A composition for diagnosing colorectal cancer, rectal cancer, or colorectal adenoma utilizing changes in CpG methylation of the GLRB gene, and its use.

Abstract

The present invention provides a diagnostic composition for colon cancer, rectal cancer, or colorectal adenoma, comprising a specific methylation marker capable of predicting the risk of colorectal cancer, rectal cancer, and colorectal adenoma having similar characteristics, and an agent for measuring the methylation level of CpG sites. [Solution] The present invention provides a composition, kit, nucleic acid chip, and method for diagnosing colon cancer, rectal cancer, or colorectal adenoma, which include an agent for measuring the methylation level of the CpG site of the GLRB gene. This enables not only accurate and rapid diagnosis of colon cancer, rectal cancer, or colorectal adenoma, but also early diagnosis.

Description

Technical Field

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[0001] This application claims priority based on Korean Patent Application No. 10-2020-0042726, filed on April 8, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a composition, a kit, a nucleic acid chip, and a method for diagnosing colorectal cancer, rectal cancer, or colonic adenoma by detecting the methylation level of CpG sites of the GLRB gene.

Background Art

[0004] Colorectal cancer can occur in any part of the colon / rectum, but the incidence is highest at approximately 40% when it occurs in the rectum, followed by approximately 30% when it occurs in the sigmoid colon adjacent to the rectum. Due to changes in eating habits, the incidence and mortality rate of colorectal cancer have increased significantly in Korea, and it is also a major factor in cancer-related deaths in the United States and Europe (Non-Patent Document 1).

[0005] The diagnosis of colorectal cancer is simply performed by a fecal occult blood reaction test during a health check, but additional examinations and tests are required to actually confirm colorectal cancer, and the prognosis varies greatly depending on the stage of cancer at the time of diagnosis. Therefore, early detection of colorectal cancer patients is very important for improving the survival rate of patients.

[0006] On the other hand, epigenetics is the field that studies the regulation of gene expression that occurs without altering the DNA base sequence. Epigenetics studies gene expression regulation through epigenetic mutations such as DNA methylation, miRNA or histone acetylation, methylation, phosphate, and ubiquitination.

[0007] Among these, DNA methylation is the most extensively studied epigenetic mutation. Epigenetic mutations can lead to changes in gene function and transformation into tumor cells. Therefore, DNA methylation is associated with the expression (or suppression and induction) of intracellular disease-regulating genes, and recently, cancer diagnostic methods using DNA methylation measurement have been proposed. In particular, cancer-specific methylation often occurs in precancerous tissues, and the detection of cancer-specific methylation has high potential for use in cancer diagnosis.

[0008] Therefore, there is a need to develop effective colon cancer, rectal cancer, or colorectal adenoma-specific methylation markers that can predict the risk of colon cancer, rectal cancer, and colorectal adenoma, which have similar characteristics. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] American Cancer Society statics for 2009 [Overview of the project]

[0010] [Disclosure of the Invention] [Technical issues] The inventors have discovered that in colon cancer, rectal cancer, or colorectal adenoma, the CpG site of a specific gene is hypermethylated, and have developed a composition, kit, nucleic acid chip, and method that can diagnose colon cancer, rectal cancer, or colorectal adenoma by detecting the methylation level, thereby completing the present invention.

[0011] Therefore, the object of the present invention is to provide a diagnostic composition for colorectal cancer, rectal cancer, or colorectal adenoma that includes an agent for measuring the methylation level of a specific gene's CpG site.

[0012] Furthermore, an object of the present invention is to provide a diagnostic composition for colorectal cancer, rectal cancer, or colorectal adenoma, comprising an agent for measuring the methylation level of a specific gene's CpG site.

[0013] Furthermore, an object of the present invention is to provide a diagnostic composition for colorectal cancer, rectal cancer, or colorectal adenoma, which essentially consists of an agent for measuring the methylation level of a specific gene's CpG site.

[0014] Another object of the present invention is to provide a diagnostic kit for colorectal cancer, rectal cancer, or colorectal adenoma, comprising a PCR primer pair for amplifying a fragment containing a CpG site of a specific gene, and a sequencing primer for pyrosequencing the PCR product amplified by the primer pair.

[0015] Another object of the present invention is to provide a nucleic acid chip for diagnosing colorectal cancer, rectal cancer, or colorectal adenoma, on which a probe that can be hybridized under strict conditions with a fragment containing a CpG site of a specific gene is immobilized.

[0016] Another object of the present invention is to provide a method for providing information for the diagnosis of colon cancer, rectal cancer, or colorectal adenoma, comprising the step of measuring the methylation level of a specific gene's CpG site from a patient's sample and using this as a reference to determine the presence or absence of colon cancer, rectal cancer, or colorectal adenoma.

[0017] Another object of the present invention is to provide an agent for measuring the methylation level of the CpG site of the GLRB gene for the production of diagnostic preparations for colorectal cancer, rectal cancer, or colorectal adenoma.

[0018] Furthermore, another object of the present invention is a) The step of obtaining a sample from an individual, b) measuring the methylation level of the CpG site of the GLRB gene from the sample; c) providing a method for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level.

[0019] [Technical Solution] To achieve the above object, the present invention provides a composition for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising an agent for measuring the methylation level of the CpG site of the GLRB gene.

[0020] The present invention also provides a composition for diagnosing colon cancer, rectal cancer, or colorectal adenoma, consisting of an agent for measuring the methylation level of the CpG site of the GLRB gene.

