WO2024080731A1 - Methylation marker genes for pancreatic cancer diagnosis and use thereof - Google Patents
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Definitions
- the present invention relates to methylation marker genes and uses thereof for diagnosing pancreatic cancer. More specifically, the present invention relates to a methylation marker gene that enables early diagnosis of pancreatic cancer using biological samples, such as blood samples, and its use.
- Pancreatic cancer is the second most common cancer among digestive tract carcinomas in Korea, following stomach cancer, liver cancer, and colon cancer, and ranks 8th in overall cancer incidence and 5th among causes of cancer death.
- the 5-year relative survival rate of pancreatic cancer patients is 12.2%, ranking last among the top 10 cancer types. This is because the pancreas is surrounded by several organs, making it difficult to detect cancer, and because there are no special symptoms in the early stages, most cases are discovered in an advanced state and are in a terminal state where curative surgery cannot be performed. Therefore, early detection of pancreatic cancer is very important to improve survival rate.
- DNA methylation is an epigenetic modification that plays a central role in regulating gene expression.
- DNA is transformed into 5-methylcytosine by attaching a methyl group (-CH3) to carbon 5 of cytosine.
- DNA methylation mainly occurs at the cytosine group of CpG dinucleotides and occurs frequently in DNA regions where CpGs are concentrated, called CpG islands.
- DNA methylation is recognized as playing an important role in the carcinogenesis of various solid cancers, including pancreatic cancer. It is known that during the course of cancer, DNA hypomethylation occurs throughout the genome, and local hypermethylation occurs frequently in some regions of CpG islands.
- DNA methylation shows tissue-specific patterns, and for this reason, it is generally accepted that DNA methylation patterns are clinically meaningful information in the diagnosis of various cancers. For example, in cancer cells, if the CpG region of the promoter region of a tumor suppressor gene is abnormally hypermethylated, the expression of this gene may be suppressed, causing cancer.
- NGS Next-Generation Sequencing
- a biomarker that has been widely used in pancreatic cancer screening is the serum CA19-9 indicator, a cancer-specific antigen.
- 10% of the population does not produce CA19-9 protein, so its level cannot be measured, and this protein can mostly be screened for late-stage pancreatic cancer.
- CA19-9 protein has an elevated index value in many other diseases and has low specificity, so it is not recommended to use this value itself as a screening test for pancreatic cancer or as a basis for recurrence. Therefore, there is a need to discover biomarkers that can diagnose pancreatic cancer at an early stage.
- the purpose of the present invention is to solve the problems of the prior art described above.
- Another object of the present invention is to provide a methylation marker gene for diagnosing pancreatic cancer.
- Another object of the present invention is to provide a method of providing information for diagnosing pancreatic cancer or a method of diagnosing pancreatic cancer.
- Another object of the present invention is to provide a composition or kit for diagnosing pancreatic cancer.
- Another object of the present invention is to provide a gene chip or gene panel for diagnosing pancreatic cancer.
- Another object of the present invention is to provide a method for detecting methylation markers in individuals suspected of having pancreatic cancer or at risk of developing pancreatic cancer.
- a representative configuration of the present invention to achieve the above object is as follows.
- MIRLET7BHG MIRLET7B Host Gene
- XXYLT1 Xyloside Xylosyltransferase 1
- ZNF879 Zinc Finger Protein 879
- EPS8L2 EPS8 Like 2
- TRPC6 Transient Receptor Potential Cation Channel Subfamily C Member 6
- genes or CpGs thereof selected from the group consisting of AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) genes A new marker gene for pancreatic cancer diagnosis containing a region is provided.
- MIRLET7BHG MIRLET7B Host Gene
- XXYLT1 Xyloside Xylosyltransferase 1
- ZNF879 Zinc Finger Protein 879
- EPS8L2 EPS8 Like 2
- TRPC6 Transient Receptor Potential Cation Channel Subfamily C Member 6
- AVPR1A Argonal Vasopressin Receptor 1A
- GSC Goosecoid Homeobox
- TBCD Tubulin Folding Cofactor D
- MAFB MAF BZIP Transcription Factor B
- TCEA2 Transcription Elongation Factor A2 genes.
- a method of providing information for diagnosing pancreatic cancer is provided, which includes measuring the methylation status of one or more genes or CpG regions thereof selected from the group consisting of.
- one or more genes may include MIRLET7BHG.
- the one or more genes may further include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1.
- the one or more genes may further include one or more genes selected from the group consisting of EPS8L2 and BNC1.
- the one or more genes may further include TRPC6, AVPR1A, or TBCD.
- the step of measuring the methylation status includes measuring the methylation status of a marker gene or a CpG region thereof of a combination of (i), (ii), (iii), (iv), (v), or (vi) below. It may include: (i) BNC1, TRPC6 and MIRLET7BHG; (ii) BNC1, AVPR1A, and MIRLET7BHG; (iii) BNC1 and MIRLET7BHG; (iv) BNC1, MIRLET7BHG, and EPS8L2; (v) MIRLET7BHG and EPS8L2; or (vi) MIRLET7BHG, EPS8L2, and TBCD.
- the one or more genes may include one or more genes selected from the group consisting of TBCD and EPS8L2.
- the one or more genes may further comprise BNC1.
- the biological sample can be blood, plasma, or serum.
- the nucleic acid isolated from a biological sample may be cfDNA.
- measuring the methylation status includes i) methylation of the gene or CpG region thereof selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, salts thereof, and combinations thereof. It may include the step of treating the gene with an agent that detects the condition and ii) a primer that specifically amplifies the one or more genes.
- the step of measuring methylation status includes methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, methylation DNA-specific polymerase chain reaction. It can be performed by PCR using a binding protein, quantitative PCR, pyrosequencing, or bisulfite sequencing.
- determining the methylation status may further include comparing a control methylation profile generated from the corresponding methylation measurement results of the corresponding marker in the control sample.
- control methylation profile constitutes a model constructed by machine learning the methylation patterns of one or more genes obtained above, and may be used to identify pancreatic cancer patient-specific methylation patterns that are different from normal controls.
- machine learning may be performed by one or more algorithms selected from the group consisting of random forests, logistic regression, support vector machines, decision trees, association rule mining, neural networks, and deep learning.
- control sample may be a sample from a normal individual.
- the pancreatic cancer may be early pancreatic cancer, metastatic pancreatic cancer, or recurrent or refractory pancreatic cancer.
