JP2019000017A - Method for identifying individuals with sensitivity or resistance to deamidated wheat protein allergy - Google Patents

Abstract

To provide useful indicators for determining sensitivity or resistance to deamidated wheat protein.SOLUTION: Disclosed is a detection method of sensitivity or resistance to a deamidated wheat protein comprising detecting HLA-DQ genotype of a nucleic acid sample collected from a subject, the detection of any genotype selected from the group consisting of HLA-DQB1*03:03, HLA-DQB1*03:02, HLA-DQB1*05:01, HLA-DQB1*06:09, and HLA-DQB1*02:01 being an indicator of sensitivity, and the detection of any genotype selected from the group consisting of HLA-DQB1*06:01, HLA-DQB1*03:01, HLA-DQB1*06:02, and HLA-DQB1*04:02 being an indicator of resistance.SELECTED DRAWING: None

Description

  The present invention relates to an index (marker) useful for testing sensitivity or resistance to deamidated wheat protein allergy and use thereof.

  The prevalence of food allergies has been reported to be about 5-10% for infants in Japan, about 5% for infants, and 1.5-3% for schoolchildren and beyond (Non-patent Document 1). Globally, food allergies have been on the rise in recent years, especially in developed countries, and it is an urgent task to deal with it because it suddenly develops one day even in adults who have never had any symptoms until now. Wheat is the third largest after food eggs and milk as the cause of food allergies (Non-patent Document 2), and is in an increasing trend as wheat consumption increases. Among wheat allergies, wheat-dependent exercise-induced anaphylaxis (WDEIA) is a serious disease that presents an anaphylactic shock by exercising after ingesting wheat-containing foods and can be life-threatening. In patients with WDEIA, an increase in specific IgE antibody level against ω-5 gliadin, one of the components of wheat allergen, is observed in many patients.

  Previous studies have suggested an IgE epitope (Non-Patent Document 4), but details on the mechanism by which it develops are not clear.

  Allergic diseases are multifactorial diseases that develop by involving multiple genes and environmental factors. In recent years, genome-wide association analysis (GWAS), a technique for identifying genes associated with diseases, has been performed by determining hundreds of thousands to millions of gene polymorphisms present on all chromosomes. Asthma, atopic It contributes to the identification of novel disease susceptibility genes for many multifactorial diseases such as dermatitis and autoimmune diseases. GWAS is a hypothesis-free experimental technique and has recently been actively used to identify disease susceptibility genes for multifactorial diseases such as diabetes and cancer. Use hundreds of thousands of SNPs covering the entire human genome to statistically determine whether there is a difference in the frequency of polymorphisms between patients (cases) and individuals not affected by the disease (controls) To test. There are many GWAS reports on asthma and atopic dermatitis, but for food allergies, Hong et al. Conducted a GWAS for food allergies in 2015, and reported the relationship between HLA-DR and DQ SNP and peanut allergy. (Non-patent paper 5) and the same 2017 peanut allergy paper (non-patent document 6).

  There are various intolerances to wheat, such as celiac disease, wheat allergy, non-celiac gluten sensitivity, but the mechanism of its development has not been clearly elucidated. Particularly in Europe and the United States, the prevalence of celiac disease and non-celiac gluten sensitivity is higher than in Japan, which is a social problem. Dietary therapy for gluten contained in wheat and gluten-free diet are generally penetrating in Europe and the United States, and have been attracting attention in Japan in recent years. With regard to these intolerances, it is difficult to notice that they are intolerant to wheat because of various symptoms such as gastrointestinal symptoms, fatigue, and headaches.

  In recent years, cases of severe wheat allergy caused by ingestion of wheat have been reported in Japan when using hydrolyzed wheat-containing soap (Non-Patent Document 3), which has become a major social problem. This case is a type of wheat allergy that is completely different from the above WDEIA due to transdermal sensitization caused by the use of the soap, and has attracted worldwide attention. The causal wheat-derived protein is the trade name “Glupearl 19S”, and Western blotting using serum IgE antibody collected from patients against the protein also shows reactivity different from WDEIA. The wheat-derived product “Glupearl 19S” is a product obtained by hydrolyzing and deamidating wheat protein with an enzyme, hydrochloric acid or the like, and has high moisture retention and excellent water solubility. Shampoo, food, etc.). In this study, it is considered that the wheat-derived product has acquired antigenicity due to deamidation by hydrochloric acid treatment at high temperature (Non-patent Document 7).

