Genetic diagnosis of inflammatory bowel disease

Abstract

The present invention provides a method for predicting the likelihood or low likelihood of progression of inflammatory bowel disease (IBD) in a subject, and a method for diagnosing inflammatory bowel disease (IBD) in a subject. Furthermore, it provides a method for identifying genes / locuses associated with disease conditions such as IBD, CD, and / or UC. [Solution] A method for predicting whether a subject is likely or unlikely to develop inflammatory bowel disease, the method comprising: a step of identifying the subject's genotype with respect to a risk allele of a gene / locus; a step of predicting, after detecting the risk allele, that the subject is likely to develop IBD; or, if the risk allele is not detected, a step of predicting that the subject is unlikely to develop IBD.

Description

Technical Field

[0001] Statement Regarding Federally Sponsored Research The present invention was made with government support under grant numbers DK108140 and DK062413 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0002] The present invention relates to genetics and medicine.

Background Art

[0003] All publications herein referenced are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The following description contains information that is useful in understanding the present invention. It is not admitted that any of the information provided herein is prior art, or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Crohn's disease (CD) and ulcerative colitis (UC), two common forms of idiopathic inflammatory bowel disease (IBD), are chronic, relapsing inflammatory diseases of the gastrointestinal tract. Each has a peak age of onset between 20 and 40 years of age, and an average prevalence of approximately 100–150 per 100,000 people in the European ancestral population (DK Podolsky, N Engl J Med 347, 417 (2002); EV Loftus, Jr., Gastroenterology 126, 1504 (2004)). While the precise etiology of IBD remains unclear, a widely accepted hypothesis is that ubiquitous, symbiotic gut bacteria trigger an inappropriate, hyperactive, and ongoing mucosal immune response that mediates intestinal tissue damage in genetically sensitive individuals (DK Podolsky, N Engl J Med 347, 417 (2002)). Genetic factors play a crucial role in the pathogenesis of IBD, as evidenced by the increased rate of IBD in Ashkenazi Jews, the familial clustering of IBD, and the increased incidence of IBD in monozygotic twin pairs compared to dizygotic twin pairs (S. Vermeire, P. Rutgeerts, Genes Immun 6, 637 (2005)). CD and UC are considered related disorders that share several genetic susceptibility loci but are distinct from each other.

[0005] Therefore, there is a need in the art to determine other genes, allele variants, and / or haplotypes that can assist in explaining, diagnosing, and / or predicting susceptibility to or protection against inflammatory bowel disease, including but not limited to CD and / or UC. [Overview of the project]

[0006] Various embodiments of the present invention provide a method for predicting whether a subject is likely or unlikely to develop inflammatory bowel disease (IBD), the method comprising: identifying the subject's genotype for a risk allele of a gene / locus; predicting, after detection of the risk allele, that the subject is likely to develop IBD; or predicting, if no risk allele is detected, that the subject is unlikely to develop IBD.

[0007] In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, the HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof. In various embodiments, the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. In various embodiments, the gene / locus includes one or more of SEQ ID NO:1-SEQ ID NO:341.

[0008] In various other embodiments, the step of identifying the genotype of a subject includes the steps of obtaining a sample from the subject and identifying the genotype of the sample with respect to a risk allele at a gene / locus. In yet another embodiment, the step of identifying the genotype of a sample includes contacting the sample with an oligonucleotide probe specific to the risk allele; generating an allele-specific hybridization complex between the oligonucleotide probe and the risk allele; detecting the risk allele after detecting the allele-specific hybridization complex; or not detecting the risk allele if the allele-specific hybridization complex is not detected. In some embodiments, the oligonucleotide probe is labeled with a fluorescent dye, where the detection of the allele-specific hybridization complex includes detecting a fluorescent signal from the oligonucleotide probe. In another embodiment, the oligonucleotide probe includes a reporter dye and a quencher dye.

[0009] In various embodiments, the method further includes the step of performing PCR amplification after forming an allele-specific hybridization complex.

[0010] Various embodiments of the present invention provide a method for diagnosing inflammatory bowel disease (IBD) in a subject, the method comprising: identifying the genotype of a subject sample for a risk allele at a gene / locus; diagnosing IBD in the subject after detection of the risk allele; and administering IBD treatment to the subject diagnosed with IBD, thereby treating the subject's IBD. In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, the HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof. In various embodiments, the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. In various embodiments, the gene / locus includes one or more of SEQ ID NO:1-SEQ ID NO:341. In various embodiments, the method further includes the step of providing IBD treatment to a subject. In some embodiments, the IBD treatment includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof.

[0011] Various embodiments of the present invention provide a method comprising the steps of: identifying the genotype of a subject sample for risk alleles at a gene / locus; diagnosing IBD in the subject after detection of the risk allele; and administering IBD treatment to the subject diagnosed with IBD, thereby treating the subject's IBD. In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or combinations thereof. In various embodiments, the gene / locus includes ETS1, HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or combinations thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. In various other embodiments, the gene / locus includes one or more of SEQ ID NO:1-SEQ ID NO:341. In various embodiments, the method further includes the step of providing IBD treatment to a subject. In some embodiments, the IBD treatment includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof.

[0012] Various embodiments of the present invention provide a method for identifying disease-associated genes / locuses, the method comprising: obtaining genetic data from a sample of a disease cohort; performing GLS conversion on the genetic data to decorrelate the genetic data; performing gene-based analysis on the GLS-converted genetic data; and identifying disease-associated genes / locuses. In various embodiments, the disease is IBD, CD, or UC, or a combination thereof. In some embodiments, the cohort includes correlated subjects or family subjects. In other embodiments, the genetic data includes SNP genotypes. In yet another embodiment, the GLS conversion step is performed using the following function

[0013]

number

[0014] In various embodiments, the step of performing a gene-based analysis includes the step of applying gene-based tests based on the assumption of independent or uncorrelated subjects. In various embodiments, the step of performing a gene-based analysis includes the step of applying C-alpha, SKAT, SKAT-CommonRare, CMC, WSS, variable threshold, or a comprehensive approach, or a combination thereof. [Brief explanation of the drawing]

[0015] Typical embodiments are illustrated in the reference drawings. The embodiments and drawings disclosed herein are intended to be considered illustrative, not limiting. [Figure 1] A and B represent single-SNP-based and gene-based analyses according to various embodiments of the present invention. [Figure 2A] The Manhattan Plot is represented according to various embodiments of the present invention. [Figure 2B] The Manhattan Plot is represented according to various embodiments of the present invention. [Figure 3] A Manhattan plot is shown according to various embodiments of the present invention. The Jostin region is excluded. [Figure 4] The following is a list of novel genes / regions identified in our analysis according to various embodiments of the present invention: SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, and TET2. [Figure 5A] According to various embodiments of the present invention, this represents an increased possibility of histone marks compared to non-IBD regions. [Figure 5B] According to various embodiments of the present invention, an increased likelihood of finding a histone mark compared to known IBD SNPs is represented. [Figure 6A] Detailed examination of the TET2 region according to various embodiments of the present invention: local plots are shown. rs17035289 is SEQ ID NO: 333, and rs2726518 is SEQ ID NO: 241. All other rs numbers are found in Table 1. [Figure 6B] Detailed examination of the TET2 region according to various embodiments of the present invention: Represents SNPs. rs17035289 is SEQ ID NO: 333, and rs2726518 is SEQ ID NO: 241. All other rs numbers are found in Table 1. [Figure 6C] Detailed inspection of the TET2 region according to various embodiments of the present invention: representing fine mapping. rs17035289 is SEQ ID NO: 333, and rs2726518 is SEQ ID NO: 241. All other rs numbers are found in Table 1. [Figure 6D] Detailed inspection of the TET2 region according to various embodiments of the present invention: representing function. rs17035289 is SEQ ID NO: 333, and rs2726518 is SEQ ID NO: 241. All other rs numbers are found in Table 1. [Figure 7A]According to various embodiments of the present invention, detailed examination of the LRRC16 region: represents local plots. [Figure 7B] According to various embodiments of the present invention, detailed examination of the LRRC16 region: represents local plots. [Figure 7C] According to various embodiments of the present invention, detailed examination of the LRRC16 region: represents fine mapping with four independent signals. [Figure 8A] According to various embodiments of the present invention, eQTL results: represented by SeeQTL. rs9358858 is SEQ ID NO:335. All other rs numbers are found in Table 1. [Figure 8B] According to various embodiments of the present invention, eQTL results: represented by Scandb. rs9358858 is SEQ ID NO:335. All other rs numbers are found in Table 1. [Figure 8C] According to various embodiments of the present invention, eQTL results: represented by GeneVar. rs9358858 is SEQ ID NO:335. All other rs numbers are found in Table 1. [Figure 9A] According to various embodiments of the present invention, detailed examination of the first part in the HIST1 cluster: represents genes. rs2071303 is SEQ ID NO:336, rs13198474 is SEQ ID NO:337, rs198846 is SEQ ID NO:338, and rs198854 is SEQ ID NO:339. [Figure 9B] According to various embodiments of the present invention, detailed examination of the first part in the HIST1 cluster: represents local signals. rs2071303 is SEQ ID NO:336, rs13198474 is SEQ ID NO:337, rs198846 is SEQ ID NO:338, and rs198854 is SEQ ID NO:339. [Figure 9C]According to various embodiments of the present invention, a detailed examination of the first portion in the HIST1 cluster represents a fine-grained mapping with three independent signals (P=2.23E-25). rs2071303 is SEQ ID NO:336, rs13198474 is SEQ ID NO:337, rs198846 is SEQ ID NO:338, and rs198854 is SEQ ID NO:339. [Figure 10A] According to various embodiments of the present invention, eQTL analysis: SCANdb is represented. rs13198474 is SEQ ID NO: 337, rs198846 is SEQ ID NO: 338, and rs198854 is SEQ ID NO: 339. [Figure 10B] According to various embodiments of the present invention, eQTL analysis: Blood eQTL is represented. rs13198474 is SEQ ID NO: 337, rs198846 is SEQ ID NO: 338, and rs198854 is SEQ ID NO: 339. [Figure 11A] Detailed examination of the second portion in the HIST1 cluster according to various embodiments of the present invention: representing genes. rs9295740 is SEQ ID NO:340, and rs9461412 is SEQ ID NO:241. [Figure 11B] Detailed examination of the second portion in the HIST1 cluster according to various embodiments of the present invention: representing local signals. rs9295740 is SEQ ID NO:340 and rs9461412 is SEQ ID NO:241. [Figure 11C] A detailed examination of the second portion in the HIST1 cluster according to various embodiments of the present invention: representing fine mapping with four independent signals (P=3.25E-29). rs9295740 is SEQ ID NO:340, and rs9461412 is SEQ ID NO:241. [Figure 12] eQTL analysis: SCANdb is represented according to various embodiments of the present invention. [Figure 13]According to various embodiments of the present invention, three independent signals are represented: LRRC16, a first portion in the HIST1 cluster, and a second portion in the HIST1 cluster. [Modes for carrying out the invention]

