Composition for diagnosing colorectal cancer using methylation level of cpg site and use thereof

Abstract

The present invention relates to a composition, a kit, and an information providing method for diagnosing colorectal cancer using the methylation levels of CpG sites. By using the composition, the kit, and the information providing method according to the present invention, colorectal cancer can be accurately and quickly diagnosed and can also be diagnosed early.

Description

Composition for diagnosing colon cancer using CpG region methylation level and use thereof

[0001] The present invention relates to a composition for diagnosing colon cancer using CpG region methylation levels and its use. This application claims priority to Republic of Korea Patent Application No. 10-2023-0148441, filed on October 31, 2023, the disclosure of which is incorporated herein by reference.

[0002] The large intestine is the final part of the digestive system, approximately 2 m long, and is divided into the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Colorectal cancer is a cancer that occurs in this part, and most cases are adenocarcinoma. It is broadly divided into colon cancer and rectal cancer by location. Colorectal cancer can occur anywhere in the large intestine or rectum, but the rectum accounts for the largest proportion at approximately 40%, followed by the sigmoid colon, which is located near the rectum, at approximately 30%.

[0003] Diagnosing colon cancer is simple: a fecal occult blood test is performed during a health checkup. However, to actually confirm colon cancer, additional examinations and tests are necessary. Since prognosis varies significantly depending on the stage of the cancer at diagnosis, early detection is crucial for increasing survival rates.

[0004] Accordingly, the inventors of the present invention discovered that a specific gene CpG region is hypermethylated in colon cancer, and developed a composition, kit, and method capable of early diagnosis of colon cancer by detecting the methylation level, thereby completing the present invention.

[0005] One aspect provides a composition for diagnosing colon cancer, comprising an agent for measuring the methylation level of a CpG region of one or more genes selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1.

[0006] Another aspect provides a colon cancer diagnostic kit comprising the composition.

[0007] Another aspect provides an information-providing method for diagnosing colon cancer, comprising the steps of: (a) measuring a CpG region methylation level of a gene in a nucleic acid isolated from a biological sample of an individual; and (b) comparing the measured CpG region methylation level of the gene with a methylation level of a normal control sample, wherein the gene is at least one selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1.

[0008] Another aspect provides a method for providing information for diagnosing colon cancer, comprising, in a computer-based system, (a) obtaining data on the methylation level of a CpG region of a gene from a nucleic acid isolated from a biological sample; (b) inputting the obtained data on the methylation level of a CpG region of a gene into a pre-trained machine learning model; and (c) generating information for diagnosing colon cancer based on the output value of the machine learning model, wherein the gene is at least one selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1.

[0009] Another aspect provides a computer-readable recording medium having a computer program for performing the above method.

[0010]

[0011] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein will be readily apparent and inferred by those skilled in the technical field of this application or similar technical fields, and therefore, their description will be omitted.

[0012] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0013]

[0014] One aspect provides a composition for diagnosing colon cancer, comprising an agent for measuring the methylation level of a CpG region of one or more genes selected from the group consisting of CNRIP1 (Cannabinoid Receptor Interacting Protein 1), FOXL1 (Forkhead Box L1), SERTM1 (Serine Rich And Transmembrane Domain Containing 1), THBD (Thrombomodulin), KCNQ5 (Potassium Voltage-Gated Channel Subfamily Q Member 5), and DBX1 (Developing Brain Homeobox 1).

[0015] The term "methylation" can mean the addition of a methyl group to a base, which alters gene expression patterns, and specifically can occur at cytosine in CpG regions of a base sequence.

[0016] The term "CpG region" is used interchangeably with "CpG site" and refers to a genomic region with a high frequency of CpG dinucleotides. In CpG, C represents cytosine, G represents guanine, and p can refer to the phosphodiester bond between cytosine and guanine. CpG regions are often found in the promoter or 5' exon region of a gene. The promoters of most human genes are located in CpG regions and are unmethylated.

[0017] The term "CpG region methylation" refers to the epigenetic methylation modification of DNA that occurs at the cytosine group of the CpG region. Mammalian DNA contains a base called 5-methylcytosine (5-mC), which has a methyl group attached to the fifth carbon of the cytosine ring. Methylation of 5-methylcytosine occurs only at the cytosine group of CpG, and methylation of the CpG region can inhibit the expression of certain genes by interfering with the binding of transcription factors. Conversely, unmethylation or hypomethylation can increase the expression of certain genes. It also suppresses the expression of transposons and genomic repetitive sequences. DNA methylation is useful for the early diagnosis of cancer because it is detected even in the early stages of cancer development before the appearance of somatic mutations.