[0021] The present invention also provides a composition for diagnosing colon cancer, rectal cancer, or colorectal adenoma, consisting essentially of an agent for measuring the methylation level of the CpG site of the GLRB gene.

[0022] To achieve another object of the present invention, the present invention provides a kit for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising a primer pair for amplifying a fragment containing the CpG site of the GLRB gene.

[0023] To achieve still another object of the present invention, the present invention provides a nucleic acid chip for diagnosing colon cancer, rectal cancer, or colorectal adenoma, on which a probe capable of hybridizing with a fragment containing the CpG site of the GLRB gene is immobilized.

[0024] To achieve still another object of the present invention, measuring the methylation level of the CpG site of the GLRB gene from a sample of a patient suspected of having colon cancer, rectal cancer, or colorectal adenoma; providing a method for providing information for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level.

[0025] To achieve still other objects of the present invention, the present invention provides the use of an agent for measuring the methylation level of a CpG site of the GLRB gene for producing a preparation for diagnosing colon cancer, rectal cancer, or colorectal adenoma.

[0026] To achieve still other objects of the present invention, the present invention a) obtaining a sample from an individual; b) measuring the methylation level of a CpG site of the GLRB gene from the sample; c) judging the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level, and provides a method for diagnosing colon cancer, rectal cancer, or colorectal adenoma.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The following references provide one of the skills having general definitions of many terms used in the specification of the present invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2th ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

[0028] Hereinafter, the present invention will be described in detail.

[0029] The present invention provides a composition for diagnosing colon cancer, rectal cancer, or colorectal adenoma, which contains an agent for measuring the methylation level of a CpG site of the GLRB gene.

[0030] In this invention, the term "methylation" refers to the attachment of a methyl group to a base that constitutes DNA. Preferably, in this invention, the presence or absence of methylation refers to the presence or absence of methylation occurring at a specific CpG site, cytosine, of a specific gene. When methylation occurs, the binding of transcription factors is interfered with, thereby suppressing the expression of the specific gene. Conversely, when demethylation or hypomethylation occurs, the expression of the specific gene increases.

[0031] In addition to A, C, G, and T, mammalian cell genomic DNA contains a fifth base called 5-methylcytosine (5-mC), which has a methyl group attached to the carbon at position 5 of the cytosine ring. Methylation of 5-methylcytosine occurs only at the carbon of a CG dinucleotide called CpG (5'-mCG-3'), and methylation of CpG suppresses the expression of alu or transposons and genomic repeat sequences. Furthermore, because the 5-mC of CpG is readily deaminated to thymine (T) spontaneously, CpG is a site where most epigenetic changes occur in mammalian cells.

[0032] In this invention, the term "measurement of methylation level" refers to the measurement of the methylation level of the CpG site of the GLRB gene, which can be measured by a bisulfite-treated detection method or a bisulfite-free detection method. The methylation level can be measured by methylation-specific PCR, such as methylation-specific polymerase chain reaction (MSP), real-time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding proteins, or quantitative PCR. Alternatively, it can be measured by automated base analysis such as pyrosequencing and bisulfite sequencing, but is not limited to these methods. Furthermore, as a bisulfite-free detection method, it can be measured using TET protein (ten-eleven translocation protein). TET protein is an enzyme that acts on DNA and is involved in the chemical change of bases. Unlike bisulfite treatment, where all Cs except methylated Cs are changed to T bases, with TET protein, only methylated Cs are changed to T bases, allowing for more efficient detection.

[0033] Preferably, the CpG site of the GLRB gene refers to a CpG site located on the DNA of the gene. The DNA of the gene is a concept that includes all the constituent units necessary for the expression of the gene and which are operably linked to one another, such as the promoter region, the protein coding region (open reading frame, ORF), and the terminator region. Therefore, the CpG site of the GLRB gene can be located in the promoter region, protein coding region (open reading frame, ORF), or terminator region of the corresponding gene.

[0034] Preferably, in the present invention, measuring the methylation level of the CpG site of the GLRB gene can mean measuring the cytosine methylation level of the CpG site of the genes listed in Table 1 below.

[0035] [Table 1]

[0036] In the present invention, the CpG region of the GLRB gene is characterized by being located between + / - 2000 bases (2kb) from the transcription start site (TSS) of the gene.

[0037] In this invention, the base sequence of the human genome chromosome region is represented by the February 2009 Human reference sequence (GRCh37). However, the specific sequence of the human genome chromosome region may be slightly altered as genome sequence research results are updated, and such alterations may result in a different representation of the human genome chromosome region in this invention. Therefore, even if the human reference sequence is updated after the filing date of this invention and the representation of the human genome chromosome region changes, it is self-evident that the scope of this invention will extend to the altered human genome chromosome region. Such alterations are easily understood by anyone with ordinary skill in the art to which this invention belongs.

[0038] In the present invention, the measuring agent for the methylation level of the CpG site may include a compound that modifies cytosine bases, a methylation-sensitive restriction enzyme, a primer specific to the methylated allele sequence of the GLRB gene, and a primer specific to the unmethylated allele sequence.