[0016] All references cited herein are incorporated in their entirety by reference as if they were fully explicitly stated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY The 2012 edition provides general guidance to those skilled in the art regarding many of the terms used in this application.For references to methods for preparing antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0017] Those skilled in the art will recognize many methods and substances similar to or equivalent to those described herein that can be used in carrying out the present invention. Other functions and advantages of the present invention will become apparent from the following detailed description, provided with the accompanying drawings, which illustrate various functions of embodiments of the present invention as an example. In fact, the present invention is not limited in any way to the methods and substances described herein. For convenience, specific terms used in the specification, examples, and appended claims are gathered hereby.

[0018] Unless otherwise specified or implicitly understood from the context, the following terms and phrases have the meanings provided below. Unless otherwise explicitly specified or evident from the context, the following terms and phrases do not exclude the meanings acquired in the art to which the term or phrase belongs. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention pertains. The invention is not limited to and therefore subject to the specific methods, protocols, and reagents described herein. The definitions and technical terms used herein are provided to aid in the description of specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited solely by the claims.

[0019] As used herein, the terms “comprising” or “comprises” are used to refer to compositions, methods, and each of their components that are useful in the embodiments, including unspecified elements, whether useful or not. It will be understood by those skilled in the art that the terms used herein are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” as “having at least,” and the term “includes” as “including but not limited to.” The open term “comprising” is used herein to describe and claim the invention as an alternative to terms such as including, containing, or having, although the invention or its embodiments may also be described using alternative terms such as “consisting of” or “substantially consisting of.”

[0020] Unless otherwise specified, the terms “a,” “an,” “the,” and similar references used in the context describing a particular embodiment of this application (especially in the context of the claims) may be interpreted as encompassing both singular and plural forms. Enumerations of value ranges in this specification are intended merely as abbreviations to refer individually to each separate value contained within that range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were separately enumerated. Unless otherwise indicated herein or expressly refuted by the context, all methods described herein may be performed in any suitable order. Any and all examples or illustrative language (e.g., “such as”) provided in reference to a particular embodiment herein are intended merely to better illustrate this application and do not impose any limitation on the scope of the application otherwise claimed. The abbreviation “eg” derives from the Latin “exempli gratia” (for example) and is used herein to indicate a non-restrictive example. Thus, the abbreviation “eg” is synonymous with the term “for example.” The language used herein should not be construed as indicating any unclaimed elements essential to the execution of the application.

[0021] As used herein, when used in relation to a disease, disorder, or medical condition, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to both therapeutic treatments and preventive or mitigating measures aimed at preventing, reversing, easing, improving, inhibiting, mitigating, slowing, or stopping the progression or severity of a symptom or condition. The term “treating” includes reducing or easing at least one side effect or symptom of a disease. Treatment is usually “effective” if one or more symptoms or clinical indicators are reduced. Alternatively, treatment is “effective” if the progression of a disease, disorder, or medical condition is reduced or stopped. That is, “treatment” includes not only improvement of symptoms or markers but also stopping or at least slowing the progression or worsening of symptoms that would be expected in the absence of treatment. Also, “treatment” may mean tracking or obtaining a useful outcome, or reducing the likelihood that an individual will progress the disease, even if the treatment is ultimately unsuccessful. Those who require treatment include not only those who already have a disease, but also those who are prone to developing a disease, or those for whom disease prevention is necessary.

[0022] “Beneficial outcomes” or “desirable outcomes” may include, but are not limited to, mitigating or reducing the severity of the disease state, preventing the worsening of the disease state, curing the disease state, preventing the progression of the disease state, reducing the likelihood of the patient progressing to the disease state, reducing morbidity and mortality, and extending the patient’s lifespan or average lifespan. As an unspecified example, “beneficial outcomes” or “desirable outcomes” may include relief of one or more symptoms, a reduction in the degree of defect, a stable state (i.e., no worsening) of intestinal inflammation and / or fibrosis, delay or slowing of intestinal inflammation and / or fibrosis, and improvement or relief of symptoms associated with intestinal inflammation and / or fibrosis.

[0023] "Disease," "illness," and "disease condition" as used herein may include, but are not limited to, any form of disease, disorder, or condition relating to inflammation of the intestines or inflammation of the intestines, such as inflammation of the intestines, intestinal fibrosis, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), colitis, acute colitis, and chronic colitis.

[0024] A method for predicting the likelihood or low likelihood of a subject developing inflammatory bowel disease (IBD) includes: identifying the subject's genotype for risk alleles of a gene / locus; predicting the likelihood of the subject developing IBD after detecting the risk allele; or predicting the likelihood of the subject developing IBD if no risk allele is detected.

[0025] As used herein, “risk variant” refers to an allele whose presence is associated with increased susceptibility to inflammatory bowel diseases, including but not limited to Crohn’s disease and ulcerative colitis, compared to individuals without the risk variant.

[0026] "High probability" as used herein refers to increased susceptibility to inflammatory bowel disease when the risk variant is present in an individual compared to an individual without the risk variant.

[0027] "Low probability," as used herein, refers to a reduced susceptibility to inflammatory bowel disease when the risk variant is not present in the individual, compared to when the risk variant is present in the individual.

[0028] As used herein, the term “administer” means the placement of a drug into a subject, such as those disclosed herein, by a method or route resulting in at least partial localization of the drug at a desired site. “Route of administration” may refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, parenteral, intestinal, topical, or local. “Pareral” refers to routes of administration generally associated with injection, including intracranial, ventricular, intrasacral, epidural, intradural, intraorbital, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspine, intrasternal, intrasternal, intrauterine, intravenous, subarachnoid, intracapsular, subcutaneous, transmucosal, or transtracheal. Via parenteral routes, compositions may be in the form of solutions or suspensions for infusion or injection, or lyophilized powders. Via the intestinal route, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microparticles or nanoparticles, or lipid vesicles or polymer vesicles, allowing for controlled release. Via the topical route, the pharmaceutical composition may be in the form of aerosols, lotions, creams, gels, ointments, suspensions, solutions, or emulsions. In accordance with the present invention, “administering” may be self-administering. For example, consumption by a subject of a composition as disclosed herein is considered “administering.”

[0029] As used herein, the terms “sample” or “biological sample” refer to a sample obtained from or isolated from a biological organism, such as a blood sample of a subject. Typical biological samples include, but are not limited to, cheek swab specimens; mucus; whole blood, blood, serum; plasma; urine; saliva; semen; lymph; fecal extracts; sputum; other body fluids or biological fluids; cell samples; and / or tissue samples. The term also includes mixtures of the above-mentioned samples. The term “sample” also includes untreated or pre-treated (or pre-processed) biological samples. In some embodiments, a sample may include one or more cells from a subject.

[0030] As used herein, “subject” means human or animal. Typically, animals are vertebrates such as primates, rodents, domestic animals, or game animals. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Rodents include mice, rats, wild mice, ferrets, rabbits, and hamsters. Domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, and canine species, e.g., dogs, foxes, and wolves. The terms “patient,” “individual,” and “subject” are used interchangeably herein. In one embodiment, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cows. In addition, the methods described herein can be used to treat domestic animals and / or pets.

