JP2009192383A - Diagnosis and medical treatment of crohn's disease using ergothioneine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel means for diagnosing and treating a Crohn's disease by elucidating the participation of OCTN1 and ergothioneine in the morbid state or sideration of the Crohn's disease. <P>SOLUTION: The level of ergothioneine in a sample isolated from a subject is used as the index to diagnose the Crohn's disease. The affection risk of the Crohn's disease is diagnosed on the basis of the detection of a diplotype 1 in the sample isolated from the subject. A preventing or treating composition contains ergothioneine or its simulant as an effective component. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the diagnosis and treatment of Crohn's disease using ergothioneine. More specifically, the present invention relates to a method for evaluating the morbidity or likelihood of Crohn's disease based on ergothioneine levels and polymorphism analysis, and a therapeutic agent for Crohn's disease containing ergothioneine or an analog thereof as an active ingredient.

  Crohn's disease is an inflammatory bowel disease of unknown cause known as an intractable disease. In Japan, Crohn's disease is recognized as a target disease for specific disease treatment research projects, and the number of patients is increasing, mainly in the 10s and 30s. Regarding the cause of Crohn's disease, various factors such as genetic predisposition, virus, environmental factors, and dietary factors have been reported, but there is no established theory.

  Because the cause of the treatment of Crohn's disease is unknown, the basic treatment has not been established, and medical treatment such as dietary therapy (nutrient therapy) or drug therapy is often selected as symptomatic treatment . Surgical treatment is performed only when medical treatment is not desired, and even in that case, medical treatment is finally taken. The diagnosis of Crohn's disease is mainly visual inspection by an intestinal camera, but since there is no specific finding, the definitive diagnosis is currently performed by the elimination method, especially ulcerative colitis, Behcet's disease and intestinal tuberculosis. Is important.

  In 2004, a Canadian research group announced that OCTN1 polymorphism (L503F), one of the carnitine transporter OCTN family, was involved in the risk of developing Crohn's disease (Non-patent Document 1). However, in 2006, a domestic research group announced that this gene polymorphism was not found in Japanese patients with Crohn's disease (Non-Patent Document 2), and the involvement of OCTN1 in the development of Crohn's disease remained unclear after all. It has become.

  OCTN1 (SLC22A4) is a novel SLC transporter discovered by the inventors in 1997. It has been confirmed in an in vitro experimental system that essential nutrients, carnitine and organic cationic drugs are transport substrates. (Non-Patent Documents 3 and 4). The inventors identified OCTN2 (SLC22A5), a homologue of OCTN1, that its physiological substrate is carnitine, and elucidated that OCTN2 is a causative gene for systemic carnitine deficiency, a fatal disease. (Non-Patent Document 5). However, the physiological role of OCTN1 has not been clarified yet.

  In 2005, a German research group reported that ergothioneine was a good transport substrate for OCTN1 using an in vitro experimental system (Non-patent Document 6). Ergothioneine is a sulfur-containing amino acid with strong antioxidant power that is abundant in fungi and mushrooms, but mammals cannot synthesize ergothioneine themselves and take it exclusively by diet. Although a large amount of ergothioneine is stored in various organs of mammals, the physiological role of ergothioneine in vivo and its relationship with OCTN are not well understood.

Peltekova VD, Wintle RF, Rubin LA, Amos CI, Huang Q, Gu X, Newman B, Van Oene M, Cescon D, Greenberg G, Griffiths AM, St George-Hyslop PH, Siminovitch KA. Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet. 2004. 36 (5): 471-5. Tosa M, Negoro K, Kinouchi Y, Abe H, Nomura E, Takagi S, Aihara H, Oomori S, Sugimura M, Takahashi K, Hiwatashi N, Takahashi S, Shimosegawa T. Lack of association between IBD5 and Crohn's disease in Japanese patients demonstrates population-specific differences in inflammatory bowel disease. Scand J Gastroenterol. 2006. 41 (1): 48-53. Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, Sai Y, Tsuji A. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations. J Pharmacol Exp Ther. 1999. 289 (2): 768-73. Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku A, Shimane M, Tsuji A. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS Lett. 1997. 419 (1): 107-11 . Nezu J, Tamai I, Oku A, Ohashi R, Yabuuchi H, Hashimoto N, Nikaido H, Sai Y, Koizumi A, Shoji Y, Takada G, Matsuishi T, Yoshino M, Kato H, Ohura T, Tsujimoto G, Hayakawa J , Shimane M, Tsuji A. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet. 1999. 21 (1): 91-4. Grundemann D, Harlfinger S, Golz S, Geerts A, Lazar A, Berkels R, Jung N, Rubbert A, Schomig E. Discovery of the ergothioneine transporter.Proc Natl Acad Sci US A. 2005. 102 (14): 5256-61 .

  It is an object of the present invention to provide a novel means for diagnosis and treatment of Crohn's disease by elucidating the involvement of OCTN1 and ergothionein in the pathology and development of Crohn's disease.

  The inventors created an OCTN1 knockout mouse and performed a metabolome analysis using this. As a result, it was revealed that ergothioneine alone was drastically reduced in OCTN1 knockout mice, and that OCTN1 was involved in ergothioneine gastrointestinal absorption, organ migration, and reabsorption in the kidney. This indicates that OCTN1 is an important molecule for ergothioneine homeostasis in vivo, and OCTN1 dysfunction leads to a decrease in ergothioneine levels in individuals.

  Furthermore, as a result of small intestinal ischemia reperfusion experiments using OCTN1 knockout mice, OCTN1 knockout mice were more vulnerable to oxidative stress than wild-type mice, which was attributed to the lack of the antioxidant ergothioneine. Was speculated to be. Therefore, when the blood ergothioneine concentration in human Crohn's disease patients was measured, it was confirmed that it was significantly lower than that in healthy individuals. On the other hand, in patients with ulcerative colitis, the same inflammatory bowel disease, there was no significant difference between healthy and patients, and it was confirmed that the decrease in ergothioneine levels was specific to Crohn's disease patients .

  Furthermore, in order to clarify the direct relationship between the onset of Crohn's disease and OCTN1, we conducted OCTN1 gene polymorphism analysis. As a result, the existence of a diplotype that appears specifically and frequently only in Crohn's disease patients was found.

  Thus, the inventors have revealed for the first time the involvement of OCTN1 and ergothioneine in the pathogenesis and development of Crohn's disease. That is, the present invention provides a novel means for diagnosis and treatment of Crohn's disease using OCTN1 and ergothioneine.

  The first embodiment of the present invention is a method for determining whether or not the subject suffers from Crohn's disease using the ergothioneine level in a body fluid isolated from the subject as an index.

  In the method, when the ergothioneine level is significantly lower than that of a healthy person, it is determined that the subject is likely to have Crohn's disease.

  The ergothioneine level can be measured by an immunological method using an ergothioneine-specific antibody or by HPLC.

  Examples of the immunological methods include immunoprecipitation methods, solid-phase immunization methods including Western blotting, dot blotting, slot blotting, ELISA, and RIA, or variations thereof.

  The present invention also provides a kit for diagnosing Crohn's disease used for the method. This kit contains an ergothioneine specific antibody as an essential component.

  In a second embodiment of the present invention, in a sample containing genomic DNA isolated from a subject, the OCTN1 gene region SNPs rs272893 and rs272879 present in chromosome 5q31 are mutant, and the OCTN2 gene region SNPs rs13180169, rs13180186, This is a method for determining the morbidity risk of Crohn's disease of the subject by detecting diplotype 1 consisting of homozygous haplotype 1 in which rs13180043, rs13180295, rs2631365, rs274558, and rs274557 are all wild type.

  In the above method, by further detecting diplotype 20, it is possible to determine the risk of suffering from Crohn's disease by distinguishing it from ulcerative colitis. Diplotype 20 is haplotype 2 in which SNPs rs272893 and rs272879 in the OCTN1 gene region present on chromosome 5q31 are wild-type, and SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, and rs274557 in the OCTN2 gene region are all mutant types. The SNPs rs272893 and rs272879 in the OCTN1 gene region are mutated, and the SNPs rs2631365 in the OCTN2 gene region are heterozygous for haplotype 11.

