KR20180125911A - Method for providing the information for predicting or diagnosing of inflammatory bowel disease using single nucleotide polymorphism to be identified from next generation sequencing screening - Google Patents

Abstract

A method for providing information for predicting or diagnosing inflammatory bowel disease by confirming single nucleotide polymorphism of the gene loci selected through next-generation sequencing is a method capable of predicting or diagnosing the occurrence of the inflammatory bowel disease among Korean patients by confirming the single nucleotide polymorphism, wherein the method is clinically meaningful because the method can predict the high risk of inflammatory bowel disease specifically in Koreans. In addition, the method is used to reduce the risk of various treatment procedures such as individual treatment, operation, surgery and the like to be expected to be effectively used to reduce the risk of inflammatory bowel disease.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for predicting or diagnosing inflammatory bowel disease caused by single nucleotide polymorphism discovered through next-generation sequence analysis screening. SCREENING}

The present invention relates to a method for providing information on the prediction or diagnosis of inflammatory bowel disease, and more particularly, to a method for predicting or diagnosing a Korean specific inflammatory bowel disease using single base polymorphism of at least one gene locus identified by a next- And to a method of diagnosing.

Recently, many parts of human genome sequences have been identified after the publication of the International HapMap Project, and research has been conducted to discover the linkage of genes and diseases to the whole genome as the measurement technology develops. Is progressing actively. Currently, the most active field in the field of field genome research is the study of whole genome single nucleotide polymorphism (SNP). Single nucleotide polymorphism refers to the polymorphism of a single nucleotide in the human nucleotide sequence, one nucleotide per 200-300 bp in average. As a result of confirming that the single nucleotide polymorphism shows differences in sensitivities to individual drugs and diseases, it has been recently shown that the sensitivity of individuals to diseases and the causes of differences in results after treatment are the main contents . In recent years, research has focused on identifying disease-related genes, which are known to have a high frequency of alleles and relatively low genetic effects, and the full-length genomic single nucleotide polymorphism It is possible to see the connection with disease by selectively producing only the place in the form of chip. Through these various studies, genes associated with various chronic diseases and various phenotypes have been revealed.

On the other hand, inflammatory bowel disease (IBD) is a chronic recurrent disease consisting clinically of ulcerative colitis (UC), Crohn's disease, and the like. In recent years, due to the increasing frequency of widespread epidemics in Korea due to the influence of westernized lifestyles, various pharmaceutical compositions and methods for treating IBD have been developed (USP 5541170). Ulcerative colitis is an unidentified chronic inflammatory bowel disease characterized by inflammation localized in the mucosa or submucosa of the large intestine. Hemorrhagic diarrhea, stomach ache, and abdominal pain are frequent recurrences and exacerbations. Ulcerative colitis is caused by a combination of genetic and environmental factors and is distributed worldwide. In recent years, the incidence is rapidly increasing in southern Europe, as well as in Asian countries including Korea and other developing countries. However, many patients suffer from ulcerative colitis and Crohn's disease, two subtypes of inflammatory bowel disease, but no definite cause or treatment of this disease has been presented. In many clinical studies, There are no problems. Therefore, it is necessary to accurately predict and diagnose the risk of ulcerative colitis and Crohn's disease in order to reduce the difference and reduce the incidence of inflammatory bowel disease.

Accordingly, the present inventors have provided a method of providing information on the prediction or diagnosis of inflammatory bowel disease caused by a single nucleotide polymorphism discovered by analyzing the ulcerative colitis susceptibility gene identified in a genome-wide association study and meta analysis by a next-generation sequence analysis method do.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for predicting or diagnosing the occurrence of inflammatory bowel disease specific to a Korean person using a single nucleotide polymorphism discovered by sequencing analysis of a gene locus, The purpose is to provide.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "biological sample " means all samples containing genomic DNA of an individual to be analyzed, and preferably includes blood, plasma, serum, bone marrow, , Saliva, sputum, hair, urine and the like, and more preferably, it is not limited to a sample which can be a body fluid, hair root, blood, plasma, serum and the like but can identify a gene locus.

In the present specification, the term " locus " refers to a position occupied by a gene on a chromosome or an associative map, but may also be used as a gene as a functional unit. Generally, the way to mark gene loci is that the first number is the chromosome number, the second English means the position on the chromosome, and the next number means the position of the band of the chromosome arm. For example, "6p21.3" means the first band of the second position of the short arm on chromosome 6, and the sub-band is 3.

