and progression.

  In recent years, attempts have been made to identify genes and single nucleotide polymorphisms (SNPs) associated with bronchial asthma by genome-wide association analysis (GWAS) (Non-Patent Documents 1 to 7). For example, GWAS related to bronchial asthma has been conducted using a European population or a population originating in Africa (Non-patent Documents 2 to 6), and a recently conducted European population as a subject. In a large-scale consortium-based GWAS, 10 regions that are strongly associated with bronchial asthma have been reported (Non-patent Document 7). It is expected that further knowledge regarding the genetic background of bronchial asthma can be obtained by performing GWAS with other ethnic groups as subjects.

  The TSLP gene present in the chromosome 5 long arm 22 region (5q22 region) is a gene involved in the Th2 immune response. Rs1837253, which is 5.7 kb upstream of the transcription start site of the TSLP gene, has been reported to be associated with bronchial asthma and airway hypersensitivity by candidate gene analysis in a Canadian population (Non-patent Document 8). . In addition, a test method for immune diseases such as bronchial asthma based on a polymorphism of the TSLP locus is known (Patent Document 1). However, it is not known whether the TSLP locus is associated with bronchial asthma at the genome-wide level.

  The major histocompatibility complex region (also referred to as MHC region or HLA locus) of the chromosome 6 short arm 21 region (6p21 region) is a region encoding an MHC antigen and is involved in the immune response. The HLA-DQ region of MHC class II has been reported to be associated with bronchial asthma and airway hypersensitivity by studies with white population as subjects (Non-patent Documents 7 and 9). However, no association between the MHC region other than the HLA-DQ region and bronchial asthma has been reported, and no association between the MHC region and bronchial asthma in the Asian population has been reported.

  In addition, there is no known association between the long arm 31 region (4q31 region), the short arm region 14 (10p14 region), the long arm region 13 (12q13 region) and the bronchial asthma.

JP2008-109915

Kabesch, M. Chest 137, 909-915 (2010). Moffatt, M.F. et al. Nature 448, 470-473 (2007). Himes, B.E. et al. Am. J. Hum. Genet. 84, 581-593 (2009). Sleiman PM, et al. N. Engl. J. Med. 362, 36-44 (2010). Li X, et al. J. Allergy Clin. Immunol. 125, 328-335 (2010). Mathias, R.A. et al. J. Allergy Clin. Immunol. 125, 336-346 (2010). Moffatt, M.F. et al. N. Engl. J. Med. 363, 1211-1221 (2010). He JQ. Et al. J. Allergy Clin. Immunol. 124, 222-229 (2009). Moffatt MF. Et al. Eur. J. Hum. Genet. 9, 341-346 (2001).

  An object of the present invention is to provide a method for accurately examining the onset risk and presence of an immune disease such as bronchial asthma or obstructive pulmonary disease, and a test reagent used in the method.

  As a result of intensive studies for solving the above problems, the present inventors have found that the 4th chromosome long arm 31 region (4q31 region), the 5th chromosome long arm 22 region (5q22 region), the 6th chromosome short arm 21 region (6p21). Region), chromosome 10 short arm 14 region (10p14 region), or chromosome 12 long arm 13 region (12q13 region) was identified to be associated with bronchial asthma. Then, by examining these polymorphisms, it was found that the risk of developing immune diseases such as bronchial asthma or obstructive pulmonary disease and the estimation of the onset can be accurately performed, and the present invention has been completed.

That is, the present invention is as follows.
[1]
Analyzing a single nucleotide polymorphism existing in any of the following regions (1) to (5) and examining an immune disease or obstructive pulmonary disease based on the analysis result, A method for determining the risk of onset of obstructive pulmonary disease and / or the presence or absence of onset.
(1) Chromosome long arm 31 region (2) Chromosome 12 long arm 13 region (3) Chromosome 10 short arm 14 region (4) Chromosome 5 long arm 22 region (5) Chromosome 6 short arm 21 region
[2]
The method according to [1], wherein the immune disease or obstructive pulmonary disease is bronchial asthma.
[3]
The single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 61st base of the nucleotide sequence selected from SEQ ID NOS: 1 to 24 or a base in linkage disequilibrium with the base [ The method according to [1] or [2].
[4]
A probe for examination of immune disease or obstructive pulmonary disease having the following sequence (1) or (2): (1) In a base sequence selected from SEQ ID NOs: 1 to 24, a sequence of 10 bases or more including the base at the 61st base number, or a complementary sequence thereof.
(2) A sequence of 10 bases or more including a base in a linkage disequilibrium relationship with the base corresponding to the 61st base of the base sequence selected from SEQ ID NOs: 1 to 24, or a complementary sequence thereof.
[5]
Primer for examination of immune disease or obstructive pulmonary disease capable of amplifying the following region (1) or (2).
(1) In the base sequence selected from SEQ ID NOs: 1 to 24, a region containing the base at the 61st base number.
(2) A region containing a base in a linkage disequilibrium relationship with the base corresponding to the 61st base in the base sequence selected from SEQ ID NOs: 1 to 24.