  The diplotype is RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturant concentration gradient gel electrophoresis method, RNase A cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method , MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele specific hybridization method, padlock probe method, UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, ECA method, etc. Can be detected.

The present invention also provides a kit for determining the risk of suffering from Crohn's disease used for the above method. The kit includes at least one selected from the following 1) to 3):
1) A continuous polynucleotide having a length of 16 bases or more including SNPs rs460271, rs398064, rs11568509, rs11568510, rs456392, rs11568500, rs3761659, rs270600, rs11568503, rs272893, rs2304081, rs272879, rs1050150, rs4646201, or rs1050152 on the OCTN1 gene region, Alternatively, in order to specifically amplify the polynucleotide that is complementary to a part of a continuous polynucleotide having a length of 16 bases or more including SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, or rs274557 on the OCTN2 gene region An oligonucleotide having a length of 15 to 30 bases or a labeled product thereof,
2) A continuous polynucleotide having a length of 16 to 500 bases including SNPs rs460271, rs398064, rs11568509, rs11568510, rs456392, rs11568500, rs3761659, rs270600, rs11568503, rs272893, rs2304081, rs272879, rs1050150, rs4646201, or rs1050152 on the OCTN1 gene region Or a continuous polynucleotide having a length of 16 to 500 bases containing SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, or rs274557 on the OCTN2 gene region (however, when the polynucleotide is RNA, The base symbol “t” should be read as “u”),
3) A solid-phased sample on which the polynucleotide of 2) above is immobilized.

  The third embodiment of the present invention is a composition for preventing or treating Crohn's disease containing ergothioneine or an analog thereof as an active ingredient. The composition is administered with the expectation of preventing the onset or improving the symptoms by improving the decrease in the level of ergothioneine that is noticeably observed in Crohn's disease.

  Conventionally, the diagnosis of Crohn's disease has been mainly visual inspection with an intestinal camera, and the treatment has mainly been symptomatic diet therapy, pharmacotherapy with immunosuppressants, and the like. According to the present invention, it is possible to easily and noninvasively diagnose Crohn's disease using a sample isolated from a subject. Further, by detecting a specific diplotype, the risk of developing Crohn's disease (risk of onset) can be evaluated before onset. Furthermore, according to the present invention, by administering (or ingesting) ergothioneine and its analogs, the low ergothioneine state can be improved, and inflammatory bowel diseases including Crohn's disease can be fundamentally prevented or treated. .

1. Definition 1.1 Crohn's disease Diseases of unknown cause that cause chronic inflammation and ulcers in the mucosa of the large and small intestine are collectively referred to as "Inflammatory Bowel Disease (IBD)". “Crohn's disease” according to the present invention is one of these inflammatory bowel diseases, and was first reported in 1932 by physicians such as Brill Bernard Crohn. Crohn's disease is found mainly in young people and is characterized by discontinuous inflammation and ulceration throughout the digestive tract from the oral cavity to the anus. In Crohn's disease, abdominal pain associated with the lesion, diarrhea, bloody stool, weight loss, etc. occur, and when it progresses, intestinal obstruction due to stenosis of the intestinal tract, perforation or fistula, abscess may occur.

  Although the cause of Crohn's disease has not been elucidated, diet and environmental factors are said to be involved in genetic predisposition. Therefore, the diagnosis of Crohn's disease is mainly visual inspection with an intestinal camera, and the treatment is mainly dietary therapy as symptomatic therapy or drug therapy with immunosuppressive agents.

1.2 Ergothioneine “Ergothioneine” according to the present invention is a kind of hydrophilic amino acid isolated from ergot for the first time in 1909 by MC Tanret. Ergothioneine is a thiol histidine betaine, which has the following structural formula in the crystalline state, but exists in solution as a thiol form and a tautomer.

  Ergothioneine has a strong antioxidant action and metal ion capture ability, and is biosynthesized by molds, mushrooms and mycobacteria, but mammals can take it only from the diet. Ergothioneine is widely used in cosmetics, health and beauty foods, etc. due to its strong antioxidant action. In addition to natural products extracted and purified from mushrooms, chemical synthetic products are also commercially available.

  Ergothioneine is abundantly accumulated in organs in mammals, but its essential role in vivo has not been fully elucidated.

1.3 OCTN
OCTN is a carnitine transporter, and OCTN1, OCTN2, OCTN3, etc. are known. A transporter is a membrane protein that exists in a biological membrane, and transports low-molecular compounds such as nutrients and drugs into and out of cells. The transporter is greatly divided into the ABC transporter that transports substrates by directly using ATP hydrolysis energy and the SLC transporter that transports substrates by using the electrochemical potential of inorganic ions (concentration gradient inside and outside cells). Separately, OCTN is classified as the latter SLC transporter.

  OCTN1 and OCTN2 have been cloned by the inventors, and as described above, OCTN2 uses carnitine as a physiological substrate, and its gene abnormality has been found to cause systemic carnitine deficiency. On the other hand, OCTN1 has been reported to use carnitine, organic cationic drugs, and ergothioneine as transport substrates in vitro, but its physiological function has not been elucidated.

  The inventors have found that OCTN1 dysfunction (or ataxia) is caused by a decrease in ergothioneine levels in an individual, making the individual vulnerable to oxidative stress such as small intestine oxidative stress, by research using OCTN1 knock-aus mice, It has been found that inflammatory bowel diseases such as Crohn's disease occur.

  All of the genes of the OCTN family are present on chromosome 5q31, but there are many immune-related genes in this region, and it is a genomic region closely related to inflammatory bowel disease. The OCTN1 and OCTN2 SNPs present in this region are shown in Tables 1 and 2 below.

  Among these SNPs, the TC haplotype is derived from two SNPs that are in linkage disequilibrium (exon 7-207th G mutation to C (IGR2222), 1672nd C mutation to T (IGR3002)). Although it has been reported that it is related to the risk of Crohn's disease in Westerners, this haplotype does not exist in Japanese (see above).

  _{NT_034772.5 is GenBank Accession Number of human chromosome 5 genome sequence}

  _{NT_034772.5 is GenBank Accession Number of human chromosome 5 genome sequence}

  In order to clarify the direct relationship between the risk of developing Crohn's disease and OCTN, the inventors conducted OCTN1 and OCTN2 gene polymorphism analysis. A new diplotype that appears frequently: diplotype 1 and a diplotype that rarely appears in Crohn's disease patients and healthy individuals and frequently appear in ulcerative colitis: diplotype 20 were found (FIGS. 8 and 11). ).

2. 2. Method for diagnosing Crohn's disease 2.1 Preparation of sample The test method of the present invention is performed noninvasively using tissue or body fluid collected from a subject, for example, peripheral blood. The blood is appropriately prepared according to the subsequent detection method after removing insoluble substances by performing high-speed centrifugation as necessary.

  As a sample for ELISA / RIA (or a modified method thereof), for example, the collected serum is used as it is or a sample diluted appropriately with a buffer. The sample for Western blot (for electrophoresis) is, for example, a sample buffer (Sigma) containing 2-mercaptoethanol for SDS-polyacrylamide gel electrophoresis by using a cell extract as it is or by appropriately diluting with a buffer. Used in combination with other products. As the sample for dot / slot blotting, for example, a collected cell extract itself or a solution diluted appropriately with a buffer solution is directly adsorbed on a membrane using a blotting apparatus or the like.

2.2 Measurement of ergothioneine level The ergothioneine level used as an index can be measured using an immunological method utilizing an antigen-antibody reaction or HPLC. Here, the “level” is not limited to the amount of ergothioneine, but also includes a titer (antibody titer, etc.) indicating this indirectly.

  Examples of immunological methods include immunoprecipitation, Western blotting, dot blotting, slot blotting, ELISA, and solid-phase immunization including RIA, or known modified methods in which these are modified ( Sandwich ELISA, the method described in US Patent No. 4202875, the method of Meager et al. (Meager A., Clin Exp Immunol. 2003 Apr, 132 (1), p128-36) and the like. That is, based on these methods, a specific antibody is used for antigen detection, and a specific antigen is used for antibody detection.