[0031] As used herein, “mammals” refers to members of the mammalian classification, including, but not limited to, humans and non-human primates such as chimpanzees and other apes and monkey species; domesticated animals such as cattle, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; and laboratory animals such as rodents such as mice, rats, and guinea pigs. This term does not imply any specific age or sex. Therefore, in addition to adult and neonatal subjects, fetuses, whether male or female, are intended to be included within the scope of this term.

[0032] A subject may be someone who suffers from or has been previously diagnosed with or identified as having a disease requiring treatment (e.g., intestinal inflammation and / or fibrosis, IBD, CD, UC, colitis, acute colitis, and chronic colitis), or one or more complications associated with that disease, and who has optionally already received treatment for the disease or one or more complications associated with that disease. Alternatively, a subject may also be someone who has not previously been diagnosed with the disease or one or more complications associated with that disease. For example, a subject may present one or more risk factors for the disease or one or more complications associated with that disease, or a subject may not present any risk factors. A "subject requiring treatment" for a particular disease may be someone who is suspected of having the disease, has been diagnosed with the disease, has already received or is receiving treatment for the disease, whose disease is untreated, or who is at risk of disease progression.

[0033] The term "statistically significant" or "significantly significant" refers to statistical evidence that a difference exists. This is defined as the likelihood of rejecting the null hypothesis when the null hypothesis is actually true. This decision is frequently made using the p-value.

[0034] As used herein, the odds of a disease in a particular population ("a disease's odds" or "odds of a disease") are defined as the ratio of the likelihood of having the disease to the likelihood of not having the disease in that population (i.e., odds of disease = likelihood of having the disease / likelihood of not having the disease).

[0035] As used herein, with respect to a disease, the odds ratio (OR) of a risk allele is defined as the ratio of the odds of disease in the carrier population to the odds of disease in the non-carrier population. (i.e., OR of a risk allele = odds of disease in carriers / odds of disease in non-carriers).

[0036] Method of invention This invention provides a method for identifying genes / locuses associated with diseases such as IBD. The identification of these genes / locuses can be used for classifying the severity of a population with respect to IBD. Such tools can be used at birth to identify individuals at risk of IBD, with the aim of influencing a population through the delivery of preventive interventions that can modulate environmental epigenetic factors. This invention also provides a method for diagnosing IBD and a method for individually addressing IBD treatment plans as precise medical techniques.

[0037] prediction Various embodiments of the present invention provide a method for predicting whether a subject is likely or unlikely to develop inflammatory bowel disease (IBD), the method comprising: identifying the subject's genotype for a risk allele of a gene / locus; predicting, after detection of the risk allele, that the subject is likely to develop IBD; or predicting, if no risk allele is detected, that the subject is unlikely to develop IBD.

[0038] In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, the HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof. In various embodiments, the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. In various embodiments, the gene / locus includes one or more of SEQ ID NO:1-SEQ ID NO:341.

[0039] In various other embodiments, the step of identifying the genotype of a subject includes the steps of obtaining a sample from the subject and identifying the genotype of the sample with respect to a risk allele at a gene / locus. In yet another embodiment, the step of identifying the genotype of a sample includes contacting the sample with an oligonucleotide probe specific to the risk allele; generating an allele-specific hybridization complex between the oligonucleotide probe and the risk allele; detecting the risk allele after detecting the allele-specific hybridization complex; or not detecting the risk allele if the allele-specific hybridization complex is not detected. In some embodiments, the oligonucleotide probe is labeled with a fluorescent dye, where the detection of the allele-specific hybridization complex includes detecting a fluorescent signal from the oligonucleotide probe. In another embodiment, the oligonucleotide probe includes a reporter dye and a quencher dye.

[0040] In various embodiments, the method further includes the step of performing PCR amplification after forming an allele-specific hybridization complex.

[0041] Various embodiments of the present invention provide a method for predicting whether a subject is likely or unlikely to develop inflammatory bowel disease (IBD). The method includes the steps of: identifying the subject's genotype for a risk allele of a gene / locus; predicting, after detecting the risk allele, that the subject is likely to develop IBD; or, if the risk allele is not detected, predicting that the subject is unlikely to develop IBD.

[0042] Various embodiments of the present invention provide a method for predicting the likelihood of a subject developing inflammatory bowel disease (IBD). The method includes the steps of: identifying the subject's genotype for a risk allele of a gene / locus; and, after detecting the risk allele, predicting the likelihood of the subject developing IBD.

[0043] Various embodiments of the present invention provide a method for predicting that a subject is unlikely to develop inflammatory bowel disease (IBD). The method includes the steps of: identifying the subject's genotype for a risk allele of a gene / locus; and, after detecting the risk allele, predicting that the subject is unlikely to develop IBD.

[0044] According to the present invention, a high or low likelihood of developing IBD means that the subject is more or less likely to develop IBD compared to the general population to which the subject belongs.

[0045] diagnosis Various embodiments of the present invention provide a method for diagnosing inflammatory bowel disease (IBD) in a subject, the method comprising: identifying the genotype of a subject sample for a risk allele at a gene / locus; diagnosing IBD in the subject after detection of the risk allele; and administering IBD treatment to the subject diagnosed with IBD, thereby treating the subject's IBD. In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, the HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof. In various embodiments, the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. In various embodiments, the gene / locus includes one or more of SEQ ID NO:1-SEQ ID NO:341. In some embodiments, IBD treatment includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof.

[0046] Various embodiments of the present invention include the steps of: identifying the genotype of a subject sample for risk alleles at a gene / locus; diagnosing IBD in the subject after detection of the risk allele; and administering IBD treatment to the subject diagnosed with IBD, thereby treating the subject's IBD. In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or combinations thereof. In various embodiments, the gene / locus includes ETS1, HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or combinations thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. In various other embodiments, the gene / locus includes one or more of SEQ ID NO:1-SEQ ID NO:341. In various embodiments, the method further includes the step of providing IBD treatment to a subject. In some embodiments, the IBD treatment includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof.

[0047] Various embodiments of the present invention provide a method for identifying a subject's susceptibility to or protection against inflammatory bowel disease (IBD). The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; identifying the subject's susceptibility to IBD after detection of the risk allele; or identifying the subject's protection against IBD after detection of the risk allele.

[0048] Various embodiments of the present invention provide a method for identifying a subject's susceptibility to inflammatory bowel disease (IBD). The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; and identifying the subject's susceptibility to IBD after the detection of the risk allele.

[0049] Various embodiments of the present invention provide a method for identifying protection against inflammatory bowel disease (IBD) in a subject. The method includes the steps of: identifying the subject's genotype for a risk allele of a gene / locus; and, after the detection of the risk allele, identifying the subject's protection against IBD.

[0050] According to the present invention, susceptibility to IBD means that the subject is more likely to develop IBD compared to the general population to which the subject belongs. According to the present invention, protection from IBD means that the subject is less likely to develop IBD compared to the general population to which the subject belongs.

[0051] Various embodiments of the present invention provide a method for diagnosing inflammatory bowel disease (IBD) in a subject. The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; diagnosing IBD in the subject after detection of the risk allele; or not diagnosing IBD in the subject if no risk allele is detected.

[0052] Various embodiments of the present invention provide a method for diagnosing inflammatory bowel disease (IBD) in a subject. The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; detecting the risk allele; and diagnosing IBD in the subject.

[0053] Various embodiments of the present invention provide a method for treating inflammatory bowel disease (IBD) in a subject. The method includes the step of administering IBD treatment to a subject diagnosed with IBD according to a method such as that described herein, thereby treating the subject's IBD. In various embodiments, the method further includes the step of providing IBD treatment to a subject.

[0054] Various embodiments of the present invention provide a method for treating inflammatory bowel disease (IBD) in a subject. The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; administering IBD treatment to the subject after detection of the risk allele; or, if no risk allele is detected, not administering IBD treatment to the subject. In various embodiments, the method further includes the step of providing IBD treatment to the subject.

[0055] Various embodiments of the present invention provide a method for treating inflammatory bowel disease (IBD) in a subject. The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; detecting the risk allele; and administering IBD treatment to a subject diagnosed with IBD, thereby treating the subject's IBD. In various embodiments, the method further includes the step of providing IBD treatment to the subject.

[0056] Various embodiments of the present invention provide a method for administering treatment for inflammatory bowel disease (IBD) to a subject. The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; administering IBD treatment to the subject after detection of the risk allele; or, if no risk allele is detected, not administering IBD treatment to the subject.

[0057] Various embodiments of the present invention provide a method for administering treatment for inflammatory bowel disease (IBD) to a subject. The method includes the steps of: identifying the subject's genotype with respect to a risk allele of a gene / locus; detecting the risk allele; and administering IBD treatment to the subject.

[0058] In various embodiments, IBD treatment includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof. In some embodiments, IBD treatment is an anti-TNF antibody. In some embodiments, IBD treatment is an anti-TL1A antibody. In some embodiments, IBD treatment is colectomy.

[0059] In various embodiments, the subject is human. In some embodiments, the subject is a child. In some embodiments, the subject is a teenager. In other embodiments, the subject is an adult. In various embodiments, IBD is Crohn's disease (CD) or ulcerative colitis (UC).