[0039] The compounds that modify the cytosine base are compounds that modify unmethylated cytosine or compounds that modify methylated cytosine, and may be, but are not limited to, bisulfite or a salt thereof that modifies unmethylated cytosine, preferably sodium bisulfite, or TET protein that modifies methylated cytosine. Methods for detecting the presence or absence of methylation of the CpG site by modifying such cytosine bases are widely known in the art (WO01 / 26536, US2003 / 0148326A1).

[0040] Furthermore, methylation-sensitive restriction enzymes may be restriction enzymes that contain CG at their recognition site, as they are restriction enzymes that can specifically detect methylation at the CpG site. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, and NotI. Methylation or demethylation at the C of the restriction enzyme recognition site changes whether or not cleavage occurs by the restriction enzyme, and this can be detected by PCR or Southern blotting analysis. Other methylation-sensitive restriction enzymes other than those mentioned above are well known in the industry.

[0041] The primers may include primers specific to the methylated allele sequence of the GLRB gene and primers specific to the unmethylated allele sequence.

[0042] In this invention, the term "primer" refers to a short nucleic acid sequence having a short free 3' terminal hydroxyl group that can form base pairs with a complementary template and serve as a starting point for template strand copying. A primer can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four nucleotide triphosphates at a suitable buffer solution and temperature. Furthermore, primers can incorporate additional features as sense and antisense nucleic acids having 7 to 50 nucleotide sequences, without altering the fundamental properties of the primer acting as a starting point for DNA synthesis.

[0043] The primers of the present invention may preferably be designed by the sequence of a specific CpG site to be analyzed for the presence or absence of methylation, and more preferably one or more selected from the group consisting of a primer pair that can specifically amplify cytosine that is methylated and not modified by bisulfites, a primer pair that can specifically amplify cytosine that is not methylated and modified by bisulfites, a primer pair that can specifically amplify cytosine that is methylated and modified by TET series proteins, and a primer pair that can specifically amplify cytosine that is not methylated and not modified by TET series proteins.

[0044] Therefore, the present invention provides a diagnostic kit for colorectal cancer, rectal cancer, or colorectal adenoma, comprising a primer pair for amplifying a fragment containing the CpG region of the GLRB gene.

[0045] In addition to the aforementioned agent, the composition and kit may also contain polymerase agarose, a buffer solution necessary for electrophoresis, and other similar components.

[0046] Furthermore, the present invention provides a nucleic acid chip for diagnosing colorectal cancer, rectal cancer, or colorectal adenoma, on which a probe capable of hybridizing with a fragment containing the CpG region of the GLRB gene is immobilized.

[0047] In the present invention, the term "nucleic acid" means oligonucleotides, nucleotides, polynucleotides, or fragments thereof, single-stranded or double-stranded DNA or RNA of genomic or synthetic origin, sense or antisense stranded DNA or RNA of genomic or synthetic origin, PNA (peptide nucleic acid), or DNA-like or RNA-like substances of natural or synthetic origin. If the nucleic acid is RNA, it will be obvious to those with ordinary skill in the art that deoxynucleotides A, G, C, and T are replaced with ribonucleotides A, G, C, and U, respectively.

[0048] Since methylation begins on the outer edge of gene regulatory sites and progresses inward, detecting methylation on the outer edge of regulatory sites allows for early diagnosis of genes involved in cellular recombination.

[0049] Therefore, early diagnosis of cells that may form colon cancer, rectal cancer, or colorectal adenoma is possible using the aforementioned methylation gene markers. When genes that have been confirmed to be methylated in cancer cells are also methylated in cells that appear clinically or morphologically normal, these normal-looking cells are actually undergoing cancerous transformation. Thus, by confirming the methylation of specific genes for colon cancer, rectal cancer, or colorectal adenoma in cells that appear normal, early diagnosis of colon cancer, rectal cancer, or colorectal adenoma is possible.

[0050] Furthermore, the present invention includes the step of measuring the methylation level of the CpG site of the GLRB gene from a sample of a patient suspected of having colon cancer, rectal cancer, or colorectal adenoma, The present invention provides a method for providing information for the diagnosis of colon cancer, rectal cancer, or colorectal adenoma, comprising the step of determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level.

[0051] The method for measuring the methylation level is selected from, but is not limited to, the group consisting of PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR using methylated DNA-specific binding proteins, methylation detection using methylation-sensitive restriction enzymes, quantitative PCR, DNA chips, pyrosequencing, and bisulfite sequencing.

[0052] Specifically, methylation-specific PCR involves treating the sample DNA with bisulfite, and then designing and using different primers depending on whether or not the CpG dinucleotide is methylated. If the primer binding site is methylated, the PCR proceeds using the methylated primer; if it is not methylated, the PCR proceeds using the normal primer. In other words, after treating the sample DNA with bisulfite, PCR is performed using two types of primers simultaneously, and the results are then compared.

[0053] Real-time methylation-specific PCR is a method that switches the methylation-specific PCR technique to a real-time measurement method. After treating genomic DNA with bisulfite, PCR primers corresponding to methylation are designed, and real-time PCR is performed using these primers. There are two methods for detection: one using a TaqMan probe complementary to the amplified base sequence, and the other using SYBRgreen. Therefore, real-time methylation-specific PCR can selectively quantitatively analyze only methylated DNA. In this method, a standard curve is created using an in vitro methylated DNA sample, and a gene without a 5'-CpG-3' sequence in its base sequence is amplified together as a negative control group for standardization, and the methylation level is quantitatively analyzed.