Food Allergy Guidelines 2012 2008 Ministry of Health, Labor and Welfare Science Research “Study on the prevention of onset, progression and severity of allergic diseases” Fukutomi Y. et al. Jpn J Allergol 2009; 58: 1325 Yokooji T. et al. Allergol Int 2013; 62: 435-45 Hong X. et al. Nat Commun, 2015 Feb 24; 6: 6304 Martino DJ et al. Clin Exp Allergy. 2017 Feb; 47 (2): 217-223. Masashi N. et al. Contact Dermatitis 2016; 74: 346-352

  The above allergy cases reaffirm the importance of transdermal sensitization in food allergies. Hereinafter, allergy developed in this patient group is referred to as immediate-type wheat allergy caused by a specific HWP (HWP-IWA). It should be noted that the incidence rate (about 0.05% of deamidated wheat-containing soap users) is not high relative to the number of products sold and used in general, and the wheat-derived products are used in the same way. It is also a feature of this case that there were those who stayed with mild allergic symptoms and those who did not complain of allergic symptoms. (Yagami A. et al., Outbreak of immediate-type hydrolyzed wheat protein allergy due to a facial soap in Japan., Journal of Allergy and Clinical Immunology.in press)

In this case, the inventors considered that the incidence was not high, and that some of the users developed, suggesting the presence of a genetic predisposition that defines the onset. If the genetic susceptibility or resistance of each individual can be determined, preventive measures can be taken to prevent their onset (for example, alerting sensitive individuals in advance), dietary restrictions (gluten Determining the need for free food based on objective indicators (eg, actively recommending dietary restrictions to sensitive individuals, while requiring dietary restrictions to highly resistant individuals) Presenting a low degree of sexuality), etc., and can bring great benefits in terms of preventive medicine, medical economy, and quality of life.
Then, this invention makes it a subject to find out a useful index | index for determination of the sensitivity or resistance with respect to a deamidated wheat protein among allergy onset cases, and to aim at the utilization / application.

  The present inventors aimed at elucidating the pathology of severe wheat allergy by skin sensitization and establishing a preventive method, and confirmed food allergy with Gulpearl 19S having deamidated wheat protein (details are described in the Examples section below). A genome-wide association analysis was performed using the genomic DNA of the patient (see Fig. 4) and detailed genotyping was performed.

As a result of detailed examination, the present inventors have succeeded in identifying a gene (HLA genotype) exhibiting sensitivity or resistance present in the HLA class II region. Some of the genotypes identified were markedly sensitive. Since this disease was thought to have developed due to transcutaneous and transmucosal sensitization, as candidate gene analysis, 22 areas of 17 areas that have been reported to be associated with atopic dermatitis to date, and the barrier function of the skin The loss of filaggrin function associated with the decrease was also genotyped and analyzed for association, but no association was found with the disease, and no association between the disease and skin vulnerability could be confirmed.
The following invention is mainly based on the above results and considerations.
[1] A step of detecting an HLA-DQ genotype of a nucleic acid sample collected from a subject, comprising HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, The detection of any genotype selected from the group consisting of HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 is an indicator of sensitivity,
Any genotype selected from the group consisting of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02 may be detected Including steps that are indicative of resistance,
A test for sensitivity or resistance to deamidated wheat protein allergy.
[2] In the above step, the presence or absence of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 or HLA-DQB1 * 04: 02 in a nucleic acid sample collected from a subject The inspection method according to [1], including determining.
[3] From the group consisting of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 The method according to [1] or [2], further comprising determining the presence or absence of any genotype selected.
[4] The inspection method according to any one of [1] to [3], wherein sensitivity or resistance is determined according to the following criteria (a) or (b):
(a) If at least one of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 or HLA-DQB1 * 02: 01 is detected Sensitive;
(b) High resistance if HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 or HLA-DQB1 * 04: 02 is detected;
[5] Any selected from the group consisting of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 Or any resistance selected from the group consisting of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02 A reagent for testing sensitivity or resistance to deamidated wheat protein allergy, which comprises a nucleic acid for detecting a genotype.
[6] Nucleic acid reagent for detecting HLA-DQB1 * 03: 03, Nucleic acid reagent for detecting HLA-DQB1 * 03: 02, Nucleic acid reagent for detecting HLA-DQB1 * 05: 01, HLA- Nucleic acid reagent for detecting DQB1 * 06: 09, Nucleic acid reagent for detecting HLA-DQB1 * 02: 01, Nucleic acid reagent for detecting HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01 One or more nucleic acid reagents selected from the group consisting of a nucleic acid reagent for detecting HLA-DQB1 * 06: 02, and a nucleic acid reagent for detecting HLA-DQB1 * 04: 02 A kit for testing sensitivity or resistance to deamidated wheat protein allergy.
[7] Selected from the group consisting of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01. A susceptibility marker for deamidated wheat protein allergy consisting of any HLA genotype.
[8] Consists of any HLA genotype selected from the group consisting of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02 , A resistance marker against deamidated wheat protein allergy.
[9] The test method according to any one of [1] to [4], wherein a genotype in linkage disequilibrium is used instead of each genotype, the test reagent according to [5], Alternatively, the test kit according to [6].