[0018] The term "measurement of methylation level" refers to measuring the degree of methylation of a nucleic acid sequence, specifically measuring the level of methylation occurring at cytosine groups in CpG regions.

[0019] In one specific example, the methylation level of the CpG region of the gene may be increased, i.e., hypermethylated, compared to a normal control. For example, the methylation level may include a similar level or an increase of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, and 1000% or more compared to a normal control. The hypermethylation may be specific to colon cancer.

[0020] In one embodiment, the methylation level of the CpG region in the CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1 genes of colorectal cancer patients was quantified, and hypermethylated regions specifically for colorectal cancer were identified through comparison with normal controls, confirming that the CpG region of the genes can be utilized as a biomarker for colorectal cancer diagnosis.

[0021] In one specific example, the CpG region of the CNRIP1 gene may be from 68546746 to 68546851 of chromosome 2.

[0022] In one specific example, the CpG region of the FOXL1 gene may be from 86612749 to 86612884 of chromosome 16.

[0023] In one specific example, the CpG region of the SERTM1 gene may be from 37248276 to 37248390 of chromosome 13.

[0024] In one specific example, the CpG region of the THBD gene may be at least one selected from the group consisting of 23029352 to 23029422 and 23029115 to 23029274 of chromosome 20.

[0025] In one specific example, the CpG region of the KCNQ5 gene may be from 73331077 to 73331174 of chromosome 6.

[0026] In one specific example, the CpG region of the DBX1 gene may be from 20184932 to 20185036 of chromosome 11.

[0027] In the present invention, the base sequence of the human genome chromosome region is expressed according to The February 2009 Human reference sequence (GRCh37). However, the specific sequence of the human genome chromosome region may be somewhat changed in expression as the genome sequence research results are updated, and the expression of the human genome chromosome region of the present invention may be different according to such changes. Therefore, even if the human genome chromosome region expressed according to The February 2009 Human reference sequence (GRCh37) of the present invention is changed from the current expression due to an update of the human reference sequence after the filing date of the present invention, it will be apparent that the scope of the present invention extends to the changed human genome chromosome region. Any person having ordinary skill in the art to which the present invention pertains can easily understand such changes.

[0028] In one embodiment, the formulation may comprise a pair of primers for amplifying a CpG region or a CpG region specific probe.

[0029] The term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group that can form base pairs with a template complementary to a specific base sequence and serves as a starting point for copying the template strand. The term "amplification" refers to increasing the number of copies of a target sequence or its complementary sequence, and specifically increasing the number of copies of a sequence containing a methylated CpG region of said gene.

[0030] The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art. The primer can have 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 50, or 20 to 30 nt.

[0031] The term "probe" refers to a nucleic acid fragment, such as RNA or DNA, that can specifically bind to a nucleic acid, and specifically bind to a CpG region of a target gene.

[0032] The probe can be labeled to confirm the presence or absence of a specific nucleic acid sequence. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art. The probe may have 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 50, or 20 to 30 nt.

[0033] The above primers and probes can be preferably designed according to the sequence of the CpG region for measuring the methylation level, and the primers and probes can be designed to be methylation specific.

[0034] The above primers or probes can be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. In addition, such nucleic acid sequences can be modified using various methods known in the art. Examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, or modification between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0035] Additionally, the probe may be modified with a label that can directly or indirectly provide a detectable signal. Examples of the label may include a radioisotope, a fluorescent substance, or biotin. Examples of the fluorescent substance include, but are not limited to, FAM (6-Carboxyfluorescein), TET (2',7'-dichloro-6-carboxy-4,7-dichlorofluorescein), Texas Red, 6-JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), Cy3 (Cyanine 3), Cy5, rhodamine, or VIC. In addition, the probe may be labeled with a fluorescent substance at the 5' end and a quencher at the 3' end for visualization using the principle of Fluorescent Resonance Energy Transfer (FRET). As the quencher, 6-TAMRA (6-carboxytetramethylrhodamine), BHQ (Black Hole Quencher)-1, BHQ-2, and BHQ-3 may be applied, but this is not limited thereto. In one specific example, the 5' of the probe may be labeled with Cy5 and FAM, and the quenchers BHQ-1 and BHQ-2 may be used to quantify the methylation level of the CpG region.

