JP2019033738A - Method of predicting risk of food allergy based on gene polymorphism and kit for examination - Google Patents

Abstract

To provide novel examination methods for predicting the onset risk of food allergy, and to provide reagents (probes and primers) used in the examination methods.SOLUTION: The above subject is solved by analyzing the allele profile of rs2071471, rs2395166, rs2670873, or rs77083409, or an SNP in linkage disequilibrium with the SNP(allergy to a shrimp component), rs2303444, rs3129860, or rs518636, or an SNP in linkage disequilibrium with the SNP(allergy to a crab component), rs11994717, or an SNP in linkage disequilibrium with the SNP.SELECTED DRAWING: None

Description

  The present invention relates to a test method for predicting the risk of developing food allergy and a reagent used in the test method.

  Food allergy is an adverse reaction caused by an antigen-specific immune response after eating food. In recent years, patients with food allergies are increasing mainly in developed countries. In Japan, it is estimated that 5-10% of infants, 5% of children, and 4.5% of school children have food allergies. ing.

  Many food allergies are mediated by IgE (immunoglobulin E), and an allergic reaction is caused within 2 hours after ingestion of the food. In the diagnosis of food allergy, IgE antibodies specific to food allergens are detected and illuminated by blood tests and skin tests. However, these tests are auxiliary to diagnosis and are invasive tests such as blood sampling and puncture. Eventually, food allergies are diagnosed by a food removal test or a food oral challenge test, which may cause harmful symptoms such as anaphylaxis.

  The risk factors for food allergies include family history, genetic tendency, skin barrier function, birth season, and the presence of atopic dermatitis is considered an important risk factor.

  In recent years, genome-wide association analysis (GWAS) has been reported to identify loci associated with peanut allergy by genome-wide association study (hereinafter also referred to simply as “GWAS”). Patent Document 1).

  However, there are no reports on food allergies that are common in Asian humans, especially Japanese, especially allergies to shrimp, crabs and buckwheat, and the risk of onset was determined by conventional diagnostic methods. It was determined by sex tests and tests that could cause serious systemic symptoms.

  As a result of exhaustive genome-wide association analysis (GWAS) in the Japanese mainland population to solve the above problems, the present inventors have found the risk of developing allergies to food components, particularly components contained in shrimps, crabs and buckwheat. The inventors have found a single nucleotide mutation (SNP) that can more accurately predict the above, and have completed the present invention.

Nat Commun. Author manuscript; available in PMC 2015 August 24.

  It is an object of the present invention to provide a method for predicting more accurately the risk of developing allergies to food components, particularly components contained in shrimp, crab, buckwheat, and a test reagent, probe and primer used in the method. To do.

  One or two or more oligonucleotides or polynucleotides (hereinafter simply referred to as “oligo (or poly) nucleotides”) including the markers for predicting the risk of developing food allergy of the present invention, ie, rs2071471, rs2395166, rs2670873 or rs77083409. The risk of developing allergies to shrimp components can be predicted. Further, by using a marker composed of oligo (or poly) nucleotides containing rs2303444, rs3129860 or rs518636, it is possible to predict the risk of developing allergy to crab components. Furthermore, the risk of allergy to buckwheat components can be predicted by using a marker composed of oligo (or poly) nucleotides containing rs11994717.

  Specifically, by analyzing the allele profile of SNP contained in each marker, it is possible to predict the onset risk of allergy to each component more reliably.