  Antibodies used in the immunological methods described above can be prepared according to known methods, or commercially available antibodies may be used. The antibody can be obtained by immunizing an animal with ergothionein as an antigen or a part thereof, and collecting and purifying the antibody produced in the animal body by a conventional method. Further, according to a known method (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p.365-367, 1980, Prenum Press, NY) Hybridomas can be established by fusing the antibody-producing cells and myeloma cells to produce monoclonal antibodies.

  The antigen used for detection or the antigen for producing the above antibody is ergothionein or a part thereof (epitope portion), or an arbitrary carrier (for example, keyhole limpet hemocyanin added at the N-terminus) added thereto. Derivatives can be mentioned.

  Anti-ergothioneine antibody can be directly labeled, or it can be detected as a primary antibody, and the primary antibody can be specifically recognized (recognizes an antibody derived from the animal from which the antibody was produced) in cooperation with a labeled secondary antibody. Used for.

Preferred examples of the type of label include an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation for binding an enzyme-labeled streptavidin to biotin of a secondary antibody is added), but is not limited thereto. Various types of pre-labeled antibodies (or streptavidin) are commercially available as labeled secondary antibodies (or labeled streptavidin). In the case of RIA, an antibody labeled with a radioisotope such as ¹²⁵ I is used, and the measurement is performed using a liquid scintillation counter or the like.

  By detecting the activity of these labeled enzymes, the expression level of the antigen is measured. In the case of labeling with alkaline phosphatase or horseradish peroxidase, a substrate that develops a color or a substrate that emits light is commercially available.

  When a substrate that develops color is used, it can be detected visually using Western blotting or dot / slot blotting. In the ELISA method, it is preferable to measure and quantify the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. In addition, by preparing a dilution series of the antigen used for antibody production described above, using this as a standard antigen sample and performing detection simultaneously with other samples, creating a standard curve in which the standard antigen concentration and measured values are plotted, It is also possible to quantify the antigen concentration in each sample.

  On the other hand, when a substrate that emits light is used, it can be detected by autoradiography using an X-ray film or an imaging plate or by taking a picture using an instant camera in Western blotting or dot / slot blotting. . Further, quantification using a densitometry, a molecular imager Fx system (manufactured by Bio-Rad) or the like is also possible. Furthermore, when a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).

  When using high performance liquid chromatogram (HPLC), a normal phase or reverse phase column is pumped with a sample containing ergothioneine to separate other impurities from ergothioneine, and then passed to a UV (ultraviolet) detector. Thus, ergothioneine can be quantified by measuring absorption at 268 nm, which is the absorption wavelength of ergothioneine, or in the vicinity thereof. In addition, it can be quantified by measuring ionized ergothioneine by installing a mass spectrometer (MS or MS / MS) instead of the UV detector.

2.3 Judgment Judgment is performed using the ergothioneine level contained in the sample as an index. That is, when a significant decrease (p <0.05) in D ergothioneine levels is observed in the subject's sample (peripheral blood (serum)) compared to healthy subjects, the subject has Crohn's disease. It can be determined that there is a high possibility of being.

2.4. The present invention also provides a kit for diagnosing Crohn's disease, which is capable of diagnosing the presence or absence of Crohn's disease in a subject. The kit of the present invention contains an anti-ergothioneine antibody as an essential component.
The origin of the ergothioneine antibody is not particularly limited as long as it can be used for human ergothioneine detection, but an anti-human ergothioneine antibody is desirable. The antibody may be labeled with an appropriate label (for example, enzyme label, radioactive label, fluorescent label, etc.), or may be appropriately modified with biotin or the like. In addition, the support may be immobilized on a suitable support, or the support may be included separately so that the support can be immobilized. Examples of such supports include synthetic resins capable of attaching proteins such as polyethylene, polypropylene, polybutylene, polystyrene, polymethacrylate, and polyacrylamide, glass, nitrocellulose, cellulose, and agarose supports, or gel-type supports. Can be used. Although the form of the support is not particularly limited, it is provided in the form of microspheres or microparticles such as beads (for example, “latex” beads), tubes (inner walls) such as microcentrifuge tubes, microtiter plates (wells) and the like.

  In addition to the above-described components, the kit of the present invention contains other elements necessary for the practice of the present invention, such as reagents for detecting the label, buffer for reaction, enzyme, substrate, etc., if necessary. May be.

3. Method for diagnosing Crohn's disease risk In order to clarify the direct relationship between the risk of Crohn's disease (risk of onset) and OCTN1, the inventors conducted OCTN1 gene polymorphism analysis. Found new diplotype 1 and diplotype 20 that appear 10 times more frequently than healthy individuals (FIG. 8). This diplotype 1 is a mutation of SNPs rs272893 (I306T) with OCTN1 amino acid substitution and rs272879 (T394T) without amino acid substitution and OCTN2 SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, rs274557 are all wild type Haplotype 1 homozygote consisting of In addition, the inventors found diplotype 20 different from diplotype 1 and analyzed this diplotype 20 in combination with diplotype 1 to distinguish the risk of developing Crohn's disease from ulcerative colitis. I found out that I can do it. By the way, diplotype 20 is haplotype 2 in which OCTN1 SNPs rs272893 (I306T) and rs272879 (T394T) are wild type, and OCTN2 SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, and rs274557 are all mutant types. OCTN1 rs272893 (I306T) and rs272879 (T394T) are mutants, and OCTN2 rs2631365 (L95L) is a heterozygote of haplotype 11 (FIG. 11).

3.1 Preparation of Sample Containing Genomic DNA “Sample containing genomic DNA” used in the method of the present invention refers to any cell (excluding germ cells), tissue, organ, etc. isolated from subjects and clinical specimens. Prepared as a material. As the material, leukocytes or mononuclear cells separated from cells of the oral mucosa and peripheral blood are suitable. These materials are isolated according to methods commonly used in clinical laboratory tests.

  For example, when leukocytes are used as a material, leukocytes are first separated from peripheral blood isolated from a subject according to a conventional method. Subsequently, proteinase K and sodium dodecyl sulfate (SDS) are added to the obtained white blood cells to degrade and denature the protein, and then phenol / chloroform extraction is performed to obtain genomic DNA (including RNA). RNA can be removed by RNase as necessary. However, the extraction of genomic DNA is not limited to the method described above, and is a method well known in the art (for example, Sambrook, J. et al. (1989): “Molecular Cloning: A Laboratory Manual (2nd Ed.)”). Cold Spring Harbor Laboratory, NY) or a commercially available DNA extraction kit may be used.

3.2 Detection of gene polymorphism Next, a novel gene polymorphism that is highly related to Crohn's disease or ulcerative colitis, which has been elucidated by the present inventors, is detected from the obtained sample containing human genomic DNA. The following methods can be used as gene polymorphism detection methods, but are not limited thereto.