[0060] In various embodiments, the sample is a cheek swab; mucus; whole blood; blood; serum; plasma; urine; saliva; semen; lymph; fecal extract; sputum; other body fluids or biofluids; cell samples; or tissue samples; or a combination thereof. In various embodiments, the sample contains nucleic acids of an individual. In some embodiments, the nucleic acids contain genomic DNA. In various embodiments, the subject is a body fluid. In some embodiments, the body fluid is whole blood, plasma, saliva, mucus, or a cheek swab. In various embodiments, the sample is cells or tissue. In some embodiments, the cells are blood cells. In some embodiments, the cells are a blood cell line (e.g., a lymphoblastoid cell line) obtained from the subject and converted with Epstein-Barr virus.

[0061] In various embodiments, the gene / locus includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more, or all of them, from the genes / locuses listed in Table 1 as SEQ ID NO: 1-341. In various embodiments, the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, the HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof. In various embodiments, the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof. In various embodiments, the gene / locus includes ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A. Each gene can contain the following sequences: SLC26A4 (SEQ ID NO: 1-6); DLG4 (SEQ ID NO: 7); GIPR (SEQ ID NO: 8-27); ZHX3 (SEQ ID NO: 28-30); TNRC6B (SEQ ID NO: 31-38); CDK6 (SEQ ID NO: 39-40); PRR5L (SEQ ID NO: 41-54); WNT2B (SEQ ID NO: 55-58); LRRC16A (SEQ ID NO: 59-75, 335); HIST1 cluster (all histone cluster 1 genes - SEQ ID NO: 76-173, 338, 339); GTF2IRD2B (SEQ ID NO: 174-180); ETS1 (SEQ ID NO: 181-325); SLC5A1 (SEQ ID NO: 326-327); and TET2 (SEQ ID NO:328-332, 334).

[0062] In various embodiments, the risk allele includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or all of them, from the risk alleles listed in Table 1 as SEQ ID NO: 1-341. In a particular embodiment, the risk allele includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, This includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 99, 100, or more, or all of them. In various embodiments, the risk allele includes N risk alleles listed in Table 1 as SEQ ID NO: 1-341, where N is a natural number less than or equal to 341 (i.e., 1 ≤ N ≤ 341). In various embodiments, the risk alleles include 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 from the risk alleles listed in Table 1 as SEQ ID NO: 1-341. In various embodiments, the risk alleles include 100-105, 105-110, 110-115, 115-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180, 180-185, 185-190, 190-195, or 195-200 from the risk alleles listed in Table 1 as SEQ ID NO: 1-341.In various embodiments, the risk alleles include 200-205, 205-210, 210-215, 215-220, 220-225, 325-230, 230-235, 235-240, 240-245, 245-250, 250-255, 255-260, 260-265, 265-270, 270-275, 275-280, 280-285, 285-290, 290-295, or 295-300 from the risk alleles listed in Table 1 as SEQ ID NO: 1-341. In various embodiments, the risk allele includes 300-305, 305-310, 310-315, 315-320, 320-325, or 330-341 from the risk alleles listed in Table 1 as SEQ ID NO: 1-341.

[0063] In some embodiments, the subject's genotype can be obtained from previous genetic or genomic tests performed on the subject, which were not performed for IBD or any other disease. For example, the subject's genotype can be obtained from an analysis of the subject's genome sequencing results, or from a database storing the subject's individual genetic or genomic information. In these embodiments, identifying the subject's genotype does not require performing a clinical trial on the subject, as it involves acquiring and analyzing already available data. In other embodiments, for example, when an individual's genetic or genomic information is not available, or when the subject or physician desires a new clinical trial, identifying the subject's genotype requires performing a clinical trial on the subject.

[0064] In various embodiments of the present invention, the step of identifying the genotype of a subject includes the step of obtaining a sample from the subject; and the step of identifying the genotype of the sample with respect to risk alleles at a gene / locus.

[0065] In some embodiments, the step of identifying the genotype of a sample includes contacting the sample with an oligonucleotide probe specific to a risk allele; forming an allele-specific hybridization complex between the oligonucleotide probe and the risk allele; and detecting the risk allele after detecting the allele-specific hybridization complex; or, if the allele-specific hybridization complex is not detected, not detecting the risk allele. In various embodiments, the oligonucleotide probe is labeled with a fluorescent dye, where the detection of the allele-specific hybridization complex includes detecting a fluorescent signal from the oligonucleotide probe. In various embodiments, the oligonucleotide probe includes a reporter dye and a quencher dye. In certain embodiments, the method further includes performing PCR amplification after forming the allele-specific hybridization complex. In various embodiments, the step of detecting the allele-specific hybridization complex includes detecting the electrophoretic mobility of the allele-specific hybridization complex.

[0066] In various embodiments, the step of identifying the genotype of a sample includes contacting the sample with a detection agent that specifically binds to the allele of the SNP; and detecting the allele of the SNP in the sample by detecting the binding level between the detection agent and the allele of the SNP. The allele can be detected by techniques such as genotyping assays, PCR, reverse transcription PCR, real-time PCR, microarrays, DNA sequencing, and RNA sequencing.

[0067] Various embodiments of the present invention provide compositions. In various embodiments, a composition comprises one or more detection agents that specifically bind to one or more alleles at one or more genes / locuses. This composition may be used to identify genes / locuses associated with disease, and / or to predict low or high likelihood of progression of IBD, and / or to predict susceptibility to or protection against IBD, and / or to diagnose IBD, and / or to treat IBD, and / or to administer IBD treatment.

[0068] In various embodiments, the detection agent is an oligonucleotide probe, nucleic acid, DNA, RNA, aptamer, peptide, protein, antibody, avimer, or small molecule, or a combination thereof. In some embodiments, the detection agent is an allele-specific oligonucleotide probe that targets the allele of the SNP. In various embodiments, the allele of the SNP is detected using a microarray. In some embodiments, the microarray is an oligonucleotide microarray, DNA microarray, cDNA microarray, RNA microarray, peptide microarray, protein microarray, or antibody microarray, or a combination thereof.

[0069] In various embodiments, the step of detecting an allele of an SNP includes: contacting a sample with one or more allele-specific oligonucleotide probes that target the allele of the SNP; generating a double-stranded hybridization complex via allele-specific binding between the allele of the SNP and the allele-specific oligonucleotide probes; and detecting the newly generated double-stranded hybridization complex via allele-specific binding between the allele of the SNP and the allele-specific oligonucleotide probes. In some embodiments, the method further includes PCR amplification of the double-stranded hybridization complex.

[0070] In various embodiments, the present invention provides allele-specific oligonucleotide probes for each of the alleles (e.g., major alleles, minor alleles, risk alleles, and non-risk alleles listed in Table 1). According to the present invention, the allele-specific oligonucleotide probes described above may contain approximately 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 nucleotides; they are identical or complementary to the sequence segment encompassing the polymorphic position of the SNP as disclosed herein; and are specific to one or other alleles at the polymorphic position. In a non-limiting example, rs10247487 has either a T or C allele (in the context of the preceding strand) at its polymorphic position (e.g., the "Y" at nucleotide 501 of the following example sequence (SEQ ID NO:1)).

[0071] [Table 1]

[0072] Therefore, an allele-specific oligonucleotide probe for the T allele in rs10247487 may, as an unrestricted example, contain 21 nucleotides; these 21 nucleotides are either identical or complementary to sequence segments 481-501, 482-502, 483-503, 484-504, 485-505, 486-506, 487-507, 488-508, 489-509, 490-511, 491-511, 492-512, 493-513, 494-514, 495-515, 496-516, 497-517, 498-518, 499-519, 500-520, or 501-521 of the sequence in the above example where nucleotide 501 is set as the T allele. Conversely, an allele-specific oligonucleotide probe for the C allele in rs10247487 may, as an unrestricted example, contain 21 nucleotides; these 21 nucleotides are either identical or complementary to sequence segments 481-501, 482-502, 483-503, 484-504, 485-505, 486-506, 487-507, 488-508, 489-509, 490-511, 491-511, 492-512, 493-513, 494-514, 495-515, 496-516, 497-517, 498-518, 499-519, 500-520, or 501-521 of the sequence in the above example where nucleotide 501 is set as the C allele.

[0073] In various embodiments, the allele-specific oligonucleotide probe is labeled with one or more fluorescent dyes, where the detection of the double-stranded hybridization complex includes detecting a fluorescent signal from the fluorescent dyes. In some embodiments, the allele-specific oligonucleotide probe is labeled with a reporter dye and a quencher dye. In some embodiments, the step of detecting the double-stranded hybridization complex includes detecting the electrophoretic mobility of the double-stranded hybridization complex.

[0074] Various methods can be used to detect the presence or absence of variant alleles or haplotypes. For example, enzymatic amplification of nucleic acids from an individual can be used to obtain nucleic acids for later analysis. The presence or absence of variant alleles or haplotypes can also be determined directly from nucleic acids of an individual without enzymatic amplification.

[0075] The detection of the presence or absence of a variant allele or haplotype may involve amplification of an individual's nucleic acid by polymerase chain reaction. The use of polymerase chain reaction for nucleic acid amplification is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).