  According to the present invention, it is possible to accurately and simply predict an onset risk (morbidity risk) of an immune disease such as bronchial asthma or obstructive pulmonary disease that has been difficult to predict. Moreover, the onset of immune diseases such as bronchial asthma or obstructive pulmonary diseases can be determined accurately and simply. Therefore, the present invention contributes to prevention and early treatment of immune diseases such as bronchial asthma or obstructive pulmonary diseases.

The figure which shows the result of the related analysis of a 6p21 area | region based on the result of GWAS, and a linkage disequilibrium map. The figure which shows the result of the related analysis of a 5q22 area | region based on the result of GWAS, and a linkage disequilibrium map. The figure which shows the result of the related analysis of a 10p14 area | region based on the result of GWAS, and a linkage disequilibrium map. The figure which shows the result of a 12q13 area | region related analysis based on the result of GWAS, and a linkage disequilibrium map. The figure which shows the result of the association analysis of a 4q31 area | region based on the result of GWAS, and a linkage disequilibrium map.

<1> Method of the Present Invention The method of the present invention comprises human long chromosome 4 long arm 31 region (4q31 region), long chromosome 5 long arm 22 region (5q22 region), and short chromosome 6 short arm 21 region (6p21 region). Analyzing SNP present in the short arm 14 region (10p14 region) or the long arm region 13 (12q13 region) of chromosome 12, and examining immune disease or obstructive lung disease based on the analysis result Is a method for determining the risk of onset of immune disease or obstructive pulmonary disease and / or the presence or absence of onset. That is, in the present invention, “test” includes a test for the risk of developing an immune disease or obstructive pulmonary disease and a test for the presence or absence of an immune disease or obstructive pulmonary disease. In the method of the present invention, the analysis result of SNP is associated with the risk of developing immune disease or obstructive pulmonary disease and / or the presence or absence of onset.

  Although it does not specifically limit as an immune disease, For example, allergic diseases, such as an airway allergic disease and atopic dermatitis, are preferable, and an airway allergic disease is more preferable. As the airway allergic disease, for example, bronchial asthma is preferable. The obstructive pulmonary disease is not particularly limited. For example, COPD (chronic obstructive pulmonary disease), bronchial asthma, chronic bronchitis, pulmonary emphysema, diffuse panbronchiolitis and the like are preferable. More preferred. Bronchial asthma includes adult bronchial asthma, childhood bronchial asthma, atopic bronchial asthma, etc., but adult bronchial asthma is preferred.

  Specific SNPs present in the 4q31 region can include human rs7686660 and rs3805236. Here, the rs number indicates the registration number of the dbSNP database (http // www.ncbi.nlm.nih.gov / projects / SNP /) of National Center for Biotechnology Information. These SNPs are located in linkage disequilibrium blocks containing the USP38 gene and GAB1 gene of the 4q31 region. Thus, in particular, immune diseases or obstructive pulmonary diseases can be examined by analyzing SNPs present in the linkage disequilibrium block. Specific examples of the USP38 gene include the region from 144106070 to 144143141 of GenBank Accession No. NC_000004.11. Specific examples of the GAB1 gene include the region from 144257983 to 144395718 of GenBank Accession No. NC_000004.11.

rs7686660 means a polymorphism of thymine (T) / guanine (G) at the 68551306th base of GenBank Accession No. NT_016354.18. When this base is T, it may indicate an immune disease or obstructive lung disease or Risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of TT>TG> GG.

  rs3805236 means a polymorphism of adenine (A) / guanine (G) in the 68905884th base of GenBank Accession No. NT_016354.18, and when this base is G, it may be an immune disease or obstructive lung disease or Risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of GG> GA> AA.