Results of whole genome association analysis (GWAS) and principal component analysis (PCA).

1. Test Method for Sensitivity or Resistance to Deamidated Wheat Protein Allergy The first aspect of the present invention relates to a test method for sensitivity or resistance to deamidated wheat protein allergy. “Deamidated wheat” obtained by deamidation of wheat protein includes cosmetics (basic cosmetics, makeup cosmetics, hair care products, body care products) using its characteristics (mainly high moisturizing properties). ), Widely used as an additive for toiletries, foods, etc. Specific examples of products to which deamidated wheat is applied include soaps, face wash, body cleansers, shampoos, conditioners, treatments, hair packs, lotions, serums, emulsions, makeup bases, face creams, massages, Hand cream, mascara, eyeliner, hair conditioner, hair tonic, perfume, toothpaste, mouthwash, bathing agent. Note that general methods for producing deamidated wheat are roughly classified into an acid decomposition method (typically using hydrochloric acid) and an enzyme decomposition method (protease).

  The “deamidated wheat protein” in the present invention is not particularly limited as long as the deamidated wheat protein is included. For example, wheat proteins including those obtained by converting glutamine in wheat protein to glutamic acid correspond to “deamidated wheat protein”. For example, when wheat protein is treated with an acidolysis method (typically treated with hydrochloric acid at a high temperature), glutamine becomes glutamic acid. Such treatment is one of the causes of deamidated wheat protein allergy.

  In the present invention, “sensitivity” and “resistance” are terms representing the possibility of developing a deamidated wheat protein allergy. For example, if sensitivity is high, allergy is likely to develop, and if resistance is high, allergy is difficult to develop. However, factors other than “sensitivity” and “resistance” are thought to affect the onset of allergies. Therefore, it is usually diagnosed by a person having specialized knowledge such as a doctor whether or not allergies are actually developed.

  In the test method of the present invention, a step of detecting the HLA-DQ genotype of a nucleic acid sample collected from a subject is performed, and the sensitivity or resistance of the subject to deamidated wheat protein allergy is determined based on the detection result. judge.

In the determination in the present invention, the genotype (referred to as “susceptibility genotype”) associated with the susceptibility described in Table 1 and the genotype associated with resistance (“ "Resistant genotype") is used as an indicator. Detection of a susceptibility genotype is an indicator of sensitivity, and detection of a resistance genotype is an indicator of resistance. The determination according to the present invention can be automatically / mechanically performed without depending on the determination of a person having expertise such as a doctor or a laboratory technician, as is apparent from the determination index or reference.

  Sequence information regarding each genotype is registered in a public database (for example, IMGT / HLA database). HLA-DQB1 * 03: 03 etc. have multiple registration numbers (Accession Numbers). For example, the registration number of HLA-DQB1 * 03: 03: 02: 01 included in HLA-DQB1 * 03: 03 is The IPD-IMGT / HLA Acc No is HLA00629. That is, the genotypes in the present specification belong to HLA-DQB1 * 03: 03 (different, for example, HLA-DQB1 * 03: 03: 02: 01 included in HLA-DQB1 * 03: 03) Base sequences) and those with different notations (same amino acid sequences).

Instead of the genotypes (1) to (9) described above, genotypes that are in linkage disequilibrium with these may be detected. This is because a specific genotype and a genotype of linkage disequilibrium (r ² value> 0.8) show similar behavior and are considered to be useful as a marker for sensitivity or resistance. A genotype in a linkage disequilibrium state can be identified by analysis software such as ARLEQUIN (Molecular Ecology Resources. 10: 564-567.). Moreover, the genotype of linkage disequilibrium with a specific genotype is not particularly limited, but an existing database (for example, http://hla.or.jp/med/frequency_search/en/haplo/) may be referred to. it can. The genotypes showing linkage disequilibrium with respect to the genotypes (1) to (6) and (8) above are HLA-DQA1 genotype (allele), HLA-DRB1 genotype genotype (allele) or HLA- It is found in DPB1 genotype (allele). Specific examples include the genotypes listed in Table 2.