(1) RFLP (restriction fragment length polymorphism) method
(2) PCR-SSCP method (single-stranded DNA conformation polymorphism analysis) [Biotechniques, 16,296-297 (1994), Biotechniques, 21, 510-514 (1996)]
(3) ASO (Allele Specific Oligonucleotide) hybridization method [Clin. Chim. Acta, 189, 153-157 (1990)]
(4) Direct sequencing method (Biotechniques, 11, 246-249 (1991))
(5) ARMS (Amplification Refracting Mutation System) method [Nuc. Acids. Res., 19, 3561-3567 (1991), Nuc. Acids. Res., 20,4831-4837 (1992)]
(6) Denaturing Gradient Gel Electrophoresis (hereinafter referred to as “DGGE”) method [Biotechniqus, 27, 1016-1018 (1999)]
(7) RNase A cleavage method [DNA Cell. Biol., 14, 87-94 (1995)]
(8) Chemical cutting method (Biotechniques, 21, 216-218 (1996))
(9) DOL (Dye-labeled Oligonucleotide Ligation) method [Genome Res., 8, 549-556 (1998)]. (10) TaqMan PCR method [Genet. Anal., 14, 143-149 (1999), J. Clin. Microbiol., 34, 2933-2936 (1996)]
(11) Invader method (Science, 5109, 778-783 (1993), J. Biol. Chem., 30, 21387-21394 (1999), Nat. Biotechnol., 17, 292-296 (1999))
(12) MALDI-TOF / MS (Matrix Assisted Laser Desorption-time of Flight / Mass Spectrometry) method [Genome Res., 7, 378-388 (1997), Eur. J. Clin. Chem. Clin. Biochem., 35, 545-548 (1997))
(13) TDI (Template-directed Dye-terminator Incorporation) method [Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)]
(14) Molecular Beacons method [Nat. Biotechnol., 1, p49-53 (1998), Gene medicine, 4, p46-48 (2000)]
(15) Dynamic Allele-Specific Hybridization (DASH) [Nat. Biotechnol., 1, p87-88, (1999), Gene Medicine, 4, p47-48 (2000)]
(16) Padlock Probe method [Nat. Genet., 3, p225-232 (1998), Gene medicine, 4, p50-51 (2000)]
(17) UCAN method [see Takara Shuzo Co., Ltd. website (http://www.takara.co.jp)]
(18) DNA chip or DNA microarray
(19) ECA method [Anal. Chem., 72, p1334-1341, 2000]

  The above are typical polymorphism detection methods, but are not limited to these, and other known gene polymorphism detection methods can be used for the determination method of the present invention. Moreover, in the method of this invention, these gene polymorphism detection methods may be used independently, or may be used in combination of 2 or more. As one of the preferred embodiments of the present invention, a determination method using a direct sequence method is shown in the examples described later.

3.3 Analysis and determination of detection results According to the method of the present invention, if diplotype 1 is detected as a result of examination of genetic polymorphism in a sample containing isolated human genomic DNA, the subject is diagnosed with Crohn's disease or ulcer. It can be determined that the risk of developing ulcerative colitis is relatively high. On the other hand, when diplotype 1 is detected but diplotype 20 is not detected, it can be determined that the subject has a relatively high risk of developing Crohn's disease (not ulcerative colitis). . Thus, by combining the two diplotypes, it is possible to evaluate with high accuracy the risk of suffering from Crohn's disease that has been difficult to differentiate from ulcerative colitis.

3.4 Kit for diagnosing Crohn's disease risk In the determination method of the present invention, in order to detect a gene polymorphism associated with the onset of Crohn's disease or ulcerative colitis, A probe or a solid-phased sample for detecting a nearby sequence, or a primer for specifically amplifying the sequence is used. These probes, primers and immobilized samples are useful as reagents or kits for carrying out the determination method of the present invention.

  That is, the present invention provides a kit for diagnosing a Crohn's disease risk comprising a probe, a primer and a solid phase sample for detecting a gene polymorphism on the OCTN1 gene, and at least one of these.

(1) Probe The probe used in the method of the present invention is a polynucleotide for specifically hybridizing to a site containing SNPs (single nucleotide polymorphism) on the OCTN1 or OCTN2 gene region and detecting the polymorphic site It is a probe.

  Such probes are 16-500 bases in length, including SNPs rs460271, rs398064, rs11568509, rs11568510, rs456392, rs11568500, rs3761659, rs270600, rs11568503, rs272893, rs2304081, rs272879, rs1050150, rs4646201, or rs1050152 on the OCTN1 gene region A continuous polynucleotide or a continuous polynucleotide having a length of 16 to 500 bases including SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, or rs274557 on the OCTN2 gene region (provided that the polynucleotide is RNA, The base symbol “t” in the sequence listing shall be read as “u”).

(2) Primer The primer used in the method of the present invention is an oligonucleotide primer for specifically amplifying a site containing SNPs (single nucleotide polymorphism) on the OCTN1 or OCTN2 gene region.

  Such primers are continuous over 16 bases in length, including SNPs rs460271, rs398064, rs11568509, rs11568510, rs456392, rs11568500, rs3761659, rs270600, rs11568503, rs272893, rs2304081, rs272879, rs1050150, rs4646201, or rs1050152 on the OCTN1 gene region Or a portion of a continuous polynucleotide having a length of 16 bases or more including SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, or rs274557 on the OCTN2 gene region, and the polynucleotide is specific. It is designed as an oligonucleotide having a length of 15 to 30 bases, preferably about 18 to 25 bases.

  The length of the polynucleotide to be amplified is appropriately set according to the detection method used, but is generally 16 to 1000 bases, preferably 20 to 500 bases, more preferably 20 to 200 bases .

  From the above, a polynucleotide (or oligonucleotide) consisting of a continuous nucleotide sequence of 15 bases or more contained in the sequence near the polymorphic site can be used as the primer or probe.

  The primer or probe of the present invention includes those to which an appropriate label for detection, for example, a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin or the like is added at the end of the sequence. .

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for quantification and detection of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ', 7'-hexachloro-6-carboxyfluorescein (green fluorescent dye), fluorescein, NED (Applied Biosystems brand name, yellow fluorescent dye), or 6-FAM (Applied Biosystems brand name, yellow green) Fluorescent dye), rhodamine or derivatives thereof (for example, tetramethylrhodamine (TMR)) and the like, but are not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one among known labeling methods can be used [see Nature Biotechnology, 14, p303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).

  In addition, the primer of the present invention includes those to which a linker sequence for polymorphism detection is added at the end. Examples of such a linker sequence include a flap (a sequence that is completely unrelated to the sequence near the polymorphism) added to the 5 ′ end of the oligonucleotide used in the invader method described above.

  In the present specification, “polynucleotide” is synonymous with nucleic acid and includes both DNA and RNA. Further, it may be double-stranded or single-stranded, and therefore, a polynucleotide having a certain sequence always includes a polynucleotide having a complementary sequence thereto (the same applies to oligonucleotides). . Furthermore, when the polynucleotide is RNA, the base symbol “t” shown in the sequence listing shall be read as “u”.

  The polynucleotide that can be used as the primer or probe can be obtained by chemical synthesis based on a method well known in the art. Polynucleotides used as these probes or primers can be used as reagents for diagnosing the risk of Crohn's disease.

(3) Solid-phased sample The solid-phased sample of the present invention is obtained by fixing the polynucleotide used as the probe described in (1) to a solid phase such as a glass plate, nylon membrane, microbead, silicon chip, or capillary. It is produced by. The immobilization on the solid phase may be a method of placing a pre-synthesized polynucleotide on the solid phase, or a method of synthesizing the target polynucleotide on the solid phase. As the immobilization method, for example, in the case of a DNA microarray, a method suitable for a target solid-phased sample is known, such as using a commercially available spotter (manufactured by Amersham, etc.). Examples of such a solid phase sample include a gene chip, a cDNA microarray, an oligo array, a membrane filter, and the like.

(4) Kit The kit of the present invention comprises at least one selected from the polynucleotide that can be used as the probe or primer and a solid-phased sample. In addition to the above components, the kit of the present invention appropriately includes other reagents and instruments necessary for carrying out the determination method of the present invention, such as hybridization, probe labeling, and detection of label bodies, as necessary. Also good.

  According to the present invention, for the subjects who have been found from the above criteria to have a relatively high risk of developing Crohn's disease or ulcerative colitis (morbidity risk), this is notified in advance and Crohn's disease or ulcer Measures can be taken to prevent the development of ulcerative colitis. Therefore, the present invention is extremely useful as a test method for preventing Crohn's disease or ulcerative colitis.

4). Composition for preventing or treating Crohn's disease In the present invention, an ergothioneine analog is a part of the basic structure of ergothioneine (may be a plurality of positions) substituted with other atomic groups or the like. Or a compound having a structure different from the basic structure at least partially by modification such as addition of another molecule.