- MIRLET7BHG MIRLET7B Host Gene
- XXYLT1 Xyloside One or more marker genes or their CpG region selected from the group consisting of Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2)
- a composition for diagnosing pancreatic cancer using nucleic acids isolated from a biological sample containing one or more agents selected from a specifically amplifying agent and an agent for detecting the methylation status of the one or more marker genes or CpG regions thereof is provided.
- the one or more marker genes may include MIRLET7BHG.
- the one or more marker genes may further include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1.
- the one or more marker genes may further include one or more genes selected from the group consisting of EPS8L2 and BNC1.
- the one or more marker genes may further include TRPC6, TBCD, or AVPR1A.
- the composition comprises an agent that amplifies a marker gene or a CpG region thereof of a combination of (i), (ii), (iii), (iv), (v) or (vi) and the marker gene or CpG region thereof.
- It may include one or more agents selected from agents that detect the methylation status of a region: (i) BNC1, TRPC6, and MIRLET7BHG; (ii) BNC1, AVPR1A, and MIRLET7BHG; (iii) BNC1 and MIRLET7BHG; (iv) BNC1, MIRLET7BHG, and EPS8L2; (v) MIRLET7BHG and EPS8L2; or (vi) MIRLET7BHG, EPS8L2, and TBCD.
- agents selected from agents that detect the methylation status of a region: (i) BNC1, TRPC6, and MIRLET7BHG; (ii) BNC1, AVPR1A, and MIRLET7BHG; (iii) BNC1 and MIRLET7BHG; (iv) BNC1, MIRLET7BHG, and EPS8L2; (v) MIRLET7BHG and EPS8L2; or (vi) MIRLET7BHG,
- the one or more marker genes may include one or more genes selected from the group consisting of TBCD and EPS8L2.
- the one or more marker genes may further include BNC1.
- the CpG region of a gene may have the following characteristics:
- the amplifying agent may include a primer, probe, or antisense nucleotide that binds complementary to the gene.
- the composition may include an agent that specifically amplifies one or more marker genes or CpG regions thereof and an agent that detects the methylation status of the one or more marker genes or CpG regions thereof.
- the agent that measures methylation status can be a bisulfite, hydrogen sulfite, disulfite, or a combination thereof.
- kits for diagnosing pancreatic cancer using nucleic acids isolated from a biological sample of an individual including a composition for diagnosing pancreatic cancer.
- the kit may include a diagnostic nucleic acid chip on which a probe capable of hybridizing with a gene to be detected including the one or more marker genes or a CpG region thereof or a fragment including a CpG region thereof is immobilized. there is.
- pancreatic cancer there is a method for treating pancreatic cancer, wherein (i) the above-described methylation marker gene or its CpG region in nucleic acid isolated from a biological sample of an individual is hypomethylated or hypermethylated compared to the normal control group. detecting; and (ii) administering therapy for pancreatic cancer in said individual.
- a method for detecting methylation markers in an individual suspected of having or at risk of developing pancreatic cancer. Specifically, the method includes measuring the methylation status of one or more methylation marker genes in a biological sample obtained from the individual, wherein the one or more methylation marker genes include MIRLET7B Host Gene (MIRLET7BHG) and Xyloside Xylosyltransferase 1 (XXYLT1).
- MIRLET7B Host Gene MIRLET7BHG
- XXYLT1 Xyloside Xylosyltransferase 1
- ZNF879 Zinc Finger Protein 879
- EPS8L2 EPS8 Like 2
- TRPC6 Transient Receptor Potential Cation Channel Subfamily C Member 6
- AVPR1A Arginine Vasopressin Receptor 1A
- GSC Goosecoid Homeobox
- TBCD Tubulin Folding Cofactor D
- MAFB MAF BZIP Transcription Factor B
- TCEA2 Transcription Elongation Factor A2
- a new methylation marker gene whose methylation level was differentially changed in a group of pancreatic cancer patients was discovered.
- pancreatic cancer can be diagnosed with excellent sensitivity, specificity and/or accuracy.
- the methylation marker gene is useful in that it enables early diagnosis of pancreatic cancer using blood-based biological samples.
- Figure 1 is a diagram showing a process for establishing a pancreatic cancer identification model according to an embodiment of the present invention.
- Figure 2 is a diagram showing a color chart showing the difference in methylation levels between pancreatic cancer patient group (33 cases) and normal control (42 cases) samples in the DMR of a new methylation marker gene.
- Figure 3 shows the sensitivity values obtained by performing a leave-one-out test on samples of the patient group (33 cases) and the normal control group (42 cases) based on the combination of MIRLET7BHG and BNC1 according to an embodiment of the present invention, by cancer stage. This is a drawing showing the organized results.
- pancreatic cancer As a result of research to secure blood-based methylation marker genes for diagnosing pancreatic cancer, the present inventors discovered new genes or gene combinations whose methylation levels were differentially changed in pancreatic cancer patient groups, and when using these marker genes or combinations of marker genes The present invention was completed by confirming that pancreatic cancer can be diagnosed with excellent sensitivity, specificity, and/or accuracy.
- MIRLET7BHG MIRLET7B Host Gene
- XXYLT1 Xyloside Xylosyltransferase 1
- ZNF879 Zinc Finger Protein 879
- EPS8L2 EPS8 Like 2
- TRPC6 Transient Receptor Potential Cation Channel Subfamily C Member 6
- genes or CpGs thereof selected from the group consisting of AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) genes A marker gene for pancreatic cancer diagnosis containing a region is provided.
- the marker gene for diagnosing pancreatic cancer may include each of the above genes or their CpG regions alone, or may include a combination of two or more genes or their CpG regions selected from the above genes. Additionally, the marker gene for diagnosing pancreatic cancer can be used in combination with other marker genes known in the art for diagnosing pancreatic cancer, such as BNC1.
- the gene MIRLET7BHG is an RNA belonging to the lncRNA class, and diseases related to it include brachydactyly type B2 disease.
- the protein encoded by the gene XXYLT1 is an alpha-1,3-xylosyltransferase enzyme that extends O-linked xylose-glucose disaccharides attached to EGF-like repeats in the extracellular domain of target proteins.
- the protein encoded by gene ZNF879 is a transcriptional repressor containing an N-terminal kruppel-associated box (KRAB) domain and 13 C-terminal C2H2-type zinc finger domains.
- KRAB N-terminal kruppel-associated box
- EPS8L2 encodes a protein related to epidermal growth factor receptor pathway substrate 8 (EPS8), a substrate for the epidermal growth factor receptor, and the EPS8L2 protein is known to function involved in actin cytoskeleton remodeling.