That is, the present invention is as follows.
[1] One or more SNPs selected from rs2071471, rs2395166, rs2670873, and rs77083409, or an allele profile of a SNP that is in linkage disequilibrium with the SNP is determined, and allergic onset of shrimp components is obtained from the obtained allele profile Inspection methods for risk prediction,
[2] For determining the allele profile of one or more SNPs selected from rs2071471, rs2395166, rs2670873, and rs77083409, and predicting the risk of developing allergy to shrimp components from the obtained allele profile [1] Inspection method,
[3] When the base of rs2071471 is T, the test method according to [1] or [2], comprising predicting that the risk of developing allergy to shrimp components is high,
[4] When the base of rs2395166 is C, the test method according to [1] or [2], comprising predicting that the risk of developing allergy to shrimp components is high,
[5] When the base of rs2670873 is C, the test method according to [1] or [2], comprising predicting that the risk of developing allergy to shrimp components is high,
[6] When the base of rs77083409 is A, the test method according to [1] or [2], comprising predicting that the risk of developing allergy to shrimp components is high,
[7] One or more SNPs selected from rs2303444, rs3129860 and rs518636 or an allele profile of SNP in linkage disequilibrium with the SNP is determined, and from the obtained allele profile, the risk of allergy to crab components is determined. Inspection method for making predictions,
[8] [7] Description for determining an allele profile of one or more SNPs selected from rs2303444, rs3129860, and rs518636, and predicting the risk of developing allergies to crab components from the obtained allele profiles Inspection method,
[9] When the base of rs2303444 is C, the test method according to [7] or [8], comprising predicting that the risk of developing allergy to a crab component is high,
[10] When the base of rs3129860 is A, the test method according to [7] or [8], comprising predicting that the risk of developing allergy to crab components is high,
[11] When the base of rs518636 is T, the test method according to [7] or [8], comprising predicting that the risk of developing allergy to a crab component is high.
[12] A test method for determining the allele profile of rs11994717 or SNP in linkage disequilibrium with the SNP, and predicting the risk of developing allergy to buckwheat components from the obtained allele profile,
[13] The test method according to [12], wherein the allele profile of rs11994717 is determined, and the risk of developing allergy to buckwheat components is predicted from the obtained allele profile,
[14] When the base of rs11994717 is A, the test method according to [12] or [13], comprising predicting that the risk of developing allergy to buckwheat components is high,
[15] A probe for use in the test method according to [1], which has a sequence of 15 bases or more including the 61st base in a base sequence selected from SEQ ID NOs: 1 to 3 and 8, or a complementary sequence thereof,
[16] A probe for use in the test method according to [7], which has a sequence of 15 bases or more including the 61st base in the base sequence of SEQ ID NO: 4 or 5, or a complementary sequence thereof,
[17] The probe for use in the test method according to [7], which has a sequence of 15 bases or more including the 56th base in the base sequence of SEQ ID NO: 6, or a complementary sequence thereof,
[18] The probe for use in the test method according to [12], which has a sequence of 15 bases or more including the base at position 61 in the base sequence of SEQ ID NO: 7, or a complementary sequence thereof,
[19] A primer for use in the test method according to [1], which can amplify a region containing the base at position 61 in the base sequence selected from SEQ ID NOs: 1 to 3 and 8.
[20] A primer for use in the test method according to [7], which can amplify a region containing the base at position 61 in the base sequence of SEQ ID NO: 4 or 5.
[21] A primer for use in the test method according to [7], which can amplify a region containing the base at position 56 in the base sequence of SEQ ID NO: 6,
[22] A primer for use in the test method according to [12], which can amplify a region containing the base at position 61 in the base sequence of SEQ ID NO: 7.

  According to the present invention, the risk of developing allergy can be predicted accurately and simply.

  In the present invention, “gene”, “oligonucleotide” and “polynucleotide” are purine bases such as adenine (A) and guanine (G), thymine (T), uracil (U) and cytosine (C). Such as pyrimidine bases and their modified bases as constituent elements, and single-stranded or double-stranded DNA, single-stranded or double-stranded RNA, and a hybrid comprising single-stranded DNA and single-stranded RNA Body, or any one or a plurality of chimeras in which RNA and DNA are combined to form a single strand. DNA also includes cDNA.

  In the present specification, the hybridizing oligo (or poly) nucleotide refers to an oligo (or poly) nucleotide that can bind completely or partially to the target oligo (or poly) nucleotide. . For example, it refers to an oligo (or poly) nucleotide that can bind completely or partially complementarily to any oligo (or poly) nucleotide as a marker constituting the marker group of the present invention. The term refers to a complementary sequence, or an oligo (or poly) nucleotide consisting of a sequence in which one to a plurality of bases are substituted, deleted, added or inserted from the complementary sequence, and a sequence that can be bound. Such oligo (or poly) nucleotides can be used as primers and probes.