  Representative examples of the analog in the present invention include derivatives in which a part of ergothioneine is substituted with another atom or atomic group. Examples of other atoms or atomic groups herein include hydroxyl groups, halogens (fluorine, chlorine, bromine, iodine, etc.), alkyl groups (methyl groups, ethyl groups, n-propyl groups, isopropyl groups, etc.), hydroxyalkyl groups ( Examples thereof include hydroxymethyl group, hydroxyethyl group, etc., alkoxy groups (methoxy group, ethoxy group, etc.), acyl groups (formyl group, acetyl group, malonyl group, benzoyl group, etc.) and the like.

  The ergothioneine analogs of the present invention include those whose functional group is protected by a suitable protecting group. As the protecting group used for such a purpose, an acyl group, an alkyl group, a monosaccharide, an oligosaccharide, a polysaccharide and the like can be used. Such a protecting group is linked by an amide bond, an ester bond, a urethane bond, a urea bond, or the like according to the peptide site to which the protecting group is bound, the kind of the protecting group to be used, or the like.

  Further examples of the ergothioneine analog of the present invention include those that have been modified by the addition of sugar chains. In addition, various derivatives classified into alkylamine, alkylamide, sulfinyl, sulfonylamide, halide, amide, aminoalcohol, ester, aminoaldehyde, etc. by replacing the N-terminal or C-terminal with other atoms or the like are also included in the present invention. Of ergothioneine analogues. The various modification methods described above can be combined as appropriate.

  Specific examples of the ergothioneine analog include stachydrine and betonicine.

  The ergothioneine of the present invention or an analog thereof includes a salt of ergothioneine or a salt of the ergothioneine analog or a hydrate thereof. The salt of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and is a salt (inorganic acid salt) with hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, boric acid or the like, formic acid, acetic acid, lactic acid, fumaric acid Examples thereof include salts (organic acid salts) with maleic acid, tartaric acid, citric acid and the like. These salts can be prepared by conventional means.

  Examples of the preventive or therapeutic agent for ulcerative colorectal disease in the present invention include pharmaceutical preparations and dietary supplements. Examples of dietary supplements include oral preparations that have the same shape as pharmaceuticals but do not belong to pharmaceuticals under the Pharmaceutical Affairs Law, or those obtained by adding the active ingredient of the present invention to foods.

  That is, the present invention corresponds to a pharmaceutical composition or dietary supplement nutrition composition containing ergothioneine or an analog thereof as an active ingredient.

  In the present invention, the pharmaceutical preparation means a carrier usually used as a pharmaceutical preparation base for the active ingredient of the present invention, such as an excipient, a disintegrant, a lubricant, a buffering agent, a binder, an emulsifier, a suspending agent, and a soothing agent. Represents additives, stabilizers, preservatives, preservatives, physiological saline, etc., and dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and Suppositories and the like can be mentioned. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the binder, pullulan, gum arabic, gelatin, starch or the like can be used. As the lubricant, magnesium stearate, methylcellulose, or magnesium silicate can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used.

  The dietary supplement oral preparation in the present invention is a carrier that is usually used as a base for a nutritional food preparation for the active ingredient of the present invention, such as an excipient, a disintegrant, an emulsifier, a stabilizer, a lubricant, a buffer, a fragrance. The dosage form includes tablets, powders, fine granules, granules, and capsules.

  Examples of the dietary supplement in which the active ingredient of the present invention is added to foods include nutritional drinks, soft drinks, and jelly produced by a conventional method using the active ingredient of the present invention.

  Ergothioneine can be produced by extraction and purification from molds, mushrooms and the like containing the ergothioneine by a known method. Alternatively, it can be chemically synthesized by a known method. Such ergothioneine (both natural and synthetic) is already on the market and is readily available.

  A pharmaceutical preparation or oral dietary supplement preparation containing a modified analog as an active ingredient can be produced according to a conventional method using the obtained ergothioneine or an analog thereof as a main active ingredient.

  In the case of formulation, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, binders, lubricants, suspending agents, soothing agents, stabilizers, Preservatives, preservatives, physiological saline, etc.) can be incorporated into ergothioneine or its analogs to produce the desired formulation. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the binder, pullulan, gum arabic, gelatin, starch or the like can be used. As the lubricant, magnesium stearate, methylcellulose, or magnesium silicate can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  Using these preparation bases, desired dosage forms such as tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories can be produced by conventional methods.

  Moreover, ergothioneine or its analog can be added and nutritive foods, such as a nutrient drink, a soft drink, and a jelly, can be manufactured by a conventional method.

  The prophylactic and therapeutic agent for ulcerative colorectal disease comprising the ergothioneine of the present invention or an analog thereof as an active ingredient thus formulated is administered orally or parenterally (intravenous, intraarterial, subcutaneous, muscle, depending on the form) , Intraperitoneal injection, etc.). The content of the active ingredient (peptide or the like) in the drug of the present invention generally varies depending on the dosage form, but in the case of a liquid preparation such as an injection, it is about 0.001% to about 10% by weight, preferably 0.01% to about 3% Particularly preferably, it is 0.1% by weight to about 1% by weight, and in the case of solid preparations such as tablets, it is 0.1% by weight to about 90% by weight, preferably 1% by weight to about 50% by weight, particularly preferably 3% by weight to about 1% by weight. 30% by weight.

  According to the present invention, there is provided a prophylactic or therapeutic agent for ulcerative colon diseases including Crohn's disease, which comprises administering a preparation containing ergothioneine or an analog thereof as an active ingredient. The preventive or therapeutic agent of the present invention includes a step of administering a preparation containing ergothioneine or an analog thereof as an active ingredient to a living body. The administration route is not particularly limited, and examples thereof include oral, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, and transmucosal. The dose of the drug varies depending on symptoms, patient age, sex, weight, and the like, but those skilled in the art can appropriately set an appropriate dose. For example, when using a preparation containing ergothioneine or an analog thereof as an active ingredient, the amount of active ingredient per day for an adult (body weight of about 60 kg) is about 0.1 μg to about 3000 mg, preferably about 0.1 mg The dose can be set to be about 2000 mg, particularly preferably about 0.1 mg to about 1000 mg. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted.

  In the present invention, a method for preventing ulcerative colorectal disease, which comprises administering a preparation containing ergothioneine or an analog thereof as an active ingredient, refers to an ulcerative colorectal disease symptom caused by stress in an adult who has not developed symptoms of ulcerative colorectal disease. In order to prevent the onset of the disease, it is shown that a preparation containing a prophylactic ergothioneine or an analog thereof is taken as an active ingredient, and a method for treating ulcerative colon disease, which comprises administering a preparation containing an ergothioneine or an analog thereof as an active ingredient, and Indicates administration to a patient who has developed symptoms of ulcerative colorectal disease in order to suppress or improve their progression. In setting the administration schedule, it is possible to take into account the patient's medical condition and the duration of the drug effect.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail, referring an Example, this invention is not limited to these Examples.

1. Experimental Method 1.1 Reagents / Equipment and Experimental Animals Mice were raised and bred at the Experimental Animal Research Facility, Kanazawa University Interdisciplinary Science Experiment Center, according to the Kanazawa University Animal Experiment Guidelines. Expand high fidelity PCR system was purchased from Roche. LA Taq with GC buffer was purchased from TaKaRa. MinElute ^™ PCR purification kit was purchased from QIAGEN. PUREGENE buccal cell DNA purification kit was purchased from Gentra SYSTEMS. All the reagents other than the above used commercially available reagent grades.

1.2 Quantification of ergothioneine concentration in human blood 26 blood samples from Japanese Crohn's disease patients (19 men, 7 women), Japanese ulcerative colitis patients blood samples (9 men, 6 women), Nine Japanese control blood samples (8 males and 1 female) are from the Department of Homeostasis Control (Prof. Shuichi Kaneko and Naofumi Kagaya), Department of Environmental Medicine, Graduate School of Medicine, Kanazawa University. Provided by 22 volunteers (16 men and 6 women) at the Graduate School of Natural Sciences, Kanazawa University. A 10 μL blood sample collected with a heparin-treated vacuum blood collection tube was diluted 10-fold with sterilized MiliQ in which 50 ng / μL of cephaloridine (internal standard substance) was dissolved, and hemolyzed. 50 μL of 10-fold diluted blood was added to 100 μL of acetonitrile and 10 μL of sterile MiliQ, centrifuged at 15,000 rpm, 4 ° C. for 15 minutes, and the supernatant was measured by HPLC.