- EPS8L2 epidermal growth factor receptor pathway substrate 8
- TRPC6 acts as a transient receptor potential channel of the TRPC subfamily, and diseases associated with it include depression, anxiety, and focal segmental glomerulosclerosis.
- the protein encoded by the gene AVPR1A acts as a receptor for arginine vasopressin. This receptor mediates cell contraction and proliferation, platelet aggregation, release of clotting factors, and glycogenolysis.
- the gene GSC encodes a member of the bicoid subfamily of the paired homeobox protein family, and the GSC protein acts as a transcription factor and can be self-regulated.
- the gene TBCD is responsible for capturing and stabilizing intermediates of beta tubulin, and diseases associated with TBCD include encephalopathy and seborrheic dermatitis.
- the gene MAFB is a basic leucine zipper (bZIP) transcription factor that plays an important role in the regulation of lineage-specific hematopoiesis, and the encoded nuclear protein represses ETS1-mediated transcription of erythroid-specific genes in myeloid cells.
- bZIP basic leucine zipper
- the protein encoded by the gene TCEA2 functions as a SII class transcription elongation factor in the nucleus and interacts with the basic transcription factor, general transcription factor IIB.
- the protein encoded by the gene BNC1 is a zinc finger protein present in the basal cell layer of the epidermis and hair follicles, is abundantly expressed in germ cells, and is known to play a role in regulating the proliferation of keratinocytes.
- nucleotide sequences of the genes can be found in the database provided by the National Center for Biotechnology Information (NCBI) maintained by the National Institutes of Health, the UniProt Knowledge Base (UniProtKB) and the Swiss-Prot database provided by the Swiss Bioinformatics Institute. It can be obtained from various DB engines, including , and can be appropriately selected by those skilled in the art.
- NCBI National Center for Biotechnology Information
- UniProtKB UniProt Knowledge Base
- Swiss-Prot database provided by the Swiss Bioinformatics Institute. It can be obtained from various DB engines, including , and can be appropriately selected by those skilled in the art.
- the marker gene for diagnosing pancreatic cancer may include one or more genes selected from the group consisting of MIRLET7BHG, EPS8L2, TRPC6, AVPR1A, and TBCD, or a CpG region thereof.
- a marker gene for diagnosing pancreatic cancer may include each of the above genes or their CpG regions alone, or may include a combination of two or more marker genes or their CpG regions selected from the above genes.
- the marker gene for diagnosing pancreatic cancer can be used in combination with other marker genes known in the art for diagnosing pancreatic cancer, such as a pancreatic cancer diagnostic marker gene such as BNC1.
- the marker gene for diagnosing pancreatic cancer may be or include MIRLET7BHG or its CpG region.
- the marker gene for pancreatic cancer diagnosis may be or include EPS8L2 or its CpG region.
- the marker gene for diagnosing pancreatic cancer may be or include TRPC6 or its CpG region.
- the marker gene for diagnosing pancreatic cancer may be or include AVPR1A or its CpG region.
- the marker gene for diagnosing pancreatic cancer may be or include TBCD or its CpG region.
- the marker gene for diagnosing pancreatic cancer includes MIRLET7BHG or a CpG region thereof, and may include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1, or a CpG region thereof.
- the marker gene for diagnosing pancreatic cancer includes MIRLET7BHG or a CpG region thereof, and may include one or more genes selected from the group consisting of EPS8L2 and BNC1 or a CpG region thereof.
- the marker gene for diagnosing pancreatic cancer may further include TRPC6 or its CpG region, TBCD or its CpG region, or AVPR1A or its CpG region.
- the marker gene for diagnosing pancreatic cancer may be or include a combination of MIRLET7BHG or a CpG region thereof and EPS8L2 or a CpG region thereof.
- the marker gene for diagnosing pancreatic cancer may be or include a combination of MIRLET7BHG or a CpG region thereof and TRPC6 or a CpG region thereof.
- the marker gene for diagnosing pancreatic cancer may be or include a combination of MIRLET7BHG or a CpG region thereof and AVPR1A or a CpG region thereof.
- the marker gene for pancreatic cancer diagnosis may be or include a combination of MIRLET7BHG or a CpG region thereof, EPS8L2 or a CpG region thereof, and TBCD or a CpG region thereof.
- a marker gene for diagnosing pancreatic cancer can be used for diagnosing pancreatic cancer in combination with BNC1 or its CpG region.
- the marker gene for diagnosing pancreatic cancer may include the following combination of marker genes or CpG regions thereof:
- the marker gene for diagnosing pancreatic cancer may include TBCD or a CpG region thereof, EPS8L2 or a CpG region thereof, or both. These marker genes can be used in combination with other marker genes known in the art for diagnosing pancreatic cancer, such as BNC1.
- the marker gene for diagnosing pancreatic cancer may include the following combination of marker genes or CpG regions thereof:
- the methylation marker gene can diagnose pancreatic cancer with excellent sensitivity, specificity, and/or accuracy based on a biological sample isolated from an individual, such as a liquid sample, or provide information regarding the diagnosis of pancreatic cancer.
- the methylation marker gene enables non-invasive diagnosis in that it shows excellent diagnostic accuracy when using liquid samples such as blood, plasma, and serum.
- methylation refers to the attachment of a methyl group to the bases that make up DNA.
- the methylation status may mean whether or not methylation occurs at a cytosine in a specific CpG region of a specific gene or the degree of methylation. If methylation occurs, it may interfere with the binding of transcription factors and suppress the expression of the gene in question. Expression suppression may occur relative to the degree of methylation. Conversely, when unmethylation or hypomethylation occurs, the expression of certain genes may increase. For this reason, DNA methylation is an epigenetic modification that plays a central role in regulating gene expression. A methyl group (-CH3) is attached to the 5th carbon of cytosine, transforming it into 5-methylcytosine. DNA methylation mainly occurs at the cytosine group of CpG dinucleotides and occurs in DNA regions where CpGs are concentrated, called CpG islands or CpG regions.
- CpG region refers to a region of the DNA of a gene or a portion thereof where CpGs are concentrated, called a CpG island.
- the CpG region may exist in a transcriptional control region such as a promoter region, a protein coding region (open reading frame, ORF), or a terminator region. It is known that during the course of cancer, DNA hypomethylation occurs throughout the genome, and local hypermethylation occurs frequently in some regions of CpG islands. For example, in cancer cells, if the CpG region of the promoter region of a cancer suppressor gene is abnormally hypermethylated, the expression of this gene may be suppressed, causing cancer. Methylation of CpG sites occurs early in cancer development, so it is useful for early diagnosis of cancer.