In the present specification, "Next Generation Sequencing (NGS)" means a method of dividing the genome into numerous pieces and decoding the genetic information of each piece and then analyzing the entire nucleotide sequence. High-throughput sequencing, massive parallel sequencing, or second-generation sequencing is an advantage of high-speed analysis of the nucleotide sequence of the genome. For example, a small-size canned panel of NGS equipment can scan up to 40 genes and up to 400 large-size panels. Compared to NGS, it is possible to read the entire human genome by luteal sequencing, but only known genes can be targeted, so it is limited to testing and repeated experiments are required to see several genes. For example, next-generation sequence analysis is a time- and cost-effective analytical method compared to singlet sequencing, which requires about three million divisions.

As used herein, the term "single nucleotide polymorphism" (SNP) refers to the case where an allele is present in a single base of a single gene locus. The allele genotype is generally represented by "[]" within the nucleotide sequence. SNP is the location where two or more alleles occur in more than 1% of the population and is called common polymorphism when the allele genotype is more than 5%, and rare polymorphism when 1 to 5% do. SNV (single nucleotide variants).

In the present specification, the term " method for providing information on the prediction or diagnosis of inflammatory bowel disease "is meant to broadly refer to a method for confirming the possibility or incidence of inflammatory bowel disease in an individual. The method can be used to predict or diagnose the occurrence of inflammatory bowel disease by confirming the possibility of inflammatory bowel disease or the incidence of inflammatory bowel disease in a particular individual or patient during daily life or in a procedure such as prescribed treatment, It can be used as a reference.

As used herein, the term "kit " means a device for examining or diagnosing the occurrence of inflammatory bowel disease, and is not limited as long as it can confirm the presence of a single nucleotide polymorphism from a biological sample. Preferably, rs41417449 as the SNP of the BTNL2 gene, rs10035653 as the SNP of the C5orf55 gene, rs3117099 as the SNP of the HCG23 gene, rs7192 (p.L242V) of the HLA-DRA gene, rs3744246 as the SNP of the ORMDL3 gene, and SNP of the IL17REL gene Or a probe or primer that specifically binds to or has a sequence complementary to the at least one single base polymorphism selected from the group consisting of the single nucleotide polymorphism, Or an antibody that specifically binds to the polymorphism or an aptamer. The term "probe or primer" means an oligonucleotide having a sequence complementary to a single nucleotide polymorphism, and may be 8 to 52 nucleotides, 10 to 50 nucleotides, or 20 to 40 nucleotides, It does not.

As used herein, the term "inflammatory bowel disease (IBD)" refers to a disorder in which abnormal chronic inflammation in the intestine recurs and recurs, including Crohn's disease (CD), ulcerative colitis (UC), collagenous colitis Constituting colitis, ischemic colitis, conversion colitis, Behcet's disease or uncertain colitis. IBD may be Crohn's disease or ulcerative colitis. IBD may be Crohn's disease, collagenous colitis, lymphoid colitis, ischemic colitis, conversion colitis, Behcet's disease or indeterminate colitis. IBD may be an IBD that is not typically limited to inflammation in the colon and rectum. IBD may be an IBD that does not affect only the lining of the colon. It is not limited to a disease in which inflammation of the intestines occurs.

As used herein, "ulcerative colitis (UC)" is a chronic inflammatory disease characterized by diffuse mucosal inflammation of the large intestine. Among other features, the disease is characterized by bloody diarrhea, often with symptoms of bowel desensitization and cramping. The term "ulcerative colitis " as used herein means a disease or condition selected from the group consisting of diverticulitis, pouchitis, rectalitis, mucositis, diverticulitis, ischemic colitis, infectious colitis, chemical colitis, radiation-induced colitis, Inflammatory bowel disease, colitis, and colitis), atypical colitis, pseudomembranous colitis, fulminant colitis, autistic small intestine colitis, noncrystalline colitis, Bechet's disease, acute ileitis, ileitis, . The present invention contemplates those described herein for the prediction or diagnosis of any of the above diseases. Ulcerative colitis can occur in part of the large intestine, or substantially in the entire large intestine. Ulcerative colitis may be ulcerative rectal arthritic colitis. "Ulcerative rectal colitis" refers to ulcerative colitis, which is confined to the rectum and esophagus. Ulcerative colitis may be left ulcerative colitis. "Left colitis" refers to ulcerative colitis located in the distal portion of the spleen, particularly ulcerative colitis extending through the rectum to the proximal portion of the spleen. Ulcerative colitis may be broadly ulcerative colitis, which occurs substantially throughout the large intestine. "Broad ulcerative" or "all colitis" refers to ulcerative colitis that extends to the proximal portion of the spleen (i.e., extends through the spleen and extends to the caecal junction).

