KR20210044441A - Composition for diagnosing colorectal cancer or adenoma using CpG methylation status of specific gene and uses thereof - Google Patents

Abstract

The present invention relates to a composition, a kit, a nucleic acid chip and a method capable of diagnosing colorectal cancer, rectal cancer or colorectal adenoma by detecting the methylation level of the CpG region of one or more genes selected from a group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2. The present invention can diagnose colorectal cancer, rectal cancer or colorectal adenoma accurately, quickly, and early.

Description

Composition for diagnosing colorectal cancer or adenoma using CpG methylation status of specific gene and uses thereof

The present invention detects the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2, thereby diagnosing colorectal cancer, colorectal cancer, or colorectal adenoma composition, kit, nucleic acid It relates to a chip and a method.

The large intestine is the last part of the digestive tract, about 2m long, and is divided into cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Colorectal cancer is cancer that occurs in this area, and is mostly adenocarcinoma, and is largely divided into colon cancer and rectal cancer by site.

Colorectal cancer can occur in any part of the colon/rectal, but it occurs in the rectum, which is the highest at about 40%, followed by about 30% in the S colon, which is close to the rectum. Changes in dietary habits have significantly increased the incidence of colon cancer and the resulting mortality in Korea, and it is also a major factor in cancer-related deaths in the United States and Europe (American Cancer Society statics for 2009).

The diagnosis of colorectal cancer is simply a fecal occult blood reaction test at the time of health checkup, but additional examinations and tests are required to actually confirm colorectal cancer. In addition to the rectal resin test, S-phase colonoscopy, a picture of the colon (barium enema), and a colonoscopy, the examination is mainly performed, but the prognosis varies greatly depending on the progression of the cancer at the time of diagnosis, so the early stage of colorectal cancer patients. Discovery is very important in increasing the patient's survival rate.

On the other hand, epigenetics is a field that studies the regulation of gene expression in a state where the nucleotide sequence of DNA is not changed. Epigenetics studies the regulation of gene expression through epigenetic mutations such as DNA methylation, miRNA or histone acetylation, methylation, phosphorylation and ubiquitination.

Of these, DNA methylation is the most studied epigenetic mutation. Epigenetic mutations can lead to mutations in gene function and changes to tumor cells. Therefore, DNA methylation is associated with the expression (or inhibition and induction) of disease-regulating genes in cells, and recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. In particular, since cancer-specific methylation may occur in advance even in pre-cancer stage tissues, detection of cancer-specific methylation is highly likely to be used for diagnosis of cancer.

Therefore, there is a need to develop an effective colorectal cancer, colorectal cancer, or colorectal adenoma specific methylation marker capable of predicting the risk of colorectal cancer and colorectal cancer and colorectal adenoma having similar characteristics.

Accordingly, the present inventors found that a specific gene CpG site in colon cancer, rectal cancer or colon adenoma is in a hypermethylated state, and by detecting the methylation level, a composition, kit, capable of diagnosing colon cancer, rectal cancer or colon adenoma The present invention was completed by developing a nucleic acid chip and method.

Accordingly, an object of the present invention is to provide a composition for diagnosing colon cancer, rectal cancer or colon adenoma, including an agent for measuring the methylation level of the CpG site of a specific gene.

In addition, another object of the present invention is 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. It is to provide a kit for diagnosis of adenoma.

Another object of the present invention is to provide a nucleic acid chip for diagnosing colon cancer, rectal cancer or colon adenoma in which a fragment including a CpG site of a specific gene and a probe capable of hybridizing under strict conditions are immobilized.

In addition, another object of the present invention is to provide a method of providing information for diagnosing colorectal cancer, colorectal cancer or colorectal adenoma, comprising measuring and comparing the methylation level of the CpG region of a specific gene from different samples.

In order to achieve the above object, the present invention comprises an agent for measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2. It provides a composition for diagnosing colon adenoma.

