KR102010899B1 - 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

The method of confirming single nucleotide polymorphism of the locus selected through the next-generation sequencing analysis according to the present invention and providing information on the prediction or diagnosis of inflammatory bowel disease is to confirm the occurrence of inflammatory bowel disease among Korean patients. As it can predict the high risk group of inflammatory bowel disease specifically for Koreans as a predictive or diagnosable method, it is clinically significant, and it is used to reduce the risk of various treatments such as individual treatment, procedures, and surgery. It is expected that it can be effectively used to reduce the risk of intestinal diseases.

Description

METHODO FOR PROVIDING THE INFORMATION FOR PREDICTING OR DIAGNOSING OF INFLAMMATORY BOWEL DISEASE USING SINGLE NUCLEOTIDE POLYMORPHISM TO BE IDENTIFIED FROM NEXT GENERATION SENERATION SCREENING}

The present invention relates to a method for providing information regarding the prediction or diagnosis of inflammatory bowel disease, and more specifically, to predict the occurrence of specific inflammatory bowel disease in Korean by using a single nucleotide polymorphism of one or more loci identified by the next generation sequencing method. It is about a method of diagnosis.

Since the recent publication of the International HapMap Project, many of the genome sequences of humans have been identified, and as the measurement technology advances, studies on the whole genome of the whole genome are used to identify the relationship between genes and diseases. Is actively underway. Currently, the most active area of research in the field of genomes is the study of whole genome Single Nucleotide Polymorphism (SNP). Single nucleotide polymorphism refers to the polymorphism of a single nucleotide sequence (single nucleotide) that is present in an average of every 200-300bp in the human base sequence. As it was confirmed that a single nucleotide polymorphism caused a difference in sensitivity to individual drugs, diseases, and the like, in recent years, it is important to identify the cause of the individual's sensitivity to disease and the difference in outcome after treatment. It is coming true. In recent years, research has focused on identifying disease-related genes that are known to have a high frequency of alleles and relatively low genetic effects, and full-length single nucleotide polymorphisms are already common among DNA sequence mutations. Only places are selectively made in the form of chips to see their association with disease. In addition, genes associated with various chronic diseases and various phenotypes are revealed through these various studies.

On the other hand, inflammatory bowel disease (IBD) is a chronic recurrent disease consisting of clinically ulcerative colitis (UC), Crohn's disease and the like. Recently, due to the influence of westernized lifestyle, the incidence is increasing in Korea, and various pharmaceutical compositions and methods for treating IBD have been developed (US Patent No. 5551170). In particular, ulcerative colitis is an unexplained chronic inflammatory bowel disease characterized by inflammation localized in the mucosa or submucosa of the large intestine. The main symptoms include bleeding diarrhea, stool urge and abdominal pain. Ulcerative colitis is caused by a combination of genetic and environmental factors and is distributed worldwide. In recent years, the incidence rate has increased rapidly in southern Europe, Asian countries including Korea, and other developing countries. However, many patients suffer from ulcerative colitis and Crohn's disease, two subgroups of inflammatory bowel disease, but there are no clear causes or treatments for the disease, and many clinical studies have received only expert opinions and opinions. There are no problems. Therefore, there may be individual differences in the treatment or treatment methods of patients, and in order to reduce such differences and lower the incidence of inflammatory bowel disease, accurate prediction and diagnosis of ulcerative colitis and Crohn's disease are required.

Therefore, the present inventors provide a method for providing information on the prediction or diagnosis of inflammatory bowel disease by univariate polymorphism, which is obtained by analyzing ulcerative colitis susceptibility genes identified in genome-wide association studies and meta-analysis by next-generation sequencing method. do.

An object of the present invention is to solve the above-mentioned problems in the prior art, a method for predicting or diagnosing the occurrence of specific inflammatory bowel disease in Korea using a single nucleotide polymorphism discovered by the next generation sequence analysis of the locus Its purpose is to provide.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details are set forth, such as specific forms, compositions, processes and the like. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in particular detail in order to not 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 one or more embodiments of the invention. Thus, the context of “in one embodiment” or “embodiment” expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. In addition, particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined, 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" refers to all samples containing genomic DNA of an individual to be analyzed, preferably blood, plasma, serum, bone marrow, tissues, cells. , Saliva, sputum, hair, urine, and the like, and more preferably body fluid, hair root, blood, plasma, serum, and the like, but is not limited thereto.