  As a specific SNP existing in the 5q22 region, human rs1837253 can be mentioned. This SNP is located in a linkage disequilibrium block containing the TSLP gene and WDR36 gene of the 5q22 region. Thus, in particular, immune diseases or obstructive pulmonary diseases can be examined by analyzing SNPs present in the linkage disequilibrium block. Specific examples of the TSLP gene include the 110407390 to 110413722 region of GenBank Accession No. NC_000005.9. Specific examples of the WDR36 gene include the region of 110427870 to 110466200 of GenBank Accession No. NC_000005.9. In particular, the TSLP gene is likely to be a bronchial asthma-related gene in this region.

  rs1837253 means a polymorphism of thymine (T) / cytosine (C) in the 12816885th base of GenBank Accession No. NT_034772.5, and when this base is C, the possibility of immune disease or obstructive lung disease or Risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of CC> CT> TT.

  Specific SNPs present in the 6p21 region include human rs204995, rs204993, rs404860, rs443198, s3130299, rs412657, rs570963, rs10484560, rs3129943, rs3117098, rs10947262, rs3806156, rs3129890, rs7775228, rs6457617, rs9275898, rs9275698, rs92756989 be able to. These SNPs are located in the major histocompatibility complex region (MHC region) of the 6p21 region. Therefore, in particular, an immune disease or obstructive pulmonary disease can be examined by analyzing a SNP present in the MHC region.

  rs204995 means a polymorphism of adenine (A) / guanine (G) in the 23012535th base of GenBank Accession No. NT — 007592.14, and when this base is A, there is a possibility of immune disease or obstructive lung disease Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs204993 means a polymorphism of adenine (A) / guanine (G) in the 23013831st base of GenBank Accession No. NT — 007592.14, and when this base is A, there is a possibility of immune disease or obstructive lung disease Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs404860 means a polymorphism of adenine (A) / guanine (G) in the 23042595th base of GenBank Accession No. NT_007592.14, and when this base is A, it may be an immune disease or obstructive lung disease. Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs443198 means a polymorphism of adenine (A) / guanine (G) in the 23048656th base of GenBank Accession No. NT — 007592.14, and when this base is A, it may be an immune disease or obstructive lung disease Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs3130299 means a polymorphism of adenine (A) / guanine (G) in the 23061787th base of GenBank Accession No. NT — 007592.14, and when this base is G, there is a possibility of immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of GG> GA> AA.

  rs412657 means a polymorphism of adenine (A) / cytosine (C) in the 23069335th base of GenBank Accession No. NT — 007592.14, and when this base is A, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of AA> AC> CC.

  rs570963 means a polymorphism of adenine (A) / guanine (G) at the 23147844th base of GenBank Accession No. NT — 007592.14, and when this base is A, it may be an immune disease or obstructive lung disease Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs10484560 means a polymorphism of thymine (T) / cytosine (C) at the 23156387th base of GenBank Accession No. NT — 007592.14. When this base is T, it may be an immune disease or obstructive pulmonary disease. Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of TT> TC> CC.

  rs3129943 means a polymorphism of thymine (T) / cytosine (C) in the 23196945th base of GenBank Accession No. NT — 007592.14. When this base is T, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of TT> TC> CC.

  rs3117098 means a polymorphism of adenine (A) / guanine (G) in the 23231663th base of GenBank Accession No. NT — 007592.14, and when this base is G, there is a possibility of immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of GG> GA> AA.

  rs10947262 is a polymorphism of thymine (T) / cytosine (C) at the 23231562th base of GenBank Accession No. NT — 007592.14. When this base is C, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of CC> CT> TT.

rs3806156 means a polymorphism of thymine (T) / guanine (G) at the 23231948th base of GenBank Accession No. NT — 007592.14. When this base is G, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of GG>GT> TT.

  rs3129890 means a polymorphism of thymine (T) / cytosine (C) in the 23232523rd base of GenBank Accession No. NT — 007592.14. When this base is T, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of TT> TC> CC.

  rs7775228 means a polymorphism of adenine (A) / guanine (G) in the 23516329th base of GenBank Accession No. NT_007592.14, and when this base is A, it may be an immune disease or obstructive lung disease Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs6457617 means a polymorphism of adenine (A) / guanine (G) in the 23522101st base of GenBank Accession No. NT — 007592.14, and when this base is A, it may be an immune disease or obstructive lung disease Or the risk of onset is high. In addition, when the analysis is performed in consideration of the genotype, the possibility or risk of developing immune disease or obstructive pulmonary disease is high in the order of AA> AG> GG.

  rs2858308 means a polymorphism of thymine (T) / guanine (G) in the 23528250th base of GenBank Accession No. NT_007592.14, and when this base is G, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of GG> GT> TT.

  rs9275698 means a polymorphism of thymine (T) / cytosine (C) at the 23546223rd base of GenBank Accession No. NT — 007592.14, and when this base is T, it may be an immune disease or obstructive lung disease Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of TT> TC> CC.

  rs9500927 means a polymorphism of thymine (T) / cytosine (C) in the 23819611th base of GenBank Accession No. NT — 007592.14. When this base is T, it may be an immune disease or obstructive pulmonary disease. Or the risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of TT> TC> CC.