  The term “linkage disequilibrium” (LD) is used to describe the statistical correlation between two adjacent polymorphic genotypes. In general, LD refers to the correlation between random gamete alleles at two loci, assuming that there is a Hardy-Weinberg equilibrium (statistical independence) between the gametes. LD is quantified by either Lewontin's relevant parameter (D ') or Pearson's correlation coefficient (r) (Devlin and Risch, 1995). Two loci with an LD value of 1 are said to be in full LD. In the other extreme case, the two loci with an LD value of 0 are called in chain equilibrium. Linkage disequilibrium is calculated according to the application of the expectation maximization algorithm (EM) to estimate haplotype frequencies (Slatkin and Excoffier, 1996). The LD value according to the present invention for adjacent genotype / locus is 0.1 or more, preferably 0.2 or more, more preferably 0.5 or more, more preferably 0.6 or more, further preferably 0.7 or more, preferably 0.8 or more, more preferably 0.9 or more, ideally about 1.0 is selected.

  “SNP” is a single nucleotide polymorphism, and indicates a polymorphism of a base present at a specific position on the human genome. “SNP” includes mutations due to insertion and deletion in addition to single-base mutations.

  “Allyl profile” refers to an individual's genotype in a particular SNP.

  The analysis of the allele profile (SNP analysis) 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.

1. Probe of the present invention The present invention provides a probe for examining the risk of developing food allergy.

  The probe of the present invention is an oligo (or poly) nucleotide containing the above SNP site, and the type of SNP site base is determined depending on the presence or absence of hybridization to any oligo (or poly) nucleotide as the marker of the present invention. Examples include probes that can be determined.

  The base sequence of the probe of the present invention is most preferably a sequence that completely forms a complementary pair with the target base sequence, but 1 to a plurality of nucleotides is sufficient to hybridize with the target base sequence. It may be a substitution, deletion, addition and / or introduced sequence. Chimeric probes can also be used.

  Specifically, (1) In order to predict the risk of developing allergies to shrimp components having a sequence containing the 61st base in a base sequence selected from SEQ ID NOS: 1 to 3 and 8 or a complementary sequence thereof (2) Prediction of the risk of developing allergy to a crab component having a sequence containing the 61st base in the base sequence selected from SEQ ID NO: 4 or 5 or a complementary sequence thereof A probe for use in a testing method for performing, (3) for predicting the risk of developing allergy to a crab component having a sequence containing the 56th base of the base sequence of SEQ ID NO: 6, or a complementary sequence thereof A probe for use in an inspection method, (4) a sequence containing the 61st base in the base sequence of SEQ ID NO: 7 or a complementary sequence thereof; Probes can be cited for use in the inspection method for performing a prediction of the risk of developing allergies to components of server. The length of the probe varies depending on the oligo (or poly) nucleotide to be detected, and cannot be generally specified, but is preferably 15 to 35 bases, more preferably 20 to 35 bases.

2. Primer of the present invention The present invention provides a primer for examining the risk of developing food allergy.

  Examples of the primer of the present invention 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 region containing the 61st base of the base sequence selected from SEQ ID NOS: 1 to 5 and 7 and 8 or the 56th base of the base sequence of SEQ ID NO: 6 is amplified or sequenced. The primer which can do is mentioned. 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 5′-side region of the base, preferably 30 to 100 bases upstream, or a 3′-side region of the base, preferably 30 to 100 bases downstream Primers having a sequence complementary to this region can be used. 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, A primer containing a base complementary to the above base on the 3 ′ side is used.

  In addition to these primers and probes, the test reagent of the present invention may contain PCR polymerases, buffers, hybridization reagents, and the like.

3. Test method of the present invention The method of the present invention analyzes a specific SNP and, based on the analysis result, develops an allergy (sometimes simply referred to as food allergy) to a food component, particularly a component contained in shrimp, crab or buckwheat. This is an inspection method for predicting risk. That is, in the present invention, “test” includes a test of the risk of developing food allergy. The method of the present invention is performed by associating the analysis result of SNP with the risk of developing food allergy.