[0036] The above probe can measure and quantify the methylation level of a CpG region from a PCR amplification product through the above primer pair, and the methylation level of a gene CpG region can be measured with the above primer pair and the above probe.

[0037] The above primer pair can measure the methylation level of the CpG region by amplifying, for example, from 68546746 to 68546851 on chromosome 2 for CNRIP1, from 86612749 to 86612884 on chromosome 16 for FOXL1, from 37248276 to 37248390 on chromosome 13 for SERTM1, from 23029352 to 23029422 or 23029115 to 23029274 on chromosome 20 for THBD, from 73331077 to 73331174 on chromosome 6 for KCNQ5, and from 20184932 to 20185036 on chromosome 11 for DBX1.

[0038] The above probe can specifically bind to, for example, chromosome 2 from 68546773 to 68546798 for CNRIP1, chromosome 16 from 86612781 to 86612805 for FOXL1, chromosome 13 from 37248324 to 37248352 for SERTM1, chromosome 20 from 23029377 to 23029398 or 23029168 to 23029192 for THBD, chromosome 6 from 73331109 to 73331131 for KCNQ5, and chromosome 11 from 20184958 to 20184984 for DBX1, thereby measuring the methylation level of the CpG region. Specific information on the CpG regions measuring the methylation level of the above genes is listed in Tables 3 and 4.

[0039] The polynucleotide of the above primer pair can be defined as a polynucleotide comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 for CNRIP1, SEQ ID NO: 3 and SEQ ID NO: 4 for FOXL1, SEQ ID NO: 5 and SEQ ID NO: 6 for SERTM1, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 15 and SEQ ID NO: 16 for THBD, SEQ ID NO: 11 and SEQ ID NO: 12, or SEQ ID NO: 17 and SEQ ID NO: 18 for KCNQ5, or SEQ ID NO: 13 and SEQ ID NO: 14 for DBX1. The polynucleotide of the above probe can be defined as a polynucleotide comprising the sequence of SEQ ID NO: 19 for CNRIP1, SEQ ID NO: 20 for FOXL1, SEQ ID NO: 21 for SERTM1, SEQ ID NO: 22 or SEQ ID NO: 23 for THBD, SEQ ID NO: 24 for KCNQ5, or SEQ ID NO: 25 for DBX1. Specific information on the primer pairs specific to the CpG region of the gene and the polynucleotides of the probe are listed in Tables 1 and 2.

[0040] The polynucleotide of each of the primer pair and the probe may have a sequence having a sequence identity of 60% or more, for example, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100%, with the polynucleotide sequence defined above.

[0041] Additionally, the preparation may include at least one selected from the group consisting of a compound or a salt thereof that selectively modifies a CpG region to measure the methylation level, a methylation-sensitive restriction enzyme, a methylation-specific binding protein, a methylation-specific binding antibody or aptamer, a methylation-sensitive restriction endonuclease, a sequencing primer, a sequencing-by-synthesis primer, and a sequencing-by-ligation primer.

[0042] Compounds that selectively modify the above CpG region can modify either unmethylated cytosine or methylated cytosine. For example, bisulfite, which modifies unmethylated cytosine, induces a deamination reaction that converts only unmethylated cytosine to uracil, leaving methylated cytosine unaffected. TET proteins (ten-eleven translocation proteins), which modify methylated cytosine, convert only methylated cytosine to uracil, enabling efficient detection.

[0043] The above methylation-sensitive restriction enzyme is a restriction enzyme that can specifically detect methylation of a CpG region and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, etc. Depending on methylation or unmethylation of the cytosine in the above restriction enzyme recognition site, whether or not the restriction enzyme cuts is different, 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.

[0044] In one specific example, it may utilize nucleic acids isolated from blood, plasma or serum.

[0045] The nucleic acid used to detect CpG methylation is, but is not limited to, DNA. Samples containing DNA or RNA, including DNA and mRNA, can be used. The DNA or RNA can be single-stranded or double-stranded, or samples containing DNA-RNA hybrids can be used. Mixtures of nucleic acids can also be used. The nucleic acid sequence to be detected need not be a pure nucleic acid; the nucleic acid can also be a small fraction of a larger molecule, such as a portion of total genomic DNA.

[0046] In one specific example, the isolated nucleic acid may be cfDNA.