[0054] In methods that measure the presence or absence of methylation using methylation-sensitive restriction enzymes, the methylation-sensitive restriction enzymes target CpG dinucleotides, and if this site is methylated, the enzyme cannot function. Therefore, if a sample DNA is treated with a methylation-sensitive restriction enzyme and then amplified by PCR to include the enzyme target site, the restriction enzyme will not act on methylated regions, resulting in PCR amplification. However, normal, unmethylated regions will be cleaved by the restriction enzyme and will not be amplified by PCR, thus allowing for the measurement of the presence or absence of methylation in specific DNA regions.

[0055] PCR or DNA chip methods using methylated DNA-specific binding proteins allow for the selective separation of methylated DNA by mixing the DNA with a protein that specifically binds only to methylated DNA. Genomic DNA is mixed with the methylated DNA-specific binding protein, and then only the methylated DNA is selectively separated. This separated DNA is then amplified using PCR primers corresponding to intron regions, and the presence or absence of methylation is measured by agarose electrophoresis. Alternatively, quantitative PCR can also measure the presence or absence of methylation; methylated DNA separated with the methylated DNA-specific binding protein can be hybridized onto a DNA chip containing complementary probes labeled with fluorescent dyes to measure methylation. Note that the methylated DNA-specific binding protein is not limited to MBD2bt.

[0056] Furthermore, pyrosequencing of bisulfite-treated DNA is based on the following principle: When methylation occurs at the CpG dinucleotide site, 5-methylcytosine (5-mC) is formed, and this modified base is converted to uracil by sodium bisulfite treatment. When DNA extracted from a sample is treated with bisulfite, methylated CpG dinucleotides are preserved as cytosine, while the remaining unmethylated cytosine is converted to uracil. Sequence analysis of bisulfite-treated DNA can preferably be performed using pyrosequencing. Detailed explanations of pyrosequencing are publicly available in prior literature. [Ronaghi et al, Science 1998 Jul 17, 281(5375), 363-365; Ronaghi et al, Analytical Biochemistry 1996 Nov 1, 242(1), 84-9; Ronaghi et al. Analytical Biochemistry 2000 Nov 15, 286(2): 282-288; Nyr, P. Methods Mol Biology 2007, 373, 114].

[0057] On the other hand, in a bisulfite-free detection method using TET proteins, only methylated C is converted to T, making it possible to detect bases in the methylated region. (See LIU, Yibin, et al., Nature Biotechnology volume 37, pages 424-429 (2019)).

[0058] When methylation occurs at the CpG dinucleotide site, forming 5-methylcytosine (5-mC) from cytosine, treatment with a TET (ten-eleven translocation) protein causes the methylated CpG dinucleotide to change to uracil, while the unmethylated cytosine is conserved. Sequence analysis of TET-treated DNA is not limited to pyrosequencing; it can also be analyzed using methods such as methylation-sensitive PCR (MSP), microarray, and next-generation sequencing (NGS).

[0059] Preferably, the method for providing information for the diagnosis of colorectal cancer, rectal cancer, or colorectal adenoma according to the present invention can be carried out by a method characterized by including a) a step of obtaining a sample from an individual; b) a step of obtaining genomic DNA from the sample; c) a step of treating the obtained genomic DNA with a compound that modifies unmethylated cytosine bases; d) a step of obtaining a PCR product by amplifying the treated DNA by PCR using a pyrosequencing primer capable of amplifying the promoter of the GLRB gene; and e) a step of measuring the methylation level by pyrosequencing the PCR product using a sequencing primer.

[0060] The acquisition of genomic DNA in step b) above can be carried out using phenol / chloroform extraction, SDS extraction, CTAB separation, or commercially available DNA extraction kits, which are commonly used in the industry.

[0061] In the present invention, "determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level" may mean measuring the methylation level of the CpG site of the GLRB gene from a sample of a patient suspected of having colon cancer, rectal cancer, or colorectal adenoma, and simultaneously, or sequentially, measuring the methylation level of the same gene's CpG site from a sample of a normal control group, and then comparing the two. Alternatively, the methylation level of the CpG site of the GLRB gene from a sample of a normal control group may be measured in advance to set a threshold, and then the determination may be made by comparing that threshold with the value of the methylation level measured from the patient's sample. If the methylation level of the CpG site of the GLRB gene from a sample of a normal control group is measured in advance to set a threshold, the methylation level of the CpG site of the GLRB gene can be measured from the patient's sample without having to measure the methylation level of the normal control group each time for each diagnosis, and information for diagnosing the presence or absence of colon cancer, rectal cancer, or colorectal adenoma in the corresponding patient can be rapidly provided.

[0062] In this invention, the term "sample" refers to a broad range of bodily fluids, including all biological bodily fluids obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of analysis performed. Methods for obtaining bodily fluids and tissue biopsies from mammals are generally well known, and in this invention, the sample can preferably be selected from the group consisting of human-derived substances, including tissues, cells, blood, plasma, serum, feces, and urine. Abnormal methylation changes in cancerous tissue show considerable similarity to methylation changes in genomic DNA obtained from biological samples such as cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine. Therefore, the use of the markers of this invention has the advantage of enabling easy diagnosis of colon cancer, rectal cancer, or colorectal adenoma development through blood, bodily fluids, etc.

[0063] The present invention provides the use of a measuring agent for the methylation level of the CpG site of the GLRB gene for the production of diagnostic preparations for colorectal cancer, rectal cancer, or colorectal adenoma.