  Although not particularly limited, in the test method of the present invention, a specific 1 in the group of genotypes (5 susceptibility genotypes, 4 resistance genotypes and genotypes in linkage disequilibrium with them) One or more (eg 2, 3, 4 or 5 (all) in susceptible genotypes, or 3 or 4 (all) in resistant genotypes, or alternatively linked The detection operation may be performed by targeting two, three, four, or five or more of the genotypes in equilibrium. Moreover, you may decide to perform detection operation targeting either one of a susceptibility genotype or a resistance genotype. Preferably, a detection operation capable of detecting all of the group of genotypes is performed in order to increase test accuracy.

  In carrying out the test method of the present invention, first, a nucleic acid sample collected from a subject is prepared. In the present invention, a nucleic acid sample derived from a person (subject) who needs to determine sensitivity or resistance is used. As long as the nucleic acid sample includes the MHC class II region where the HLA-DQ gene is present, any length of genomic DNA (including fragments of the genomic DNA) or samples containing these DNAs can be nucleic acid. It can be used as a specimen, for example, a nucleic acid specimen using a known extraction method or purification method from a biological sample containing nucleic acids such as blood, saliva, lymph, urine, sweat, skin cells, mucosal cells, hair, etc. Can be prepared.

  The scope of application of the present invention is not limited to Japanese. In other words, there is no intention to exclude the application of the present invention to non-Japanese mongoloids and other races (such as Caucasian).

  The genotype detection method (analysis method) is not particularly limited. For example, PCR-RFLP method, PCR-SSCP method, PCR-SSO method, PCR-rSSO method, PCR-SSP method, PCR-SBT method, TaqMan (registered trademark, Roche Molecular Systems) -PCR method, Invader (registered trademark, Third Wave Technologies) method, method using FRET (Heller, Academic Press Inc, pp. 245-256 (1985), etc.), ASP-PCR method, MALDI-TOF / MS (matrix) method using primer extension method (Haff LA, Smirnov IP, Genome Res 7,378 (1997)), RCA method, method using DNA chip or microarray (Wang DG et al. al., Science 280, 1077 (1998), etc.), primer extension method, Southern blot hybridization method, dot hybridization method and the like can be employed. Furthermore, a polymorphic site characteristic of the genotype to be detected may be directly sequenced. Commercially available kits (for example, WAKFlow (registered trademark) HLA typing reagent (Yunaga Pharmaceutical)) can also be used. In addition, you may detect a genotype combining these methods arbitrarily.

  In the above methods, nucleic acids such as probes and primers according to each method (also referred to as “genotype detection nucleic acid” in the present invention) are used. Examples of the genotype detection nucleic acid used as a probe include a nucleic acid that specifically hybridizes to a chromosomal region (partial chromosomal region) containing a polymorphic position characteristic of the genotype to be detected. The length of the “partial chromosome region” here is, for example, 16 to 500 bases, preferably 18 to 200 bases. The nucleic acid preferably has a sequence complementary to a partial chromosomal region, but may have some mismatch as long as specific hybridization is not hindered. The degree of mismatch is 1 to 10, preferably 1 to 5, and more preferably 1 to 3. The term “specific hybridization” as used herein means hybridization to a target nucleic acid (partial chromosomal region) under hybridization conditions (preferably stringent conditions) usually employed for detection by a probe. On the other hand, it means that cross-hybridization does not occur significantly with other nucleic acids. A person skilled in the art can easily set hybridization conditions with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).

  An example of a nucleic acid for detecting a genotype used as a primer has a sequence complementary to a certain region (partial region of a chromosome) containing a polymorphic position characteristic of the genotype to be detected. Mention may be made of nucleic acids designed to specifically amplify DNA fragments containing the type part. As another example of a genotype detection nucleic acid used as a primer, a DNA fragment containing the polymorphic site only when the polymorphic site characteristic of the genotype to be detected is of the genotype to be detected And a nucleic acid set for detecting a genotype designed to specifically amplify. More specifically, a nucleic acid set for genotype detection designed to specifically amplify a DNA fragment containing a polymorphic site characteristic of the genotype to be detected, wherein the polymorphic site is the target of detection. A sense primer that specifically hybridizes to a certain region (partial region of the chromosome) containing the polymorphic site of the antisense strand of genotype, and a part of the sense strand (part of the polymorphic site) A genotype detection nucleic acid set comprising an antisense primer that specifically hybridizes to a neighboring region) can be exemplified. Here, the length of the DNA fragment to be amplified is appropriately set within a range suitable for the detection, and is, for example, 15 to 1000 bases long, preferably 20 to 500 bases long, more preferably 30 to 200 bases long. As in the case of the probe, the nucleic acid for detecting the genotype used as a primer is a template sequence as long as it can specifically hybridize to the amplification target (template) and amplify the target DNA fragment. There may be some mismatch. The degree of mismatch is 1 to 10, preferably 1 to 3, and more preferably 1 to 2.