If ulcerative colitis is suspected in the patient, initial diagnosis is usually performed by using an aneurysm test, an urine test, a stool culture test, an ESR as an inflammatory index, a liver and kidney function test, and an electrolyte test . However, the markers alone may not be sufficient to clearly diagnose ulcerative colitis. Suitably, endoscopy is generally the most accurate diagnostic tool for ulcerative colitis. Flexible sigmoidoscopy is usually sufficient to diagnose ulcerative colitis, but full colonoscopy can be performed if the diagnosis is uncertain. The procedure may involve a superficial ulceration, mucosal erythema or vulnerability, loss of the vascular appearance of the large intestine, and examination of the presence of polyps. Biopsies can also be performed to differentiate between Crohn's disease and ulcerative colitis. Biopsy samples are typically taken at endoscopy and are examined for crypt structure twitches, inflammation of the yin, ulnar abscesses, and bleeding or inflammation in the mucosal lamina.

In the present specification, the term " diagnosis "means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the presence or absence of inflammatory bowel disease and metastasis. The diagnosis of inflammatory bowel disease consists of clinical symptoms, endoscopic and histopathologic findings, hematologic findings, and imaging findings. However, it is difficult to differentiate it from acute infectious enteritis, intestinal tuberculosis, or irritable bowel syndrome. However, it is difficult to differentiate between acute infectious enteritis, intestinal tuberculosis, or irritable intestinal syndrome. , Capsule endoscopy, and balloon assisted small bowel endoscopy may be helpful in diagnosis. Inflammatory bowel disease can be diagnosed by visual or cytological examination of tissues from patients with inflammatory bowel disease, and specimens of inflammatory bowel disease (clinically, cells, blood, sap, pleural fluid, ascites, A method for directly detecting an inflammatory bowel disease-related protein in the specimen, a method for detecting an inflammatory bowel disease-related protein in the specimen, a method for detecting an inflammatory bowel disease-related protein in the specimen, Inflammatory bowel disease can be diagnosed by directly detecting a nucleic acid encoding a disease-related protein. Examples of diagnostic means for directly detecting an antigen-antibody binding or inflammatory bowel disease-related protein include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Fluorescence Activated Cell Sorter (RT-PCR), competitive RT-PCR (reverse-transcription-polymerase chain reaction), and the like, as well as methods for directly detecting a nucleic acid encoding an inflammatory bowel disease- PCR), real-time RT-PCR, RNase protection assay (RPA), northern blotting, or DNA chip And the like, but the present invention is not limited thereto.

In the present invention, "Odds" is another expression of probability. If the probability of occurrence of an event is p, the odds for the event can be obtained as p / (1-p). Odds are especially used to indicate the probability of disease outbreaks, and the odds ratio, or odds ratio, can be used to confirm the association of two properties. In particular, the Odds Ratio of a specific disease in a person with a particular gene, as in this study, can be used to assess the association of a particular gene with a particular disease.

As used herein, "causal variant" generally refers to a case where genetic variants have a significant association (statistical significance with "odd ratio> 1") in the direction of increased susceptibility to the disease . In other words, the inclusion of the causative mutant of the present specification means that the possibility of the disease is high, and the onset of the disease can be predicted through this.

As used herein, the term "protective variant" refers to a mutation in the genetic variant that has a statistically significant (i. E., Odd ratio < If there is. That is, the inclusion of the protective variant of the present specification means that the possibility of disease development is low.

The present invention includes a step of confirming single nucleotide polymorphisms from one or more genes selected from the group consisting of BTNL2 gene, C5orf55 gene, HCG23 gene, HLA-DRA gene, ORMDL3 gene and IL17REL gene from a biological sample A method for providing information about inflammatory bowel disease prediction or diagnosis.