- the CpG region of the gene may be a continuous sequence of 200 bp or more in which C and G are linked by phosphate, and may have any size ranging from 1 kb to 9 kb.
- the CpG region of the gene may be located between +/- 2000 bases (2 kb) from the transcription start site (TSS) of the gene.
- the CpG region of the gene may be the CpG region of the gene listed in Table 1 below or the methylation state of a portion of the cytosine contained therein. In Table 1 below, the CpG region is indicated as DMR (Differentially Methylated Region).
- the base sequence of the human genome chromosome region is expressed according to The February 2009 Human reference sequence (GRCh37), but the specific sequence of the human genome chromosome region may change somewhat as the results of genome sequence research are updated. , depending on these changes, the expression of the human genomic chromosomal region of the present invention may be different. Accordingly, the human genome chromosomal region expressed according to The February 2009 Human reference sequence (GRCh37) of the present invention has been updated as a human reference sequence after the filing date of the present invention, so that the expression of the human genomic chromosomal region is the same as now. Even if it is changed differently, it will be obvious that the scope of the present invention extends to the changed human genome chromosomal region. These changes can be easily known by anyone with ordinary knowledge in the technical field to which the present invention pertains.
- a method for providing information for diagnosing pancreatic cancer or a method for diagnosing pancreatic cancer comprising measuring the methylation status of the above-described marker gene or its CpG region in nucleic acid isolated from a biological sample of an individual. do.
- a method for detecting a methylation marker in an individual suspected of having or at risk of developing pancreatic cancer comprising determining the methylation status of the marker gene or CpG region thereof in a sample obtained from the individual. do.
- subject refers to mammals in need of a diagnosis of pancreatic cancer, such as primates (e.g. humans), companion animals (e.g. dogs, cats, etc.), domestic animals (e.g. cattle, pigs, horses, sheep, etc.) goats, etc.) and laboratory animals (e.g. rats, mice, guinea pigs, etc.).
- primates e.g. humans
- companion animals e.g. dogs, cats, etc.
- domestic animals e.g. cattle, pigs, horses, sheep, etc.
- laboratory animals e.g. rats, mice, guinea pigs, etc.
- the individual is a human.
- the biological sample may be, but is not limited to, blood, plasma, or serum.
- the nucleic acid isolated from the biological sample may be cfDNA.
- cfDNA refers to cell free DNA among genomic DNA.
- Methods for separating or isolating genomic DNA or fragments thereof from the biological sample include phenol/chloroform extraction and SDS extraction (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990) commonly used in the art. ), CTAB separation method (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980), or a commercially available DNA extraction kit.
- the biological sample may be subjected to a process of destruction and dissolution by enzymatic, chemical, or mechanical means.
- Proteins and other contaminants are then removed from the DNA solution, for example by digestion with proteinase K, and genomic DNA is recovered from the solution.
- Purification of DNA can be performed by a variety of methods, including salting out, organic extraction, or binding of DNA to a solid phase support. DNA isolation and purification methods can be selected by those skilled in the art taking into account several factors including time, cost, and required amount of DNA.
- sample DNA when the sample DNA is not surrounded by a membrane (e.g., circulating or free DNA from a blood sample), standard methods in the art for isolation and/or purification of DNA can be used.
- the method includes protein denaturing reagents, such as chaotropic salts such as guanidine hydrochloride or urea; or the use of detergents such as sodium dodecyl sulfate (SDS), cyanogen bromide.
- chaotropic salts such as guanidine hydrochloride or urea
- detergents such as sodium dodecyl sulfate (SDS), cyanogen bromide.
- Alternative methods include, but are not limited to, ethanol precipitation or propanol precipitation, especially vacuum concentration by centrifugation.
- filter devices e.g., ultrafiltration, silica surfaces or membranes, magnetic particles, polystyrol particles, polystyrol surfaces, positively charged surfaces and positively charged membranes, charged A charged film, a charged surface, a charged conversion film, or a charged conversion surface can be used.
- DNA separated or isolated from the biological sample can be used as a method to measure the methylation status of the marker gene described above.
- the step of measuring the methylation status comprises i) an agent that detects the methylation status of one or more genes or CpG regions thereof described above and/or ii) an agent that specifically detects the methylation state of one or more genes or CpG regions thereof described above. It may include the step of treating the DNA with a primer to amplify.
- the agent that detects (or measures) the methylation status may be a compound that modifies a cytosine base or a methylation-sensitive restriction enzyme.
- the compound that modifies the cytosine base may be a compound that modifies unmethylated cytosine or methylated cytosine. Specifically, it may be any one selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, salts thereof, and combinations thereof. Additionally, it may be a TET protein that modifies methylated cytosine, but is not limited thereto. Methods for detecting methylation of a CpG region by modifying cytosine bases are well known in the art.
- the methylation-sensitive restriction enzyme is a restriction enzyme that can specifically detect methylation of the CpG region and may be a restriction enzyme that contains CG as a recognition site.
- a restriction enzyme that contains CG as a recognition site.
- SmaI, SacII, EagI, HpaII, MspI, BssII, BstUI, NotI, etc. but are not limited thereto.
- cleavage by restriction enzymes varies and 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 primers may include primers specific to the methylated allele sequence and/or primers specific to the unmethylated allele sequence of the one or more aforementioned marker genes.
- the step of measuring the methylation status includes methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, methylation It may be performed by, but is not limited to, PCR using a DNA-specific binding protein, quantitative PCR, pyrosequencing, or bisulfite sequencing. Other examples of PCR or sequencing listed above include MethyLight PCR, MehtyLight digital PCR, EpiTYPER, CpG island microarray, etc. Additionally, it can be measured using a detection method using TET protein (ten-eleven translocation protein).
- TET protein ten-eleven translocation protein.
- the TET protein is an enzyme that acts on DNA and is involved in the chemical change of bases. When treated with bisulfite, all Cs except methylated C are changed to T bases, but in TET protein, only methylated C is changed to T, making it efficient. Detection is possible.
- the method may further include comparing the measured methylation level with the methylation level of the same marker gene or CpG region thereof in a control sample.
- the step of measuring the methylation status may further include comparing the methylation profile with a control methylation profile generated from the corresponding methylation measurement result of the corresponding marker gene in the control sample.
- control methylation profile constitutes a model constructed by machine learning the methylation patterns of one or more genes obtained, and may be used to identify pancreatic cancer patient-specific methylation patterns that are different from normal controls.