In order to achieve another object of the present invention, the present invention is a large intestine comprising a primer pair for amplifying a fragment containing a CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 It provides a kit for diagnosis of cancer, rectal cancer or colon adenoma.

In addition, in order to achieve another object of the present invention, the present invention is a probe capable of hybridizing with a fragment containing a CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2. It provides a nucleic acid chip for diagnosing colon cancer, rectal cancer or colon adenoma on which is fixed.

In addition, in order to achieve another object of the present invention, the present invention is any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 from samples of patients suspected of developing colon cancer, rectal cancer or colon adenoma Measuring the methylation level of the CpG site of; And

Comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample, comprising, providing a method of providing information for diagnosing colorectal cancer, colorectal cancer or colorectal adenoma.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. The following references provide one of the skills with general definitions of several 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.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for diagnosing colorectal cancer, rectal cancer, or colon adenoma comprising an agent measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2.

In the present invention, the term "methylation" refers to attaching a methyl group to a base constituting DNA. Preferably, methylation in the present invention means whether methylation occurs in cytosine of a specific CpG site of a specific gene. When methylation occurs, the binding of transcription factors is prevented, thereby inhibiting the expression of a specific gene. Conversely, when unmethylation or hypomethylation occurs, the expression of a specific gene increases.

In the genomic DNA of mammalian cells, in addition to A, C, G, and T, there is a fifth base called 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. do. The methylation of 5-methylcytosine occurs only in the C of the CG dinucleotide (5'-mCG-3') called CpG, and the methylation of CpG inhibits the expression of alu or transposon and genome repeats. In addition, since 5-mC of CpG is naturally deaminated to become thymine (T), CpG is a site where most epigenetic changes occur frequently in mammalian cells.

In the present invention, the term "measurement of methylation level" refers to measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2, and detection method according to bisulfite treatment Alternatively, it can be measured through a bisulfite independent detection method. The methylation level is measured by methylation-specific PCR, such as methylation-specific polymerase chain reaction (MSP), real time methylation-specific polymerase chain reaction, and methylated DNA-specific binding protein. It can be measured through used PCR or quantitative PCR. Alternatively, it may be measured by automatic base analysis such as pyro sequencing and bisulfite sequencing, but is not limited thereto. In addition, as a bisulfite-independent detection method, it can be measured using a detection method using a ten-eleven translocation protein (TET) (see Nature Biotechnology, volume 37, pages 424 to 429 (2019)). The TET protein is an enzyme that acts on DNA and is involved in the chemical change of the base, and when bisulfite is treated, all C except methylated C is changed to T base. In the Tet protein, only methylated C is changed to T. Efficient detection is possible.

Preferably, the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 refers to a CpG site present on the DNA of the gene. The DNA of the gene is a concept including all of a series of structural units required for expression of the gene and operably linked to each other, for example, a promoter region, a protein coding region (open reading frame, ORF), and a terminator. Includes the area. Therefore, the CpG region of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 may be present in the promoter region, protein coding region (open reading frame, ORF) or terminator region of the corresponding gene.

Preferably, in the present invention, measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 is the methylation of cytosine at the CpG site of the gene shown in Table 1 below. It can mean measuring your level.

Symbol Genome Build Chromosome Region CHL1 GRCh37 3 238391-240140 GRIN2A GRCh37 16 10273943-10277424 CLIP4 GRCh37 2 29337983-29338909 KCNQ5 GRCh37 6 73330942-73333109 KRBA1 GRCh37 7 149410858-149412351 ATP8B2 GRCh37 One 154298205-154298544

In the present invention, the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 is +/- 2000 bases from the transcription start site (TSS) of the gene. ) It is characterized in that it is located between (2kb).