[0064] The present invention a) The step of obtaining a sample from an individual, b) A step of measuring the methylation level of the CpG site of the GLRB gene from the sample, c) A method for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising the step of determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level.

[0065] In one embodiment, the present invention provides a method for diagnosing and treating colon cancer, rectal cancer, or colorectal adenoma in an individual, comprising the following steps: i) The step of obtaining a sample from an individual, ii) A step of measuring the methylation level of the CpG site of the GLRB gene from the sample, iii) A step of determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level, iv) The step of administering therapeutic drugs to the individual determined to have bladder cancer, or treating colon cancer, rectal cancer, or colorectal adenoma through surgery.

[0066] The method including steps i) through iv) described above is understood in accordance with the method including steps a) through c) described above.

[0067] The iv) step is the step of administering therapeutic drugs to individuals diagnosed with the disease in the iii) step, or treating the disease through means such as surgery.

[0068] The “treatment” of the present invention comprehensively refers to improving colon cancer, rectal cancer, or colorectal adenoma, or the symptoms of the said disease, which may include curing, substantially preventing, or improving the condition of the said disease, and may include, but is not limited to, alleviating, curing, or preventing one or most symptoms originating from colon cancer, rectal cancer, or colorectal adenoma.

[0069] The “therapeutic drug” is not particularly limited in type, as long as it is a drug commonly used to treat colon cancer, rectal cancer, or colorectal adenoma. Furthermore, the therapeutic drug is administered to the individual in a “therapeutically effective amount,” and the therapeutically effective amount can be determined by a person skilled in the art by considering a variety of factors, including not only the inherent properties of the drug, the route of administration, and the number of treatments, but also the patient’s age, weight, health condition, sex, severity of the disease, diet, and excretion rate. The route of administration of the therapeutic drug is not particularly limited and may be administered orally or parenterally, and includes not only local administration but also all systemic routes of administration. Parenteral administration is not limited to this, but may include, for example, intranasal application of the drug or subcutaneous injection, or, for example, intramuscular injection or intravenous injection.

[0070] The “sample” of the present invention is, but is not limited to, a sample isolated and obtained from an individual suspected of having a disease, and is selected from the group consisting of cells, tissues, blood, serum, plasma, saliva, sputum, mucosal fluid, and urine. The “individual” may be an animal, preferably a mammal, and in particular an animal including humans, and may also be cells, tissues, organs, etc. derived from an animal. The individual may be a patient who requires the aforementioned therapeutic effect.

[0071] In this specification, the term “comprising” is used synonymously with “including” or “characterized by,” and does not exclude additional components or steps of a composition or method according to the present invention that are not specifically mentioned. The term “consisting of” means excluding additional elements, steps, or components that are not otherwise described. The term “essentially consisting of” means that, within the scope of a composition or method, it may include substances or steps that, together with the described substances or steps, do not substantially affect its fundamental properties. [Effects of the Invention]

[0072] [Advantageous effect] As described in detail above, hypermethylation of the CpG site of the GLRB gene specifically occurs in colon cancer, rectal cancer, or colorectal adenoma. Therefore, the composition, kit, chip, or method according to the present invention can be used to accurately and rapidly diagnose colon cancer, rectal cancer, or colorectal adenoma, and to enable early diagnosis. [Brief explanation of the drawing]

[0073] [Figure 1] Figure 1 shows the results of examining the methylation information of the GLRB gene in all 32 types of cancer. [Figure 2] Figure 2 shows the results of confirming the accuracy of the GLRB gene selection method used in the diagnosis of colorectal cancer. [Figure 3] Figure 3 shows the results of examining the difference in methylation between the colorectal cancer tumor tissue (tumor) cell line group and the non-colorectal cancer tumor tissue (others) cell line group. [Figure 4] Figure 4 shows the results of examining the differences in methylation between tumor tissue (cancer), adenomatous tissue (adenoma), and normal tissue (normal) in adenomas of the large intestine. [Figure 5] Figure 5 shows the results of examining the difference in qMSP-based methylation between tumor tissue (cancer) and surrounding normal tissue (nontumor) in colorectal adenomas. [Figure 6] Figure 6 shows the results of examining the methylation information of the OPLAH gene as a comparative example. [Figure 7] Figure 7 shows the results of examining the differences in methylation based on droplet digital PCR (ddPCR) in adenoma, tumor tissue, and normal surrounding tumor tissue. [Figure 8] Figure 8 shows the results of analyzing differences in methylation using droplet digital PCR (ddPCR) in plasma from normal humans and colorectal cancer patients. [Modes for carrying out the invention]

[0074] [Best mode for carrying out the invention] The following are desirable embodiments to aid in understanding the present invention. However, the following embodiments are provided only to make the present invention easier to understand and do not limit the scope of the present invention.

[0075] Example 1: Selection of colorectal cancer-specific methylated genes To select methylated genes specifically found in colorectal cancer, a large-scale methylation comparison study was conducted using data from two large-scale methylation microarray chips to compare cancerous tissue obtained from cancer surgery in colorectal cancer patients with normal tissue (see Table 2). In the corresponding studies, tumor tissue refers to cancerous tissue from colorectal cancer, while non-tumor tissue refers to tissue other than cancerous tissue, including normal tissue.