- the machine learning may be performed by one or more algorithms selected from the group consisting of random forest, logistic regression, support vector machine, decision tree, association rule mining, neural network, and deep learning.
- control sample may be a sample from a normal individual. Additionally, the control sample may be a sample from a pancreatic cancer patient, and the pancreatic cancer patient may be a patient with various pancreatic cancer stages.
- the pancreatic cancer may be early pancreatic cancer, metastatic pancreatic cancer, or recurrent or refractory pancreatic cancer. At this time, each pancreatic cancer may have various stages.
- the method for diagnosing pancreatic cancer or providing information for diagnosing pancreatic cancer according to the present invention includes (i) isolating nucleic acid from a biological sample of an individual, (ii) from the isolated nucleic acid, bisulfite, Processing any one selected from the group consisting of hydrogen sulfite, disulfite, salts thereof, and combinations thereof, (iii) amplifying the nucleic acid using primers specific for the marker gene described above. , and (iv) the marker genes described above by methylation-specific polymerase chain reaction, real-time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, or bisulfite sequencing. It may be implemented including measuring the methylation level of one or more CpG regions.
- pancreatic cancer there is a method of treating pancreatic cancer, wherein (i) the above-described marker gene or its CpG region in the nucleic acid isolated from the biological sample of the individual is in a hypomethylated or hypermethylated state compared to the normal control group. detecting; and (ii) administering therapy for pancreatic cancer in said individual.
- the method of treatment includes confirming that the subject has pancreatic cancer through additional analysis or diagnostic methods, such as tissue biopsy, before step (ii) when a hypomethylated state or a hypermethylated state is detected. Additional items may be included.
- the treatment for pancreatic cancer may include surgery, radiation therapy, chemotherapy, targeted therapy, or a combination thereof, depending on the stage.
- each marker gene such as each marker gene, “biological sample,” “CpG region,” “methylation,” and method for detecting methylation status, please refer to the above.
- composition for diagnosing pancreatic cancer Composition for diagnosing pancreatic cancer
- a composition comprising an agent for detecting or measuring the methylation status of one or more genes selected from the group consisting of the above-described marker genes or CpG regions thereof.
- it is a composition for diagnosing pancreatic cancer using nucleic acids isolated from biological samples, and is an agent capable of specifically amplifying or hybridizing one or more genes or their CpG regions selected from the group consisting of the above-mentioned marker genes, and
- a composition comprising one or more agents selected from agents that detect the methylation status of the one or more marker genes or CpG regions thereof is provided.
- hybridization can be understood as the binding of an oligonucleotide to a complementary sequence similar to Watson-Crick base pairs in sample DNA, forming a duplex structure.
- the biological sample may be, but is not limited to, blood, plasma, or serum.
- the nucleic acid isolated from the biological sample may be cfDNA.
- the agent may include one or more oligonucleotides, such as primers, probes, or antisense nucleotides, that bind complementary to the gene.
- primer used in the present invention refers to a nucleic acid sequence that can form a base pair with a complementary template of a gene in a sample and serves as a starting point for copying the template strand.
- the sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but just needs to be sufficiently complementary to hybridize with the template.
- Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization at appropriate buffer solutions and temperatures. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.
- probe used in the present invention refers to a substance that can specifically bind to a gene to be detected in a sample, and can specifically confirm the presence of the gene in the sample through this binding.
- Probes may be manufactured in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
- antisense nucleotide used in the present invention refers to a nucleic acid-based molecule that has a complementary sequence to the target gene and can form a dimer with the target gene, and can be used to detect the target gene.
- the antisense nucleotide may be of an appropriate length to increase detection specificity.
- oligonucleotides such as primers, probes or antisense nucleotides can be preferably designed using known knowledge and techniques known in the art according to the sequence of a specific CpG region whose methylation status is to be analyzed.
- the composition may include an agent for detecting the methylation status of the CpG region of the gene.
- agent for detecting the methylation status please refer to the previous description.
- the agent may be bisulfite, hydrogen sulfite, disulfite, a salt thereof, or a combination thereof.
- the agent is not limited thereto.
- Compounds that modify unmethylated cytosine bases, such as bisulfite, hydrogen sulfite, and disulfite, and methods for detecting methylation of genes by modifying unmethylated cytosine residues using the same are well known in the art. (WO 01/26536; US 2003/0148326 A1).
- NGS can be used to determine the presence and extent of methylation by confirming the degree of conversion of the base sequence of the target gene region. Sequence analysis by NGS was performed using Illumina's MiSeq, NextSeq500, NextSeq550, Hiseq 2500, Hiseq 4000, Hiseq It can be performed using MGISEQ-T7, etc., but is not limited to this.
- compositions for diagnosing pancreatic cancer using nucleic acids isolated from biological samples including an agent for detecting the methylation status of one or more genes selected from the group consisting of the above-described marker genes or a CpG region thereof. .
- kits for diagnosing pancreatic cancer using nucleic acid isolated from a biological sample of an individual including the composition is provided.
- a kit containing an agent for detecting or measuring the methylation status of one or more genes selected from the group consisting of the above-mentioned marker genes or their CpG region is provided.
- the kit includes (i) one or more oligonucleotides, such as primers, that can hybridize under stringent or moderately stringent conditions to one or more of the pancreatic cancer-specific genes described above that are methylated in cancer but not methylated in non-cancerous tissue; Probe or antisense nucleotide; (ii) a container suitable for containing one or more oligonucleotides, such as primers, probes or antisense nucleotides, capable of hybridizing to the gene and a biological sample of interest (e.g., a nucleic acid isolated from the biological sample); (iii) means for detecting hybridization of (ii); and/or optionally (iv) instructions for use and interpretation of kit results.
- one or more oligonucleotides such as primers, that can hybridize under stringent or moderately stringent conditions to one or more of the pancreatic cancer-specific genes described above that are methylated in cancer but not methylated in non-cancerous tissue
- the kit may also contain other components, including hybridization solution, packaged in separate containers.
- hybridization solution the nucleic acid of the gene and a plurality of primers, probes, or antisense nucleotides may hybridize.
- the kit consists of one or more different component compositions, solutions, or devices suitable for the analysis method.
- the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, or a rapid kit.
- RT-PCR reverse transcription polymerase chain reaction
- ELISA enzyme-linked immunosorbent assay
- the kit may additionally include polymerase agarose, a buffer solution required for electrophoresis, etc.