In the present specification, the term "locus" refers to a position occupied by a gene in a gene map on a chromosome or a related map, but may also be used synonymously with a gene as a functional unit. In general, the method of marking the locus is the first number is the chromosome number, the second English means the position on the chromosome, the next number is 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 three.

In the present specification, "Next Generation Sequencing (NGS)" refers to a method of dividing a genome into a myriad of fragments, and then analyzing the entire nucleotide sequence after deciphering and combining the genetic information of each fragment. It has the advantage of being able to analyze genome sequences at high speed. It is also called high-throughput sequencing, massively parallel sequencing or second-generation sequencing. For example, a small sized canceler panel in an NGS device can test up to 40 genes and up to 400 large sized panels. Compared with NGS, Sanger sequencing can read the entire human genome, but only known genes can be targeted, limiting testing and requiring repeated experiments to see multiple genes. For example, next-generation sequencing is a significant improvement in time and cost compared to singer sequencing, which requires about 3 million times.

In the present specification, "single nucleotide polymorphism (SNP)" refers to a case where an allele exists at a single base at a single locus, and the allele is generally expressed as "[]" in the nucleotide sequence. The location where two or more alleles occur in the population at 1% or more is called SNP, and when the allele is present at 5% or more, it is called common polymorphism, and in the case of 1-5%, it is classified as rare polymorphism. do. It may be used in the same sense as single nucleotide variants (SNV).

In the present specification, the "method of providing information on inflammatory bowel disease prediction or diagnosis" refers to a method of identifying whether or not an individual develops inflammatory bowel disease or whether it occurs. The method may be performed by predicting or diagnosing the occurrence of inflammatory bowel disease by identifying the likelihood, occurrence, etc. of inflammatory bowel disease in any particular individual or patient during daily life or during a course of treatment such as a treatment, procedure, surgery, or the like. May be used.

In the present specification, "kit" means a screening device capable of predicting or diagnosing the occurrence of inflammatory bowel disease, and there is no limitation as long as it is a form capable of confirming the presence of a single base polymorphism from a biological sample. Preferably rs41417449 SNP of BTNL2 gene, rs10035653 SNP of C5orf55 gene, rs3117099 SNP of HCG23 gene, rs7192 (p.L242V) SNP of HLA-DRA gene, rs3744246 of SNP of ORMDL3 gene, and SNP of IL17REL gene Or a probe or primer specifically binding to one or more single polymorphisms selected from the group consisting of rs713669 or having a sequence complementary to the single nucleotide polymorphism, or the single base It may be in a form including an antibody or aptamer that specifically binds to polymorphism. The term "probe or primer" refers to an oligonucleotide having a sequence complementary to monobasic polymorphism, and may be 8 to 52 nucleotides, 10 to 50 nucleotides, or 20 to 40 nucleotides in length. It doesn't work.

In the present specification, "inflammatory bowel disease (IBD)" is a disease in which abnormal chronic inflammation in the intestine is repeated and recurred, Crohn's disease (CD), ulcerative colitis (UC), collagen colitis, lymph Constitutive colitis, ischemic colitis, converting colitis, Behcet's disease or indeterminate colitis. IBD can be Crohn's disease or ulcerative colitis. IBD can be Crohn's disease, collagen colitis, lymphocytic colitis, ischemic colitis, converting colitis, Behcet's disease or indeterminate colitis. IBD can be IBD, which is not typically limited to inflammation in the colon and rectum. IBD may be IBD that does not only affect the lining of the colon. Intestinal inflammation is not limited to this disease.