[0076] [Table 2]

[0077] To select colorectal cancer-specific methylated genes, DNA was extracted from various tissues, and the methylation levels of the gene regions were confirmed using an Infinium Human Methylation 450 Beadchip microarray.

[0078] DNA extracted from each tissue is transformed through bisulfite treatment. This process modifies cytosine bases depending on the presence or absence of methylation in the DNA region. The probes used in the corresponding microarray analysis are designed specifically for methylation and unmethylation to confirm the presence or absence of cytosine base modification in the methylated regions of the gene.

[0079] This microarray test measures the methylation level of genes through approximately 450,000 (450k) probes, each representing a methylated region of a gene. The results for each probe derived through the test are presented as a beta value. The beta value ranges from 0 to 1, with values ​​closer to 1 indicating a higher methylation level in the corresponding gene region.

[0080] To identify differentially methylated regions (DMRs) between the tumor and non-tumor groups, we used the Limma (Linear Models for Microarray Data) empirical Bayes t-test to identify gene sites showing statistically significant methylation differences between the groups.

[0081] The Limma method is known to be the least affected by outliers among many methylation statistical analysis methods for identifying differences between groups. Therefore, it is a suitable method for searching for cancer-specific markers because it is less influenced by abnormal measurements from some samples. In this experiment, a smaller adjusted p-value derived through the Limma method was considered to indicate a significant methylation difference between the two groups.

[0082] In particular, to search for tumor-specific methylation regions, we selected gene regions that showed even higher methylation in tumor tissue than in non-tumor tissue, among those with a significant difference in beta values ​​between tumor and non-tumor groups, as candidate cancer-specific biomarkers.

[0083] As a result, in each of the two datasets, gene regions that showed significantly lower p-values ​​(top 10% with the lowest p-values) when comparing Limma analysis results between the tumor group and the non-tumor group, and that showed a large difference of 0.2 or more in beta values ​​between the groups, were selected as tumor-specific hypermethylated regions. Through this process, 3,878 gene regions that showed tumor-specific hypermethylation in both datasets were selected as candidate biomarkers from among approximately 450,000 gene regions.

[0084] Example 2: Selection of hypermethylated genes specific to colorectal cancer In Example 1, we examined and compared the methylation levels of the 3878 gene regions of the biomarkers identified in tumors other than colorectal cancer, and identified gene regions specific to colorectal cancer or colorectal adenoma among the biomarkers. By analyzing the DNA methylation 450k array test results from TCGA (The Cancer Genome Atlas), a public cancer gene database, we were able to confirm gene region methylation information corresponding to 32 cancer types. Among these, we identified gene regions that showed significantly higher beta values ​​in colorectal cancer, rectal cancer, or colorectal adenoma compared to 30 other cancers. As a result, we were able to confirm that, among the 3878 gene regions, the gene region of the GLRB gene undergoes specific methylation in colorectal cancer, rectal cancer, or colorectal adenoma.

[0085] The methylation levels of the aforementioned genes, obtained through microarray testing on tumor tissue (cancer tissue of colorectal cancer) and non-tumor tissue (tissue other than cancer tissue, including normal tissue), are shown in Figure 1. The methylation levels are shown as beta values, representing the results of each probe derived through the testing. Beta values ​​range from 0 to 1, with values ​​closer to 1 indicating a higher methylation level in the corresponding gene region.

[0086] On the other hand, in gene regions where methylation differences are observed when comparing tumor tissue from colon cancer, rectal cancer, or colorectal adenoma with non-tumor tissue, methylation may also occur in other cancers besides colon cancer, rectal cancer, or colorectal adenoma. In other words, no colon cancer, rectal cancer, or colorectal adenoma-specific methylation was confirmed.

[0087] For example, in the case of the OPLAH (5-oxoprolinase, ATP-hydrolysing) gene, it was one of the regions among the 3878 gene regions identified in Example 1 that showed the largest difference in methylation between tumor and non-tumor tissue. As can be seen from Figure 5, high methylation was confirmed to occur in all types of cancer except for acute myeloid leukemia, ocular melanoma, pheochromocytoma / ganglioma, thymoma, and thyroid cancer.

[0088] The preceding 32 types of cancer are: Acute Myeloid Leukemia, Adrenocortical Cancer, Bile Duct Cancer, Breast Cancer, Cervical Cancer, Colon Cancer, Endometrioid Cancer, Esophageal Cancer, Glioblastoma, Head and Neck Cancer, Kidney Chromophobe, Kidney Clear Cell Carcinoma, Kidney Papillary Cell Carcinoma, Liver Cancer, Lower Grade Glioma, and Lung Adenocarcinoma. adenocarcinoma, melanoma, mesothelioma, ocular melanomas, ovarian cancer, pancreatic cancer, phochromocytoma / adrenal nodule, prostate cancer, rectal cancer, sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, and uterine carcinosarcoma.

[0089] Among these gene regions, those where the corresponding region is located in a CpG island rather than a pseudogene, within + / - 2000 bases (2kb) of the gene's transcription start site (TSS), and on an autosome were selected as colorectal cancer-specific hypermethylated genes. As a result, one gene was selected, as shown in Table 3 below (see Figure 1).

[0090] [Table 3]

[0091] Example 3: Confirmation of colorectal cancer specificity of selected genes in cell lines To confirm whether the selected GLRB genes exhibit colorectal cancer or rectal cancer-specific methylation that distinguishes them from other cancers, we utilized public databases to analyze methylation patterns in 1022 cancer cell lines derived from 14 major tissues. The corresponding data are the results of Infinium Human Methylation 450 Beadchip microarray testing of DNA extracted from each cell line according to the manufacturer's standardized methylation analysis test procedure.