  As the genotype detection nucleic acid (probe, primer), DNA, RNA, peptide nucleic acid (PNA), etc. are used as appropriate according to the detection method. The base length of the nucleic acid for genotype detection should just be the length by which the function is exhibited. When used as a probe, examples of the base length are 15 to 150 bases, preferably 16 to 100 bases. Moreover, when used as a primer, as an example of base length, it is 10-70 base length, Preferably it is 15-60 base length, More preferably, it is 20-50 base length.

  The nucleic acid (probe, primer) for genotype detection can be synthesized by a known method such as a phosphodiester method. Regarding the design and synthesis of nucleic acids for detecting genotypes, published documents (eg, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York, Current protocols in molecular biology (edited by Frederick M. Ausubel et al. 1987) ) Can be helpful.

  The nucleic acid for genotype detection in the present invention can be labeled with a labeling substance in advance. By using such a labeled genotype-detecting nucleic acid, for example, the genotype can be detected using the labeling amount of the amplification product as an index. In addition, if multiple types of primers designed to specifically amplify partial DNA regions in genes of each genotype that make up the polymorphism are labeled with different labeling substances, they can be detected from amplification products. The genotype of the nucleic acid sample can be discriminated by the labeling substance and the labeling amount. As a specific example of the detection method using such a labeled primer, two types of nucleic acid primers (allele-specific sense primers) that specifically hybridize to the sense strand of each genotype constituting the polymorphism are separately provided. Each fluorescent substance in the amplified product obtained by amplifying a partial DNA region containing a polymorphic site using these labeled primers and an antisense primer that specifically hybridizes to the antisense strand And a method for detecting a polymorphism by measuring the amount of the label. If the antisense primer here is labeled with, for example, biotin, amplification products can be separated using specific binding between biotin and avidin.

Labeling substances used for labeling nucleic acids for genotype detection include 7-AAD, Alexa Fluor (registered trademark) dye (for example, Alexa Fluor488, PE-Alexa Fluor700, APC-Alexa Fluor750, etc.), Cy (registered trademark) dye ( For example, Cy2, PE-Cy5, APC -Cy7 , etc.) DsRED, EGFP, EYFP, FITC , PerCP TM, R-Phycoerythrin, Propidium Iodide, AMCA, DAPI, ECFP, MethylCoumarin, Allophycocyanin ,, Rhodamine-123, Tetramethylrhodamine, Texas Red (Registered trademark), PE, Oregon Green, FAM ^™ , VIC (registered trademark), ABY (registered trademark), carboxyfluorescein, carboxyfluorescein diacetate, fluorescent dyes such as quantum dots, ³² P, ¹³¹ I, ¹²⁵ I, etc. Examples include radioisotopes and biotin. Labeling methods include 5 'end labeling using alkaline phosphatase and T4 polynucleotide kinase, 3' end labeling using T4 DNA polymerase and Klenow fragments, and nick trans. Shon method, random primer method (Molecular Cloning, Third Edition, Chapter 9, Cold Spring Harbor Laboratory Press, New York) and the like can be exemplified.

  Moreover, it can also be used in the state which fixed the nucleic acid for genotype detection to the insoluble support body. If the insoluble support used for immobilization is processed into a chip shape, a bead shape or the like, each genotype can be detected more easily using these immobilized nucleic acids.

As described above, in the present invention, the sensitivity or resistance of a subject to deamidated wheat protein allergy is determined using detection of a susceptible genotype or resistant genotype as an index. The following (a) and (b) can be adopted as specific determination criteria.
(a) If at least one of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 or HLA-DQB1 * 02: 01 is detected Assume that sensitivity is high.
(b) If HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 or HLA-DQB1 * 04: 02 is detected, the resistance is high.

  Note that typically, when one or more sensitive alleles are detected, the sensitivity becomes high. Similarly, it is typically assumed that the resistance increases when one or more resistive alleles are detected. In the case of heterozygotes of sensitive and resistant alleles, the risk is assessed to be no different from the general population.