  In the present specification, the rs number indicates a registration number in the dbSNP database (http // www.ncbi.nlm.nih.gov / projects / SNP /) of the National Center for Biotechnology Information (NCBI).

  rs2071471 means a polymorphism of cytosine (C) / thymine (T) at the 32784645th base of GenBank Accession No. NT — 007592.15. When this base is T, the risk of developing allergy to shrimp components is high. Moreover, when analyzing in consideration of the genotype, the risk of developing allergy to shrimp components is high in the order of TT> CT> CC.

  rs2395166 means a polymorphism of cytosine (C) / thymine (T) in the 32388275th base of GenBank Accession No. NT — 007592.15. When this base is C, the risk of developing allergy to shrimp components is high. Moreover, when analyzing in consideration of the genotype, the risk of developing allergy to shrimp components is higher in the order of CC> CT> TT.

  rs2670873 means a polymorphism of cytosine (C) / thymine (T) at the 131328302th base of GenBank Accession No. NT — 008046.16. When this base is C, the risk of developing allergy to shrimp components is high. Moreover, when analyzing in consideration of the genotype, the risk of developing allergy to shrimp components is higher in the order of CC> CT> TT.

  rs77083409 means a polymorphism of adenine (A) / guanine (G) in the 28180810th base of GenBank Accession No. NT — 01109.16, and when this base is A, the risk of developing allergy to shrimp components is high. Moreover, when analyzing in consideration of the genotype, the risk of developing allergy to shrimp components is higher in the order of AA> AG> GG.

  For rs2071471, rs2395166, rs2670873, and rs77083409, sequences having a total length of 121 bp including the SNP base and a region of 60 bp before and after that are shown in SEQ ID NOs: 1 to 3 and 8 (SEQ ID NO: 8: rs2071471, SEQ ID NO: rs2395166, SEQ ID NO: 2: rs2670873 and SEQ ID NO: 3: rs77083409). The 61st base has a polymorphism.

  rs2303444 means a polymorphism of cytosine (C) / thymine (T) in the 131226752 base of GenBank Accession No. NT — 008046.16. When this base is C, there is a high risk of developing allergy to crab components. Moreover, when analyzing in consideration of the genotype, the risk of developing allergy to the crab component is high in the order of CC> CT> TT.

  rs3129860 means a polymorphism of adenine (A) / guanine (G) in the 32401079th base of GenBank Accession No. NT — 007592.15, and when this base is A, there is a high risk of developing allergies to crab components. In addition, when analyzing in consideration of the genotype, the risk of developing allergy to crab components is high in the order of AA> AG> GG.

  rs518636 means a polymorphism of thymine (T) / cytosine (C) in the 116363146th base of GenBank Accession No. NT — 008470.19, and when this base is T, there is a high risk of developing allergy to crab components. Moreover, when analyzing in consideration of the genotype, the risk of developing allergy to crab components is high in the order of TT> TC> CC.

  With regard to rs2303444 and rs3129860, sequences having a total length of 121 bp including the SNP base and the region of 60 bp before and after that are shown in SEQ ID NOs: 4 and 5, respectively (SEQ ID NO: 4: rs2303444, SEQ ID NO: 5: rs3129860). The 61st base has a polymorphism.

  Regarding rs518636, a sequence having a total length of 116 bp including the SNP base and its 5'-side 55 bp and 3'-side 60 bp regions is shown in SEQ ID NO: 6. The 56th base has a polymorphism.

rs11994717 means a polymorphism of adenine (A) / guanine (G) in the 57160043 base of GenBank Accession No. NT — 008183.19. When this base is A, the risk of developing allergy to buckwheat components is high. In addition, when the analysis is performed in consideration of the genotype, the risk of developing allergy to buckwheat components is high in the order of AA>AG> GG.
Regarding rs11994717, a sequence having a total length of 121 bp including the SNP base and a region of 60 bp before and after it is shown in SEQ ID NO: 7. The 61st base has a polymorphism.

  An SNP in linkage disequilibrium with rs2071471 is rs12207915 (further possible examples are shown in Table 1). An SNP in linkage disequilibrium with rs2395166 is rs4335021 (further possible examples are shown in Table 2) and linkage disequilibrium with rs2670873. SNPs in rs2791344 (further possible examples are shown in Table 3), SNPs in linkage disequilibrium with rs77083409 are rs75479720 (further possible examples are shown in Table 4), SNPs in linkage disequilibrium with rs2303444 Rs74403851 (an additional possible example is shown in Table 5), rs59642557 as an SNP in linkage disequilibrium with rs3129860 (an additional possible example is shown in Table 6), and rs72763856 as an SNP in linkage disequilibrium with rs518636 (further Possible examples are shown in Table 7), and SNPs in linkage disequilibrium with rs11994717 include rs13271434 (further possible examples are shown in Table 8).