In the present invention, the nucleotide sequence of the human genomic chromosome region was expressed according to The February 2009 Human reference sequence (GRCh37), but the specific sequence of the human genomic chromosome region may be slightly changed as the genomic sequence study results are updated. , According to these changes, the expression of the human genome chromosomal region of the present invention may be different. Therefore, the human genomic chromosome region expressed according to The February 2009 Human reference sequence (GRCh 37) of the present invention has been updated since the filing date of the present invention, so that the expression of the human genomic chromosome region is now Even if it is changed differently, it will be apparent that the scope of the present invention extends to the modified human genomic chromosome region. These changes can be easily recognized by anyone of ordinary skill in the art to which the present invention belongs.

In the present invention, the agent for measuring the methylation level of the CpG site is a compound that modifies a cytosine base or a methylation-sensitive restriction enzyme, CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1, and ATP8B2 methylation of any one or more genes selected from the group consisting of Primers specific for the allelic sequence that have been formed, and primers specific for the unmethylated allele sequence.

The compound that modifies the cytosine base is an unmethylated cytosine or a compound that modifies a methylated cytosine, and a bisulfite or a salt thereof that modifies an unmethylated cytosine, preferably sodium bisulfite, or a methylated cytosine It may be a TET protein, but is not limited thereto. A method for detecting methylation of the CpG site by modifying such a cytosine base is well known in the art (WO01/26536; US2003/0148326A1).

In addition, the methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of a CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. For example, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, and the like, but are not limited thereto. Depending on the methylation or non-methylation at C of the restriction enzyme recognition site, whether or not cleavage by the restriction enzyme is changed, and this can be detected through PCR or Southern Blot analysis. Other methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

The primer may include a primer specific for the methylated allele sequence of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2, and a primer specific for the unmethylated allele sequence.

In the present invention, the term "primer" refers to a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free 3'terminal hydroxyl group and serving as a starting point for template strand copying. . Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. In addition, the primers are sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides, and may incorporate additional features that do not change the basic properties of the primers serving as an initiation point for DNA synthesis.

The primer of the present invention may be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and more preferably, it can specifically amplify cytosine that is methylated and has not been modified by bisulfite. A primer pair that is not methylated, a primer pair capable of specifically amplifying a cytosine modified by bisulfite, a primer pair that is methylated and capable of specifically amplifying a cytosine modified by a Tet family protein, and a non-methylated primer pair. It may be any one or more selected from the group consisting of primer pairs capable of specifically amplifying cytosine that has not been modified by the Tet family of proteins.

Accordingly, the present invention provides a kit for diagnosing colon cancer, rectal cancer or colon adenoma comprising a primer pair for amplifying a fragment containing the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2. to provide.

In addition to the above formulations, the composition and kit may further include a polymerase agarose, a buffer solution required for electrophoresis, and the like.

In addition, the present invention is a fragment containing a CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1, and ATP8B2, and a probe capable of hybridizing with a fixed colon cancer, rectal cancer or colon adenoma Provides a diagnostic nucleic acid chip.

In the present invention, the term "nucleic acid" refers to oligonucleotides, nucleotides, polynucleotides, fragments thereof, DNA or RNA of genomic origin or synthetic origin of single-stranded or double-stranded DNA, or DNA of genomic origin or synthetic origin of the sense or antisense strand Or RNA, PNA (peptide nucleic acid), or a DNA amount or RNA-amount material of natural or synthetic origin. It is apparent to those of ordinary skill in the art that if the nucleic acid is RNA, it is replaced with ribonucleotides A, G, C and U, respectively, in place of deoxynucleotides A, G, C and T.

Since methylation starts from the outside of the regulatory site of the gene and proceeds to the inside, detecting methylation at the outside of the regulatory site allows early diagnosis of genes involved in cell transformation.

Therefore, early diagnosis of cells capable of forming colon cancer, rectal cancer or colon adenoma is possible by using the methylated gene marker. When a gene confirmed to be methylated in cancer cells is methylated in cells that appear to be clinically or morphologically normal, the cells that appear to be normal are undergoing cancer. Therefore, colon cancer, rectal cancer or colon adenoma can be diagnosed early by confirming the methylation of a colon cancer, rectal cancer or colon adenoma-specific gene in cells that appear to be normal.