[0076] The analysis of nucleic acids from organisms may be performed using any of a variety of techniques, with or without amplification. Useful techniques include, but are not limited to, polymerase chain reaction-based analysis, sequence analysis, and electrophoretic analysis. As used herein, the term “nucleic acid” means polynucleotides, such as single-stranded or double-stranded DNA or RNA molecules, including, for example, genomic DNA, cDNA, and mRNA. The term nucleic acid also includes molecules of linear, cyclic, or branched configurations that represent the sense strand or antisense strand of a natural nucleic acid molecule, or both, in addition to nucleic acid molecules of natural and synthetic origin.

[0077] The Taqman B allele discrimination assay, available from Applied Biosystems, may be useful for determining the presence or absence of a mutant allele. In the Taqman B allele discrimination assay, probes specifically labeled with fluorescent dyes are constructed for each allele. The probes contain different fluorescent reporter dyes, such as FAM and VICTM, to distinguish the amplification of each allele. In addition, each probe has a quencher dye at one end that quenches fluorescence by fluorescence resonance energy transfer (FRET). During PCR, each probe is specifically annealed to a complementary sequence in the nucleic acid from the individual. Using the 5' nuclease activity of Taq polymerase, only the probes that hybridize to the allele are cleaved. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye. Therefore, the fluorescent signal generated by PCR amplification indicates which allele is present in the sample. A mismatch between the probe and the allele reduces the efficiency of both probe hybridization and cleavage by Taq polymerase, resulting in little to no fluorescence signal. Improved specificity in allele discrimination assays can be achieved by conjugating DNA probes with minor groove binders (MGBs), as described, for example, by Kutyavin et al. in "3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperature, Nucleic Acids Research 28:655-661 (2000)". Minor groove binders include, but are not limited to, compounds such as dihydrocyclopyrroloindole tripeptides (DPIs).

[0078] Sequence analysis can also be useful in determining the presence or absence of variant alleles or haplotypes.

[0079] Restriction fragment length polymorphism (RFLP) analysis can also be useful in determining the presence or absence of a specific variant allele or haplotype (Jarcho et al. in Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1-2.7.5, John Wiley & Sons, New York; Innis et al., (Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990)). As used herein, restriction fragment length polymorphism is a method for identifying genetic polymorphisms using restriction enzymes, which are endonucleases that catalyze the degradation of nucleic acids and recognize specific nucleotide sequences, generally palindromes or reverse repeat sequences. Those skilled in the art will understand that the use of RFLP analysis relies on enzymes that can distinguish between two alleles at a polymorphic site.

[0080] Allele-specific oligonucleotide hybridization can also be used to detect mutant alleles or haplotypes. Allele-specific oligonucleotide hybridization is based on the use of labeled oligonucleotide probes having sequences that are perfectly complementary to the sequences containing the mutant allele or haplotype, for example. Under appropriate conditions, the allele-specific probe hybridizes to nucleic acids containing the mutant allele or haplotype but not to other alleles or haplotypes that have one or more nucleotide mismatches compared to the probe. If desired, a second allele-specific oligonucleotide probe matching an alternative allele can be used. Similarly, allele-specific oligonucleotide amplification techniques can be used to selectively amplify mutant alleles or haplotypes by using allele-specific oligonucleotide primers that are perfectly complementary to the nucleotide sequences of the mutant allele or haplotype but have one or more mismatches compared to other alleles or haplotypes (Mullis et al., (1994) above). Those skilled in the art understand that one or more nucleotide mismatches that distinguish a mutant allele or haplotype from another allele or haplotype are preferably located at the center of an allele-specific oligonucleotide primer to be used in allele-specific oligonucleotide hybridization. In contrast, allele-specific oligonucleotide primers to be used for PCR amplification preferably contain one or more nucleotide mismatches at the 3' end of the primer that distinguish a mutant allele or haplotype from another allele.

[0081] Heteroduplex mobility assays (HMAs) are another well-known assay that can be used to detect mutant alleles or haplotypes. HMAs are useful for detecting the presence of pleomorphic sequences because DNA double strands with mismatches exhibit reduced mobility on polyacrylamide gels compared to perfectly base-paired double strands (Delwart et al., Science 262:1257-1261 (1993); White et al., Genomics 12:301-306 (1992)).

[0082] Single-stranded conformational polymorphism (SSCP) techniques can also be used to detect the presence or absence of variant alleles or haplotypes (see Hayashi, K., Methods Applic. 1:34-38 (1991)). This technique allows for the detection of mutations based on differences in the secondary structure of single-stranded DNA that generate altered electrophoretic mobility during non-denaturing gel electrophoresis. Polymorphic fragments are detected by comparing the electrophoretic patterns of a test fragment with those of a corresponding standard fragment containing a known allele.

[0083] Denaturing gradient gel electrophoresis (DGGE) can also be used to detect mutant alleles or haplotypes. In DGGE, double-stranded DNA is subjected to electrophoresis in a gel containing increasing concentrations of denaturing agents; double-stranded fragments composed of mismatched alleles dissolve more rapidly than perfectly complementary sequences and have segments that move differently (Sheffield et al., “Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrophoresis” in Innis et al., supra, 1990).

[0084] Other molecular methods useful for determining the presence or absence of mutant alleles or haplotypes are known in the art and are useful in the method of the present invention. Other well-known methods for determining the presence or absence of mutant alleles or haplotypes include automated sequencing and RNAase mismatch techniques (Winter et al., Proc. Natl. Acad. Sci. 82:7575-7579 (1985)). Furthermore, those skilled in the art will understand that when the presence or absence of multiple alleles or haplotypes is to be determined, individual alleles or haplotypes can be detected by any combination of molecular methods. See, for example, Birren et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press (1997). In addition, those skilled in the art will understand that multiple alleles can be detected in individual reactions or single reactions ("multiplex" assays). In consideration of the foregoing, those skilled in the art will understand that the method of the present invention can be carried out using one or any combination of the well-known assays described above or other gene assays recognized in the art.

[0085] Gene identification Various embodiments of the present invention provide a method for identifying disease-associated genes / locuses, the method comprising: obtaining genetic data from a sample of a disease cohort; performing GLS conversion on the genetic data to decorrelate the genetic data; performing gene-based analysis on the GLS-converted genetic data; and identifying disease-associated genes / locuses. In various embodiments, the disease is IBD, CD, or UC, or a combination thereof. In some embodiments, the cohort includes correlated subjects or family subjects. In other embodiments, the genetic data includes SNP genotypes. In yet another embodiment, the GLS conversion step is performed using the following function

[0086]

number

[0087] In various embodiments, the step of performing a gene-based analysis includes the step of applying gene-based tests based on the assumption of independent or uncorrelated subjects. In various embodiments, the step of performing a gene-based analysis includes the step of applying C-alpha, SKAT, SKAT-CommonRare, CMC, WSS, variable threshold, or a comprehensive approach, or a combination thereof.

[0088] Various embodiments of the present invention provide a method for identifying disease-associated genes / locuses. The method includes the steps of: obtaining genetic data from a sample of a disease cohort; performing GLS transformation on the genetic data to decorrelate the genetic data; performing gene-based analysis on the GLS-transformed genetic data; and identifying disease-associated genes / locuses. In various embodiments, the disease is IBD, CD, or UC, or a combination thereof.

[0089] In various embodiments, the cohort includes correlated or familial subjects. In some embodiments, the cohort includes case subjects diagnosed with a disease. In some embodiments, the cohort includes control subjects who are healthy or not diagnosed with a disease. In various embodiments, the genetic data includes SNP genotypes.

[0090] In various embodiments, the GLS conversion step includes the step of converting genetic data according to the functions (5)-(8) described above. In various embodiments, the gene-based analysis step includes the step of applying gene-based tests based on the assumption of independent or uncorrelated subjects. In various embodiments, the gene-based analysis step includes the step of applying C-alpha, SKAT, SKAT-CommonRare, CMC, WSS, variable threshold, or comprehensive approach, or a combination thereof.

[0091] Invention kit Various embodiments of the present invention also provide kits. A kit may consist of, substantially consist of, or include, one or more detection agents for detecting one or more alleles at one or more genes / locuses; instructions for using agents for identifying genes / locuses associated with disease, and / or predicting low or high likelihood of progression of IBD, and / or predicting susceptibility to or protection against IBD, and / or diagnosing IBD, and / or treating IBD, and / or administering IBD treatment. In some embodiments, one or more alleles are risk alleles associated with IBD.

[0092] Various embodiments of the present invention also provide kits. A kit may consist of, substantially consist of, or include, one or more detection agents for detecting one or more alleles at one or more genes / locuses; and instructions for using agents to identify genes / locuses associated with a disease. In various embodiments, the kit further includes samples obtained from a cohort of the disease. In various embodiments, the disease is IBD, Crohn's disease (CD), or ulcerative colitis (UC).

[0093] Various embodiments of the present invention also provide kits. A kit consists of, substantially consists of, or includes, one or more detection agents for detecting one or more risk alleles at one or more genes / locuses; instructions for using agents for predicting low or high likelihood of progression of IBD, and / or predicting susceptibility to or protection against IBD, and / or diagnosing IBD, and / or treating IBD, and / or administering IBD treatment. In various embodiments, the risk alleles are associated with IBD. In various embodiments, the kit further includes a sample obtained from a subject for whom prognosis, diagnosis, and / or treatment of IBD is desired. In various embodiments, IBD is Crohn's disease (CD) or ulcerative colitis (UC).