- the kit is a diagnostic nucleic acid chip (CHIP) on which a probe capable of hybridizing with a target gene to be detected including the one or more marker genes or a CpG region thereof or a fragment including a CpG region thereof is immobilized, or May include a genetic panel.
- CHIP diagnostic nucleic acid chip
- a diagnostic nucleic acid chip or gene on which a probe capable of hybridizing with a detection target gene containing the above-described one or more marker genes or a CpG region thereof or a fragment containing a CpG region thereof is immobilized is provided.
- the nucleic acid chip may be characterized in that an array of oligonucleotides and/or PNA (Peptide Nucleic Acid)-oligomers are bound to a solid phase and arranged on a solid phase, for example, in the form of a rectangular or hexagonal lattice.
- the solid surface may be made of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold. Additionally, plastics such as nitrocellulose and nylon, which may exist in the form of pellets or also as a resin matrix, may also be used.
- the oligonucleotide can be interpreted as a concept that includes all primers, probes, or antisense nucleotides, and the “PNA” refers to an artificially synthesized oligonucleotide.
- Fluorescently labeled probes can also be used for scanning immobilized DNA chips, and the simple binding of Cy3 and Cy5 dyes to the 5'-OH of specific probes is particularly suitable for fluorescent labeling. Fluorescence detection of hybridized probes can be performed, for example, by confocal microscopy.
- pancreatic cancer was classified from stage 0 to stage 4 by gastroenterologists according to the TNM staging system and the American Joint Committee on Cancer (AJCC) staging system.
- AJCC American Joint Committee on Cancer
- each sample was centrifuged at 3000 rpm for 15 minutes to separate plasma.
- Extraction of cell-free DNA from separated plasma was performed using the MagMAXTM Cell-Free DNA Isolation Kit (Thermo fisher scientific). The extraction method was performed according to the manufacturer's manual.
- Extracted cell-free DNA was quantified using a fluorescence intensity meter (Qubit Flex, Invitrogen), and DNA status was checked for DNA degradation and size using automated electrophoresis equipment (4150 Tapestation system, Agilent). .
- Example 2 The cell-free DNA extracted in Example 2 was treated with bisulfite using the EZ DNA methylation-LightningTM kit (Zymo Research, USA).
- Accel-NGS® Methyl-Seq DNA Library Kit (Swift Biosciences, USA) was used as a preparation reagent for library production. This process includes methylation adapter ligation, indexing of the library, and pure isolation of ligated DNA using AMPure XP beads.
- the bisulfite-library was designed as a single mixing reaction using the fabricated panel, and the library was completed using the SureSelectXT Human Methyl Seq Kit (Agilent, USA). This process includes library hybridization, hybrid capture using streptavidin beads, library amplification, and pure separation using AMPure XP beads.
- the length and amount of the library were measured using an Agilent Bioanalyzer 2200 (Agilent, USA) instrument and a high sensitivity chip, and the quality control (QC) conditions suggested by the manufacturer were measured. Satisfactory libraries were used for sequence analysis.
- Bisulfite sequencing was performed on the library produced in Example 3 using next-generation sequencing equipment (NovaSeq 6000, Illumina). Quantity of bases produced from sequencing equipment, quality score (Q30), on-target ratio, average number of reads required for analysis by target region, Unique Molecular Identifier (UMI) duplication ratio, mapping The quality of the generated data was evaluated by checking the amount of data generated. Afterwards, a total of 3.7 million CpG sites out of 84 million bases of cell-free DNA were targeted and their methylation levels were analyzed.
- next-generation sequencing equipment NovaSeq 6000, Illumina. Quantity of bases produced from sequencing equipment, quality score (Q30), on-target ratio, average number of reads required for analysis by target region, Unique Molecular Identifier (UMI) duplication ratio, mapping
- UMI Unique Molecular Identifier
- the degree of DNA methylation was measured for a total of 3.7 million CpG loci, which were the target regions captured in Example 4.
- the calculated degree of methylation is expressed as a value ranging from 0 to 1, where a value of 0 means that the corresponding CpG locus is not completely methylated, and a value of 1 means that the corresponding CpG locus is completely methylated.
- DMRs differentially methylated regions
- the evaluation was conducted on a gene and DMR basis, and was conducted as a leave-one-out test using several machine learning algorithms.
- the machine learning algorithms used were Support Vector Machine, Random Forest, Neural Network, and Deep Learning.
- the 11 genes whose methylation levels were differentially changed in the pancreatic cancer patient group and the DMRs contained in each gene were selected (Table 2).
- the 11 genes consisted of existing genes (BNC1) and novel genes (XXXLT1, ZNF879, EPS8L2, TRPC6, AVPR1A, GSC, TBCD, MAFB, TCEA2, and MIRLET7BHG).
- Methylation levels in the DMRs of the above 11 genes using samples from 33 pancreatic cancer patients and 42 normal controls were analyzed through target region-based bisulfite sequencing, and the results are shown in Figure 2.
- a pancreatic cancer identification model was established using a machine learning algorithm based on the methylation level in the DMR region present in the marker gene discovered in Example 5.
- a typical modeling program is created through explicit programming in which the developer pre-programs it to output the final result when certain conditions are met for the initial input values.
- machine learning trains the computer to find conditions under which the result will be a specific value when the initial input value is received. This learning process is a process of finding optimal parameters so that the result value can be properly derived for the input value, and the result of learning is the optimal parameter or weight value.
- a model established using machine learning algorithms such as deep learning or neural networks can identify pancreatic cancer patient-specific methylation patterns that are different from normal controls by learning the methylation patterns of marker gene regions obtained from multiple normal control and pancreatic cancer patient samples. It can be said to be a mathematical model created for this purpose. Using the model created in this way, it is possible to infer and predict new patterns of data. Accordingly, the established model evaluates whether a given test sample shows a methylation pattern that appears in a pancreatic cancer patient sample, and derives a determination result as to whether the subject from which the test sample is derived is normal or has pancreatic cancer.
- Deep learning algorithms are a type of machine learning that performs learning using multiple layers of artificial neural networks.
- data analysts must be directly involved in determining which features should be extracted from the training data, while in the case of deep learning, the computer automatically extracts features from the training data and performs self-learning.
- Table 3 The results of these deep learning tests are shown in Table 3.
- Table 3 shows the sensitivity, specificity, and accuracy values calculated for the model established based on 2 to 3 of the 11 marker genes discovered in the present invention, as well as the corresponding values calculated when these genes are applied as single markers. The figures are presented together.