[0047] The term "cfDNA (cell-free DNA)" refers to genomic fragments of various lengths present in blood, primarily truncated portions of chromatin not protected by histone proteins. In cancer patients, ctDNA (circulating tumor DNA), DNA fragments derived from tumor cells, are mixed with cfDNA. The size of cfDNA is not limited thereto, but may range from about 80 bp to about 10 kbp, from about 100 bp to about 1 kbp, or from about 120 bp to about 500 bp. Furthermore, cfDNA may range from about 150 bp to about 200 bp.

[0048] In one embodiment, the methylation level of CpG regions of genes in cfDNA was measured based on a liquid sample isolated from an individual, and the methylation markers provided information regarding the diagnosis of colon cancer with excellent sensitivity, specificity, and / or accuracy. Thus, the use of liquid samples enables noninvasive diagnosis, demonstrating excellent diagnostic accuracy.

[0049] In one embodiment, the composition may comprise a plurality of agents for measuring the CpG region methylation level of at least two genes selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1. The composition may comprise, for example, agents for measuring the CpG region methylation level of the CNRIP1 and FOXL1 genes. The composition may comprise, for example, agents for measuring the CpG region methylation level of the genes of CNRIP1, FOXL1, and SERTM1. The composition may comprise, for example, agents for measuring the CpG region methylation level of the genes of CNRIP1, FOXL1, SERTM1, and THBD. The composition may comprise, for example, agents for measuring the CpG region methylation level of the genes of CNRIP1, FOXL1, SERTM1, THBD, and KCNQ5. The composition may include a formulation for measuring the methylation level of the CpG region of the genes CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1. Specific information about the genes in the composition is as listed in Tables 3 and 4.

[0050] In one embodiment, the combination of the aforementioned methylation markers improved the sensitivity and accuracy of colorectal cancer diagnosis compared to single methylation markers. Therefore, combining colorectal cancer methylation markers allows for more accurate and rapid colorectal cancer diagnosis.

[0051]

[0052] Another aspect provides a colon cancer diagnostic kit comprising the composition.

[0053] In the above colon cancer diagnostic kit, the same elements of the terminology mentioned above as those already mentioned are as described above.

[0054] The term "diagnosis" may refer to the identification of the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis may refer to the identification of the presence of colon cancer.

[0055] The above colon cancer diagnostic kit is effective in diagnosing colon cancer by measuring the level of methylation of a gene CpG region including the above composition.

[0056] The kit may further include a primer pair, probe, or antisense oligonucleotide for diagnosing colon cancer, as well as one or more other component compositions, solutions, or devices suitable for the analytical method.

[0057] Another aspect comprises the steps of (a) measuring the level of methylation of a CpG region of a gene in a nucleic acid isolated from a biological sample of an individual; and

[0058] (b) A method for providing information for diagnosing colon cancer, comprising a step of comparing the methylation level of the CpG region of the measured gene with the methylation level of a normal control sample,

[0059] The above gene may be one or more selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1.

[0060] In the method for providing information for the above colon cancer diagnosis, the same elements of the terminology mentioned above as those already mentioned are as described above.

[0061] The term "subject" refers to an individual for whom the presence or absence of colon cancer is to be determined or predicted. The individual may be a vertebrate, specifically a mammal, amphibian, reptile, bird, etc., and more specifically a mammal, such as a human (Homo sapiens).

[0062] In one specific example, the biological sample may be blood, plasma or serum, and the isolated nucleic acid may be cfDNA.

[0063] In one specific embodiment, step (a) may comprise a step of treating an agent for measuring the level of methylation of a CpG region of a gene, wherein the agent may be a pair of primers for amplifying a CpG region or a CpG region-specific probe.

[0064] In one specific embodiment, the step of measuring the methylation level (a) may be performed by a method selected from the group consisting of PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylated DNA specific binding protein, DNA microarray, pyrosequencing, bisulfite sequencing, and next generation sequencing (NGS).

[0065] In one specific example, the step of comparing the methylation levels (b) may be performed by an algorithm selected from the group consisting of random forest, logistic regression analysis, support vector machine, decision tree, association rule mining, neural network, and deep learning.

[0066] In one specific example, if the methylation level of the CpG region of the measured gene is increased, i.e., hypermethylated, compared to the methylation level of a normal control sample, it can be determined that the person is more likely to develop colon cancer or has colon cancer.