[0094] In various embodiments, one or more genes / locuses include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more, or all of them, from the genes / locuses listed in Table 1 as SEQ ID NO: 1-341. In various embodiments, one or more genes / locuses include SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof. In various embodiments, one or more genes / locuses include ETS1, HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof. In various embodiments, the gene / locuses include ETS1. In various embodiments, the gene / locus includes the HIST1 cluster (all histone cluster 1 genes). In various embodiments, the gene / locus includes CDK6. In various embodiments, the gene / locus includes LRRC16A.

[0095] In various embodiments, the kit further comprises an IBD therapeutic agent. Examples of IBD therapeutic agents include, but are not limited to, anti-TNF and anti-TL1A therapeutic agents. In some embodiments, the IBD therapeutic agent is an anti-TNF antibody. In some embodiments, the IBD therapeutic agent is an anti-TL1A antibody.

[0096] A kit is a construct of a substance or components comprising at least one of the inventive elements or modules. In various embodiments, one or more detection agents specifically bind to one or more SNP alleles. In some embodiments, one or more SNP alleles may be major alleles, minor alleles, or both. In some embodiments, one or more SNP alleles may be risk alleles, non-risk alleles, or protection alleles, or a combination thereof.

[0097] In some embodiments, one or more detection agents specifically bind to one or more risk alleles listed in Table 1 as SEQ ID NO: 1-341. In some embodiments, one or more detection agents specifically bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or all of the risk alleles listed in Table 1 as SEQ ID NO: 1-341. In certain embodiments, one or more detection agents bind to SEQ ID Among the risk alleles listed in Table 1 as NO:1-341, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 5 The agents specifically bind to 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 99, or 100, or more, or all of them. In some embodiments, one or more detection agents specifically bind to N alleles from a single allele listed in Table 1, where N is a natural number less than or equal to 341 (i.e., 1 ≤ N ≤ 341). In various embodiments, one or more detection agents specifically bind to risk alleles 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 among the risk alleles listed in Table 1 as SEQ ID NO: 1-341.In various embodiments, one or more detection agents specifically bind to risk alleles 100-105, 105-110, 110-115, 115-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180, 180-185, 185-190, 190-195, or 195-200 from the list of risk alleles in Table 1 as SEQ ID NO: 1-341. In various embodiments, one or more detection agents specifically bind to risk alleles 200-205, 205-210, 210-215, 215-220, 220-225, 325-230, 230-235, 235-240, 240-245, 245-250, 250-255, 255-260, 260-265, 265-270, 270-275, 275-280, 280-285, 285-290, 290-295, or 295-300 among the risk alleles listed in Table 1 as SEQ ID NO: 1-341. In various embodiments, one or more detection agents specifically bind to risk alleles 300-305, 305-310, 310-315, 315-320, 320-325, 325-330, 330-335, 335-340, and 340-341 listed in Table 1 as SEQ ID NO: 1-341.

[0098] In various embodiments, one or more detection agents are applied to contact a biological sample obtained from a subject; the level of binding between one or more detection agents and one or more alleles is detected. In some embodiments, one or more detection agents are oligonucleotide probes, nucleic acids, DNA, RNA, peptides, proteins, antibodies, aptamers, or small molecules, or combinations thereof. In various embodiments, the level of binding is detected using a microarray. In some embodiments, the microarray is an oligonucleotide microarray, a DNA microarray, a cDNA microarray, an RNA microarray, a peptide microarray, a protein microarray, or an antibody microarray, or a combination thereof.

[0099] In various embodiments, one abnormality detection agent is an oligonucleotide probe specific to one or more alleles. In various embodiments, the oligonucleotide probe is labeled with a fluorescent dye. In various embodiments, the oligonucleotide probe includes a reporter dye and a quencher dye. In various embodiments, the kit further includes a module configured to detect a fluorescent signal from one or more detection agents. In various embodiments, the kit further includes a module configured to perform PCR amplification.

[0100] The exact nature of the components comprising an inventive kit depends on its intended use. Instructions for use may be included in the kit. Instructions for use typically include tactile language describing the techniques to be used when using the components of the kit to influence the desired outcome. Optionally, the kit may also include other useful components such as spray bottles or cans, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators (e.g., applicators for creams, gels, or lotions), pipetting or measuring instruments, bandaging materials, or other useful tools readily recognizable to those skilled in the art.

[0101] The substances or components assembled in the kit may be stored in a convenient and appropriate manner to maintain their operability and usefulness and provided to the operator. For example, detection agents may be in dissolved, dehydrated, or freeze-dried form; they may be provided at room temperature, refrigerated, or frozen temperature. Components are typically contained in appropriate packaging materials. As used herein, the phrase “packaging materials” refers to one or more physical structures used to contain the contents of the kit, such as inventive compositions. Packaging materials are preferably constructed in a well-known manner to provide a sterile, contaminant-free environment. Packaging materials used in kits are those conventionally used in assays and treatments. As used herein, the term “packaging” refers to an appropriate solid matrix or material, such as glass, plastic, paper, or foil, that can hold the components of the individual kits. Thus, for example, packaging may be a glass vial used to contain an appropriate amount of a composition as described herein. Packaging materials typically have an external label indicating the contents and / or the purpose of the kit and / or its components.

[0102] Novel genes / regions, SNPs, and risk alleles Table 1 provides information on genes / regions, SNPs, SEQ ID NOs (SEQ ID NO: 1-341), and risk alleles according to various embodiments of the present invention. "Dis" represents the disease; "gene.i" represents the gene ID; "SNP" represents the single nucleotide polymorphism; "rsID" represents the reference SNP cluster ID (rs number); "chr" represents the chromosome; "pos_hg19" represents the location in human genome version 19; "pos_hg19" represents the location in human genome version 18; "A1" represents the minor allele; "A2" represents the major allele; "risk.allele" represents the allele that leads to increased disease risk; "OR.risk.""allele" represents the odds ratio in the meta-analysis of risk alleles; "F_A_cedars" represents the frequency of minor alleles in cases affected by Cedars; "F_U_cedars" represents the frequency of minor alleles in controls not affected by Cedars; "OR_cedars" represents the odds ratio in the Cedars cohort; and "SE_cedars" represents the standard error of the logarithm (OR) in the Cedars cohort. "L95_cedars" represents the lower limit of the 95% confidence interval for the OR in the Cedars cohort; "U95_cedars" represents the upper limit of the 95% confidence interval for the OR in the Cedars cohort; "STAT_cedars" represents the test statistic (Z-value) in the Cedars cohort; "P_cedars" represents the P-value in the Cedars cohort; "F_A_iibdgc" represents the minor allergic reaction in cases affected by IIBDGC. "F_U_iibdgc" represents the frequency of the minor allele in the control unaffected by IIBDGC; "OR_iibdgc" represents the odds ratio in the IIBDGC cohort; "SE_iibdgc" represents the standard error of the logarithm (OR) in the IIBDGC cohort; "L95_iibdgc" represents the lower limit of the 95% confidence interval for the OR in the IIBDGC cohort; "U95_iibdgc" represents the frequency of the minor allele in the IIBDGC cohort. This represents the upper limit of the 95% confidence interval for the OR in the meta-analysis; "STAT_iibdgc" represents the test statistic (Z-statistic) in the IIBDGC cohort; "P_iibdgc" represents the P-value in the IIBDGC cohort; "beta_meta_fixed" represents the logarithm (OR) in the meta-analysis; "se_meta_fixed" represents the standard error of the logarithm (OR) in the meta-analysis; and "P_meta_fixed" represents the P-value in the meta-analysis.

[0103] [Table 2-1]

[0104] [Table 2-2]

[0105] Table 2-3

[0106] Table 2-4

[0107] Table 2-5

[0108] Table 2-6

[0109] Table 2-7

[0110] Table 2-8

[0111] Table 2-9

[0112] Table 2-10

[0113] Table 2-11

[0114] Table 2-12

[0115] Table 2-13

[0116] Table 2-14

[0117] Table 2-15

[0118] Table 2-16

[0119] Table 2-17

[0120] Table 2-18

[0121] Table 2-19

[0122] Table 2-20

[0123] Table 2-21

[0124] Table 2-22

[0125] Table 2-23

[0126] Table 2-24

[0127] Table 2-25

[0128] Table 2-26

[0129] Table 2-27

[0130] Table 2-28

[0131] Table 2-29

[0132] Table 2-30

[0133] Table 2-31

[0134] Table 2-32

[0135] Table 2-33

[0136] Table 2-34

[0137] Table 2-35

[0138] Table 2-36

[0139] Table 2-37

[0140] Table 2-38

[0141] Table 2-39

[0142] Table 2-40

[0143] Table 2-41

[0144] Table 2-42

[0145] Table 2-43

[0146] Table 2-44

[0147] Table 2-45

[0148] Table 2-46

[0149] Table 2-47

[0150] Table 2-48

[0151] Table 2-49

[0152] Table 2-50

[0153] Table 2-51

[0154] Table 2-52

[0155] Table 2-53

[0156] [Table 2-54]

[0157] [Table 2-55]

[0158] [Table 2-56]

[0159] [Table 2-57]

[0160] [Table 2-58]

[0161] Many variations and alternative elements have been disclosed in embodiments of the present invention. Further variations and alternative elements will be obvious to those skilled in the art. These variations include, but are not limited to, inventive methods, compositions, kits, and systems, as well as selections of constituent modules for various diseases, disorders, and disabilities that can be diagnosed, assumed, or treated. Various embodiments of the present invention may specifically include or exclude any of these variations or elements.