In addition, the present invention comprises the steps of measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 from a sample of a patient suspected of developing colon cancer, rectal cancer or colon adenoma; And

Comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample, comprising, providing a method of providing information for diagnosing colorectal cancer, colorectal cancer or colorectal adenoma.

The method of measuring the methylation level is PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylated DNA specific binding protein, and methylation using a methylation sensitive restriction enzyme. , Quantitative PCR, DNA chip, pyro-sequencing, and bisulfite sequencing may be selected from the group consisting of, but is not limited thereto.

Specifically, the methylation-specific PCR method is a method of designing and using different types of primers depending on whether the CpG dinucleotide is methylated or not as a primer for PCR after treatment with bisulfite on sample DNA. If the primer binding site is methylated, PCR proceeds with methylated primers, and if methylation is not performed, PCR proceeds with normal primers. In other words, this is a method of comparing the results after performing PCR using two types of primers simultaneously after treatment with bisulfite on the sample DNA.

Real-time methylation-specific PCR is a conversion of the methylation-specific PCR method to a real-time measurement method. After treating genomic DNA with bisulfite, a PCR primer corresponding to methylation is designed, and real-time using these primers. It is to perform PCR. At this time, there are two methods of detection using the amplified nucleotide sequence and the complementary TanMan probe and the detection using Sybergreen. Therefore, real-time methylation-specific PCR can selectively quantitatively analyze only methylated DNA. At this time, a standard curve is prepared using an in vitro methylated DNA sample, and for standardization, a gene without a 5'-CpG-3' sequence in the nucleotide sequence is amplified together as a negative control group to quantitatively analyze the degree of methylation.

In the method of measuring methylation using a methylation-sensitive restriction enzyme, the methylation-sensitive restriction enzyme uses CpG dinucleotide as a site of action, and when this site is methylated, it cannot act as an enzyme. Therefore, if the sample DNA is treated with a methylation-sensitive restriction enzyme and then amplified by PCR to include the enzyme target site, in the case of methylation, the restriction enzyme does not work and is amplified by PCR, but the non-methylated normal site is cleaved by the restriction enzyme and PCR Since it is not amplified, it is possible to determine whether a specific DNA site is methylated.

In the PCR or DNA chip method using methylated DNA-specific binding protein, if a protein that specifically binds only to methylated DNA is mixed with DNA, only methylated DNA can be selectively separated because the protein is specifically bound to only methylated DNA. . After mixing the genomic DNA with a methylated DNA-specific binding protein, only methylated DNA is selectively isolated. This is a method of amplifying these isolated DNAs using PCR primers corresponding to the intron site, and then measuring methylation by agarose electrophoresis. In addition, methylation can be measured by quantitative PCR method. The methylated DNA isolated with a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a DNA chip in which a complementary probe is integrated to measure the methylation status. I can. Here, the methylated DNA specific binding protein is not limited to MBD2bt.

In addition, bisulfite 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 when treated with bisulfite. When the DNA extracted from the sample is treated with bisulfite, if the CpG dinucleotide is methylated, it is preserved as cytosine, and the remaining unmethylated cytosine is converted to uracil. The sequencing of the bisulfite-treated DNA can be preferably performed using a pyrosequencing method. A detailed description of pyrosequencing is known 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].

On the other hand, it is also possible to detect the base of the methylation site by converting only C methylated using Tet protein into T by a bisulfite independent detection method using Tet protein (LIU, Yibin, et al., Nature Biotechnology volume 37, pages 424). -429 (2019)).

When methylation occurs at the CpG dinucleotide site, resulting in the formation of 5-methylcytosine (5-mC) in cytosine.When the Cpg dinucleotide is methylated when processing the Tet (ten-eleven translocation) protein, it changes to uracil, and is not methylated. Is preserved. The sequencing of Tet-treated DNA is not limited only to the pyro-sequencing method, but using methods such as methylation-sensitive PCR (MSP), microarray, and next generation sequencing (NGS). Can be analyzed.