[0092] The results of the conducted tests were similar to those in Example 1, where gene methylation levels were measured through approximately 450,000 probes, and the methylation value for each probe was presented as a beta value. The beta value ranges from 0 to 1, with values ​​closer to 1 indicating a higher methylation level in the corresponding gene region.

[0093] The aforementioned 14 tissues are as follows: aerodigestive tract, blood, bone, breast, digestive system, kidney, lung, nervous system, pancreas, skin, soft tissue, thyroid, urogenital system, and other tissue.

[0094] To confirm the colorectal cancer, rectal cancer, or colorectal adenoma-specific methylation of the selected GLRB gene, methylation data from 1022 cell lines were broadly classified into a colorectal cancer cell line group (n=51) and a non-colorectal cancer cell line group (n=971).

[0095] To identify differentially methylated regions (DMRs) between the two classified groups, we used the Limma (Linear Models for Microarray Data) empirical Bayes t-test to identify gene regions showing statistically significant methylation differences between the groups.

[0096] [Table 4]

[0097] Analysis using cell lines also confirmed that the GLRB gene has a significantly lower adjusted p-value in colorectal cancer and rectal cancer cell lines compared to other cancer cell lines, indicating its specificity to colorectal cancer.

[0098] Example 4: Evaluation of diagnostic performance of candidate diagnostic markers for colon cancer, rectal cancer, or colorectal adenoma. To confirm the usefulness of selected genes as diagnostic markers for colorectal cancer, the accuracy of colorectal cancer diagnosis based on methylation levels was evaluated.

[0099] Sensitivity and specificity are used to evaluate the accuracy of a diagnosis. By calculating sensitivity and specificity values ​​for a given cut-off value of a continuous diagnostic test measurement, a Receiver Operating Characteristic (ROC) curve can be presented, showing how sensitivity and specificity change with respect to the cut-off value. Diagnostic accuracy can be measured by the area under the ROC curve (AUC). The AUC value ranges from 0.5 to 1, with a higher value indicating higher diagnostic accuracy. For example, an AUC value of 1 means the diagnostic result is perfectly accurate, while an AUC value of 0.5 is considered equivalent to a random result.

[0100] The accuracy of cancer classification based on methylation levels between non-tumor and tumor tissues using selected genes was analyzed using the collected methylation dataset. As shown in Figure 2, all selected genes had an AUC (Area Under Curve) value of 0.920 or higher, indicating high diagnostic accuracy and confirming that the selected genes are useful for the diagnosis of colorectal cancer.

[0101] Example 5: Confirmation of methylation of selected genes in adenomas. Adenoma is a precancerous condition that can progress to colorectal cancer, and most colorectal cancers originate from adenomas. Therefore, early detection of adenomas is essential for the early diagnosis of colorectal cancer. To confirm whether hypermethylation biomarkers selected through previous studies also exhibit hypermethylation characteristics in adenomas, we evaluated the hypermethylation properties of genes selected from 64 colorectal cancer tumor tissues, 42 colorectal adenoma tissues, and 41 non-tumor tissues.

[0102] Analysis of methylation data derived through the Human Methylation 450 Beadchip microarray test confirmed, as shown in Figure 4, that the selected genes exhibited significant hypermethylation compared to non-tumor tissue, in a similar manner not only in colorectal cancer but also in colorectal adenomas.

[0103] These results indicate that the selected genes can be used not only for diagnosing colorectal cancer but also for diagnosing colorectal adenomas.

[0104] Example 6: qMSP-based methylation measurement of selected genes in tissues To confirm colorectal cancer and adenoma-specific methylation of the GLRB gene in cancer tissue, methylation-specific PCR (quantitative methylation-specific PCR, qMSP) was used to measure the methylation difference between cancerous and non-cancerous tissue. For this purpose, genomic DNA was isolated from pairs of cancerous and surrounding tissues in 12 colorectal cancer patients and 4 adenoma patients. After bisulfite treatment, amplification and methylation levels of the GLRB-specific gene region were observed using a generalized qMSP method.

[0105] Furthermore, we used the ACTB gene, which specifically binds to the gene region modified and amplified by bisulfite, and does not participate in methylation to standardize the amplified value of the corresponding region.

[0106] The methylation level obtained by PCR amplification of the bisulfite-modified DNA is expressed as ΔCt, which is a value corrected by the ACTB Ct (Cycle of Throshold) value used as an internal control. ΔCt is defined as follows: ΔCt = Ct value of the ACTB gene - Ct value of the target gene

[0107] As shown in Figure 5, GLRB gene methylation was associated with relatively high ΔCt values ​​in colorectal cancer tissue compared to normal tissue surrounding the cancer, regardless of disease stage. In particular, very high ΔCt values ​​were observed in adenoma, a precancerous stage, confirming that the GLRB gene was hypermethylated in colorectal cancer and colorectal adenoma. This result demonstrates that the selected GLRB gene methylation is effective as a biomarker for the diagnosis of colorectal cancer, especially for early diagnosis.

[0108] These results indicate that the selected genes can be used not only for diagnosing colorectal cancer but also for diagnosing colorectal adenomas.