  Surrogate markers for SNP rs2071471 are shown in Table 1. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, r2 and D 'values for rs2071471, and the position of the surrogate marker in GRCh 37 are shown.

  Surrogate markers for SNP rs2395166 are shown in Table 2. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The name of the correlated SNP, the r2 and D 'values for rs2395166, and the position of the surrogate marker in GRCh 37 are shown.

  The surrogate markers for SNP rs2670873 are shown in Table 3. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, r2 and D 'values for rs2670873, and the position of the surrogate marker in GRCh 37 are shown.

  Surrogate markers for SNP rs77083409 are shown in Table 4. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, r2 and D 'values for rs77083409, and the position of the surrogate marker in GRCh 37 are shown.

  Surrogate markers for SNP rs2303444 are shown in Table 5. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, r2 and D 'values for rs2303444, and the position of the surrogate marker in GRCh 37 are shown.

  Surrogate markers for SNP rs3129860 are shown in Table 6. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, the r2 and D 'values for rs3129860, and the location of the surrogate marker in GRCh 37 are shown.

  Surrogate markers for SNP rs518636 are shown in Table 7. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, the r2 and D 'values for rs518636, and the position of the surrogate marker in GRCh 37 are shown.

  The surrogate markers for SNP rs11994717 are shown in Table 8. The top 20 marker SNPs with r2 higher than 0.05 for this marker were selected. The names of the correlated SNPs, the r2 and D 'values for rs11994717, and the position of the surrogate marker in GRCh 37 are shown.

  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 SNP due to a difference in race. .

  By examining the type of the SNP base and associating the obtained result with food allergy based on the above criteria, food allergy can be examined. The SNP may be analyzed alone, or a plurality of SNPs (SNPs) including at least one of the SNPs may be collectively analyzed (haplotype analysis).

  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.

Alternatively, the type of polymorphism can be analyzed 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 food allergy can be obtained.

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

  Genome-wide association analysis (GWAS) was performed to identify genetic mutations associated with the development of food allergies. 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, statistically testing whether there is a difference due to combination of polymorphisms (genotype) in the distribution of the mean value of a certain trait By doing so, you can find genetic mutations associated with the disease. In this example, whether or not there was food allergy was determined by the questionnaire results for the subjects.

1. Genotype Data Quality Control 1-1: Integration of Genotyping Platform The Genotype data used in this study used two array platforms: HumanCore-v12 Bead Chip (illumina) and HumanCore-v24 Bead Chip (illumina). We extracted 296,675 SNPs that were commonly installed on both array platforms, and performed subsequent data processing.

1-2: Confirm gender match
The gender was estimated from the genotype data, and it was confirmed whether it matched with the gender obtained by the questionnaire survey. Genotype data of 52 people whose genders did not match were removed from subsequent data processing.

1-3: Confirmation of blood relationship
PLINK (v1.9, https://www.cog-genomics.org/plink2, Purcell, Shaun, et al. "PLINK: a tool set for whole-genome association and population-based linkage analyzes." The American Journal of Human genetics 81.3 (2007): 559-575.) Pairwise IBD estimation was performed to estimate the relationship between subjects. Genotype data of 89 pairs and 164 persons, presumed to be related (p (Z0) <0.05), were removed from the subsequent data processing.

1-4: Quality control of SNP
When performing GWAS, Genotype data for subjects with call rates less than 95%, SNPs with call rates less than 90%, SNPs with minor allele frequencies less than 5%, Hardy-Weinberg equilibrium fit test p <0.05 SNPs were deleted. In addition, SNPs used for the subsequent analysis were limited to SNPs located on the autosome.

1-5: Representatives from around the world collected and published by the International HapMap Project (Gibbs, Richard A., et al. "The international HapMap project." Nature 426.6968 (2003): 789-796.) Genotype data of four typical populations (CEU, YRI, CHB, JPT) were acquired, and the data of 163,708 SNPs measured in common with the Genotype data of this study were integrated. SNPs were narrowed down using linkage disequilibrium (LD) from the integrated Genotype data, and principal component analysis was performed using the narrowed Genotype data. The obtained top two principal components were clustered by the k-means method, and Genotype data of two persons belonging to clusters other than the Asian population (CHB, JPT) were removed from the subsequent analysis.
Similarly, Genotype data of Asian population of International HapMap project and Genotype data of this study were integrated and narrowed down to perform principal component analysis. The top two principal components were clustered by the k-means method, and 11,409 people belonging to the cluster considered to be a Japanese mainland population were extracted. The following analysis was conducted on this Japanese mainland group.