Preferably, the method of providing information for diagnosing colorectal cancer, colorectal cancer or colorectal adenoma of the present invention comprises: a) obtaining a sample from an individual, b) obtaining a genomic DNA from the sample, c) the obtained genomic DNA Treating a compound that modifies an unmethylated cytosine base, d) converting the treated DNA to a pie capable of amplifying the promoter of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2. A step of obtaining a PCR product by amplifying by PCR using a primer for low sequencing, and e) measuring the degree of methylation by pyrosequencing the PCR product using a sequencing primer. It can be carried out by the method characterized by.

Obtaining the genomic DNA of step b) is a phenol/chloroform extraction method commonly used in the art, SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB separation method (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or a commercially available DNA extraction kit.

In the present invention, the term “sample” refers to a wide range of bodily fluids including all biological fluids obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of analysis to be performed. Methods for obtaining bodily fluids and tissue biopsies from mammals are generally widely known, and in the present invention, the sample is preferably a group consisting of human derivatives including tissues, cells, blood, plasma, serum, feces, and urine. Can be chosen from. Abnormal methylation changes in cancer tissues show considerable similarity to changes in methylation of genomic DNA obtained from biological samples such as cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine. For predicting the occurrence of colon cancer, rectal cancer or colon adenoma, there is an advantage in that easy diagnosis is possible through blood or body fluids.

As described above, since the CpG site hypermethylation of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 appears specifically in colon cancer, rectal cancer or colon adenoma, according to the present invention By using a composition, kit, chip or method, it is possible to accurately and quickly diagnose colon cancer, rectal cancer or colon adenoma, and in addition, it can be diagnosed early.

1 is a result of confirming the methylation information of the CHL1 gene in a total of 33 cancer types.
2 is a result of confirming the methylation information of the GRIN2A gene in a total of 33 cancer types.
3 is a result of confirming the methylation information of the CLIP4 gene in a total of 33 cancer types.
4 is a result of confirming the methylation information of the KCNQ5 gene in a total of 33 cancer types.
5 is a result of confirming the methylation information of the KRBA1 gene in a total of 33 cancer types.
6 is a result of confirming the methylation information of the ATP8B2 gene in a total of 33 cancer types.
7 is a result of confirming the accuracy of colorectal cancer diagnosis of a total of eight genes selected according to the present invention.
8 is a result of confirming the difference in methylation between the group of tumor tissue (tumor) cell line and the group of non-tumor tissue (others) cell line.
9 is a result of confirming the difference in methylation in tumor tissue (cancer), colorectal adenoma tissue (adenoma), and normal tissue (normal) in colon adenoma.
10 is a comparative example, the result of confirming the methylation information of the OPLAH gene.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited thereto.

Example 1: Colorectal cancer specific methylation gene selection

In order to select methylation genes specifically found in colorectal cancer, a large-scale methylation comparison study of cancerous and normal tissues obtained from cancer surgery in colorectal cancer patients was conducted using data from two large-scale methylation microarray chips. (See Table 2). Tumor tissue used in this study refers to cancerous tissues of colorectal cancer, and non-tumor tissue refers to tissues other than cancerous tissues including normal tissues.

dataset#1 dataset#2 total Tumor 112 pcs 395 pcs 507 pcs Non-tumor
(non-tumor) 149 pieces 45 pcs 194 pcs

In order to select colorectal cancer-specific methylation genes, DNA was extracted from each tissue, and the degree of methylation of the gene site was confirmed using Infinium Human Methylation 450 Beadchip microarray.

DNA extracted from each tissue is transformed through bisulfite treatment. Through this, the cytosine base is modified depending on whether or not the DNA region is methylated. The probe used in the microarray experiment was specifically designed for methylation and unmethylation to check whether the cytosine base at the methylation site of the gene was modified.