- the sensitivity values obtained by performing a leave-one-out test using samples from 33 patients and 42 normal controls for the combination model of the methylation marker genes BNC1 and MIRLET7BHG are summarized by pancreatic cancer stage in Figure 3. Shown. As can be seen from Figure 3, the model showed a sensitivity of 50% for stage 1 and 100% for stages 2 to 4.
- Non-patent Document 1 Eissa, Maryam AL, et al. Clinical epigenetics 11.1 (2019): 1-10; and Non-patent Document 2: Yi, Joo Mi, et al. al.
- non-patent document 1 measured diagnostic markers for pancreatic cancer by performing quantitative methylation specific PCR (Quantitative Methylation Specific PCR). Specifically, in the case of non-patent document 1, a TaqMan probe was used, and in the case of non-patent document 2, SYBR was used.
- Non-patent Document 1 Non-patent Document 2 genetic combination BNC1 ADAMTS1 BNC1+ ADAMTS1 BNC1 ADAMTS1 BNC1+ ADAMTS1 responsiveness(%) 64.1% 87.2% 97.3% 79.0% 48.0% 81.0% Specificity (%) 93.7% 95.8% 91.6% 89.0% 92.0% 85.0%
- pancreatic cancer markers and their combinations identified in Examples 1 to 6 of the present invention were proven to be superior in terms of sensitivity, specificity, and accuracy (Table 3).
- the combination of marker genes including BNC1, an existing marker gene, and MIRLET7BHG, a new marker was confirmed as an excellent marker in stages 2 to 4 pancreatic cancer, and also showed effective results in stage 1 pancreatic cancer (Figure 3), making it suitable for early diagnosis of pancreatic cancer. It can be useful.
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Abstract
The present invention relates to a methylated marker gene for the diagnosis of pancreatic cancer and a use thereof. As an alternative to the challenging early diagnosis of pancreatic cancer, the present invention aims to enhance the sensitivity, specificity, and accuracy of diagnosis by introducing a methylation analysis method using circulating free DNA (cfDNA) in plasma. Identified according to the present invention have been a methylated marker gene of which the methylation level is differentially changed in the patient group with pancreatic cancer. Thus, the methylation marker gene of the present invention can be advantageously used in the diagnosis of pancreatic cancer.
Description
본 출원은 2022년 10월 11일자로 출원된 한국특허출원 제2022-0130106호에 대한 우선권을 주장하며, 이의 개시 내용은 전체적으로 본 명세서에 참고로 포함된다.This application claims priority to Korean Patent Application No. 2022-0130106, filed on October 11, 2022, the disclosure of which is incorporated herein by reference in its entirety.
본 발명은 췌장암 진단을 위한 메틸화 마커 유전자 및 이의 용도에 관한 것이다. 보다 구체적으로, 본 발명은 생물학적 시료, 예컨대 혈액 시료를 이용하여 췌장암의 조기 진단이 가능한 메틸화 마커 유전자 및 이의 용도에 관한 것이다.The present invention relates to methylation marker genes and uses thereof for diagnosing pancreatic cancer. More specifically, the present invention relates to a methylation marker gene that enables early diagnosis of pancreatic cancer using biological samples, such as blood samples, and its use.
췌장암은 국내 소화기 암종 중 위암, 간암, 대장암 다음으로 흔한 암으로, 전체 암 발병률 8위, 암 사망 원인 중 5위를 차지하는 질환이다. 2020년 한국 중앙 암 등록사업 연례보고서에 따르면 췌장암 발생자의 5년 상대 생존율은 12.2% 로 10대 암종 중 최하위를 차지하고 있다. 이는 췌장이 여러 장기로 둘러싸여 암의 발견이 어렵다는 점과 초기에 특별한 증상이 없어 대부분 진행된 상태로 발견되어 근치적 수술을 할 수 없는 말기 상태인 경우가 많기 때문이다. 따라서 췌장암을 조기에 발견하는 것은 생존율 개선을 위해 매우 중요하다고 할 수 있다. Pancreatic cancer is the second most common cancer among digestive tract carcinomas in Korea, following stomach cancer, liver cancer, and colon cancer, and ranks 8th in overall cancer incidence and 5th among causes of cancer death. According to the 2020 Korea Central Cancer Registration Project annual report, the 5-year relative survival rate of pancreatic cancer patients is 12.2%, ranking last among the top 10 cancer types. This is because the pancreas is surrounded by several organs, making it difficult to detect cancer, and because there are no special symptoms in the early stages, most cases are discovered in an advanced state and are in a terminal state where curative surgery cannot be performed. Therefore, early detection of pancreatic cancer is very important to improve survival rate.
최근, 암 조기 진단을 위해, 침습적인 진단 및 검사 방법의 대안으로 액체 생검을 이용한 분자진단법이 활발히 연구되고 있으며, 유전자의 발현 조절 기전을 연구하는 후성유전학 분야의 기술 발전으로 혈장 내 유리 DNA(cfDNA)에서 메틸화 정도를 측정하는 방법이 가능해졌다.Recently, for the early diagnosis of cancer, molecular diagnostic methods using liquid biopsies have been actively researched as an alternative to invasive diagnostic and testing methods, and due to technological advancements in the field of epigenetics, which studies gene expression regulation mechanisms, free DNA (cfDNA) in plasma ), a method for measuring the degree of methylation has become possible.
DNA 메틸화는 유전자 발현 조절에 중추적인 역할을 하는 후성유전적 변형으로서, 사이토신(cytosine)의 5번 탄소에 메틸기(-CH3)가 붙어 5-메틸사이토신 형태로 변형되는 것이다. DNA 메틸화는 주로 CpG 디뉴클레오티드의 사이토신 기에서 발생하며, CpG 섬(island)이라고 불리는 CpG 가 집중적으로 몰려있는 DNA 영역에서 많이 나타난다. 특히, DNA 메틸화는 췌장암을 비롯한 각종 고형암의 발암 과정에서 중요한 역할을 하는 것으로 인식되고 있다. 암이 진행되는 과정 동안 게놈 전반적으로는 DNA 저메틸화가 나타나고, CpG 섬의 일부 영역에서 국소적인 과메틸화가 빈번하게 일어나는 것으로 알려져 있다. 또한, DNA 메틸화는 조직 특이적인 패턴을 보이는 것이 확인되었는데, 이러한 이유로 DNA 메틸화 패턴은 각종 암의 진단에 있어 임상적으로 의미 있는 정보라는 사실이 일반적으로 받아들여 지고 있다. 예를 들어, 암세포에서는 암 억제 유전자(tumor suppressor gene)의 프로모터 영역의 CpG 부위가 비정상적으로 과메틸화(hypermethylation)되면 이 유전자의 발현이 억제되어 암을 일으킬 수 있다.DNA methylation is an epigenetic modification that plays a central role in regulating gene expression. DNA is transformed into 5-methylcytosine by attaching a methyl group (-CH3) to carbon 5 of cytosine. DNA methylation mainly occurs at the cytosine group of CpG dinucleotides and occurs frequently in DNA regions where CpGs are concentrated, called CpG islands. In particular, DNA methylation is recognized as playing an important role in the carcinogenesis of various solid cancers, including pancreatic cancer. It is known that during the course of cancer, DNA hypomethylation occurs throughout the genome, and local hypermethylation occurs frequently in some regions of CpG islands. In addition, it has been confirmed that DNA methylation shows tissue-specific patterns, and for this reason, it is generally accepted that DNA methylation patterns are clinically meaningful information in the diagnosis of various cancers. For example, in cancer cells, if the CpG region of the promoter region of a tumor suppressor gene is abnormally hypermethylated, the expression of this gene may be suppressed, causing cancer.