[0067] Additionally, it may be possible to determine whether a patient has colorectal cancer by pre-measuring the methylation level of the CpG region of a gene from a normal control sample, setting a threshold, and then comparing that threshold with the methylation level measured from a patient sample. By pre-measuring the methylation level of the CpG region of a gene from a normal control sample and setting a threshold, information for quickly diagnosing whether a patient has colorectal cancer can be provided simply by measuring the methylation level of the CpG region of a gene from a patient sample, without having to measure the methylation level of the normal control group every time for each diagnosis.

[0068] In one embodiment, methylated CpG regions were subjected to subsequent methylation levels using various measurement methods, such as multiplex PCR and real-time PCR-based analysis. Thus, PCR and sequencing can be performed to sufficiently amplify signals even with small amounts of biological samples, providing information on the methylation levels of CpG regions of a gene with excellent sensitivity and / or accuracy for the diagnosis of colorectal cancer.

[0069] Another aspect is that in computer-based systems,

[0070] (a) a step of obtaining data on the methylation level of a CpG region of a gene from a nucleic acid isolated from a biological sample;

[0071] (b) a step of inputting the data of the acquired CpG region methylation level into a pre-trained machine learning model; and

[0072] (c) A method for providing information for diagnosing colon cancer, comprising a step of generating information for diagnosing colon cancer based on the output value of the machine learning model,

[0073] The method of providing information is provided, wherein the above gene is at least one selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1.

[0074] In the above method for providing information for diagnosing colon cancer, the same elements of the terminology mentioned above as those already mentioned are as described above.

[0075] In the step (a), the step of obtaining data on the CpG region methylation level may be performed by a method selected from the group consisting of PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylated DNA specific binding protein, DNA microarray, pyrosequencing, bisulfite sequencing, and next generation sequencing (NGS). The method may be performed by single-end sequencing in which sequencing is performed at one end or paired-end sequencing in which sequencing is performed at both ends.

[0076] The data on the methylation level of the above CpG region refers to data on the methylation level of a specific individual or entity.

[0077] The above step (b) is a step of inputting the data on the CpG region methylation level obtained in the above step (a) into a machine learning model.

[0078] In the present invention, the machine learning model may use any one of various types. Alternatively, the machine learning model may be a combination of multiple models. The machine learning model may be a variety of models, such as random forests, logistic regression analysis, support vector machines, decision trees, association rule mining, neural networks, and deep learning, or a combination thereof. The machine learning model may be configured to compare the input CpG region methylation level with a reference methylation level of a non-cancer patient. The reference methylation level may be selected according to the purpose, and may be, but is not limited to, a reference methylation level derived from a normal person.

[0079] Machine learning models can be trained using hypermethylation rate information obtained by comparing methylation levels at CpG regions. These hypermethylated regions may be specific to colon cancer.

[0080] The above step (c) is a step for generating information for diagnosing colon cancer based on the output value output by the machine learning model based on the methylation level input in the above step (b), and if the methylation level of the CpG region is more hypermethylated than the reference methylation level, the patient can be determined to be a colon cancer patient.

[0081]

[0082] Another aspect provides a computer-readable recording medium having a computer program for performing the above method.

[0083] In the above recording medium, the same elements of the terminology mentioned above as those already mentioned are as mentioned above.

[0084] The above method can be implemented in the form of readable software via various computer means and recorded on a computer-readable recording medium. Here, the recording medium may include program commands, data files, data structures, etc., either singly or in combination. The program commands recorded on the recording medium may be those specifically designed and configured for the above method, or may be known and usable by those skilled in the art of computer software.

[0085] For example, the recording medium includes magnetic media such as hard disks, floppy disks, and magnetic tapes, optical media such as CDROMs (Compact Disk Read Only Memory) and DVDs (Digital Video Disks), magneto-optical media such as floptical disks, and hardware devices specifically configured to store and execute program instructions such as ROMs, RAMs (Random Access Memory), and flash memories. Examples of the program instructions may include not only machine language codes generated by a compiler, but also high-level language codes that can be executed by a computer using an interpreter, etc. These hardware devices may be configured to operate as one or more software modules to perform the operations of the method according to the above, and vice versa.

[0086] The composition, kit, and information providing method according to the present invention include a step of measuring the methylation level of a CpG region of one or more genes selected from the group consisting of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1, thereby enabling accurate and rapid diagnosis of colon cancer.