1.3 Measurement conditions by HPLC Column: Atlantis HILIC silica column 5.0 μm, 4.6 x 250 mm (Waters, USA)
Guard column: Atlantis HILIC silica guard column 5.0 μm, 4.6 x 10 mm (Waters, USA)
Mobile phase: Methanol / 100 mM ammonium formate (95/5)
Detection wavelength: 268 nm
Flow rate: 1.0 mL / min Sample injection volume: 20 μL
Pump: PU-2080 Plus (Jasco, Tokyo, Japan)
Detector: UV-2075 Plus (Jasco, Tokyo, Japan)
Autosampler: AS-2057 Plus (Jasco, Tokyo, Japan)
Column oven: Co-2065 Plus (Jasco, Tokyo, Japan)
Degasser: DG-2080-54 (Jasco, Tokyo, Japan)
Integrator: C-R7Ae Plus CHROMATOPAC (SHIMADZU)

1.4 Genomic DNA Samples Analysis using human genomic DNA samples is a research plan approved by the Human Genome / Gene Analysis Research Ethics Review Committee at the Graduate School of Medicine of Kanazawa University [problem name: Onset of inflammatory bowel disease. Study on drug response and drug transporter gene polymorphism ”(Examination number: No. 96, 2005), title of project“ Study on single nucleotide polymorphism of organic cation transporter gene in Japanese patients with Crohn's disease ”(Review) No. 97, 2005), Project name: “Study on single nucleotide polymorphisms of transporter genes and regulators in healthy Japanese individuals” (Examination number: No. 109, 2006)] did.

  Of the genomic DNA samples from Japanese patients with Crohn's disease, 63 samples are from Kanazawa University Graduate School of Medicine (Prof. Shuichi Kaneko and Dr. Kazuya Kitamura), and 193 samples are Graduate School of Medicine, Kyushu University (Professor Mamoru Wada; currently Nagasaki International University) Provided by Professor of Pharmacy).

  Of the genomic DNA samples from Japanese patients with ulcerative colitis, 15 samples were provided by the Graduate School of Medicine, Kanazawa University (Prof. Shuichi Kaneko and Assistant Professor Naofumi Kagaya), and 30 samples were provided by Prof. Mamoru Wada.

  Control samples were provided by Prof. Shuichi Kaneko and Naofumi Kagaya, 8 samples, 33 volunteers at the Graduate School of Natural Sciences, Kanazawa University, and 35 from the Faculty of Pharmaceutical Sciences, Kanazawa University.

1.5 Extraction of genomic DNA of control sample Oral mucosal cells were collected, and genomic DNA was extracted according to the standard protocol of PUREGENE buccal cell DNA purification kit (Gentra SYSTEMS).

1.6 Direct sequence
Primers specific to OCTN1 and OCTN2 were designed based on the NCBI database (http://www.ncbi.nlm.nih.gov/), and synthesis was requested from Fasmac (Kanagawa, Japan). The primer sequences of OCTN1 exon5, 7 and OCTN2 5'UTR, exon1, 4 are shown in Table 3.

  In PCR reactions using Expand High Fidelity (OCTN1 exon5, 7, OCTN2 exon4), the following reaction solutions were prepared.

  In a PCR reaction (OCTN2 5′UTR, exon1) using LA Taq with GC Buffer, the following reaction solution was prepared.

  Each 24 μL of the above reaction solution was divided into PCR tubes, 1 μL of template (5 ng / μL) was added, and the mixture was lightly centrifuged, and PCR was performed with a thermal cycler according to the following conditions.

Purification of DNA after the PCR reaction was performed using MinElute ^™ PCR purification kit (QIAGEN). A mixture obtained by adding 500 μL of buffer PB to the PCR reaction solution was applied to a Minuteute column, and the DNA was bound to the column and centrifuged for 1 minute. The flow-through solution was discarded, and 750 μL of Buffer PE was added to the column for washing, followed by centrifugation for 1 minute. The flow-through solution was discarded, and the mixture was further centrifuged for 1 minute in order to completely remove residual ethanol derived from Buffer PE. The Minelute column was placed on a new 1.5 mL tube, 20 μL of buffer EB was added to the center of the membrane surface of the column, allowed to stand for 1 minute, and then centrifuged for 1 minute. The flow-through solution was added again to the center of the membrane surface of the column, left for 1 minute, and then centrifuged for 1 minute.

  In the sequencing reaction using purified DNA as a template, the following reaction solution was prepared.

  The above reaction mixture was divided into 19 μL aliquots into PCR tubes, 1 μL of purified template DNA was added, and the mixture was lightly centrifuged, and PCR was performed with a thermal cycler according to the following conditions.

  2 μL of 3 M sodium acetate and 50 μL of 99.99% ethanol were added to the sequencing reaction solution, and centrifuged at 15,000 rpm for 20 minutes. The supernatant was removed, 100 μL of 70% ethanol was added, and the mixture that had been vortexed for several seconds was centrifuged at 15,000 rpm for 10 minutes. The supernatant was completely removed and ethanol was removed, and the sample was placed at 90 ° C. for 3 minutes and at 4 ° C. for 3 minutes, and then measured with an ABI PRISM 310 Genetic Analyzer.

1.7 Analysis of sequence data
In Genotype analysis, in each SNP registered in the NCBI database (http://www.ncbi.nlm.nih.gov/), the SNP that is the contig reference is the wild type, and other SNPs are used. It was defined as mutant. Further, nonsynonymous SNPs with amino acid mutations and synonymous SNPs without amino acid mutations are represented by amino acid sequence numbers and amino acid symbols. Other SNPs are represented by NCBI dbSNP ID numbers.

  In Haplotype and diplotype frequency analysis, frequency analysis of haplotype and diplotype was performed using SNPAlyze software ver. 6.0 (DYNACOM Co., Ltd) based on the obtained genotype data of each SNPs. SPSS software ver. 15.0 (SPSS Japan Inc.) was used for statistical analysis between disease-control and disease-disease of each haplotype and diplotype.

1.8 Intravenous administration test (mouse)
Mice fasted for 16-24 hours were anesthetized by intraperitoneal administration of 25 μL / body of somnopentyl and fixed on their back. The left and right jugular veins were exposed, and a bladder cannula was applied using a polyethylene tube (SP31), and then a drug solution ([ ³ H] ergothioneine 100 μg, [ ¹⁴ C] inulin 7 mg / 3 mL / kg body ) Was administered. 1, 5, 15, 30, 60, 90, 120, 180, 240 minutes after administration, collect approximately 30 μL of blood from the right jugular vein, place it in a heparin-coated 1.5 mL tube, and centrifuge at 10,000 rpm for 10 minutes. Later, 10 μL of plasma was collected. Further, after 15, 30, 60, 120, and 240 minutes, 200 μL of physiological saline was poured from the bladder cannula to collect urine. After blood collection for 240 minutes after administration, the head was decapitated and the pancreas, spleen, liver, intestine, kidney, testis, heart, thymus, lung, skin, muscle, femur, and brain were removed and washed with ice-cold saline. Then, the moisture was wiped off and weighed.