In one embodiment of the invention, the single nucleotide polymorphism is rs41417449 of the BTNL2 gene, rs10035653 of the C5orf55 gene, rs3117099 of the HCG23 gene, rs7192 of the HLA-DRA gene, and rs3744246 of the ORMDL3 gene. In another embodiment of the present invention, the single base polymorphism is predicted to result in a high incidence of inflammatory bowel disease. In another embodiment of the present invention, the single nucleotide polymorphism is rs713669 of the IL17REL gene. In another embodiment of the present invention, the single nucleotide polymorphism is predicted to have a low incidence of inflammatory bowel disease. In another embodiment of the present invention the inflammatory bowel disease is selected from the group consisting of ulcerative colitis (UC), Crohn's disease, collagenous colitis, lymphoid colitis, ischemic colitis, conversion colitis, ≪ / RTI > In another embodiment of the present invention, the method for providing information on the prediction or diagnosis of inflammatory bowel disease is characterized in that it is applied to a Korean person. In another embodiment of the present invention, the biological sample is characterized by body fluids, hair follicles, blood, plasma or serum. In yet another embodiment of the present invention, the step of identifying may comprise one or more of the following steps: Dipylysequencing analysis, microarray hybridization, allele specific PCR, dynamic allele- Specific hybridization, DASH), TaqMan technique, SnapShot technique, and qRT-PCR analysis.

The present invention also relates to a primer that specifically binds to a single nucleotide polymorphism of one or more genes selected from the group consisting of BTNL2 gene, C5orf55 gene, HCG23 gene, HLA-DRA gene, ORMDL3 gene and IL17REL gene Lt; RTI ID = 0.0 > inflammatory bowel disease < / RTI >

In one embodiment of the invention, the single nucleotide polymorphism is rs41417449 of the BTNL2 gene, rs10035653 of the C5orf55 gene, rs3117099 of the HCG23 gene, rs7192 of the HLA-DRA gene, and rs3744246 of the ORMDL3 gene. In another embodiment of the present invention, the single nucleotide polymorphism is predicted to have a high incidence of inflammatory bowel disease. In another embodiment of the present invention, the single nucleotide polymorphism is rs713669 of the IL17REL gene. In another embodiment of the present invention, the single nucleotide polymorphism is predicted to have a low incidence of inflammatory bowel disease. In another embodiment of the present invention the inflammatory bowel disease is selected from the group consisting of ulcerative colitis (UC), Crohn's disease, collagenous colitis, lymphoid colitis, ischemic colitis, conversion colitis, ≪ / RTI > In another embodiment of the present invention, the method for providing information on the prediction or diagnosis of inflammatory bowel disease is characterized in that it is applied to a Korean person.

The present invention also provides a kit for predicting or diagnosing inflammatory bowel disease, comprising the above composition for the prediction or diagnosis of inflammatory bowel disease.

Hereinafter, the present invention will be described in detail.

A method for providing information on the prediction or diagnosis of inflammatory bowel disease by confirming the single nucleotide polymorphism of the gene locus according to the present invention is a method for predicting or diagnosing the incidence of inflammatory bowel disease among Korean patients by confirming single nucleotide polymorphism . This is clinically meaningful because it can predict the high risk of inflammatory bowel disease specifically in Koreans. By using this, it is possible to reduce the risk of inflammatory bowel disease by decreasing the risk of various treatments such as treatment, It is expected that it can be used effectively.

1 is a flowchart illustrating a next generation sequence analysis according to an embodiment of the present invention.
FIG. 2 is a view showing immunohistochemical staining results of BTNL2 and C5orf55 according to an embodiment of the present invention.
3 is a graph showing mRNA levels of BTNL2 and C5orf55 in inflammatory bowel tissue according to an embodiment of the present invention.
4 is a graph showing mRNA expression of BTNL2 and C5orf55 according to an embodiment of the present invention.
5 is a graph showing mRNA expression of TNF-? According to an embodiment of the present invention.
6 is a diagram showing a result of Western blot according to an embodiment of the present invention.
7 is a diagram showing LC3B spot accumulation and cell-killing nucleation according to an embodiment of the present invention.
FIG. 8 is a diagram showing a linkage disequilibrium (LD) structure of BTNL2, IL23R, HLA, and ZPBP2 SNV in Korean according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Target group

The group according to the present example was a Korean, consisting of patients with ulcerative colitis and normal controls. Patients with ulcerative colitis were diagnosed and followed up regularly at the Severance Hospital of Yonsei University College of Medicine from June 2006 to August 2014 according to conventional clinical, colonoscopy, radiology, and histopathological criteria. Crohn's disease, indeterminate colitis (IC), which can not be classified as inflammatory bowel disease, was excluded in this example. A control group without specific disease was also recruited. This control was randomly selected from those recruited from the Yonsei Cardiovascular Genomic Center. This study was approved by the Institutional Review Board of Yonsei University Severance Hospital and all participants or guardians received written consent. All procedures and protocols were carried out in accordance with relevant guidelines and regulations.