As used herein, "ulcerative colitis (UC)" is a chronic inflammatory disease characterized by diffuse mucosal inflammation of the large intestine. The disease is characterized by bloody diarrhea, among other features, often with symptoms of stool urgency and sequelae. As used herein, the term "ulcerative colitis" refers to diverticulitis, pouchitis, rectal analitis, mucositis, converting colitis, ischemic colitis, infectious colitis, chemical colitis, radiation-induced colitis, microscopic colitis (collagen Colitis and lymphocytic colitis), atypical colitis, gastric colitis, blunt colitis, autistic colitis, nondeterministic colitis, Bechet's disease, colitis, ileitis, colitis, granulomatous colitis Include. The present invention contemplates what is described herein for the prediction or diagnosis of any such disease. Ulcerative colitis can occur in part of the large intestine or substantially in the entire colon. Ulcerative colitis can be ulcerative rectal colitis. "Ulcerative colitis colitis" refers to ulcerative colitis confined to the rectum and S phase colon. Ulcerative colitis can be left ulcerative colitis. "Left colitis" refers to ulcerative colitis, which is confined to the large intestine part of the distal chole, especially ulcerative colitis that extends beyond the rectum and as far as proximal as the spleen. Ulcerative colitis can be widespread ulcerative colitis that develops substantially throughout the colon. "Wide ulcer" or "pronitis" means ulcerative colitis that extends to the proximal spleen (ie, extends past the spleen and into the ileal splice).

If ulcerative colitis is suspected in a patient, the initial diagnosis usually includes whole blood counts, urinalysis, stool culture tests, erythrocyte sedimentation rate (ESR) as an indicator of inflammation, liver and kidney function tests, and electrolyte studies to test for anemia. It includes. However, these labels alone may not be sufficient to clearly diagnose ulcerative colitis. Appropriately, endoscopy is generally the most accurate diagnostic tool for ulcerative colitis. Flexible sigmoidoscopy is usually sufficient to diagnose ulcerative colitis, but a full colonoscopy may be performed if the diagnosis is uncertain. The procedure may involve investigation of superficial ulcers, erythema or fragility of the mucous membranes, loss of the vascular appearance of the large intestine, and the presence of the gapolyps. Biopsies can also be performed to distinguish Crohn's disease from ulcerative colitis. Biopsy samples are generally taken during endoscopy and examined for distortion of the crypt structure, inflammation of the crypts, ulceral abscesses, and bleeding or inflammation in the mucosa.

In the present specification, "diagnosis" means confirming the presence or characteristics of a pathological state, and for the purpose of the present invention, the diagnosis is to confirm the presence and metastasis of inflammatory bowel disease. Inflammatory bowel disease is diagnosed by combining clinical symptoms, endoscopic and histopathologic findings, hematologic findings, and imaging findings. However, there are cases where it is difficult to differentiate between acute infectious enteritis, tuberculosis, or irritable bowel syndrome, but imaging tests such as blood and serum tests, stool tests, abdominal computed tomography (CT), magnetic resonance imaging (MRI), and small intestine angiography For example, capsule endoscopy or balloon-assisted intestinal endoscopy may be helpful. Inflammatory bowel disease can be diagnosed by gross or cytological confirmation of tissue from patients with inflammatory bowel disease, and samples of tissues with inflammatory bowel disease (clinically, cells, blood, fluids, pleural fluid, ascites, joint fluid, pus) (Iv), secretion, bile, pharyngeal mucus, urinary tract, bile, stool, etc.) using an inflammatory bowel disease-compatible antibody, a method for directly detecting an inflammatory bowel disease-related protein in the sample or an inflammatory bowel Inflammatory bowel disease can be diagnosed by directly detecting a nucleic acid encoding a disease-related protein. Diagnostic means using direct detection of antigen-antibody binding or inflammatory bowel disease-related proteins include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (RIA), and OY. Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein Chips (protein chips), but are not limited thereto, and methods for directly detecting nucleic acid encoding proteins related to inflammatory bowel disease include reverse transcriptase (RT-PCR) and competitive reverse transcriptase (Competitive RT-). PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting or DNA chip Etc., but is not limited thereto.

In the present invention, " Odds " is another expression of probability. If p is the probability of an event occurring, odds of the event can be obtained as p / (1-p). Odds are especially used to indicate the incidence of disease, and the relationship between two properties can be confirmed by using odds ratio, that is, odds ratio. In particular, the Odds ratio of a particular disease in a person with a specific gene, as in this study, can be used to evaluate the association between a particular gene and a particular disease.