  As a specific SNP existing in the 10p14 region, human rs10508372 can be mentioned. rs10508372 is a polymorphism of thymine (T) / cytosine (C) at the 3334914th base of GenBank Accession No. NT_077569.2. When this base is C, it may be an immune disease or obstructive lung disease or Risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of CC> CT> TT.

Specific SNPs present in the 12q13 region can include human rs2069408 and rs1701704. These SNPs are located in linkage disequilibrium blocks including the IKZF4 gene (Eos gene) and the like in the 12q13 region. Thus, in particular, immune diseases or obstructive pulmonary diseases can be examined by analyzing SNPs present in the linkage disequilibrium block. IKZF
Specific examples of the 4 gene (Eos gene) include the region of 56414689 to 56432219 of GenBank Accession No. NC — 000012.11.

  rs2069408 means a polymorphism of thymine (T) / cytosine (C) in the 18507627th base of GenBank Accession No. NT — 029419.11. When this base is C, it may indicate an immune disease or obstructive lung disease or Risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of CC> CT> TT.

  rs1701704 means a polymorphism of thymine (T) / guanine (G) at the 18555793 base of GenBank Accession No. NT — 029419.11. When this base is G, it may indicate an immune disease or obstructive lung disease or Risk of onset is high. Moreover, when analyzing in consideration of the genotype, there is a higher possibility or risk of developing immune disease or obstructive pulmonary disease in the order of GG> GT> TT.

  Regarding the 24 SNPs, sequences having a total length of 121 bp including SNP bases and a region of 60 bp before and after that are shown in SEQ ID NOs: 1 to 24. The 61st base has a polymorphism. Details of the 24 SNPs are also shown in Table 2 described later.

  In the present invention, a base corresponding to the above base is analyzed. The “base corresponding to the above base” means the corresponding base in the above region. That is, “analyzing the base corresponding to the base” includes analyzing the base in the region even if the sequence is slightly changed at a position other than the SNP due to a difference in race. .

In addition, the base to be analyzed in the present invention is not limited to the above, and a polymorphism of a base in linkage disequilibrium with the above base may be analyzed. Here, the “base in linkage disequilibrium with the above-mentioned base” satisfies the relationship of r ² > 0.5, preferably r ² > 0.8, more preferably r ² > 0.9 with the above-mentioned base. Say base. r ² is a linkage disequilibrium coefficient. In addition, a base in linkage disequilibrium with the above base can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja). Alternatively, DNA collected from a plurality of people (usually about 20-40 people) can be identified by sequence analysis using a sequencer and searching for SNPs in linkage disequilibrium. Bases that are in linkage disequilibrium with the above bases, when analyzed in consideration of genotype, are homozygotes for risk alleles> heterozygotes for risk and non-risk alleles> homozygotes for non-risk alleles In turn, there is a higher probability or risk of developing immune or obstructive pulmonary disease.

  By examining the base type of the SNP and associating the obtained result with the immune disease or obstructive pulmonary disease based on the above criteria, the immune disease or obstructive pulmonary disease can be examined. The SNP may be analyzed alone, or a plurality of SNPs including at least one of the SNPs may be collectively analyzed (haplotype analysis). For example, a plurality of the SNPs may be analyzed together, or at least one of the SNPs, known SNPs (Non-Patent Documents 1 to 9) related to immune diseases or obstructive pulmonary diseases, and the known SNPs. You may analyze combining SNPs in linkage disequilibrium. Specifically, for example, at least two or more SNPs selected from the group consisting of rs7686660, rs1837253, rs404860, rs10508372, and rs1701704 may be combined and analyzed. If a plurality of SNPs associated with an immune disease or obstructive pulmonary disease are collectively analyzed, the accuracy of the examination of the immune disease or obstructive pulmonary disease is improved. Any SNP may analyze either strand of the double-stranded DNA. For example, the sequence of the MHC region gene or TSLP gene may be analyzed for the sense strand or the antisense strand.