1.9 Oral administration test (mouse)
Mice fasted for 16-24 hours were anesthetized by intraperitoneal administration of 25 μL / body of somnopentyl and fixed on their back. The right jugular vein was exposed, and a bladder cannula was applied using a polyethylene tube (SP31), followed by an oral sonde ([ ³ H] ergothioneine 330 μg, [ ¹⁴ C] inulin 23 mg / 10 mL / kg body) was orally administered. At 5, 15, 30, 60, 90, 120, 180, 240 minutes after administration, blood was collected from the right jugular vein approximately 30 μL at a time, placed in a heparin-coated 1.5 mL tube, centrifuged at 10,000 rpm for 10 minutes, 10 μL of plasma was collected. Further, after 15, 30, 60, 120, and 240 minutes, 200 μL of physiological saline was poured from the bladder cannula to collect urine. After blood collection for 240 minutes after administration, decapitated, then removed pancreas, spleen, liver, intestine, kidney, testis, heart, thymus, lung, skin, muscle, femur, brain, ice-cold saline Then, the water was wiped off and weighed. The intestine was divided into three equal parts, and 5 mL of physiological saline was poured into each with a syringe, and the drug solution remaining in the intestinal lumen was collected.

1.10 Long-term oral administration test (mouse)
Mice fasted for 16-24 hours in metabolic cages were anesthetized by intraperitoneal administration of 25 μL / body of somnopentyl, and then a solution ([ ³ H] ergothioneine 330 μg / 10 mL / kg body) was administered using an oral sonde. Orally administered. One hour later, the tail vein was cut with a razor, and about 40 μL of blood was collected using a heparinized capillary tube (Fisher Scientific). After recovering from anesthesia, the animals were raised in metabolic cages, and food and water were freely consumed, and urine was collected. 1, 2, 3, 5, 7, 10, 14 days after administration, the metabolic cage was washed and urine was collected. 10 μL of blood was collected, the remaining blood was placed in a heparin-coated 1.5 mL tube, centrifuged at 10,000 rpm for 10 minutes, and 10 μL of plasma was collected. The collected urine was weighed and 0.5 mL was used for the measurement. 14 days after administration, decapitation, pancreas, spleen, liver, intestine, kidney, testis, heart, thymus, lungs, skin, muscles, femur, brain were removed, washed with ice-cold saline, and water Were wiped off and weighed. The intestine was divided into three equal parts, and 5 mL of physiological saline was poured into each with a syringe, and the drug solution remaining in the intestinal lumen was collected.

1.11 Radioactivity measurement of plasma, blood, urine and intestinal luminal collected fluid (mouse)
3 mL of clear sol I (nacalai tesque) was added to 10 μL of plasma. After diluting 10 μL of blood 1,000 times with distilled water and leaving it overnight at room temperature, 50 μL of H ₂ O ₂ was added to decolorize, and 3 mL of clear sol I was added. Clear Sol I 3 mL was added to the total amount of urine collected and 1 mL of the intestinal lumen recovery solution. After standing overnight, each radioactivity was measured with a liquid scintillation counter.

1.12 Measurement of organ radioactivity (mouse)
Each organ was solubilized by adding 2 mL of SOLUENE-350 (Packerd) and shaking at 50-60 ° C, 3 hr or 40 ° C, over night, 100 rpm. Add 600 μL of H ₂ O _{2 and} leave it for 1-2 hr to decolorize, neutralize by adding 200 μL of 5 M HCl, add 10 mL of clear sol I (nacalai tesque), and measure the radioactivity with a liquid scintillation counter did.

1.13 Determination of ergothioneine concentration in organs (mouse)
The mice were fasted overnight and then anesthetized with somnopentyl and fixed on the back. After collecting urine from the bladder and blood from the vena cava, the organs (liver, kidney, lung, heart, small intestine, large intestine, brain, spleen, pancreas, thymus, muscle, skin) are quickly removed and ice-cooled physiological saline Washed with water. Thereafter, each organ was cut into a size of about 0.050 to 0.15 g and the weight was measured. Four times as much sterilized distilled water was added to each weighed organ, and the organ was sufficiently crushed with an ultrasonic homogenizer microson XL-200 (Waken Pharmaceutical Co., Ltd.) on ice. For the liver, kidney, lung, heart, middle small intestine, lower small intestine, spleen and pancreas, the homogenate solution was further diluted 2-fold with sterile distilled water. A homogenate solution of 50 μL of each organ was added to 100 μL of acetonitrile and 10 μL of sterilized MiliQ, stirred, centrifuged at 15,000 rpm, 4 ° C. for 10 minutes, and the supernatant was measured by HPLC.
A blood sample (10 μL) was collected with a heparin-treated vacuum blood collection tube, and then diluted 10-fold with cephaloridine (internal standard) 50 ng / μL for hemolysis. 50 μL of 10-fold diluted blood was added to 100 μL of acetonitrile and 10 μL of sterile MiliQ, centrifuged at 15,000 rpm, 4 ° C. for 15 minutes, and the supernatant was measured by HPLC. The blood sample was centrifuged at 3,000 rpm at 4 ° C. for 10 minutes to separate red blood cells and plasma. 20 μL of erythrocytes were diluted 5 times with sterile MiliQ and hemolyzed. 20 μL of urine was diluted 5-fold with sterile MiliQ. Add 100 μL of acetonitrile, sterile MiliQ 10 μL, cephaloridine 250 ng / μL 10 μL, 5 times diluted erythrocytes, plasma, and 5 times diluted urine 50 μL each and centrifuge at 15,000 rpm at 4 ° C. for 15 minutes. The supernatant was measured by HPLC.

1.14 Small intestinal ischemia reperfusion (mouse)
Both mice were fasted for 1 day and then anesthetized by intraperitoneal administration of somnopentyl to expose the superior mesenteric artery, and the arterial clamp (bent, grooved, extremely small; Natsume Seisakusho, Tokyo, Japan) for 60 minutes I was ligated. Thereafter, reperfusion of the blood flow was started by slowly removing the clamp, and the survival time was determined. The p value of the survival curve of both mice was determined by Graphpad Prism 4. Thirty minutes after reperfusion, both mouse small intestine tissues were quickly removed and fixed by immersion in 4% paraformaldehyde / PBS at 4 ° C. for 4 hours. Thereafter, the tissue was immersed in order of ehhanol and xylene to embed paraffin, and a paraffin section having a thickness of 5 μm was prepared with a microtome. Paraffin sections were deparaffinized with xylene, immersed in ethanol and distilled water in that order, and stained with HE according to a conventional method. That is, the sections were immersed in a hemtoxyolin solution for 30-60 seconds and immersed in running water for 3 minutes. Next, it was immersed in eosin solution for 30-60 seconds, sealed with ethanol and xylene, and sections were observed using an optical microscope (Axiovert S 100 microscope; Carl Zeiss).

2. Experimental results 2.1 Role of OCTN1 in ergothioneine homeostasis Plasma, blood cells and organs were collected from normal mice and OCTN1 knockout mice, and the extract was analyzed by metabolomic analysis using a combination of capillary electrophoresis and mass spectrometry. As a result of simultaneous quantification, it was revealed that only ergothionein was drastically decreased in the body of OCTN1 knockout mice (FIG. 1). This result could be reproduced when similar analysis was performed using HPLC (FIG. 2).

In order to clarify the absorption, distribution, and excretion of ergothioneine, plasma and blood cell concentration, urinary excretion, and organ residual were measured after oral administration of [ ³ H] -labeled ergothioneine. in while ^[3 H] ergothioneine is maintained in plasma for two weeks, once plasma concentration at OCTN1 knockout mice were then rapidly reduced to what is raised (Figure 3). On the other hand, [ ³ H] ergothionein accumulated in blood cells in normal mice, but rapidly decreased in OCTN1 knockout mice (FIG. 3). This was consistent with the large renal excretion in the knockout mice (FIG. 4). Furthermore, the amount of [ ³ H] ergothioneine remaining in the gastrointestinal tract was slightly higher in OCTN1 knockout mice (FIG. 4). From the above results, it was revealed that OCTN1 is involved in the absorption of ergothioneine in the gastrointestinal tract, organ migration, and reabsorption in the kidney (FIGS. 3 and 4). From the above, it was revealed that OCTN1 is an important molecule for ergothioneine homeostasis in living organisms, and that OCTN1 dysfunction leads to a decrease in ergothioneine in individuals.