In the present specification, "causal variant" generally refers to a case in which a genetic variant has a significant correlation ("odd ratio> 1" and statistically significant) in the direction of increasing disease susceptibility. . In other words, if the causative variant of the present specification is included, it means that the onset of the disease is high, and thus the onset of the disease can be predicted.

In addition, in the present specification, as opposed to a causative variant, a "protective variant" has a significant correlation ("odd ratio <1" and statistically significant) in the direction of decreasing disease susceptibility as analyzed by genome studies. Say it is. In other words, if the protective variant of the present specification is included, it means that the incidence of disease is low.

The present invention includes identifying a single nucleotide polymorphism in at least one gene selected from the group consisting of BTNL2 gene, C5orf55 gene, HCG23 gene, HLA-DRA gene, ORMDL3 gene, and IL17REL gene from a biological sample. It provides a method for providing information regarding the prediction or diagnosis of inflammatory bowel disease.

In one embodiment of the present invention, the monobasic polymorphism is rs41417449 of BTNL2 gene, rs10035653 of C5orf55 gene, rs3117099 of HCG23 gene, rs7192 of HLA-DRA gene, and rs3744246 of ORMDL3 gene. In another embodiment of the present invention, if the monobasic polymorphism is confirmed, it is predicted that the incidence of inflammatory bowel disease is high. In another embodiment of the invention, the monobasic polymorphism is rs713669 of the IL17REL gene. In another embodiment of the present invention, if the monobasic polymorphism is confirmed, it is predicted that the incidence of inflammatory bowel disease is low. In another embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis (UC), Crohn's disease, collagen colitis, lymphocytic colitis, ischemic colitis, converting colitis, Behcet's disease and indeterminate colitis Any one selected from the group consisting of. In another embodiment of the present invention, the method for providing information regarding the prediction or diagnosis of inflammatory bowel disease is characterized in that it is targeted to Koreans. In another embodiment of the invention, the biological sample is characterized in that the body fluid, hair root, blood, plasma or serum. In another embodiment of the invention, the step of identifying may include deep resequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele specific hybridization technique (dynamic allele- specific hybridization (DASH), TaqMan technique, SnapShot technique and qRT-PCR analysis.

In addition, the present invention is 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. Provided is a composition for predicting or diagnosing inflammatory bowel disease, comprising the set.

In one embodiment of the present invention, the monobasic polymorphism is rs41417449 of BTNL2 gene, rs10035653 of C5orf55 gene, rs3117099 of HCG23 gene, rs7192 of HLA-DRA gene, and rs3744246 of ORMDL3 gene. In another embodiment of the present invention, if the monobasic polymorphism is confirmed, it is predicted that the incidence of inflammatory bowel disease is high. In another embodiment of the invention, the monobasic polymorphism is rs713669 of the IL17REL gene. In another embodiment of the present invention, if the monobasic polymorphism is confirmed, it is predicted that the incidence of inflammatory bowel disease is low. In another embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis (UC), Crohn's disease, collagen colitis, lymphocytic colitis, ischemic colitis, converting colitis, Behcet's disease and indeterminate colitis It is characterized in that any one selected from the group consisting of. In another embodiment of the present invention, the method for providing information regarding the prediction or diagnosis of inflammatory bowel disease is characterized in that it is targeted to Koreans.

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

Hereinafter, the present invention will be described in detail step by step.

The method of confirming the single nucleotide polymorphism of the locus according to the present invention and providing information on the prediction or diagnosis of inflammatory bowel disease is a method of confirming or diagnosing the occurrence of inflammatory bowel disease among Korean patients by confirming the single nucleotide polymorphism. As it can predict the high risk group of inflammatory bowel disease in Korea, it has clinical significance, and it reduces the risk of inflammatory bowel disease by reducing the risk of various treatments such as individual treatment, procedure, and surgery. It is expected to be effectively used to

1 is a flowchart illustrating a next generation sequence analysis according to an embodiment of the present invention.
Figure 2 is a view showing the immunohistochemical staining results of BTNL2 and C5orf55 according to an embodiment of the present invention.
Figure 3 is a graph showing the mRNA levels of BTNL2 and C5orf55 in inflammatory colon 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-α according to an embodiment of the present invention.
6 is a view showing the result of performing a Western blotting according to an embodiment of the present invention.
7 is a view showing the results of LC3B spot accumulation and apoptotic nucleus formation according to an embodiment of the present invention.
FIG. 8 is a view showing a linkage disequilibrium (LD) structure of BTNL2, IL23R, HLA, and ZPBP2 SNV in Koreans according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Target group