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

  The analysis of SNP can be performed by a normal gene polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, invader method and the like.

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

  SNP analysis can be performed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base showing a polymorphism and having a 3 'end corresponding to each polymorph is prepared. PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of the amplification product. Further, the presence or absence of amplification may be examined by the LAMP method (Japanese Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Japanese Patent No. 2844386), ICAN method (Japanese Patent Laid-Open No. 2002-233379), or the like. it can. In addition, a single-strand amplification method may be used.

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

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

  It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization. That is, a probe corresponding to each base is prepared, and it is also possible to examine which base the SNP is by examining which probe hybridizes.

  Thus, by determining which base the SNP is, data for examining immune disease or obstructive pulmonary disease can be obtained.

<2> Reagent for test | inspection of this invention This invention also provides test reagents, such as a primer and a probe for test | inspecting immune diseases, such as bronchial asthma, or obstructive pulmonary disease. Examples of such a probe include a probe that includes the SNP site and can determine the type of base at the SNP site based on the presence or absence of hybridization. Specifically, a sequence containing the 61st base of the base sequence selected from SEQ ID NOs: 1 to 24, or a probe having a length of 10 bases or more having a complementary sequence thereof, or a relationship of linkage disequilibrium with the base. A probe having a length of 10 bases or more having a sequence containing a base or a complementary sequence thereof can be mentioned. The nucleotide sequences of “bases in linkage disequilibrium with the base” and the regions before and after the base are, for example, the dbSNP database (http // www.ncbi.nlm.nih.gov / projects) of National Center for Biotechnology Information. / SNP /). The length of the probe is preferably 15 to 35 bases, more preferably 20 to 35 bases.

  Examples of the primer include a primer that can be used for PCR for amplifying the SNP site, or a primer that can be used for sequence analysis (sequencing) of the SNP site. Specifically, a primer that can amplify or sequence a region containing the 61st base of the base sequence selected from SEQ ID NOS: 1 to 24, or a base that is in a linkage disequilibrium relationship with the base. Examples include primers that can amplify or sequence the contained region. The length of such a primer is preferably 10 to 50 bases, more preferably 15 to 35 bases, and even more preferably 20 to 35 bases.

  As a primer for sequencing the SNP site, a primer having a sequence 5 ′ side of the base, preferably 30 to 100 bases upstream, or a 3 ′ side region of the base, preferably 30 to 100 bases downstream A primer having a sequence complementary to this region is exemplified. As a primer used to determine polymorphism based on the presence or absence of amplification by PCR, it has a sequence containing the base, a primer containing the base on the 3 ′ side, a complementary sequence of the sequence containing the base, Examples include a primer containing a base complementary to the above base on the 3 ′ side.

  The test reagent of the present invention may contain PCR polymerase, buffer, hybridization reagent, etc. in addition to these primers and probes.

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

(1) Identification of SNPs associated with bronchial asthma In order to identify genetic mutations that determine bronchial asthma susceptibility, genome-wide association analysis (GWAS) was performed using Japanese subjects. GWAS is a genetic statistical technique for searching for genetic variation related to a phenotype such as a disease. For example, using hundreds of thousands to one million SNPs covering the entire human genome, the frequency of polymorphism between a patient with a certain disease (case) and a subject who does not have the disease (control) By statistically testing for differences, genetic variations associated with the disease can be found.

<Subject>
Table 1 shows the characteristics of the subject used in this example.

  A total of 1,532 bronchial asthma subjects (cases) used for GWAS were recruited at several hospitals. The 5,639 subjects with bronchial asthma used in the replication study were BioBank Japan (BBJ) (Nakamura, Y. The BioBank Japan Project. Clin Adv Hematol Oncol 5, 696-7 (2007)) was taken from patients with bronchial asthma. All subjects with bronchial asthma were Japanese, and bronchial asthma was diagnosed by a physician based on the American Thoracic Society (ATS) criteria.

  The 3,304 control subjects used for GWAS consisted of 2,403 patients enrolled in the BBJ and 901 healthy volunteers recruited at the Osaka Midosuji Rotary Club. The 24,608 control subjects used in the reproduction study were recruited from patients enrolled in the BBJ. Patients enrolled in the BBJ used as control subjects are bronchial asthma, allergic rhinitis, atopic dermatitis, lung fibrosis, chronic obstructive lung Patients with diseases other than diseases (Chronic Obstructive Pulmonary Disease). The control subjects are all Japanese.