[0087] Figure 1 is a heatmap showing the difference in methylation levels of cfDNA between colorectal cancer patients (n=42) and normal controls (n=29) by single methylation marker.

[0088] Figure 2 is a graph evaluating the performance of a methylation marker in terms of sensitivity, specificity, and AUC.

[0089] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0090]

[0091] Example 1. Plasma separation and cfDNA extraction

[0092] To screen for methylation markers for colorectal cancer diagnosis, cfDNA was extracted from plasma. Specifically, whole blood from colorectal cancer patients was collected in cfDNA test tubes (Cell-free DNA BCT CE, Streck). Plasma was separated from the whole blood through two centrifugations. After centrifugation at 1,600 rcf for 10 minutes, only the upper layer of plasma was separated, and then again at 3,000 rcf for 20 minutes. Only the upper layer of plasma was collected and stored in a new tube. cfDNA was then extracted using a commercially available cfDNA extraction kit (QIAamp Circulating Nucleic Acid Kit, Qiagen) according to the manufacturer's instructions. cfDNA quality was confirmed using an automated electrophoresis device (4200 Tapestation, Agilent) and a fluorometer (Qubit 4 Fluorometer, Invitrogen).

[0093]

[0094] Example 2. Bisulfite conversion

[0095] To distinguish between methylated and unmethylated cytosines, the method proposed by Frommer et al. was used using the EZ DNA Methylation-Gold Kit (Zymo Research).

[0096] The specific bisulfite conversion process was as follows. A 0.2 mL PCR tube was prepared with a total volume of 40 μl containing 20 ng of cfDNA. 110 μl of CT conversion reagent was added to the 0.2 mL PCR tube containing the sample, mixed, and spun down. The bisulfite conversion reaction was then performed in a PCR machine at 98°C for 10 minutes, 64°C for 2.5 hours, and 4°C for up to 20 hours.

[0097] Next, a desulfonation reaction was performed. Specifically, 600 μl of M-Binding Buffer was added to a Zymo-Spin IC column, 150 μl of bisulfite-treated DNA was added, and the buffer and DNA were mixed well. After centrifugation at 13,000 rpm for 1 minute, the flow-through was removed, the column was placed in the same collection tube, 100 μl of M-Wash Buffer was added again, and centrifugation was performed at 13,000 rpm for 1 minute. The flow-through was removed, 200 μl of M-Desulphonation Buffer was added, and incubation was performed at room temperature for 20 minutes. After centrifugation at 13,000 rpm for 1 minute, the flow-through was removed, 200 μl of M-Wash Buffer was added again, and centrifugation at 13,000 rpm for 1 minute was repeated twice. Finally, 21 ul of M-Elution Buffer was added, left at room temperature for 10 minutes, and centrifuged at 13,000 rpm for 1 minute.

[0098]

[0099] Example 3. Screening of colorectal cancer-specific methylation regions

[0100] 3-1. Setting the marker region

[0101] Methyl-sequencing (next-generation sequencing: NGS) was used to perform base sequence analysis of the extracted cfDNA. Subsequently, only regions with six or more CpGs within the panel designed using NGS data were designated as marker candidate regions.

[0102]

[0103] 3-2. Calculating methylation levels for marker regions

[0104] To identify methylated marker genes, the methylation level of the marker candidate region was calculated. Specifically, only reads containing all CpGs in the marker candidate region of Example 3-1 were selected. The number of reads containing either all CpGs methylated or only one CpG unmethylated was counted and the value was entered into the following equation for calculation:

[0105] Methylation level = Number of methylated reads in the marker region CpG / Number of all reads in the marker region CpG

[0106]

[0107] 3-3. Primers for methylation-specific PCR and selection of colorectal cancer-specific methylation regions

[0108] Using methylation level data from cfDNA of colorectal cancer patients (n=141) and normal controls (n=200), a total of 2,854 marker regions were initially selected through a T test between the two groups for each marker. A set of methylation-specific binding primers (qMSP primers) was designed for the surrounding regions (CpG shores) of less than 200 bp including the CpG regions of the selected marker regions.

[0109] Multiplexing PCR primers and real-time PCR primers were designed for the marker regions selected as primary candidates. Secondary primer pairs were selected based on the expected final PCR product size exceeding 60 bp and a Tm value of 55°C or higher. As a result, a total of 243 primer pairs for marker regions were selected as secondary candidates.