The microarray experiment measures the degree of methylation of a gene through approximately 450,000 (450k) probes representing the methylation site of each gene, and the result of each probe derived through the test is presented as a beta value. . The beta value ranges from 0 to 1, and the closer it is to 1, the higher the degree of methylation of the genetic site is determined.

To identify differentially methylated regions (DMRs) between tumor and non-tumor groups, we used the empirical Bayes t-test, Limma (Linear Models for Microarray Data) Gene sites showing significant differences in methylation were identified.

It is known that the Limma method is the least affected by outliers among several statistical analysis methods for methylation that identify differences between groups. Therefore, it is a suitable method for finding cancer-specific markers because it is less affected by abnormal measurements of some samples. In this experiment, it was determined that there was a significant difference in methylation between the two groups as the adjusted p-value derived through the Limma method decreased.

In particular, in order to search for tumor-specific methylation sites, among the gene sites with significant differences in beta values between tumor and non-tumor groups, higher methylation sites in tumor tissue than non-tumor groups were selected as candidates for cancer-specific biomarkers.

As a result, limma analysis in each of the two datasets showed a significantly lower p value (10 ^-30 or less) when compared between tumor groups compared to non-tumor groups, and a large difference of 0.2 or more beta values between groups was tumor-specific hypermethylation of gene sites. It was selected by hypermethyalted regions. Through this, 3,344 genetic sites that showed tumor-specific hypermethylation in all datasets among about 450,000 genetic sites were selected as biomarker candidates.

Example 2: Colorectal cancer specific hypermethylated gene selection

In the 3,344 biomarker genetic sites identified in Example 1, the degree of methylation of each corresponding site in tumors other than colorectal cancer was checked and compared to find a specific genetic site for colorectal cancer or colon adenoma among the biomarkers. As a result of analyzing the DNA methyaltion 450k array experiment results of TCGA (The Cancer Genome Atlas), a public cancer gene database, it was possible to confirm the genetic region methylation information corresponding to 33 cancer types. Among them, compared to colorectal cancer and the remaining 32 types of cancer, as a result of confirming the genetic sites with significantly higher beta values in colorectal cancer, rectal cancer or colon adenoma, 11 of the 3,344 genetic sites were specific for colorectal cancer, rectal cancer, or colon adenoma. It was confirmed that red methylation occurred.

The degree of methylation of the gene through the microarray experiment on tumor tissue (cancer tissue of colorectal cancer) and non-tumor tissue (non-tumor tissue, non-cancer tissue including normal tissue) for the gene is shown in FIG. 1. The same as in FIG. 6. As for the methylation degree, the result of each probe derived through the test was expressed as a beta value, and the beta value ranged from 0 to 1, and the closer to 1, the higher the methylation degree of the genetic site was determined.

On the other hand, in the case of a genetic site in which a methylation difference is observed when comparing tumor tissues of colon cancer, rectal cancer or colon adenoma with non-tumor tissues, methylation may also occur for cancers other than colon cancer, rectal cancer or colon adenoma. That is, colon cancer, rectal cancer or colon adenoma-specific methylation was not confirmed.

For example, in the case of the OPLAH (5-oxoprolinase, ATP-hydrolysing) gene, among the 3,344 genetic sites identified in Example 1, the largest difference in methylation between tumor tissue and non-tumor tissue was confirmed, but in FIG. 10 As you can see, the high methylation value in all types of carcinoma except Acute Myeloid Leukemia, Ocular melanomas, Pheochromocytoma & Paraganglioma, Thymoma, and Thyroid cancer. It was confirmed that it occurred.