  This study was approved by the Ethics Committee of the Institute of Medical Science at the University of Tokyo and Yokohama Institute of Physical and Chemical Research, and informed consent was obtained from all participants.

<GWAS>
The genotypes of 1,532 subjects with bronchial asthma were analyzed using Illumina HumanHap610-Quad Genotyping BeadChip (Illumina (USA)). The genotype of 3,304 control subjects was analyzed using an Illumina HumanHap550 Genotyping BeadChip (Illumina (USA)). As a quality control, one subject who was determined to be an outlier by Principal Component Analysis (PCA) was excluded, and SNPs with a minor allele frequency (MAFs) of less than 0.01, call rate SNPs having a (call rate) of less than 0.99 and SNPs having a P value in the Hardy-Weinberg equilibrium test of less than the cutoff value (P <10 ^{−6 in the} control) were excluded. A total of 458,847 SNPs that passed quality control were assessed for association with bronchial asthma by the Cochran-Armitage trend test. The genomic inflation factor (GC) was 1.07, and the λ ₁₀₀₀ corrected by the sample size was 1.03. Therefore, the possibility of obtaining a false positive relationship due to the bias of the population was considered to be low.

As a result of GWAS, rs8192565 present in the MHC class III region of 6p21 was found to have the strongest association with bronchial asthma. In addition, the two SNPs of the NOTCH4 locus and the C6orf10 locus satisfy P <5.0 × 10 ⁻⁸ set as a genome-wide significance threshold and have bronchial asthma. A significant association was shown.

  Furthermore, the reproducibility of the region already reported to be associated with bronchial asthma was confirmed using the results of the Japanese GWAS obtained here. For example, in a recent large-scale meta-analysis by a European consortium, 10 regions that have a strong genome-wide association with asthma have been reported (Non-patent Document 7). In 10 Japanese GWAS, 6p21 rs1063355 (P = 0.0010), 15q22 rs11071559 (P = 0.0036), and 15q22 rs744910 (P = 0.0095) were reproduced with bronchial asthma. .

<Reproduction test>
In order to further identify genomic regions associated with adult bronchial asthma, a replication study was performed on a total of 102 SNPs using subjects independent of those analyzed by GWAS. The 102 SNPs are in a linkage disequilibrium relationship with one of the 102 SNPs out of SNPs satisfying P <1 × 10 ⁻⁴ in GWAS Cochrane Armitage trend test and r ² > 0.9. This excludes 31 SNPs. The genotypes of 5,639 subjects with bronchial asthma were analyzed by TaqMan assay (Applied Biosystems) or multiplex-PCR invader assay (Third Wave Technologies). The genotypes of 24,608 control subjects were analyzed using Illumina HumanHap610-Quad Genotyping BeadChip (Illumina (USA)).

As a result of the reproduction test, out of 102 SNPs, a total of 24 SNPs existing in 5 regions are P <4.9 × 10 ⁻⁴ (= 0.05), which is a threshold value of significance after Bonferroni correction. / 102) and showed a significant association with bronchial asthma ("Replication" column in Table 2). Details of these 24 SNPs are shown in Table 2.

Furthermore, as a result of the combined analysis of GWAS and reproducibility tests using the Mantel-Haenszel method for these 24 SNPs, all 24 were used as genome-wide significance thresholds. The set P <5.0 × 10 ⁻⁸ was satisfied, indicating a significant association with bronchial asthma (“combined” column in Table 2). In addition, as a result of the integrated analysis, SNPs in which the strongest association with bronchial asthma was recognized in each region are as follows.
4q31 (rs7686660, P = 1.87 × 10 ^-12 , OR = 1.16)
5q22 (rs1837253, P = 1.24 × 10 ^-16 , OR = 1.17)
6p21 (rs404860, P = 4.07 × 10 ^-23 , OR = 1.21)
10p14 (rs10508372, P = 1.79 × 10 ^-15 , OR = 1.16)
12q13 (rs1701704, P = 2.33 × 10 ^-13 , OR = 1.19)

Risk: Risk Allele
RAF: Risk Allele Frequency
a: P value of GWAS and replication study was calculated by Cochrane Armitage trend test.
b: P value of GWAS indicates P _GC corrected with λ value.
c: OR; Odds Ratio and CI; Confidence Interval were calculated using a non-risk allele as a control.
d: The result of combined analysis was calculated by the Mantel-Haenszel method.