In the first 24 Korean patients with ulcerative colitis, 108 genes were subjected to the next generation sequencing analysis, followed by association analysis using data from 126 healthy controls. This variant was then verified using a two-step cloning study involving 793 patients with ulcerative colitis and 783 controls.

Example  1: Investigation of genome-wide association analysis to identify genes related to ulcerative colitis

To identify genes associated with ulcerative colitis in Koreans, next generation sequencing (NGS) was performed. The selection was initially based on nine GWAS and one GWA meta-analysis data. NGS studies were conducted in Asian populations (Japanese) and the rest in European ancestral populations. Also included are genes considered based on functions that play a role in IBD outbreaks, and by reviewing these genes, 108 genes were finally selected as targets for deep re-sequencing.

1.1 Dip Resequencing deep resequencing ) And SNV  Justice( SNV (single nucleotide variant) calling

The samples were quantitated using a sensitive Qubit system (Invitrogen, Carlsbad, Calif., USA) and assessed for integrity with agarose gels, using a DNA blood maxi kit (Qiagen, Santa Clarita, CA) Lt; / RTI > The library was prepared using Agilent's SureSelect Exome Enrichment kit (Illumina, San Diego, CA) according to the manufacturer's protocol. Using the SureSelect Target Enrichment System (Agilent Technologies, Santa Clarita, Calif., USA) according to the manufacturer's protocol, an exome capture at the 10 Kb upstream of the 5 'end of the gene and the 10 Kb downstream of the 3' Were performed on 108 genes. Captured ligation-mediated polymerase chain reaction (LM-PCR) products were analyzed with an Agilent 2100 Bioanalyzer to assess library quality. The captured library was then loaded onto an Illumina Hiseq 2000 platform (Illumina) and the sequencing of the captured library was performed at high throughput to ensure that each sample met the desired degree of average sequencing. The raw image file was processed by HiSeq Control Software version 1.4.8. Base-calling was performed using basic parameters, and each individual sequence was created with a 90 base-pair (bp) paried-end read.

After the sequence of the target site was determined, the obtained fair end sequencing data was subjected to a short-read mapping program Burrows-Wheeler Aligner (BWA, Version 0.6.2-4126; http://bio-bwabd.net/ ) Was used as the default parameter to match the reference genome (NCBI build GRCh37). We used the Genome Analysis Toolkit (GATK 1.6-11; https://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit) for SNV definition (SNV calling).

SNV confirmed by resequencing was compared to sequencing data from 126 healthy controls. Based on the association analysis, nonsynonymous and upstream SNVs with significant differences in the frequency of alternative alleles between cases and controls were selected for additional replication genotype analysis. When various SNVs were identified in one ulcerative colitis-susceptible gene, representative SNVs of coding regions with significantly lower p values than other SNVs were selected.

Using Fludigm or TaqMan probes, 106 SNVs containing 52 non-synonymous SNVs, 29 upstream SNVs of 27 genes and 13 SNVs in 5 genes from 29 genes reported in conventional GWAS studies Genotypes were analyzed in samples from 225 patients with ulcerative colitis for replication studies and in 230 healthy controls. Linkage analysis of selected SNVs in test and control groups was performed under four alternative models (dominant, recessive, dominant, and allelic). Based on this analysis, in the second cloned genotype study, 28 SNVs (BTNL2 (butyrophilin like 2), C5orf55 (Chromosome 5 Open Reading Frame 55), CCL2 The major histocompatibility complex (HLA-DRA), class II, and chemokine ligand 2, FCGR2A, G protein-coupled receptor 35, GSDMB, HCG23, DR alpha), IL17REL, interleukin 22, interleukin 23 receptor, ORMDL sphingolipid biosynthesis regulator 3, TNFSF 15, UTS2 (urotensin 2 ) And ZPBP2 (zona pellucida binding protein 2).

In a second cloning study, the 28 SNVs were genotyped in 568 independent ulcerative colitis patients and 553 control samples. A combined analysis confirmed four SNVs that were significantly associated with ulcerative colitis susceptibility (see Table 3). (P.L242V) in HLA-DRA, rs3117099 (non-coding region) in HCG23, rs3117099 (non-coding region) in HCG23, , Rs3744246 (noncoding region) in ORMDL3, and rs713669 (noncoding region) in IL17REL were novel ulcerative colitis-sensitive genetic loci (UC susceptibility loci) not previously reported. It was confirmed that this was highly correlated with the risk of ulcerative colitis in Koreans. Thus, it was confirmed that the four loci were locus related to Korean specific ulcerative colitis. In addition, it was confirmed that the risk of ulcerative colitis in Koreans, that is, the occurrence of ulcerative colitis, can be predicted or diagnosed by confirming the gene locus or single base polymorphism of the present invention.