The 33 types of cancer are as follows: Acute Myeloid Leukemia, Adrenocortical cancer, Bile Duct cancer, Breast cancer, Cervical Cancer, and colon cancer. (Colon cancer), Endometrioid Cancer, Esophageal Cancer, Glioblastoma, Head and neck cancer, Kidney chromophobe, Kidney Clear cell carcinoma), Kidney Papillary cell carcinoma, Large b-cell lymphoma, Liver cancer, Lower Grade Glioma, Lung adenocarcinoma, Melanoma, Mesothelioma, Ocular melanomas, Ovarian cancer, Pancreatic cancer, Pheochromocytoma&paraganglioma, Prostate cancer, Rectal cancer (Rectal cancer), Sarcoma, Stomach cancer, Testicular cancer, Thymoma, Thyroid cancer, Uterine carcinosarcoma.

Among these genetic sites, it is not a pseudogene, and the site is present in the CpG island site and is between +/- 2000 bases (2kb) from the transcription start site (TSS) of the gene. When present in the selected genetic site and present in an autosome, it was selected as a colorectal cancer-specific hypermethylated gene. As a result, as shown in Table 3 below, a total of 6 genes were selected (see FIGS. 1 to 6).

Symbol Name Location
(Chromosome) CpG Island CHL1 cell adhesion molecule L1 like 3 Island GRIN2A glutamate ionotropic receptor NMDA type subunit 2A 16 Island CLIP4 CAP-Gly domain containing linker protein family member 4 2 Island KCNQ5 potassium voltage-gated channel subfamily Q member 5 6 Island KRBA1 KRAB-A domain containing 1 7 Island ATP8B2 ATPase phospholipid transporting 8B2 One Island

In particular, CHL1 (FIG. 1), CLIP4 (FIG. 3), KRBA1 (FIG. 5), and ATP8B2 (FIG. 6) showed distinct specific hypermethylation for colorectal cancer and rectal cancer compared to other carcinomas.

Example 3: Confirmation of colorectal cancer specificity of a selection gene in a cell line

The methylation patterns in 1,022 cancer cell lines derived from 14 tissues were analyzed using public databases to confirm whether the selected 8 genes exhibited colorectal and rectal cancer-specific methylation distinct from other cancers. Analyzed. The corresponding data was performed in an Infinium Human Methylation 450 Beadchip microarray experiment according to the standardized manufacturer's methylation analysis test procedure for DNA extracted from each cell line.

As for the result of the experiment performed, the degree of gene methylation was measured through about 450,000 probes as in Example 1, and the methylation value of each probe was presented as a beta value. The beta value ranges from 0 to 1, and the closer it is to 1, the higher the degree of methylation of the genetic site is determined.

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

To confirm the specific methylation of colorectal cancer, colorectal cancer or colorectal adenoma of the selected 14 genes, the methylation data derived from 1,022 cell lines were largely in the colorectal cancer cell line group (n=51) and the non colorectal cancer cell line group (n=971). Classified as

To identify differentially methylated regions (DMRs) between the two classified groups, we used the empirical Bayes t-test, Limma (Linear Models for Microarray Data), to be statistically significant between groups. Gene sites showing a difference in methylation were identified.

Differences in methylation of selected genes between colorectal cancer cell line groups and non-colorectal cancer cell line groups Symbol Difference (average △β) adjusted p-value CHL1 0.45 4.76e-27 GRIN2A 0.41 3.31e-12 CLIP4 0.74 2.76e-49 KCNQ5 0.41 2.03e-15 KRBA1 0.54 2.64e-58 ATP8B2 0.54 1.31e-40

In particular, in the case of CHL1, CLIP4, KRBA1, and ATP8B2, which showed distinct specific hypermethylation for colorectal cancer compared to other carcinomas in actual patient samples, even in the analysis using cell lines, it was significantly lower in colorectal and rectal cancer cell lines than other cancer cell lines. It was confirmed to be specific with an adjusted p-value.

Example 4: Evaluation of diagnostic performance of colon cancer, rectal cancer or colon adenoma diagnostic marker candidates

In order to confirm the usefulness of the selected gene as a diagnostic marker in colorectal cancer, the accuracy of colorectal cancer diagnosis according to the degree of methylation was evaluated.