2. Selection of phenotype
The phenotype was obtained through a questionnaire survey using the Web.

2-1: Quality control of questionnaire regarding food questionnaires Consistency between questionnaires was confirmed from questionnaires obtained and defined as Pre-Control and Pre-Case, respectively. Subjects who did not satisfy both Pre-Control and Pre-Case conditions were excluded from GWAS with a phenotype of Missing.

Pre-Control definition
Answer “No” to X73 “Have you ever had a food allergy?”
X74.1-X74.28 No answer to "(Yes only) What product allergies do you have?"
X75, “(Yes only) When did you notice food allergies?”
Not responding to X76 “(Yes only) Do you still have food allergies?”
Not answering X1080.5.1 “Food Allergy”

Definition of Pre-Case
Answer “Yes” to X73 “Have you ever had a food allergy?”
X74.1 ~ X74.28 At least one answer to "(Yes only) What food allergies?"
Answer X75, “(Yes only) When did you notice food allergies?”
Answer to X76 “(Yes only) Do you still have food allergies?”
Answer X1080.5.1 “Food Allergy”

2-2: Simple
The selection criteria for Control based on the phenotypic criteria based on the history of each food
Not responding to the corresponding questionnaire from X74.1 to X74.28 ・ Case selection criteria
Answer the corresponding questionnaire from X74.1 to X74.28

2-3: No-food allergy
Subjects with no history of food allergy (= Pre-Control) were used as controls.
・ Control selection criteria
Pre-Control
・ Case selection criteria
Answer the corresponding questionnaire from X74.1 to X74.28 in Pre-Case

3. Genome-wide association analysis (GWAS)
GWAS was performed on Genotype data and phenotype obtained by the above data processing. We analyzed the relationship between phenotypes and SNPs by logistic regression assuming an additive model. We analyzed the case where no covariate was included, and the case where age, gender, BMI, smoking calendar, and the top two principal components obtained by population structuring analysis were used as covariates.

Results The phenotype GWAS results with more than 100 Cases are shown below for each food ingredient.

  Table 9 shows the number of people with and without shrimp allergy, age, and BMI. In Control and Case, the P-values tested by Student's t-test to see if there are differences in male ratio, age, and BMI are shown.

  Table 10 shows the number, age, and BMI of shrimp allergy in the case and control selection method No food allergy. In Control and Case, the P-values tested by Student's t-test to see if there are differences in male ratio, age, and BMI are shown.

Table 11 shows the GWAS results for the presence or absence of shrimp allergy in Simple and No food allergy. For SNPs satisfying P value <1 × 10 ^-5 , Top SNPs excluding linked SNPs are shown. no covariates indicates that no covariates are included, and adjusted indicates that it includes covariates. The column names are described below. SNP: SNP ID, Chr: chromosome number, Position: position, Allele: base pair (Effect Allele / Reference Allele), OR: Effect Allele odds ratio, P: P value.

  Table 12 shows the number, age, and BMI of the presence / absence of crab allergy in Simple and Control selection methods. In Control and Case, the P-values tested by Student's t-test to see if there are differences in male ratio, age, and BMI are shown.

  Table 13 shows the number, age, and BMI of the presence or absence of crab allergy in No food allergy. In Control and Case, the P-values tested by Student's t-test to see if there are differences in male ratio, age, and BMI are shown.

Table 14 shows the GWAS results for the presence or absence of crab allergy in Simple and No food allergy. For SNPs satisfying P value <1 × 10 ^-5 , Top SNPs excluding linked SNPs are shown. no covariates indicates that no covariates are included, and adjusted indicates that it includes covariates. The column names are described below. SNP: SNP ID, Chr: chromosome number, Position: position, Allele: base pair (Effect Allele / Reference Allele), OR: Effect Allele odds ratio, P: P value.

  Table 15 shows the number, age, and BMI of the presence / absence of buckwheat allergy in Simple and Control. In Control and Case, the P-values tested by Student's t-test to see if there are differences in male ratio, age, and BMI are shown.