  Information (detection result) provided by the detection method of the present invention is useful for determining the risk of developing deamidated wheat protein allergy. That is, if the detection result is used, it can be determined whether the subject has a high risk of developing deamidated wheat protein allergy or low risk of developing deamidated wheat protein allergy. Useful for discovery. It also helps improve the quality of life. Specifically, a subject who is determined to have a high risk of onset is diagnosed by a person with specialized knowledge such as a doctor with reference to the determination result, for example, alerting in advance or actively In particular, it is possible to prevent the occurrence of symptoms and reduce symptoms by recommending dietary restrictions (such as foods containing deamidated wheat and food intake / use restrictions). In addition, after onset, it becomes possible to prevent aggravation and determine a more appropriate treatment policy. On the other hand, for a subject who is determined to have a low risk of onset, for example, it can be shown that the need for dietary restrictions is low, and the quality of life can be improved.

2. Test Reagents and Kits Another aspect of the present invention provides reagents and kits used for testing sensitivity or resistance to deamidated wheat protein allergy. The reagent of the present invention (hereinafter also referred to as “nucleic acid reagent”) contains the nucleic acid for genotype detection described above. Nucleic acid reagents are specific susceptibility genotypes, namely HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 or HLA-DQB1 * 02: Genotype detection nucleic acid to detect 01, or specific resistance genotype, ie HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 or HLA-DQB1 * 04: 02 genotype detection nucleic acid to detect 02, or genotype detection nucleic acid to detect genotype in linkage disequilibrium with the above susceptibility genotype or resistance genotype, at least one kind of genotype detection Nucleic acid and / or more preferably two or more types of nucleic acids for genotype detection. The nucleic acid reagent of the present invention is appropriately designed according to the detection method (PCR-RFLP method, PCR-SSCP method, PCR-SSO method, PCR-rSSO method, PCR-SSP method, PCR-SBT method, etc.). The details of the genotype detection nucleic acid are as described above.

  The kit of the present invention contains one or more nucleic acid reagents as essential components. For example, if it is configured to include two or more types of nucleic acid reagents, the kit can detect a plurality of genotypes. Alternatively, a combination of five nucleic acid reagents, each corresponding to a specific susceptibility genotype, may be made into a kit that can detect all five susceptibility genotypes. Similarly, four types of nucleic acid reagents, each corresponding to a specific resistance genotype, may be combined into a kit capable of detecting all four types of resistance genotypes. In addition, five nucleic acid reagents each corresponding to a specific susceptibility genotype and four nucleic acid reagents each corresponding to a specific resistance genotype, including five susceptibility genotypes and four types It may be a kit that covers the resistance genotypes.

  In the kit of the present invention, in addition to the nucleic acid reagent of the present invention, reagents (DNA polymerase, restriction enzyme, buffer solution, coloring reagent, etc.) and containers necessary for using the nucleic acid reagent (that is, for genotype detection) An instrument or the like may be included in the kit of the present invention. Usually, an instruction manual is attached to the kit of the present invention.

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

<Subject>
Genomic DNA of HWP-IWA patients who developed wheat allergy after using soap containing deamidated wheat (Glupar 19S) with deamidated wheat protein was used. All patients meet the diagnostic criteria (http://www.fa.kyorin.co.jp/jsa/jsa_0528_09.pdf) of the “Special Committee on Safety of Protein Hydrolyzate in Cosmetics” shown below. ing.
[HWP-IWA patient selection criteria]
[Confirmed example]
A case satisfying all of the following 1 to 3 was determined.
1. Have used soap containing hydrolyzed wheat (Gulpearl 19S).
2. There was at least one clinical symptom:
2-1) Itching, eyelid edema, nasal discharge, wheal, etc. appeared within 30 minutes after several minutes using soap containing hydrolyzed wheat (Glupearl 19S).
2-2) Systemic symptoms such as itching, wheal, eyelid edema, nasal discharge, dyspnea, nausea, vomiting, abdominal pain, diarrhea, and blood pressure decreased within 4 hours after ingestion of wheat products.
3. The following test shows at least one positive.
3-1) The prick test is positive with Glupearl 19S 0.1% solution or thinner.
3-2) The presence of a specific IgE antibody against Glupearl 19S in blood can be proved by immunological methods such as dot blot, ELISA, and Western blot.
3-3) A basophil activation test using Glupearl 19S as an antigen is positive.
[Negative criteria]
4). Negative prick test with Glupearl 19S 0.1% solution

  Genomic DNA from 2700 people in the general population was used as a healthy control without wheat allergy. Seven ml of blood was collected from a patient's vein, and genomic DNA was extracted using QIAamp (registered trademark) DNA Blood Midi / Maxi Kit (Qiagen). The extracted genomic DNA was used as a nucleic acid sample.