1.2. Conduct statistical analysis

By running SAS version 9.3.1 (SAS Institute Inc., Carrie, NC), the chi-square test or the Cochran-Armitage Trend test and the Jonckheere-Terpstra test were used to determine the frequency of alleles of different groups of subjects And by comparing the frequency of genotypes. Allele frequencies were compared between patients with ulcerative colitis and controls using logistic regression to obtain age and gender-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) Respectively. The Benjamini and Hochberg test was applied to modify multiple tests in a combined association analysis of the first and second replication genotypes. The HaploView program (http://www.broad.mit.edu/mpg/haploview/) was used to determine the association non-equilibrium (LD) block and association disequilibrium among the SNPs. Gene expression results derived from in vitro and in vivo experiments were expressed as mean ± SD (standard deviation) or ± standard error (SEM). Prism software (GraphPad Software, Inc., San Diego, Calif.) Was used for all analyzes. Differences in conditions were assessed using ANOVA (analysis of variance) and t-test. A p-value less than 0.05 was considered statistically significant.

Example  2: ulcerative colitis SNV  Verification and cloning studies

The overall result GWAS, SNV 9 dogs, that is C5orf55 of ^{rs10035653 (P = 2.08 × 10 -3} ; OR = 1.50), rs3117099 (P = 9.98 × 10 -6; OR = 1.40) in HCG23, the BTNL2 rs41417449 (P = 1.27 ^{× 10 -2; OR = 1.32)} , rs28362675 (p.G454C, P = 1.39 × 10 -2; OR = 1.31), rs41441651 (p.D336N, P = 1.53 × 10 -2; OR = 1.31), and rs28362680 (p.A202V, P = 8.49 × 10 -3; OR = 1.24), the HLA-DRA rs7192 (P = 6.95 × 10 -9; OR = 1.57), the ORMDL3 rs3744246 (P = 2.21 × 10 -2; OR = 1.21) and rs9271366 (P = 3.34 × 10 ^-13 ; OR = 2.23) have reached significant limits, and they may be causal variants associated with the development of ulcerative colitis. That is, it can be confirmed that the SNV (SNP) is closely related to the high risk of ulcerative colitis (high incidence) by causing the degradation of gene function due to the production of abnormal protein or ribonucleic acid . Conversely, SNV 7 dogs, that is IL17REL of ^{rs713669 (P = 2.69 × 10 -2} ; OR = 0.84), rs6426833 ( non-coding ^{region, P = 1.76 × 10 -2;} OR = 0.80) of IL23R, rs4654903 (P = 2.88 ^{× 10 -3; OR = 0.76)} , rs76418789 (P = 2.31 × 10 -3; OR = 0.61), the FCGR2A rs1801274 (P = 4.50 × 10 -4; OR = 0.72), rs9268853 (P = 1.33 × 10 - ^11; OR = 0.58), and ^{rs2395185 (P = 7.74 × 10 -12} ; OR = 0.58) was identified as an important variant capable of providing protection against the onset of ulcerative colitis (defense variant, protective variant). That is, the seven SNPs are mutations that protect the inflammation by lowering the reactivity to the inflammatory reaction by mutation in the gene promoting the inflammation, and are closely related to the low risk of ulcerative colitis (low incidence) , And the incidence of mutation was lower in ulcerative colitis group.

SNV by GWAS is closely related to other SNVs in the region and may be related to the causative mutant rather than the causative mutant itself. Single - phase analysis and associative non - equilibrium structure analysis were performed among the final candidates to confirm the presence of haplotypes associated with ulcerative colitis. The results are shown in Table 4. The genotypes of 793 patients with ulcerative colitis and 783 healthy control samples were used for association non-equilibrium estimation. In the single-phase type analysis, BTNL2 (ATCAC, P = 2.84 × 10 -3; CCTGC, P = 4.34 × 10 -3), IL23R (AT, P = 1.70 × 10 -3; GT, P = 1.12 × 10 -2) , HLA (GCT, P = 2.83 × 10 -14; TTG, P = 3.91 × 10 -11), and ZPBP2 (AGAC, P = 2.42 × 10 - 2) were significantly associated with disease susceptibility to UC . These results indicate that these genes are potential causative mutants that contribute to the development of ulcerative colitis. LD analysis, variants of BTNL2 p.G454C and p.D336N (rs28362675 and rs41441651), as well as the IL23R, HLA and ZPBP2 showed that almost complete association disequilibrium (r ² = 1) with each other. These results suggest that mutants of the BTNL2 gene can be identified as major causative mutations in ulcerative colitis in Koreans, suggesting that other SNPs in the BTNL2 gene may also be associated with the risk of ulcerative colitis (Fig. 8).