[0109] Example 7: Digital PCR-based methylation measurement of selected genes in tissues We measured the methylation difference between cancerous and non-cancerous tissues using digital PCR, which exhibits superior detection sensitivity compared to general real-time PCR. For this purpose, genomic DNA was isolated from pairs of cancerous and surrounding tissue (four pairs, categorized by disease stage) from 16 colorectal cancer patients and 5 adenoma patients. After bisulfite treatment, the amplification and methylation levels of GLRB-specific gene regions were observed using Droplet digital PCR (ddPCR).

[0110] The ACTB gene was used as the internal control group for gene region modification using bisulfite and gene region amplification using PCR.

[0111] The methylation level of the cfDNA converted to bisulfite, amplified by ddPCR, was calculated by copy number.

[0112] As can be seen in Figure 7, GLRB gene methylation was relatively high in colorectal cancer tissue compared to normal tissue surrounding the cancer, regardless of disease stage. In particular, it showed a very high copy number in adenoma, a precancerous stage. As confirmed by the results using real-time PCR, hypermethylation of the GLRB gene was observed in colorectal cancer and colorectal adenoma. This result indicates that methylation of selected GLRB genes can be effectively detected not only by real-time PCR but also by digital PCR.

[0113] These results demonstrate that the selected genes can be diagnosed using a variety of molecular biological techniques.

[0114] Example 8: Digital PCR-based methylation measurement in plasma To confirm the specific methylation of colorectal cancer in blood samples, the methylation difference between colorectal cancer patients and healthy individuals was measured using the ddPCR method. For this purpose, plasma samples from 10 colorectal cancer patients and 10 healthy individuals were collected, cell-free DNA (cfDNA) was separated, treated with bisulfite, and then the amplification and methylation levels of GLRB-specific gene regions were observed using the ddPCR method.

[0115] As can be seen in Figure 8, GLRB gene methylation showed higher levels in plasma samples from colorectal cancer patients compared to plasma samples from normal individuals, and it was demonstrated that it could be measured with 100% accuracy. This result indicates that selected GLRB gene methylation is effective not only in tissue but also in the diagnosis of colorectal cancer based on blood. [Industrial applicability]

[0116] As detailed above, hypermethylation of the CpG site of the GLRB gene specifically occurs in colon cancer, rectal cancer, or colorectal adenoma. Therefore, the compositions, kits, chips, or methods of the present invention can be used not only to accurately and quickly diagnose colon cancer, rectal cancer, or colorectal adenoma, but also to enable early diagnosis.

Claims

1. A diagnostic composition for colorectal cancer, rectal cancer, or colorectal adenoma, comprising an agent for measuring the methylation level of the CpG site of the GLRB gene.

2. The composition according to claim 1, characterized in that the CpG site is located within + / - 2000 base pairs (2 kb) from the transcription start site of the gene.

3. The measuring agent for the methylation level of the CpG site of the gene is a compound that modifies a non-methylated cytosine base, or a compound that modifies a methylated cytosine base; Primers specific to the methylated sequence of the CpG region of the GLRB gene; and The composition according to claim 1, characterized in that it is selected from the group consisting of primers specific to non-methylated sequences.

4. The composition according to claim 3, characterized in that the compound modifying the unmethylated cytosine base is bisulfite or a salt thereof, and the compound modifying the methylated cytosine base is a TET protein.

5. The composition according to claim 1, characterized in that the method for measuring the methylation level is selected from the group consisting of a bisulfite-free detection method, a methylation-specific polymerase chain reaction, a real-time methylation-specific polymerase chain reaction, PCR using a methylated DNA-specific binding protein or a methylation-sensitive restriction enzyme, quantitative PCR, pyrosequencing, bisulfite sequencing, and next-generation sequencing (NGS).

6. A diagnostic kit for colorectal cancer, rectal cancer, or colorectal adenoma, comprising a primer pair for amplifying a fragment containing the CpG region of the GLRB gene.

7. A nucleic acid chip for diagnosing colorectal cancer, rectal cancer, or colorectal adenoma, with a probe immobilized that can hybridize with a fragment containing the CpG region of the GLRB gene.

8. The procedure involves measuring the methylation level of the CpG site of the GLRB gene in samples from patients suspected of having colon cancer, rectal cancer, or colorectal adenoma, and A method for providing information for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising the step of determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level.

9. The method according to claim 8, characterized in that the method for measuring the methylation level is selected from the group consisting of a bisulfite-free detection method, a methylation-specific polymerase chain reaction, a real-time methylation-specific polymerase chain reaction, PCR using a methylated DNA-specific binding protein or a methylation-sensitive restriction enzyme, quantitative PCR, pyrosequencing and bisulfite sequencing, and next-generation sequencing (NGS).

10. The method according to claim 8, characterized in that the sample is selected from the group consisting of tissue, cells, blood, plasma, serum, feces, and urine.

11. Use of a measuring agent for the methylation level of the CpG site of the GLRB gene for the manufacture of diagnostic preparations for colorectal cancer, rectal cancer, or colorectal adenoma.

12. a) The step of obtaining a sample from an individual, b) A step of measuring the methylation level of the CpG site of the GLRB gene from the sample, c) A method for diagnosing colon cancer, rectal cancer, or colorectal adenoma, comprising the step of determining the presence or absence of colon cancer, rectal cancer, or colorectal adenoma based on the measured methylation level.

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