[0110] Finally, the top 32 primer pairs were selected as final candidates based on △Ct obtained in real-time PCR experiments using methylated and unmethylated standard DNA.

[0111]

[0112] Example 4. Analysis of methylation levels in selected colon cancer-specific regions.

[0113] To quantify the methylation levels of colorectal cancer-specific regions for the selected candidates, multiplex PCR and real-time PCR were performed on samples from colorectal cancer patients (n=42) and normal controls (n=29).

[0114] Specifically, multiplex PCR was performed using DNA converted by the bisulfite conversion method of Example 2 as a template in the following steps: pre-denaturation (95°C, 2 minutes), denaturation (95°C, 30 seconds), binding (60°C, 1 minute 30 seconds), extension (72°C, 1 minute), and final extension (72°C, 10 minutes), and the denaturation-binding-extension steps were performed for 25 cycles.

[0115] To perform real-time PCR, probes were designed to specifically bind to the sequence of the above region, and the 5' of the probe was labeled with fluorescent substances FAM (6-Carboxyfluorescein) and Cy5 (Sulfo-Cyanine5), and quenchers BHQ (Black Hole Quencher)-1 and BHQ-2. The multiplex PCR product was diluted to 1 / 2 and used as a template for real-time PCR reaction. Real-time PCR was performed in the steps of pre-denaturation (95℃, 15 min), denaturation (95℃, 20 sec), and binding (60℃, 40 sec), and the denaturation-binding step was performed 40 cycles. The cycle value (Ct) when the fluorescence color development increased rapidly for each probe through real-time PCR for the region was obtained, and it was quantified using the amount of PCR product of the B2M gene. This is because the B2M primer pair recognizes a site where a CpG region does not exist and amplifies a site where the sequence does not change even with bisulfite treatment.

[0116] Figure 1 is a heatmap showing the difference in methylation levels of cfDNA between colon cancer patients (n=42) and normal controls (n=29) by single marker.

[0117] As a result, it was confirmed that the methylation level was increased specifically in colon cancer in seven markers.

[0118] Table 1 shows the primer sequences for multiplex PCR for seven markers, and Table 2 shows the primer and probe sequences for real-time PCR. Table 3 shows the chromosomal locations (0-based) and number of CpGs for the primer sequences for multiplex PCR, and Table 4 shows the chromosomal locations (0-based) and number of CpGs for the primer and probe sequences for real-time PCR.

[0119] [Table 1]

[0120]

[0121] [Table 2]

[0122]

[0123] [Table 3]

[0124]

[0125] [Table 4]

[0126]

[0127]

[0128] Example 5. Establishment of a colon cancer diagnostic model and evaluation of combined marker performance.

[0129] A model was developed to classify colon cancer patients and controls based on the cycle values ​​(Ct) of the selected candidates. The classification model used logistic regression analysis.

[0130] Specifically, a standard curve was drawn using gDNA from colon cancer cell lines with methylation levels of 100% and 0% as reference materials. The methylation level was quantified by inputting the sample results from colon cancer patients (n=42) and normal controls (n=52). After that, logistic regression analysis of machine learning was utilized to create 100 models with randomly configured training and test sets (0.5:0.5), and the predicted probability value of each model was output. The logistic regression model was created by setting the Elastic-Net penalty to a Lasso ratio of 0.5 to prevent overfitting. The predicted probability value was obtained as the average of these probability values, and the area under the curve (AUC), sensitivity, and specificity of the diagnostic model were calculated to measure the performance. The Receiver Operating Characteristic (ROC) curve, which calculates sensitivity and specificity values ​​for a given cutoff value, and the area under the curve (AUC) are presented. "Sensitivity" refers to the likelihood of correctly identifying cancer patients as having cancer, while "specificity" refers to the likelihood of correctly identifying non-cancer patients as not having cancer.

[0131] The classification model performance when seven markers were sequentially combined from a single marker is shown in Table 5 and Figure 2. Figure 2 is a graph evaluating the performance of one aspect of methylation markers.

[0132] [Table 5]

[0133]

[0134] As a result, according to Table 5, it was found that when markers were combined, a higher AUC value was observed with higher sensitivity than when methylation markers were used alone. In particular, when four or more methylation markers were combined, an AUC value of 0.9 or higher was observed.

[0135] In summary, the above results show that all six methylation markers, including CNRIP1, can be used specifically for the diagnosis of colon cancer, and their combination is useful for excellent colon cancer diagnosis.