[0162] In some embodiments, numbers representing specific amounts, concentrations, and reaction conditions of components used to describe and claim particular embodiments of the present invention should be understood as being modified in some examples by the term “approximately.” Accordingly, in some embodiments, the numerical parameters described in the specification and appended claims may vary depending on the desired properties to be obtained by the particular embodiment. In some embodiments, the numerical parameters should be interpreted in light of the number of significant figures reported and by the application of common rounding techniques. Although the numerical ranges and parameters describing a broad range of embodiments of the present invention are approximate, the numerical values ​​stated in particular embodiments are reported as accurately as possible. The numerical values ​​presented in some embodiments of the present invention may include certain errors that inevitably result from the standard deviation found in each test measurement.

[0163] The grouping of alternative elements or embodiments of the Invention disclosed herein is not to be construed as limiting. Members of each group may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or omission, this Specification shall be deemed to include the group to be qualified and thus satisfy the written description of all Markush groups used in the appended claims. [Examples]

[0164] The present invention is further illustrated by the following examples, which are intended to be purely typical of the invention and should not be considered to limit the invention in any way. The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that certain substances are mentioned, it is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive ability and without departing from the scope of the invention.

[0165] Example 1 GL-SKAT: A novel method for gene-based analysis in cohorts with family structure Gene-based analysis can be crucial for identifying novel loci for complex diseases. However, most available methods are based on the independent assumption of population-based case-control samples. Here, we propose a generalized least squares (GLS)-based analytical strategy for identifying genes using data including complex family structures. The rational numbers of this method can be explained as follows:

[0166] Assuming linear specialization of the association of one set of genetic factors X and result y,

[0167]

number

[0168] Let's assume we can write the variation of result y as follows:

[0169]

number

[0170] If the subjects in the sample are not correlated, the estimate of β can be written as follows:

[0171]

number

[0172] When samples are correlated, for example, in family-based samples, the least squares estimation (OLS) of β becomes uncertain.

[0173]

number

[0174] One solution to the obstacles of the independence assumption in linear models is to perform a generalized least-squares transformation. This involves:

[0175]

number

[0176] The GLS-transformed model can then be written as follows:

[0177]

number

[0178] And the estimation based on the transformed model can be written as follows:

[0179]

number

[0180] Clearly, this is the best linear unbiased predictor (BLUP) by structure. In other words, after GLS transformation, the data lose correlation while retaining unbiased estimators. This allows models developed under independence assumptions to be applied to GLS-transformed data. Here, for gene-based analysis, we choose to apply SKAT-CommonRare to the GLS-transformed data because it performs better in most scenarios while the independence assumption is retained. The transformation matrix G was calculated as the reciprocal of the decomposition of the kinship matrix. This method is called GLS-SKAT.

[0181] Example 2 - Numerous novel gene loci identified through gene-based analysis Single-SNP-based associations, due to their simplicity and ease, drive most GWAS findings (Figure 1A). This tests whether the frequency of a single SNP is the same in cases and controls. However, this association has several drawbacks, including the need for multiple test corrections, prohibition of increasing the number of variants, neglect of numerous weak signals, and deletion of several causative loci.

[0182] Gene-based analysis examines the entire gene instead of looking at a single SNP (Figure 1B). This tests whether the distribution of all SNPs in a given gene is the same in cases and controls. This analysis becomes more powerful when there are many causative SNPs that have a weak effect. This analysis can reduce the multiple test penalties for millions of SNPs and approximately 25,000 known genes.

[0183] Current methods for gene-based analysis include data collapsing methods (e.g., the Combining Multivariate and Collapsing method (CMC), Weighted Sum Statistics (WSS), variable thresholds, and comprehensive methods), and distribution-based methods (e.g., C-alpha, SNP Set Kernel Association Test (SKAT), and SKAT-CommonRare). Most of these methods can only be applied to population-based designs assuming subject independence.

[0184] The present invention provides a new method GLS-SKAT for gene-based analysis in families. Consider the following linear model:

[0185]

Number

[0186] For independent subjects, the following holds:

[0187]

Number

[0188] For correlated subjects, the following holds:

[0189]

Number

[0190] To transform the correlated data, do the following:

[0191]

Number

[0192] Therefore, T can be multiplied in the linear model:

[0193]

Number

[0194] After that, it becomes as follows:

[0195]

Number

[0196] [[ID=,22]]That is, the correlated data "loses its correlation" here.

[0197] OLS estimation with GLS-transformed data: <00,00964>

Number

[0199] This is an accurate estimate of the maximum likelihood of the true model:

[0200]

Number

[0201] <, GLS-SKAT is applied to the iChip data Cedars vs BBC: 4600 cases and 6800 controls. SKAT-CommonRare is applied to IIBDGC (excluding Cedars and BBC samples): 30200 cases and 29700 controls. PCA is included in the controls due to confounding factors. The gene region is defined as 100 kb upstream and downstream of each gene. The analysis focuses on IBD and genes with at least two SNPs (about 8000 genes). Therefore, the significance threshold is 0.05 / 8000 = 6.25E-{6}. Fisher's combined P-value is used for the meta-analysis of gene-level p-values.

[0202] The TET2 code for Tet methylcytosine dioxygenase 2 is involved in Foxp3 demethylation to drive regulatory T cell differentiation and maintain immunohomeostasis.

[0203] LRRC16A (a leucine-rich repeat containing 16A) is a protein-coding gene. Diseases associated with LRRC16A include acute urate nephropathy. An important paralog of this gene is LRRC16B. LR16A_HUMAN Q5VZK9 binds to CAPZA2 with high affinity and significantly reduces CAPZA2 affinity due to the actin's curved end. This increases the rate of elongation from seed in the presence of CAPZA2. However, it appears that filament nucleation is not possible. This rapidly decapitates the curved end capped by CAPZA2, enhancing curved end actin polymerization b-similarity. This may regulate actin dynamics in lamellipodia, which are necessary for cell migration.

[0204] There are binding associations within the overall HIST1 region. HIST1 cluster portion 1 (~26.2M, first portion) and HIST1 cluster portion 2 (~27.8M, second portion). After combining ~1.6M (26.2M~27.8M) into one large region, the P-value for associations based on the overall region is 1.64 x 10⁻⁷.

[0205] BTN3A1 / A2 / A3 is an interesting gene group. Butyrophyllin, subfamily 3, belongs to the B7 family and is expressed in various immune cells such as T cells and NK cells. BTN3 / CD277 includes three structurally related members: BTN3A1, BTN3A2, and BTN3A3. BTN3 / CD277 plays a role in the T cell response in adaptive immune responses and inhibits the release of IFNG from activated T cells. BTN3 / CD277 plays an important role in antigen activation of human γδ T cells. BTN3 / CD277 has different roles relative to CD277 as a co-regulator of immune signaling in T cells and NK cells (see, for example, Messal N, Mamessier E, et al. Eur J Immunol. 2011 Dec;41(12):3443-54). T cells express all BTN3 / CD277 transcriptions, while NK cells typically express BTN3A2, which lacks the B30.2 intracellular domain. Furthermore, cytokine production induced by NKp30 is reduced not by BTN3A1 triggering, but by the specific binding of BTN3A2.

[0206] Fourteen novel loci were identified through gene-based analysis of iChip data (Figure 4 and Table 1). All of these exhibited numerous weak signals, while some signals were very strong in binding models. BTN3A2 is also strongly related to the pathogenesis of IBD based on eQTL analysis of the LRRC16A region.

[0207] Example 3 - Gene-based analysis identified numerous novel IBD loci. Over 200 loci have been identified in inflammatory bowel disease (IBD), mostly through single SNP analysis. This study aims to utilize gene-based analysis, combining signals from all SNPs within a gene, to identify novel IBD loci that were deleted in single SNP analysis.

[0208] 3312 IBD cases from Cedars-Sinai Medical Center and 7154 family and population-based controls with ImmunoChip data were included as the discovery cohort. Genes with gene-level p-values ​​<0.05 were subsequently replicated in IIBDGC (30179 cases and 29678 controls, with duplicate samples from the discovery stage excluded). SKAT-CommonRare was performed to assess gene-level association. Fisher combined p-values ​​were calculated and p-values ​​were combined from the discovery and replication cohorts. A Bonferroni-adjusted significance threshold of 6.25E-6 was used for gene-based p-values ​​to count 7,924 genes with at least two SNPs on iChip.