The population according to the present example was a Korean, consisting of ulcerative colitis patients and a normal control group. Patients with ulcerative colitis were diagnosed at the Yonsei University College of Medicine Severance Hospital from June 2006 to August 2014 according to conventional clinical, colonoscopy, radiography, and histopathological criteria and regularly followed. Indeterminate colitis (IC), which cannot be classified as Crohn's disease or inflammatory bowel disease, was excluded from this example. In addition, control groups without specific diseases were recruited. This control group was randomly selected from those recruited from the Yonsei Cardiovascular Genome Center. The 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 performed in accordance with the relevant guidelines and regulations.

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

Example  1: Review of full-length genetic association analysis to identify genes related to ulcerative colitis

To identify genes related to 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 the Asian population (Japanese) and the rest in the European ancestor population. Also included were genes considered based on the function that plays a role in the development of IBD, and upon reviewing these genes, 108 genes were finally selected as targets for deep resequencing.

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

Whole blood samples using DNA blood maxi kit (Qiagen (Santa Clarita, CA)), quantitatively analyzed using a high-sensitivity Qubit system (Invitrogen, Carlsbad, CA) and assessed for integrity with agarose gel Genomic DNA was extracted from. The library was prepared using Agilent's SureSelect Exome Enrichment kit (Illumina, San Diego, California, USA) according to the manufacturer's protocol. Exome capture in the 10 Kb region upstream of the 5 'end and the 10 Kb downstream of the 3' end of the gene was performed using a SureSelect Target Enrichment System (Agilent Technologies, Santa Clarita, CA, USA) according to the manufacturer's protocol. 108 genes were carried out. Captured ligation mediated polymerase chain reaction (LM-PCR) products were analyzed by Agilent 2100 Bioanalyzer to assess the quality of the library. The captured library was then loaded onto the Illumina Hiseq2000 platform (Illumina) and sequencing of the captured library was performed at high throughput to ensure that each sample met the desired degree of average sequencing. Raw image files were processed by HiSeq Control Software version 1.4.8. Base-calling was performed using baseline parameters, and each individual sequence was generated with 90 base pair (bp) paired-end reads.

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

SNV identified by resequencing was compared with sequencing data from 126 healthy controls. According to the association analysis, nonsynonymous SNV and upstream SNV with significant differences in replacement allele frequencies between cases and controls were selected for further replication genotyping. When various SNVs were identified in one ulcerative colitis sensitive gene, a representative SNV of the coding region with a significantly lower p value than the other SNV was selected.

Using Fludigm or TaqMan probes, 106 SNVs including 52 nonsynonymous SNVs in 29 genes, 41 upstream SNVs of 27 genes, and 13 SNVs in 5 genes were reported in 29 GWAS studies. Genotypes were analyzed in a sample of 225 ulcerative colitis patients and a 230 healthy control group for a replicate study. Association analysis of SNV selected in test and control groups was performed under four alternative models (dominant, recessive, co-dominant and allele). Based on this analysis, 28 SNVs (BTNL2 (butyrophilin like 2), C5orf55 (Chromosome 5 Open Reading Frame 55), CCL2 (CC motif) in 15 genes with significantly lower p-values than other SNVs in the second clone genotyping study. chemokine ligand 2), FC fragment of IgG receptor IIa (FCGR2A), G protein-coupled receptor 35 (GPR35), gasdermin B (GSDMB), HLA Complex Group 23 (HCG23), major histocompatibility complex (HLA-DRA), class II, DR alpha), IL17REL (interleukin 17 receptor E-like), IL22 (interleukin 22), IL23R (interleukin 23 receptor), ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3), TNFSF15 (tumor necrosis factor superfamily member 15), UTS2 (urotensin 2) ), ZPBP2 (zona pellucida binding protein 2)) was selected.