  Table 16 shows the number, age and BMI of the presence or absence of buckwheat allergy in No food allergy. In Control and Case, the P-values tested by Student's t-test to see if there are differences in male ratio, age, and BMI are shown.

Table 17 shows the GWAS results for the presence or absence of buckwheat allergy in Simple and No food allergy. For SNPs satisfying P value <1 × 10 ^-5 , Top SNPs excluding linked SNPs are shown. no covariates indicates that no covariates are included, and adjusted indicates that it includes covariates. The column names are described below. SNP: SNP ID, Chr: chromosome number, Position: position, Allele: base pair (Effect Allele / Reference Allele), OR: Effect Allele odds ratio, P: P value.

The risk of developing food allergy can be easily and accurately predicted by the test method of the present invention.

Claims (22)

One or more SNPs selected from rs2071471, rs2395166, rs2670873, and rs77083409, or an allele profile of SNP in linkage disequilibrium with the SNP is determined, and prediction of allergic risk of shrimp components from the obtained allele profile Inspection method for doing. The test according to claim 1, for determining an allele profile of one or more SNPs selected from rs2071471, rs2395166, rs2670873, and rs77083409, and predicting the risk of developing allergy against shrimp components from the obtained allele profile Method. The test method according to claim 1 or 2, comprising predicting that when the base of rs2071471 is T, the risk of developing allergy to shrimp components is high. The test method according to claim 1 or 2, comprising predicting that when the base of rs2395166 is C, the risk of developing allergy to shrimp components is high. The test method according to claim 1 or 2, comprising predicting that when the base of rs2670873 is C, the risk of developing allergy to shrimp components is high. The test method according to claim 1 or 2, comprising predicting that when the base of rs77083409 is A, the risk of developing allergy to shrimp components is high. One or more SNPs selected from rs2303444, rs3129860 and rs518636, or an allele profile of a SNP in linkage disequilibrium with the SNP is determined, and the risk of allergy to crab components is predicted from the obtained allele profile. Inspection method for. The test method according to claim 7, wherein the allyl profile of one or more SNPs selected from rs2303444, rs3129860 and rs518636 is determined, and the risk of allergy to crab components is predicted from the obtained allyl profile. . The test method according to claim 7 or 8, comprising predicting that, when the base of rs2303444 is C, there is a high risk of developing an allergy to a crab component. The test method according to claim 7 or 8, comprising predicting that, when the base of rs3129860 is A, there is a high risk of developing an allergy to a crab component. The test method according to claim 7 or 8, comprising predicting that when the base of rs518636 is T, the risk of developing an allergy to a crab component is high. A test method for determining an allele profile of rs11994717 or an SNP in linkage disequilibrium with the SNP and predicting the risk of developing allergy to buckwheat components from the obtained allele profile. The test method according to claim 12, wherein the allele profile of rs11994717 is determined, and the risk of allergy to buckwheat components is predicted from the obtained allele profile. The test method according to claim 12 or 13, comprising predicting that when the base of rs11994717 is A, the risk of developing allergy to buckwheat components is high. The probe for use in the test method according to claim 1, which has a sequence of 15 bases or more including the 61st base in a base sequence selected from SEQ ID NOs: 1 to 3 and 8, or a complementary sequence thereof. The probe for use in the test method according to claim 7, wherein the base sequence of SEQ ID NO: 4 or 5 has a sequence of 15 bases or more including the base at position 61, or a complementary sequence thereof. The probe for use in the test method according to claim 7, wherein the base sequence of SEQ ID NO: 6 has a sequence of 15 bases or more including the base of the 56th base number or a complementary sequence thereof. The probe for use in the test method according to claim 12, wherein the base sequence of SEQ ID NO: 7 has a sequence of 15 bases or more including the base at position 61, or a complementary sequence thereof. The primer used for the test | inspection method of Claim 1 which can amplify the area | region containing the base of the 61st base number in the base sequence chosen from sequence number 1 thru | or 3 and 8. The primer used for the test | inspection method of Claim 7 which can amplify the area | region containing the 61st base of base number in the base sequence of sequence number 4 or 5. The primer used for the test | inspection method of Claim 7 which can amplify the area | region containing the base of the 56th base number in the base sequence of sequence number 6. The primer used for the test | inspection method of Claim 12 which can amplify the area | region containing the base of the 61st base number in the base sequence of sequence number 7.