[0209] In addition to known IBD genes such as IL23R and NOD2, we identified numerous novel genes associated with IBD. These genes include TET2 (discovery p-value 0.019, replication p-value 2.82E-9, combined p-value 1.33E-9); LRRC16A (discovery p-value 1.55E-6, replication p-value 3.43E-5, combined p-value 1.19E-8); and numerous genes at the histone cluster 1 locus (e.g., HIST1H4H, discovery p-value 2.89E-5, replication p-value 2.44E-4, combined p-value 4.24E-6; HIST1H1B, discovery p-value 1.45E-4, replication p-value 8.61E-5, combined p-value 2.41E-7). The SNPs of these genes are listed in Table 1.

[0210] Our bioinformatics analysis indicates that the top SNP (rs7752195) driving the LRRC16A signal is a potent quantitative phenotypic locus (eQTL) of BTN3A2 (p=5.96E-51 in seeQTL; p=8E-9 in SCANdb; p=0.0025 in GeneVar) that plays a crucial role in regulating adaptive immune responses. Furthermore, TET2, which encodes the translocation (Tet) methylcytosine dioxygenase 2, a top gene identified in current studies, has been reported to drive T cell differentiation via FOXP3 DNA demethylation. It has also been reported to mediate interleukin-6 (IL-6) transcription by regulating chromatin structure.

[0211] Without being bound by any particular theory, the novel loci strongly identified in current research through gene-based analysis strongly suggest that it would be beneficial to re-examine previous single-SNP-based GWAS at the gene level.

[0212] The various methods and techniques described herein provide many ways of carrying out this application. Of course, it should be understood that not all objectives or benefits can necessarily be achieved according to the specific embodiments described herein. Therefore, for example, a person skilled in the art will recognize that the methods can be carried out in a manner that achieves or optimizes one benefit or group of benefits taught herein without necessarily achieving other objectives or benefits as taught or suggested herein. Various alternatives are referred to herein. It should be understood that some preferred embodiments specifically include one, another, or a variety of features, while other embodiments specifically exclude one, another, or a variety of features, and other embodiments mitigate certain features by including one, another, or a variety of convenient features.

[0213] Furthermore, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, in addition to the various elements, features, and processes discussed above, other known equivalents for each of such elements, features, or processes can be used in various combinations by those skilled in the art to carry out the methods according to the principles described herein. Among the various elements, features, and processes, some are specifically included in various embodiments, while others are specifically excluded.

[0214] While this application has been disclosed in the context of specific embodiments and examples, it will be understood by those skilled in the art that the embodiments of this application, and the application, are disclosed in the context of certain embodiments and examples, but that the embodiments of the application extend beyond the embodiments and their equivalents specifically disclosed to use other embodiments and modifications.

[0215] Preferred embodiments of this application are described herein and include the best form known to the inventors for carrying out this application. Variations of such preferred embodiments will be obvious to those skilled in the art by reading the foregoing description. It is taken into consideration that such variations may be used as appropriate by those skilled in the art and that this application may be carried out in a manner other than that specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the content detailed in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations is incorporated herein, unless otherwise shown herein or expressly rejected by the context.

[0216] All patents, patent applications, published patent applications, and other materials such as articles, books, specifications, publications, documents, and things referenced herein are incorporated herein by reference in their entirety for all purposes, except for any history of prosecution associated therewith, any inconsistency or conflict with this document, or any limited effect on the broadest claims currently or subsequently associated with this document. For example, in the event of any inconsistency or conflict in the description, definition, and / or use of terms associated with any of the incorporated materials and terms associated with this document, the description, definition, and / or use of terms in this document shall prevail.

[0217] It should be understood that the embodiments of the present application disclosed herein are illustrative of the principles of the embodiments of the present application. Other available modifications are within the scope of the present application. Therefore, alternative configurations of the embodiments of the present application, without limitation, can be used in accordance with the teachings herein. Accordingly, the embodiments of the present application are not limited to those previously shown and described.

[0218] Various embodiments of the present invention are described above in the detailed description. While these descriptions directly illustrate the embodiments described above, it will be understood that those skilled in the art may conceive of modifications and / or variations to the specific embodiments shown and described herein. Such modifications or variations within the scope of this description are also intended to be included therein. Unless specifically noted, the words and phrases in the specification and claims are intended by the inventors to have meanings that are common and familiar to those skilled in the art in the applicable field.

[0219] The above description of various embodiments of the Invention known to the applicant at the time of filing of this application is presented and intended for illustrative and explanatory purposes. This description is not intended to be comprehensive or to limit the Invention to the exact form disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments are intended to illustrate the principles and practical applications of the Invention and to enable those skilled in the art to utilize the Invention with various modifications suitable for various embodiments and specific uses considered. Therefore, the Invention is not intended to be limited to the specific embodiments disclosed for carrying out the Invention.

[0220] While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made based on the teachings herein without departing from the present invention and its broader aspects. Therefore, the appended claims are intended to encompass all such changes and modifications within their scope, in a manner true to the spirit and scope of the present invention.

Claims

1. A method for predicting whether a subject's inflammatory bowel disease (IBD) is likely or unlikely to progress, the method being: A process for identifying the genotype of a subject with respect to a risk allele of a gene / locus; and A step of predicting that the subject is likely to develop IBD after detecting a risk allele; or a step of predicting that the subject is unlikely to develop IBD if no risk allele is detected. A method characterized by including

2. The method according to claim 1, characterized in that the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof.

3. The method according to claim 1, characterized in that the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof.

4. The method according to claim 1, characterized in that the gene / locus includes one or more from SEQ ID NO: 1 to SEQ ID NO:

341.

5. The process of identifying the genotype of the subject is: The process of obtaining a sample from a subject; and The process of identifying the genotype of a sample based on risk alleles at a gene / locus. The method according to claim 1, characterized by including the following:

6. The process of identifying the genotype of the sample is: A step of contacting a sample with an oligonucleotide probe specific to the risk allele; A step of generating an allele-specific hybridization complex between an oligonucleotide probe and a risk allele; and A step of detecting a risk allele after detecting an allele-specific hybridization complex; or a step of not detecting a risk allele if an allele-specific hybridization complex is not detected. The method according to claim 5, characterized by including the following:

7. The method according to 6, characterized in that the oligonucleotide probe is labeled with a fluorescent dye, and the detection of an allele-specific hybridization complex comprises detecting a fluorescent signal from the oligonucleotide probe.

8. The method according to 6, characterized in that the oligonucleotide probe comprises a reporter dye and a quencher dye.

9. The method according to claim 6, further comprising the step of performing PCR amplification after forming an allele-specific hybridization complex.

10. A method for diagnosing inflammatory bowel disease (IBD) in a subject, the method being: The process of identifying the genotype of a subject's sample based on risk alleles at a gene / locus; A step of diagnosing IBD in the subject after detecting the risk allele; and A process of administering IBD treatment to a subject diagnosed with IBD, thereby treating the subject's IBD. A method characterized by including

11. The method according to 10, characterized in that the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof.

12. The method according to 10, characterized in that the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof.

13. The method according to 10, characterized in that the gene / locus includes one or more from SEQ ID NO:1 to SEQ ID NO:

341.

14. The method according to 10, characterized in that the treatment of IBD includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof.

15. The process of identifying the genotype of a subject's sample based on risk alleles at a gene / locus; A step of diagnosing IBD in the subject after detecting the risk allele; and A process of administering IBD treatment to a subject diagnosed with IBD, thereby treating the subject's IBD. A method characterized by including

16. The method according to 15, characterized in that the gene / locus includes SLC26A4, DLG4, GIPR, ZHX3, TNRC6B, CDK6, PRR5L, WNT2B, LRRC16A, HIST1 cluster (all histone cluster 1 genes), GTF2IRD2B, ETS1, SLC5A1, or TET2, or a combination thereof.

17. The method according to 15, characterized in that the gene / locus includes ETS1, the HIST1 cluster (all histone cluster 1 genes), CDK6, LRRC16A, or a combination thereof.

18. The method according to 15, characterized in that the gene / locus includes one or more from SEQ ID NO:1 to SEQ ID NO:

341.

19. The method according to 15, characterized in that the treatment of IBD includes anti-TNF therapy, anti-TL1A therapy, colectomy, or a combination thereof.

20. A method for identifying a gene / locus associated with a disease, the method being: The process of obtaining genetic data from disease cohort samples; A process of performing GLS conversion on genetic data, thereby causing the genetic data to lose its correlation; A process of performing gene-based analysis on GLS-converted gene data; and The process of identifying genes / locuses associated with disease. A method characterized by including

21. The method according to 20, characterized in that the disease is IBD, CD, or UC, or a combination thereof.

22. The method according to 20, characterized in that the cohort includes correlated subjects or family subjects.

23. The method according to 20, characterized in that the genetic data includes SNP genotypes.

24. The process of performing GLS conversion is as follows: [Math 1] The method according to 20, characterized in that it includes a step of converting genetic data according to a combination thereof.

25. The method according to 20, characterized in that the step of performing gene-based analysis includes the step of applying gene-based tests based on the assumption of independent or uncorrelated subjects.

26. The method according to 20, characterized in that the step of performing gene-based analysis includes the step of applying C-alpha, SKAT, SKAT-CommonRare, CMC, WSS, variable threshold, or comprehensive approach, or a combination thereof.

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