In a second cloning study, the 28 SNVs described above were genotyped in independent samples of 568 ulcerative colitis patients and 553 controls. Combined analysis identified four SNVs that were significantly associated with ulcerative colitis susceptibility (see Table 3). 6 SNVs (or SNPs), rs10035653 (p.P50S) in C5orf55, rs41417449 (p.M295V) in BTNL2, rs3117099 (non-coding region) in HCG23, rs7192 (p.L242V) in HLA-DRA , Rs3744246 (non-coding region) in ORMDL3, and rs713669 (non-coding region) in IL17REL were new ulcerative colitis susceptibility loci not previously reported. This was confirmed to have a very high correlation with the risk of ulcerative colitis in Korean, and through these four locus was confirmed that the locus is associated with the specific ulcerative colitis in Korea. In addition, it was confirmed that the locus or monobasic polymorphism of the present invention can predict or diagnose the risk of ulcerative colitis in Koreans, that is, the occurrence of ulcerative colitis.

1.2. Statistical analysis

Allele frequency and allele frequency of subjects in different groups using the chi square test or Cochran-Armitage Trend test, and the Jonckheere-Terpstra test by running SAS version 9.3.1 (SAS institute Inc. Cary, NC). Obtained by comparing genotype frequencies. Allele frequencies were compared between ulcerative colitis patients and controls using logistic regression to determine age and gender corrected odds ratio (OR) and 95% confidence interval (CI). It was. The Benjamini and Hochberg test was applied to modify multiple tests in the combined association analysis of the first and second genotypes. Associative equilibrium (LD) blocks and associative equilibriums were determined in SNPs using the HaploView program (http://www.broad.mit.edu/mpg/haploview/). Gene expression results derived from in vitro and in vivo experiments are expressed as mean ± standard deviation (SD) or ± standard error (SEM). Prism software (GraphPad Software, Inc., San Diego, CA) was used for all assays. Differences in conditions were assessed using analysis of variance (ANOVA) and t-tests. When the p value was less than 0.05, it was determined to be statistically significant.

Example  2: ulcerative colitis On SNV  Validation and cloning research

Overall GWAS results, 9 SNVs, i.e. rs10035653 (P = 2.08 × 10 ^-3 ; OR = 1.50) for C5orf55, rs3117099 (P = 9.98 × 10 ^-6 ; OR = 1.40) for HCG23, rs41417449 (P = 1.27 for BTNL2) × 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), rs7192 (P = 6.95 × 10 ⁻⁹ ; OR = 1.57) of HLA-DRA, rs3744246 (P = 2.21 × 10 ⁻² ; OR of ORMDL3 = 1.21) and rs9271366 (P = 3.34 × 10 ⁻¹³ ; OR = 2.23) have reached significant thresholds, which may be causal variants associated with the development of ulcerative colitis. In other words, the SNV (SNP) was found to have a close relationship with the high risk of ulcerative colitis (high incidence) by causing degradation of gene function due to abnormal protein or ribonucleic acid production. . Conversely, 7 SNVs, i.e. rs713669 (P = 2.69 × 10 ⁻² ; OR = 0.84) of IL17REL, 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) have been identified as important variants that can confer protection against the development of ulcerative colitis (protective variants). That is, the seven SNPs are closely related to the low risk of developing ulcerative colitis as a mutation that reduces inflammation by preventing mutations in genes that promote inflammation. , The incidence of mutations was lower in the ulcerative colitis group.

SNVs by GWAS are closely correlated with other SNVs in the domain and may be associated with causative variants rather than causal variants themselves. To determine if there were any haplotypes associated with ulcerative colitis, a single-phase analysis and an associated non-equilibrium structural analysis were performed among the final candidates. The results are shown in Table 4. Genotypes of 793 ulcerative colitis patients and 783 healthy control samples were used to estimate the association equilibrium. In single phase 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 variants that contribute to the development of ulcerative colitis. LD analysis showed that the BTNL2 variants p.G454C and p.D336N (rs28362675 and rs41441651) were nearly complete associative equilibrium (r ² = 1) with each other as well as with IL23R, HLA and ZPBP2. Through the above results, the variant of BTNL2 gene was identified as a major causative variable for ulcerative colitis in Korea, and it can be inferred that other SNPs of BTNL2 gene may be related to the risk of developing ulcerative colitis. (FIG. 8).