To evaluate the accuracy of diagnosis, sensitivity and specificity are used. The ROC (Receiver Operating Characteristic) curve that shows the change in sensitivity and specificity according to the cut-off value can be displayed by calculating the sensitivity and specificity values for possible cut-off values of continuous diagnostic test measurements. have. The accuracy of diagnosis can be measured by the area under the ROC curve (AUC). The AUC value has a value between 0.5 and 1, and the larger the value is, the higher the diagnostic accuracy is evaluated. If the AUC value is 1, it means that the diagnosis result is completely accurate, but if it is 0.5, it is judged to be the same as the random result.

As a result of analyzing the accuracy of cancer classification according to the degree of methylation between non-tumor tissues and tumor tissues using the selected genes, using the collected methylation data set, as shown in FIG. 7, all the selected genes have an AUC (Area Under Curve) value of 0.920 or higher. It showed high diagnostic accuracy and confirmed that the selected gene was useful for the diagnosis of colorectal cancer.

Example 5: Confirmation of methylation of selected genes in adenoma

Adenoma is a disease in the pre-stage stage of colorectal cancer. Most colorectal cancers arise from adenoma. Therefore, rapid adenoma detection is essential for early diagnosis of colorectal cancer. In order to confirm that the hypermethylated biomarkers selected through the previous study show the characteristics of hypermethylation in adenomas, 64 colorectal cancer tissues, 42 colorectal adenoma tissues, and non-tumor tumor) tissues were examined for the hypermethylation of genes selected from 41 tissues.

As a result of analyzing the methylation data derived through the HumanMethylation450 Beadchip microarray experiment, as shown in FIG. 9, the selected gene showed that the characteristics of non-tumor tissue and significant hypermethylation appeared in the same pattern not only in colorectal cancer but also in colorectal adenoma. I could confirm.

As a result of this, it was found that the selected genes can be used not only for colorectal cancer but also for the diagnosis of colorectal adenoma.

As described above, since the CpG site hypermethylation of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 appears specifically in colon cancer, rectal cancer or colon adenoma, the composition according to the present invention, Using a kit, chip or method, it is possible to diagnose colon cancer, rectal cancer or colon adenoma accurately and quickly, as well as early.

Claims (9)

CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 A composition for diagnosing colon cancer, rectal cancer or colon adenoma, comprising an agent for measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of.
The composition of claim 1, wherein the CpG site is located between +/- 2000 bases (2kb) from the transcription start site of the gene.
The method of claim 1, wherein the agent for measuring the methylation level of the CpG site of the gene is
Compounds that modify unmethylated cytosine or methylated cytosine bases;
Primers specific for the methylated sequence of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2; And
A composition, characterized in that it is selected from the group consisting of primers specific to the unmethylated sequence.
The composition of claim 3, wherein the compound that modifies the unmethylated cytosine base is bisulfite, a salt thereof, and the compound that modifies the methylated cytosine base is a Tet protein.
A kit for diagnosing colon cancer, rectal cancer or colon adenoma comprising a primer pair for amplifying a fragment containing a CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2.
A nucleic acid chip for diagnosing colon cancer, rectal cancer or colon adenoma in which a probe capable of hybridizing with a fragment containing a CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1 and ATP8B2 is immobilized.
Measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of CHL1, GRIN2A, CLIP4, KCNQ5, KRBA1, and ATP8B2 from a sample of a patient suspected of developing colorectal cancer, rectal cancer or colorectal adenoma; And
Comparing the measured methylation level with the methylation level of the CpG site of the same gene of the normal control sample; Containing, a method for providing information for diagnosing colorectal cancer, colorectal cancer or colorectal adenoma.
The method of claim 7, wherein the method of measuring the methylation level is a bisulfite-free detection method, a methylation-specific polymerase chain reaction, and a real-time methylation-specific polymerase reaction. time methylation-specific polymerase chain reaction), PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing.
The method of claim 7, wherein the sample is selected from the group consisting of tissue, cells, blood, plasma, serum, feces, and urine.

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