2.2 Relationship between OCTN1 and inflammatory stress
As a result of small intestinal ischemia reperfusion experiments using OCTN1 knockout mice, many mice die more quickly than wild type mice, and knockout mice are more vulnerable to oxidative stress. Became clear (FIGS. 5 and 6). This was speculated to be vulnerable to oxidative stress due to a deficiency of the antioxidant ergothioneine in OCTN1 knockout mice.

2.3 Relationship between Crohn's disease and ergothioneine When the blood ergothioneine concentration in Crohn's disease patients was measured, it was confirmed that it was significantly lower than that of healthy individuals (FIG. 7). On the other hand, ulcerative colitis patients with the same inflammatory bowel disease were not significantly different from healthy individuals. This result indicated that ergothioneine is specifically associated with Crohn's disease (FIG. 7).

2.4 Relationship between Crohn's disease and OCTN1 gene polymorphism
As a result of OCTN1 gene polymorphism analysis, diplotype 1 was found in which the OCTN1 gene mutation was homozygous (FIG. 8). There were a total of 12 people with this diplotype1, but 11 of them were Crohn's disease patients. Therefore, in Crohn's disease patients, this diplotype1 was more than 10 times more frequent than healthy individuals. It became clear that it appeared, and it was shown that the person with this diplotype 1 has an extremely high risk of developing Crohn's disease (FIG. 9).

  Diplotype1 is a combination of haplotype1 and haplotype1. In Haplotype1, OCTN1 I306T and T394T are mutants, and OCTN2 SNPs are all wild types (FIG. 10). Since ergothioneine is mainly transported by OCTN1, diplotype1, which is a combination of haplotype1 in which a mutation exists in OCTN1, suggests an association with a decrease in blood ergothioneine concentration in patients with Crohn's disease.

   Diplotype20 is a combination of haplotype2 and haplotype11. In Haplotype2, OCTN1 SNPs are all wild type, and OCTN2 SNPs are all mutant. On the other hand, haplotype11 is OC3061 I306T and T394T and OCTN2 L95L is mutant haplotype (FIG. 11).

  The frequency of diplotype1 and diplotype20 in Control, Crohn's disease, and ulcerative colitis, and a significant difference test between each group were performed. As a result, it was confirmed that diplotype1 appeared frequently in Crohn's disease patients and diplotype20 appeared frequently in ulcerative colitis patients (FIGS. 12 and 13). From this, it was shown that the onset risk of Crohn's disease and ulcerative colitis, which had been difficult to distinguish so far, can be determined separately by detecting diplotype1 and diplotype20.

3. Discussion As a result of this experiment, it was confirmed that OCTN1 and ergothionein are deeply involved as factors in the complex pathogenesis of Crohn's disease.

  The present invention provides novel findings regarding the pathophysiology and pathogenesis of Crohn's disease. The present invention enables fundamental diagnosis of Crohn's disease and differential diagnosis from other ulcerative colon diseases, and is extremely useful in the medical field.

FIG. 1 shows the results of metabolome analysis (the amount of ergothioneine at each site) of OCTN1 knockout mice and wild type mice. FIG. 2 shows the amount of ergothioneine at each site of OCTN1 knockout mice and wild type mice. FIG. 3 shows blood (right) and plasma (left) ergothioneine levels in OCTN1 knockout mice and wild type mice. FIG. 2 shows the intestinal residual amount (right) and urinary excretion (left) of ergothioneine in OCTN1 knockout mice and wild type mice. FIG. 5 shows the resistance of OCTN1 knockout mice and wild type mice to intestinal oxidative stress. FIG. 6 shows HE-stained images 30 minutes after the start of reperfusion after ischemia for 60 minutes in the small intestine of CTN1 knockout mice and wild-type mice. FIG. 7 shows blood ergothioneine levels in Crohn's disease patients, ulcerative colitis patients, and healthy individuals. FIG. 8 shows the structure of diplotype 1. FIG. 9 shows χ ² test results between healthy subjects and Crohn's disease patients. Figure 10 shows the haplotype composition of each SNPs (top) and the diplotype composition of each haplotype (Reference (NCBI) is in NCBI database (http://www.ncbi.nlm.nih.gov/)) In each registered SNPs, it means a sequence called contig reference, and this sequence was defined as a wild type in this analysis). FIG. 11 shows the structure of diplotype 20. FIG. 12 shows the frequency of diplotype 1 in Control, Crohn's disease, and ulcerative colitis, and the results of a significant difference test between each group. FIG. 13 shows the frequency of diplotype 20 in Control, Crohn's disease, and ulcerative colitis, and the results of a significant difference test between each group.

SEQ ID NO: 1-Primer SEQ ID NO: 2-Primer SEQ ID NO: 3-Primer SEQ ID NO: 4-Primer SEQ ID NO: 5-Primer SEQ ID NO: 6-Primer SEQ ID NO: 7-Primer SEQ ID NO: 9-Primer SEQ ID NO: 9-Primer SEQ ID NO: 10-Primer

Claims (10)

  A method for determining whether or not the subject suffers from Crohn's disease using ergothioneine level in a body fluid isolated from the subject as an index.   The method according to claim 1, wherein when the ergothioneine level is significantly lower than that of a healthy person, it is determined that the subject is likely to have Crohn's disease.   The method according to claim 1, wherein the ergothioneine level is measured by an immunological method using an ergothioneine-specific antibody or HPLC.   The immunological method is an immunoprecipitation method and a solid phase immunization method including Western blotting, dot blotting, slot blotting, ELISA, and RIA, or any of these methods. The method according to claim 3.   A kit for diagnosing Crohn's disease comprising an ergothioneine-specific antibody.   In a sample containing genomic DNA isolated from a subject, the SNPs rs272893 and rs272879 of the OCTN1 gene region present on chromosome 5q31 are mutated and the SNPs of the OCTN2 gene region rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, rs274557 A method for determining the risk of suffering from Crohn's disease in a subject by detecting diplotype 1 consisting of homozygous haplotype 1 that is all wild type.   Furthermore, SNPs rs272893 and rs272879 in the OCTN1 gene region present on chromosome 5q31 are wild type, and SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, and rs274557 in the OCTN2 gene region are all mutated, and OCTN1 SNPs rs272893 and rs272879 in the gene region are mutated and SNPs rs2631365 in the OCTN2 gene region are mutated to detect diplotype 20 consisting of heterozygotes of haplotype 11 to distinguish it from ulcerative colitis and Crohn's disease The method according to claim 6, wherein the risk of morbidity is determined.   Detection of diplotype is RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturant concentration gradient gel electrophoresis method, RNase A cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader Method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele specific hybridization method, padlock probe method, UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, and ECA method The method according to claim 6 or 7, wherein the method is carried out using one or more methods selected from the group consisting of: A kit for determining the risk of suffering from Crohn's disease, comprising at least one or more selected from the following 1) to 3):
1) A continuous polynucleotide having a length of 16 bases or more including SNPs rs460271, rs398064, rs11568509, rs11568510, rs456392, rs11568500, rs3761659, rs270600, rs11568503, rs272893, rs2304081, rs272879, rs1050150, rs4646201, or rs1050152 on the OCTN1 gene region, Alternatively, in order to specifically amplify the polynucleotide that is complementary to a part of a continuous polynucleotide having a length of 16 bases or more including SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, or rs274557 on the OCTN2 gene region An oligonucleotide having a length of 15 to 30 bases or a labeled product thereof,
2) A continuous polynucleotide having a length of 16 to 500 bases including SNPs rs460271, rs398064, rs11568509, rs11568510, rs456392, rs11568500, rs3761659, rs270600, rs11568503, rs272893, rs2304081, rs272879, rs1050150, rs4646201, or rs1050152 on the OCTN1 gene region Or a continuous polynucleotide having a length of 16 to 500 bases containing SNPs rs13180169, rs13180186, rs13180043, rs13180295, rs2631365, rs274558, or rs274557 on the OCTN2 gene region (however, when the polynucleotide is RNA, The base symbol “t” should be read as “u”),
3) A solid-phased sample on which the polynucleotide of 2) above is immobilized.   A composition for preventing or treating Crohn's disease comprising ergothioneine or an analog thereof as an active ingredient.

Images