Example  3: SNV  Function evaluation

To assess the functional effects of nonsynonymous SNV, the effects of amino acid changes on protein damage in silico were investigated using the SIFT, PolyPhen2 and MutationTaster databases. P.P50S at C5orf55 and p.G454C and p.D336N at BTNL2 were found to be damaged in in silico analysis. Potential regulatory functions of candidate SNPs were assessed using RegulomeDB. The score for rs713669 (score = 1f) in IL17REL indicated a relatively high proportion of evidence that there was a potential regulatory function. The locus of ORMDL3 was significantly associated with susceptibility to ulcerative colitis. cis-eQTL analysis showed evidence that variants of HCG23, HLA-DRA, and ORMDL3 influence progenitor differentiation gene (NOTCH4) inflammation related genes (HLA-DRB, HLA-DRA, ORMDL3).

In other words, rs10035653 in the C5orf55 gene, rs41417449 in the BTNL2 gene, rs3117099 in the HCG23 gene, and HLA- in the analysis of the subject population (793 patients, 783 normal patients), which combined both the next-generation sequencing analysis and the replication step It was confirmed that the single nucleotide polymorphism of rs7192 in the DRA gene, rs3744246 in the ORMDL3 gene, and rs713669 in the IL17REL gene was highly associated with ulcerative colitis. In addition, these variants are newly discovered genetic variants and provide meaningful information for the diagnosis and prediction of ulcerative colitis. In addition, the present invention is a genetic variant research through the next generation sequence analysis conducted for Koreans, it has a significant meaning to discover important genetic variants.

Additionally, Figures 2 and 3 show gene expression in inflammatory tissues of human and mouse colon. Figure 2 is a photograph showing the immunohistochemical staining of BTNL2 and C5orf55, human colon tissue (CTL, n = 8, UC, n = 17) BTNL2 (A in Figure 2) and C5orf55 (B in Figure 2) Staining with antibody against. The graph on the right shows disease-dependent expression scores in crypt and laminar propria (LP). Sections were counterstained with hematoxylin (blue).

3 shows mRNA levels of BTNL2 and C5orf55 in inflammatory colorectal tissue. Total RNA was isolated from human and mouse colon tissue and converted to complementary DNA. Transcriptional levels of BTNL2 (left), C5orf55 (right), and Btn12 (left), C5orf55 (right) of mouse colon tissue (Fig. 3B) from human colon tissue (FIG. 3A). Measurements were made by real-time quantitative reverse transcription polymerase chain reaction and normalized to the level of transcription 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 <0.05 vs. CTL or Sham, ** P <0.01 vs. CTL or Sham.

4-7 show the functional analysis of BTNL2 and C5orf55 in human intestinal epithelial cells. 4 is a graph showing mRNA expression of BTNL2 and C5orf55. To determine whether LPS stimulation affected the expression of BTNL2 and C5orf55, protein and mRNA levels were measured in HT-29 cells treated with lipid polysaccharides (LPS, 1 μg / mL) for 4 hours. 5 to 7 show that siRNA was used to inhibit BTNL2 and C5orf55 expression in HT-29 cells. 5 is a graph showing mRNA expression of TNF-α. Real-time quantitative reverse transcriptase chain reaction (qRT-PCR) was used to analyze the transcription level of TNF-α 3 hours after LPS stimulation. 6 is an image of Western blot. Protein levels of TNF-α were analyzed 24 hours after LPS stimulation. 7 is an image showing LC3B spot accumulation and apoptotic nucleation. Cells were treated with LPS for 24 hours, stained and photographed under fluorescence microscopy (× 400). Indicators for autophagy activity indicated LC3B spots (green), and apoptosis indicators (indicated by arrows) as apoptotic nucleation indicated by staining of DAPI (blue). Data are expressed as mean ± standard error (SEM). * P <0.05 vs. Untreated, ** P <0.01 vs. Untreated, *** P <0.005 vs. Untreated, ^# P <0.05 vs. Untreated, ** P <0.01 vs. Untreated, *** P <0.005 vs. Untreated.