[0136]

[0137] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for diagnosing colon cancer, comprising an agent for measuring the methylation level of a CpG region of one or more genes selected from the group consisting of CNRIP1 (Cannabinoid Receptor Interacting Protein 1), FOXL1 (Forkhead Box L1), SERTM1 (Serine Rich And Transmembrane Domain Containing 1), THBD (Thrombomodulin), KCNQ5 (Potassium Voltage-Gated Channel Subfamily Q Member 5), and DBX1 (Developing Brain Homeobox 1).

2. In claim 1, the CpG region is at least one selected from the group consisting of CNRIP1 gene from 68546746 to 68546851 of chromosome 2, FOXL1 gene from 86612749 to 86612884 of chromosome 16, SERTM1 gene from 37248276 to 37248390 of chromosome 13, THBD gene from 23029352 to 23029422 and 23029115 to 23029274 of chromosome 20, KCNQ5 gene from 73331077 to 73331174 of chromosome 6, and DBX1 gene from 20184932 to 20185036 of chromosome 11. Composition for diagnosing colon cancer.

3. A composition for diagnosing colon cancer, comprising a preparation for measuring the methylation level of the CpG region of the CNRIP1 and FOXL1 genes in claim 1.

4. A composition for diagnosing colon cancer, comprising a preparation for measuring the methylation level of the CpG region of the CNRIP1, FOXL1, and SERTM1 genes in claim 1.

5. A composition for diagnosing colon cancer, comprising an agent for measuring the methylation level of the CpG region of the CNRIP1, FOXL1, SERTM1, and THBD genes in claim 1.

6. A composition for diagnosing colon cancer, comprising an agent for measuring the methylation level of CpG regions of CNRIP1, FOXL1, SERTM1, THBD, and KCNQ5 genes in claim 1.

7. A composition for diagnosing colon cancer, comprising an agent for measuring the methylation level of CpG regions of CNRIP1, FOXL1, SERTM1, THBD, KCNQ5, and DBX1 genes in claim 1.

8. A composition for diagnosing colon cancer, wherein the preparation according to claim 1 is a primer pair for amplifying a CpG region or a CpG region-specific probe.

9. A composition for diagnosing colon cancer, which utilizes nucleic acid isolated from blood, plasma or serum according to claim 1.

10. A composition for diagnosing colon cancer, wherein the isolated nucleic acid according to claim 9 is cfDNA.

11. A colon cancer diagnostic kit comprising the composition of claim 1. 12.(a) a step of measuring the methylation level of the CpG region of a gene in a nucleic acid isolated from a biological sample of an individual; and (b) A method for providing information for diagnosing colon cancer, comprising a step of comparing the methylation level of the CpG region of the measured gene with the methylation level of a normal control sample, A method for providing information, wherein the above gene is at least one selected from the group consisting of CNRIP1 (Cannabinoid Receptor Interacting Protein 1), FOXL1 (Forkhead Box L1), SERTM1 (Serine Rich And Transmembrane Domain Containing 1), THBD (Thrombomodulin), KCNQ5 (Potassium Voltage-Gated Channel Subfamily Q Member 5), and DBX1 (Developing Brain Homeobox 1).

13. A method for providing information according to claim 12, wherein step (a) comprises a step of processing a preparation for measuring the methylation level of a CpG region of a gene.

14. A method for providing information according to claim 13, wherein the preparation is a primer pair for amplifying a CpG region or a CpG region-specific probe.

15. In a computer-based system, (a) a step of obtaining data on the methylation level of a CpG region of a gene from a nucleic acid isolated from a biological sample; (b) a step of inputting the data of the acquired CpG region methylation level into a pre-trained machine learning model; and (c) A method for providing information for diagnosing colon cancer, comprising a step of generating information for diagnosing colon cancer based on the output value of the machine learning model, A method for providing information, wherein the above gene is at least one selected from the group consisting of CNRIP1 (Cannabinoid Receptor Interacting Protein 1), FOXL1 (Forkhead Box L1), SERTM1 (Serine Rich And Transmembrane Domain Containing 1), THBD (Thrombomodulin), KCNQ5 (Potassium Voltage-Gated Channel Subfamily Q Member 5), and DBX1 (Developing Brain Homeobox 1).

16. A computer-readable recording medium containing a computer program for performing the method of claim 15.

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