<Relationship between SNP and HWP-IWA>
Whole genome association analysis (GWAS) was performed using the nucleic acid sample prepared above. The number of subjects analyzed in GWAS was 468 HWP-IWA patients. The illumina HumanOmniExpress Exome BeadChip was used as an array for genome-wide association analysis, and 532,838 SNPs were examined. In addition, Principal component Analysis (PCA) was performed. The analysis results are shown in FIG.

  From the results of GWAS and PCA, the strongest association with HWP-IWA was inferred from SNP located in the vicinity of the HLA-DQB1 locus. Therefore, the genotype analysis of the HLA-DQB1 allele was performed.

<Genotype analysis of HLA-DQB1 allele>
The HLA-DQ allele genotype analysis used the allele frequency of the general population of the HLA laboratory as a control (public data, Allele Frequency provided by HLA Laboratory) (http://hla.or.jp/med/frequency_search/ en / allele /). The number of subjects analyzed was 466 HWP-IWA patients. HLA gene typing was carried out by PCR-rSSO method using WAKFlow (registered trademark) HLA typing reagent (Yunaga Pharmaceutical) based on the instruction manual. The allele frequency of HWP-IWA patients was determined by the following calculation method (as a representative example, the calculation formula for examining the allele frequency of DQB1 * 0302 is shown).
(DQB1 * 0302 allele count) / (DQB1 * 0302 allele count + HLA-DQB1 allele count other than DQB1 * 0302)

  The frequency of each HLA-DQB1 allele was calculated and calculated by the same calculation method. The relationship between each HLA-DQB1 allele and HWP-IWA was evaluated by logistic regression test. In addition, the regression coefficient obtained by the logistic regression test was exponentially converted into the odds ratio.

  Tables 3 and 4 show the results of genotype analysis of the HLA-DQB1 allele. In addition, it shows that it is significantly related with HWP-IWA when satisfy | filling that q value obtained by the correction | amendment of the multiple test by the False Discovery Rate (FDR) method by Benjamini & Hochberg (BH) method is 0.05 or less.

  In this example, when the q value obtained by the multiple test correction by the FDR method by the BH method is 0.05 or less and the odds ratio is less than 1.0, it is determined that the wheat allergy is resistant. On the other hand, when the q value is 0.05 or less and the odds ratio is greater than 1.0, it is determined that the wheat allergy is sensitive. Further, when the odds ratio is 5.0 or more regardless of the q value, it is determined that the wheat allergy is sensitive.

CasealleleFreq：HWP-IWA患者のアレル頻度
ContalleleFreq：一般集団のアレル頻度(コントロールのアレル頻度)
OR：オッズ比
LCI: 95％信頼区間の下限値
UCI: 95％信頼区間の上限値
P値：ロジスティック回帰分析より得られるＰ値
q値：BH法によるFDR法による多重検定の補正により得られる値

CasealleleFreq: Allele frequency of HWP-IWA patients
ContalleleFreq: Allele frequency of general population (control allele frequency)
OR: odds ratio
LCI: Lower limit of 95% confidence interval
UCI: Upper limit of 95% confidence interval
P value: P value obtained from logistic regression analysis
q value: Value obtained by correcting multiple tests using the FDR method using the BH method

The frequency of HLA-DQB1 * 06: 01 alleles was significantly lower in HWP-IWA patients compared to the general population (Table 3, q value 6.02 × 10 ⁻⁹ , odds ratio 0.484). Furthermore, the percentage of genotypes in HLA-DQB1 * 06: 01 patients was not found in homozygous HWP-IWA patients, and the genotype frequency of HLA-DQB1 * 06: 01 also deviated from Hardy Weinberg equilibrium. (Table 4, P-value 0.0095 in Hardy-Weinberg equilibrium test). From the above, it can be seen that DQB1 * 06: 01 has particularly increased resistance in homozygotes.

  The frequency of HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02, and HLA-DQB1 * 04: 02 alleles was significantly lower in HWP-IWA patients compared to the general population. Therefore, HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02 were evaluated as having high resistance to deamidated wheat protein.