Example  3: Of SNV  Functional evaluation

To evaluate the functional effects of asynchronous SNV, we used SIFT, PolyPhen2 and MutationTaster databases to investigate the effect of amino acid changes on protein damage in silico. P.P50S in C5orf55 and p.G454C and p.D336N in BTNL2 were found to be damaged in silico analysis. Potential regulatory functions of candidate SNPs were assessed using RegulomeDB. The score for rs713669 (score = 1f) in the IL17REL showed evidence of a relatively high proportion of potential modulating function. The locus of ORMDL3 was significantly associated with susceptibility to ulcerative colitis. The cis-eQTL analysis showed evidence that HCG23, HLA-DRA and ORMDL3 mutants affect the differentiation genes of the precursor cells (NOTCH4) (HLA-DRB, HLA-DRA, ORMDL3).

In the analysis of the subject group (793 patients and 783 normal subjects) that combined both the next generation sequence analysis and the replication step, rs10035653 in the C5orf55 gene, rs41417449 in the BTNL2 gene, rs3117099 in the HCG23 gene, HLA- Single nucleotide polymorphisms of rs7192 in the DRA gene, rs3744246 in the ORMDL3 gene, and rs713669 in the IL17REL gene were found to be highly associated with ulcerative colitis. These mutants are newly discovered genetic variants and provide meaningful information for the diagnosis and prediction of ulcerative colitis. Further, the present invention is of great significance in finding important genetic variants as a genetic mutant study through next generation sequence analysis conducted in Korean.

In addition, Figures 2 and 3 show gene expression in inflammatory tissues of the human and mouse colon. Fig. 2 is a photograph showing the results of immunohistochemical staining of BTNL2 and C5orf55. CTN (CTL, n = 8, UC, n = 17) was labeled with BTNL2 (Fig. 2A) and C5orf55 Lt; / RTI > antibody. The right graph shows the expression scores according to disease in the crypt and laminar propria (LP). The sections were counterstained with hematoxylin (blue).

Figure 3 shows mRNA levels of BTNL2 and C5orf55 in inflammatory bowel tissues. Total RNA was isolated from human and mouse colon tissues and transformed into complementary DNA. The transcription levels of BTNL2 (left), C5orf55 (right) and Btn12 (left) and C5orf55 (right) of mouse colon tissues (FIG. 3B) Was quantitated by real-time quantitative reverse transcription polymerase chain reaction and normalized to the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin. Data are expressed as mean ± standard error (SEM). (CTL, n = 8; UC, n = 14; Sham, n = 3; DSS, n = 4). * P < CTL or Sham, ** P < CTL or Sham.

Figures 4-7 illustrate the functional analysis of BTNL2 and C5orf55 in human intestinal epithelial cells. Figure 4 is a graph showing mRNA expression of BTNL2 and C5orf55. To determine whether LPS stimulation affects the expression of BTNL2 and C5orf55, protein and mRNA levels were measured in HT-29 cells treated with lipopolysaccharide (LPS, 1 [mu] g / mL) for 4 hours. FIGS. 5 to 7 show inhibition of BTNL2 and C5orf55 expression in HT-29 cells using siRNA. Figure 5 is a graph showing mRNA expression of TNF-a. Transcription levels of TNF-α were analyzed 3 hours after LPS stimulation using real-time quantitative RT-PCR (qRT-PCR). 6 is an image obtained by Western blotting. Protein levels of TNF-α were analyzed 24 hours after LPS stimulation. Figure 7 is an image showing LC3B spot accumulation and cell killing nucleation. Cells were treated with LPS for 24 hours, stained and photographed under a fluorescence microscope (x400). The indicator for the autophage activity represents the LC3B spot (green), and the cell death mark (indicated by the arrow) as the cell death killing nucleus is represented by DAPI (blue) staining. Data are expressed as mean ± standard error (SEM). * P < Untreated, ** P < Untreated, *** P < Untreated, ^# P < 0.05 vs. < Untreated, ** P < Untreated, *** P &lt; Unprocessed.