  Of these five regions, rs1837253, located 5.7 kb upstream of the transcription start site of the 5q22 TSLP gene, has been reported to be associated with bronchial asthma and airway hypersensitivity by candidate gene analysis in a Canadian population. However, it has not been known whether the TSLP locus is associated with bronchial asthma at the genome-wide level. In addition, an MHC class II HLA-DQ region located in the 6p21 MHC region (HLA locus) has been reported to be associated with bronchial asthma and airway hypersensitivity in studies involving white populations (non-relevant) Patent Documents 7 and 9) have not reported an association between an MHC region other than the HLA-DQ region and bronchial asthma. Neither of these two areas has been reported to be associated with bronchial asthma in Asian populations, and SNPs in these areas have been associated with immune diseases such as bronchial asthma or obstructive pulmonary disease in Asian populations such as Japanese. It is the first time found by this example that it is useful for inspection. In addition, regarding the 3 regions of 4q31, 10p14, and 12q13, a recent large-scale meta-analysis by a European consortium (Non-patent Document 7) has not reported any association with bronchial asthma. An association with asthma was found. Note that 65.4% of bronchial asthma subjects used in the large-scale meta-analysis in Europe were children with childhood-onset asthma. Only 19.6% of the respondents were adult asthmatic subjects. Since bronchial asthma is a highly diverse disease, and age-specific pathologies are also observed, the asthma traits may differ between the population analyzed in this example and the population used in a large-scale European meta-analysis There is sex.

Of the 24 SNPs that showed significant association with bronchial asthma by integrated analysis, 18 SNPs are located in the 6p21 major histocompatibility complex region (also referred to as MHC region, HLA locus). Among them, the strongest association was found in rs404860 in the MHC class III region (P = 4.07 × 10 ^-23 in integrated analysis, OR = 1.21). FIG. 1 shows the results of the association analysis of the 6p21 region based on the GWAS data and the linkage disequilibrium map.

In a recent large-scale meta-analysis by a European consortium, an association between rs9273349 located in the HLA-DQ region of MHC class II and bronchial asthma has been reported, and this SNP is childhood-onset. It is said that it influences adult-onset (later-onset) more strongly (nonpatent literature 7). Therefore, the relationship between rs9273349 and rs8192565 (not shown in Table 2 because P <5.0 × 10 ^-8 was not satisfied in the integrated analysis) that showed the strongest association with bronchial asthma in the GWAS of this example. Were examined by logistic regression analysis using GWAS data. Since the GWAS of this example does not include rs9273349 data, rs1063355 data that is completely linked with rs9273349 and r ² = 1 in the HapMap JPT and CEU databases was used instead. As a result, it became clear that even when corrected with rs1063355, SNPs in the MHC class III region are strongly associated with bronchial asthma. That is, it is considered that the relationship between the MHC class III region and bronchial asthma found in this example is independent of the previously reported relationship between the HLA-DQ region and bronchial asthma.

In addition, it has been reported that rs2070600 present in the 6p21 AGER locus is related to the 1 second rate (FEV1 / FVC). The 1-second rate is one of the indices of respiratory function, and a decrease in the 1-second rate is a characteristic of obstructive pulmonary diseases such as bronchial asthma and chronic obstructive pulmonary disease. In this example, rs2070600 did not show an association with bronchial asthma, but rs404860, which showed the strongest association with bronchial asthma in the integrated analysis, is located near rs2070600 (32.9 kb downstream), and these SNPs are weakly linked. Equilibrium is shown (r ² = 0.115, D ′ = 0.805). This suggests that there may be a common genetic factor for bronchial asthma and reduced respiratory function.

Furthermore, in order to identify genes associated with bronchial asthma in four related regions other than the MHC region, mapping of related strengths was performed using GWAS data. For mapping, SNPs with a minor allele frequency (MAF) ≧ 0.05 and r ² <0.8 based on the information of Japanese HapMap phase II + III (release 27) are tagged SNPs ( tagSNPs). The results of association analysis and linkage disequilibrium maps for each region are shown in FIGS.

  As a result of detailed mapping of the 5q22 region using 13 tag SNPs, an approximately 88 kb linkage disequilibrium block containing two genes, TSLP and WDR36, was identified as a related region of bronchial asthma, the strongest association being rs1837253 It was recognized (FIG. 2). The TSLP gene is a gene that is strongly induced in airway epithelial cells by respiratory tract virus infection, allergens with multiple protease activities, inflammatory cytokines, and tobacco, and the Th2 cell response after sensing environmental factors and exposure to environmental factors Plays an important role in the induction of. Therefore, the TSLP gene is likely to be a bronchial asthma-related gene in this region.