  DQB1 * 02: 01 does not meet the significance threshold after FDR correction in the multiple test by the FDR method by the BH method, but this is due to the fact that the allele frequency is low and the odds ratio exceeds 5. It is judged as a sensitive allele. Therefore, DQB1 * 03: 03, DQB1 * 03: 02, DQB1 * 05: 01, DQB1 * 06: 09 and DQB1 * 02: 01 were evaluated as having high sensitivity to deamidated wheat protein. In addition, comparing the general population with HWP-IWA patients, when there are multiple susceptibility / resistance alleles, if there are many resistance alleles, the risk of developing HWP-IWP tends to decrease. If there are many, the risk of developing HWP-IWP tends to increase, and in the case of heterozygotes of sensitive and resistant alleles, the risk is estimated to be the same as in the general population.

<About the owner of the evaluated allele>
In HWP-IWA patients, there were holders of alleles (DQB1 * 03: 03, DQB1 * 03: 02, DQB1 * 05: 01, DQB1 * 06: 09 and DQB1 * 02: 01) that were evaluated as highly sensitive. On the other hand, alleles (HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02, HLA-DQB1 * 04: 02) evaluated as highly resistant in HWP-IWA patients Some did not have it. In HWP-IWA patients, we identified those who had some resistant alleles and sensitive alleles. Allergic disease is a multifactorial disease, which is thought to be due to changes in genes other than “susceptibility” and “resistance” in this study, and environmental factors.

  As described above, it was possible to determine the risk of developing deamidated wheat protein allergy by using as an index (resistance marker / sensitivity marker) an allele evaluated to have high resistance / sensitivity. That is, by using the detection result, it is possible to determine whether the subject has a high risk of developing deamidated wheat protein allergy or a low risk of developing it. The determination result is useful for diagnosis by a doctor or other expert who has specialized knowledge and advice based on the diagnosis.

  According to the present invention, the sensitivity or resistance to deamidated wheat protein allergy can be examined based on an objective index. The present invention makes it possible to identify individuals with a high risk of developing deamidated wheat protein allergy and / or individuals with a low risk of development, and bring benefits in terms of preventive medicine, medical economy, quality of life, and the like.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (9)

A step of detecting an HLA-DQ genotype of a nucleic acid sample collected from a subject, which includes HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 selected from the group consisting of one of the genotypes detected is an indicator of sensitivity,
Any genotype selected from the group consisting of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02 may be detected. Including steps that are indicative of resistance,
A test for sensitivity or resistance to deamidated wheat protein allergy.   Determining whether or not HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 or HLA-DQB1 * 04: 02 is present in a nucleic acid sample collected from a subject. The test | inspection method of Claim 1 containing this.   The step is selected from the group consisting of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 The method according to claim 1, further comprising determining the presence or absence of any genotype. The test method according to any one of claims 1 to 3, wherein sensitivity or resistance is determined according to the following criteria (a) or (b):
(a) If at least one of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 or HLA-DQB1 * 02: 01 is detected Sensitive;
(b) Resistance is high if HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 or HLA-DQB1 * 04: 02 is detected.   Any sensitivity selected from the group consisting of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 Genotype or any resistant genotype selected from the group consisting of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02, A reagent for testing susceptibility or resistance to deamidated wheat protein allergy, which comprises a nucleic acid for detecting.   Nucleic acid reagent for detecting HLA-DQB1 * 03: 03, Nucleic acid reagent for detecting HLA-DQB1 * 03: 02, Nucleic acid reagent for detecting HLA-DQB1 * 05: 01, HLA-DQB1 * 06 : Nucleic acid reagent for detecting 09, Nucleic acid reagent for detecting HLA-DQB1 * 02: 01, Nucleic acid reagent for detecting HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01 A nucleic acid reagent for detecting HLA-DQB1 * 06: 02, and one or more nucleic acid reagents selected from the group consisting of a nucleic acid reagent for detecting HLA-DQB1 * 04: 02, A kit for testing sensitivity or resistance to deamidated wheat protein allergy.   Any one selected from the group consisting of HLA-DQB1 * 03: 03, HLA-DQB1 * 03: 02, HLA-DQB1 * 05: 01, HLA-DQB1 * 06: 09 and HLA-DQB1 * 02: 01 A susceptibility marker for deamidated wheat protein allergy consisting of HLA genotype.   Deamidation consisting of any HLA genotype selected from the group consisting of HLA-DQB1 * 06: 01, HLA-DQB1 * 03: 01, HLA-DQB1 * 06: 02 and HLA-DQB1 * 04: 02 Resistance marker against allergen wheat protein allergy.   The test method according to any one of claims 1 to 4, the test reagent according to claim 5, or the test reagent according to claim 5, wherein a genotype in linkage disequilibrium is used instead of each genotype. The test kit described.

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