As shown in Fig. 2, protein levels of BTNL2 and C5orf55 were significantly lower in the negative and mucosal plate of the large intestine of patients with ulcerative colitis than in the control tissues. However, as shown in Fig. 3, transcription levels of BTNL2 and C5orf55 in inflammatory bowel tissues of inflammatory bowel disease (IBD) patients and mice with colitis were significantly higher than in the healthy control group. In addition, BTNL2-positive cells were found more frequently in the mucosal plate of patients with ulcerative colitis than in healthy control mucosal plates. Thus, it can be seen that these genes are regulated after transcription as a compensatory mechanism for defects in protein function, thereby protecting them from inflammation. As shown in FIG. 4, lipopolysaccharide (LPS) induced an increase in mRNA and protein levels of BTNL2 and C5orf55 in HT-29 cells, which are epithelial cells. As shown in FIGS. 5 and 6, additional functional studies were performed on HT-29 cells using siRNA. The levels of mRNA and protein of TNF-α were significantly higher in BTNL2 and C5orf55 knockdown cells than in the control, .

The collapse of basic autophage promotes the production of LPS-induced IL-1β, and over-active autophagy leads to childhood cell death. Thus, autophagic dysregulation is associated with a variety of inflammatory diseases. As shown in Figure 7, untreated control cells showed diffuse expression of LC-3B (green), while baseline LC-3B levels were reduced in BTNL2 and C5orf55 knockdown cells compared to control cells. In addition, autophas formation and child cell death were significantly increased in these cells in response to LPS.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (17)

Comprising identifying a single nucleotide polymorphism in one or more genes selected from the group consisting of HLA-DRA gene, ORMDL3 gene, and IL17REL gene from a biological sample, How to provide. The method according to claim 1,
Wherein said single nucleotide polymorphism is rs7192 of the HLA-DRA gene or rs3744246 of the ORMDL3 gene. 3. The method of claim 2,
Wherein the polymorphism of the single nucleotide polymorphism is predicted to increase the incidence of inflammatory bowel disease. The method according to claim 1,
Wherein said single nucleotide polymorphism is rs713669 of the IL17REL gene. 5. The method of claim 4,
Wherein the polymorphism of the single nucleotide polymorphism is predicted to predict a low incidence of inflammatory bowel disease. The method according to claim 1,
The inflammatory bowel disease is any one selected from the group consisting of ulcerative colitis (UC), Crohn's disease, collagenous colitis, lymphoid constituting colitis, ischemic colitis, conversion colitis, Behcet's disease and uncertain colitis , Inflammatory bowel disease prediction or diagnosis. The method according to claim 1,
Wherein the method for providing information about the inflammatory bowel disease prediction or diagnosis is for a Korean person. The method according to claim 1,
Wherein said biological sample is information about the prediction or diagnosis of inflammatory bowel disease, which is body fluids, hair follicles, blood, plasma or serum. The method according to claim 1,
The step of confirming may be selected from the group consisting of Deep re-sequencing analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH), TaqMan technique, SnapShot technique, and qRT-PCR analysis. &Lt; Desc / Clms Page number 13 &gt; A primer set that specifically binds to a single nucleotide polymorphism of one or more genes selected from the group consisting of HLA-DRA gene, ORMDL3 gene, and IL17REL gene. 11. The method of claim 10,
Wherein said single nucleotide polymorphism is rs7192 of the HLA-DRA gene or rs3744246 of the ORMDL3 gene. 12. The method of claim 11,
A composition for predicting or diagnosing inflammatory bowel disease, wherein the single nucleotide polymorphism is identified and predicts a high incidence of inflammatory bowel disease. 11. The method of claim 10,
Wherein said single nucleotide polymorphism is rs713669 of IL17REL gene. 14. The method of claim 13,
Wherein the polymorphism predicts that the incidence of inflammatory bowel disease is low when the single nucleotide polymorphism is identified. 11. The method of claim 10,
The inflammatory bowel disease is any one selected from the group consisting of ulcerative colitis (UC), Crohn's disease, collagenous colitis, lymphoid constituting colitis, ischemic colitis, conversion colitis, Behcet's disease and uncertain colitis , A composition for predicting or diagnosing inflammatory bowel disease. 11. The method of claim 10,
Wherein the prediction or diagnosis by the composition for prediction or diagnosis of inflammatory bowel disease is intended for a Korean person. 17. A kit for predicting or diagnosing inflammatory bowel disease, comprising a composition for the prediction or diagnosis of inflammatory bowel disease according to any one of claims 10 to 16.

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