  As a result of detailed mapping of the 10p14 region using 22 tag SNPs, an approximately 112 kb linkage disequilibrium block in which rs10508372 was present was identified as a related region of bronchial asthma (FIG. 3). This region is a region where there is no known gene called a so-called gene desert, but a GATA3 gene encoding a major regulatory factor in the differentiation of Th2 cells is present 1 Mb upstream. Enhancers can also regulate the expression of genes separated by Mb units in the genome, and whether or not the gene polymorphism present on the relevant region of bronchial asthma has the activity to regulate GATA3 gene expression as cis, etc. Waiting for a new study.

  As a result of detailed mapping of the 12q13 region using 12 tag SNPs, a linkage disequilibrium block of about 201 kb in which rs1701704 is present was identified as a related region of bronchial asthma (FIG. 4). rs1701704 has been reported to be associated with type I diabetes and alopecia areata. This linkage disequilibrium block contained 13 genes, and rs1701704 was located 2 kb upstream of the IKZF4 gene (Eos gene). The IKZF4 gene is known to be involved in the differentiation of regulatory T cells as a co-regulator of FoxP3-dependent gene silencing. Regulatory T cells function to maintain lung homeostasis and suppress harmful immune responses to inhaled antigens, which are essentially harmless.

  As a result of detailed mapping of the 4q31 region using 11 tag SNPs, a linkage disequilibrium block of about 497 kb in which rs1397527 is present was identified as a related region of bronchial asthma (FIG. 5). This linkage disequilibrium block contains two genes, USP38 and GAB1. The USP38 gene encodes ubiquitin specific peptidase 38 whose function is unknown. GAB1 gene encodes scaffolding adopter protein and is activated through cytokine receptors such as IL-3, IL-6, IFN-α and IFN-γ, and B cell and T cell receptors. Plays an important role.

Furthermore, it was examined whether the association with bronchial asthma was recognized in the races other than Japanese about the five related areas of bronchial asthma identified in this Example. Using 499 adult non-Hispanic white subjects with asthma and 639 control subjects obtained from Illumina's iControl database, 5 regions of 5SNPs (as a result of integrated analysis, bronchial in each region) Rs7686660, rs1837253, rs404860, rs10508372, the strongest association with asthma
rs1701704) was analyzed. As a result, rs1837253 present in 5q22 was significantly associated with bronchial asthma (P = 0.023). This suggests that there may be common genetic factors for adult asthma between Japanese and Caucasians in the region containing TSLP on the genome.

  As described above, five regions related to bronchial asthma that meet the genome-wide level were found in the Japanese population. SNPs present in these regions are useful for examination of immune diseases such as bronchial asthma or obstructive pulmonary disease, and contribute to prevention and / or treatment of immune diseases such as bronchial asthma or obstructive pulmonary disease.

Claims (5)

Analyzing a single nucleotide polymorphism existing in any of the following regions (1) to (5) and examining an immune disease or obstructive pulmonary disease based on the analysis result, A method for determining the risk of onset of obstructive pulmonary disease and / or the presence or absence of onset.
(1) Chromosome long arm 31 region (2) Chromosome 12 long arm 13 region (3) Chromosome 10 short arm 14 region (4) Chromosome 5 long arm 22 region (5) Chromosome 6 short arm 21 region   The method according to claim 1, wherein the immune disease or obstructive pulmonary disease is bronchial asthma.   The single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 61st base of the base sequence selected from SEQ ID NOs: 1 to 24 or a base in linkage disequilibrium with the base. Item 3. The method according to Item 1 or 2. A probe for examination of immune disease or obstructive pulmonary disease having the following sequence (1) or (2): (1) In a base sequence selected from SEQ ID NOs: 1 to 24, a sequence of 10 bases or more including the base at the 61st base number, or a complementary sequence thereof.
(2) A sequence of 10 bases or more including a base in a linkage disequilibrium relationship with the base corresponding to the 61st base of the base sequence selected from SEQ ID NOs: 1 to 24, or a complementary sequence thereof. Primer for examination of immune disease or obstructive pulmonary disease capable of amplifying the following region (1) or (2).
(1) In the base sequence selected from SEQ ID NOs: 1 to 24, a region containing the base at the 61st base number.
(2) A region containing a base in a linkage disequilibrium relationship with the base corresponding to the 61st base in the base sequence selected from SEQ ID NOs: 1 to 24.