As shown in FIG. 2, the protein levels of BTNL2 and C5orf55 were significantly lower in the negative and mucosal plaques of the colon of the ulcerative colitis patient group than in the control tissues. However, as shown in Figure 3, the levels of transcription of BTNL2 and C5orf55 in inflammatory bowel disease (MDD) patients and mice with colitis were significantly higher than in healthy controls. In addition, BTNL2 positive cells were found more frequently in the stromal lamellar plate of ulcerative colitis patients than in the healthy control group. Thus, it can be seen that these genes are regulated post-transcriptional as a compensatory mechanism for deficiencies of protein function, thereby protecting them from inflammation. As shown in FIG. 4, LPS (lipid polysaccharide) induced increased expression of mRNA and protein levels of BTNL2 and C5orf55 in HT-29 cells, which are enteric epithelial cells. As shown in FIGS. 5 and 6, additional functional studies were performed in HT-29 cells using siRNA, and BTNL2 and C5orf55 knockdown cells had significantly higher mRNA and protein levels of TNF-α than controls, which was LPS stimulated. Was further increased by.

Disruption of primary autophages promotes the production of LPS-induced IL-1β, and overactivated autophages lead to child cell death. Thus, autophagy dysregulation is associated with various inflammatory diseases. As shown in FIG. 7, untreated control cells showed diffuse expression of LC-3B (green), whereas basal LC-3B levels decreased in BTNL2 and C5orf55 knockdown cells compared to control cells. In addition, in response to LPS, the formation of autopasomes and progeny cell death increased significantly in these cells.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (17)

Identifying a single nucleotide polymorphism in at least one gene selected from the group consisting of HLA-DRA gene, ORMDL3 gene, and IL17REL gene from a biological sample, wherein the single nucleotide polymorphism is A method of providing information concerning the prediction or diagnosis of ulcerative colitis (UC), which is rs7192, rs3744246 of the ORMDL3 gene, or rs713669 of the IL17REL gene. delete The method of claim 1,
When the rs7192 of the HLA-DRA gene or the rs3744246 of the ORMDL3 gene is identified during the monobasic polymorphism, the incidence of ulcerative colitis (UC) is expected to be high, and the prediction or diagnosis of ulcerative colitis (UC) How to Provide Information. delete The method of claim 1,
A method of providing information on the prediction or diagnosis of ulcerative colitis (UC) predicting that the incidence of ulcerative colitis (UC) is low when rs713669 of the IL17REL gene is identified during the monobasic polymorphism. delete The method of claim 1,
The method of providing information about the prediction or diagnosis of ulcerative colitis (UC) is for Koreans, the method of providing information about the prediction or diagnosis of ulcerative colitis (UC). The method of claim 1,
Wherein said biological sample is fluid, hair root, blood, plasma or serum, providing information regarding the prediction or diagnosis of ulcerative colitis (UC). The method of claim 1,
The checking step may include deep resequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH), TaqMan technique, A method of providing information about ulcerative colitis (UC) prediction or diagnosis performed by a technique selected from the group consisting of SnapShot technique and qRT-PCR analysis. 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, wherein the single nucleotide polymorphism is HLA-DRA gene A composition for predicting or diagnosing ulcerative colitis (UC), which is rs7192, rs3744246 of the ORMDL3 gene, or rs713669 of the IL17REL gene. delete The method of claim 10,
When the rs7192 of the HLA-DRA gene or rs3744246 of the ORMDL3 gene is confirmed during the single nucleotide polymorphism, the incidence of ulcerative colitis (UC) is high, predicting or diagnosing ulcerative colitis (UC). delete The method of claim 10,
If rs713669 of the IL17REL gene of the single nucleotide polymorphism is confirmed that the incidence of ulcerative colitis (ulcerative colitis, UC) is expected to be high, ulcerative colitis (UC) prediction or diagnostic composition. delete The method of claim 10,
The prediction or diagnosis by the composition for predicting or diagnosing ulcerative colitis (ulcerative colitis, UC) is for Koreans, ulcerative colitis (UC) predictive or diagnostic composition. 16. A kit for predicting or diagnosing ulcerative colitis (UC), comprising a composition for predicting or diagnosing ulcerative colitis (UC) according to any one of claims 10, 12, 14, and 16. .

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