차세대 염기서열 분석(Next-Generation Sequencing, NGS)이라 불리는 기술은 긴 서열을 짧은 단편으로 잘라서 대규모로 병렬 서열분석을 행함으로써 뛰어난 처리량, 확장성 및 속도를 바탕으로 이전에는 불가능했던 수준의 다양한 연구를 가능하게 만들었다. NGS 기술은 현재 게놈 전체 프로파일링을 통해 단일 뉴클레오티드 혹은 단일 세포 수준에서의 메틸화 마커의 식별을 가능하게 한다. 이는 PCR 기반의 종래 검사 방법에 비해 높은 해상도, 정량화 성능 및 마커의 확장성(여러 마커를 유연하게 적용하는 것이 가능함)을 제공함으로써, 질환 관련 마커 발굴, 진단 예후 및 치료에 대한 반응 예측, 환자 맞춤형 치료의 새로운 타겟 발굴을 위한 후성유전학 프로파일링에 널리 이용되고 있다.A technology called Next-Generation Sequencing (NGS) cuts long sequences into short fragments and performs large-scale parallel sequencing, enabling diverse research at a level that was previously impossible based on excellent throughput, scalability, and speed. made it possible NGS technology now enables the identification of methylation markers at the single nucleotide or single cell level through genome-wide profiling. Compared to conventional PCR-based testing methods, this provides higher resolution, quantification performance, and marker scalability (multiple markers can be applied flexibly), enabling discovery of disease-related markers, prediction of diagnostic prognosis and response to treatment, and personalized patient care. It is widely used in epigenetic profiling to discover new targets for treatment.
현재 췌장암 검사에 많이 사용해 온 바이오 마커로는 암 특이적인 항원인 혈청 CA19-9 지표가 있다. 하지만 인구의 10%는 CA19-9 단백질 생성이 되지 않아 수치측정이 불가능하며, 상기 단백질은 대부분 췌장암 말기에서 선별이 가능하다. 또한 CA19-9 단백질은 여러 다른 질환에서도 지표 값이 상승하여 특이도가 낮아 이 값 자체를 췌장암 선별 검사나 재발의 근거로 사용하는 것은 권장되지 않는다. 따라서, 췌장암을 조기에 진단할 수 있는 바이오마커의 발굴이 필요한 실정이다. Currently, a biomarker that has been widely used in pancreatic cancer screening is the serum CA19-9 indicator, a cancer-specific antigen. However, 10% of the population does not produce CA19-9 protein, so its level cannot be measured, and this protein can mostly be screened for late-stage pancreatic cancer. In addition, CA19-9 protein has an elevated index value in many other diseases and has low specificity, so it is not recommended to use this value itself as a screening test for pancreatic cancer or as a basis for recurrence. Therefore, there is a need to discover biomarkers that can diagnose pancreatic cancer at an early stage.
본 발명은 상술한 종래 기술의 문제점을 해결하는 것을 그 목적으로 한다. The purpose of the present invention is to solve the problems of the prior art described above.
본 발명은 췌장암 진단을 위한 메틸화 마커 유전자를 제공하는 것을 다른 목적으로 한다.Another object of the present invention is to provide a methylation marker gene for diagnosing pancreatic cancer.
본 발명은 췌장암 진단을 위한 정보를 제공하는 방법 또는 췌장암 진단 방법을 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide a method of providing information for diagnosing pancreatic cancer or a method of diagnosing pancreatic cancer.
본 발명은 췌장암 진단을 위한 조성물 또는 키트를 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide a composition or kit for diagnosing pancreatic cancer.
본 발명은 췌장암 진단을 위한 유전자 칩 또는 유전자 패널을 제공하는 것을 또 다른 목적으로 한다. Another object of the present invention is to provide a gene chip or gene panel for diagnosing pancreatic cancer.
본 발명은 췌장암을 가진 것으로 의심되거나 췌장암 발병 위험이 있는 개체에서 메틸화 마커를 검출하는 방법을 제공하는 것을 또 다른 목적으로 한다. Another object of the present invention is to provide a method for detecting methylation markers in individuals suspected of having pancreatic cancer or at risk of developing pancreatic cancer.
본 발명의 목적은 이상에서 언급한 목적으로 제한되지 않는다. 본 발명의 목적은 이하의 설명으로 보다 분명해질 것이며, 특허청구범위에 기재된 수단 및 그 조합으로 실현될 것이다.The object of the present invention is not limited to the objects mentioned above. The object of the present invention will become clearer from the following description and may be realized by means and combinations thereof as set forth in the claims.
상기 목적을 달성하기 위한 본 발명의 대표적인 구성은 다음과 같다.A representative configuration of the present invention to achieve the above object is as follows.
본 발명의 일 태양에 따르면, MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2) 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함하는 췌장암 진단을 위한 신규 마커 유전자가 제공된다. According to one aspect of the present invention, MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside Xylosyltransferase 1), ZNF879 (Zinc Finger Protein 879), EPS8L2 (EPS8 Like 2), TRPC6 (Transient Receptor Potential Cation Channel Subfamily C Member 6), One or more genes or CpGs thereof selected from the group consisting of AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) genes A new marker gene for pancreatic cancer diagnosis containing a region is provided.
본 발명의 다른 태양에 따르면, 개체의 생물학적 시료로부터 분리된 핵산에서, MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2) 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역의 메틸화 상태를 측정하는 단계를 포함하는 췌장암 진단을 위한 정보를 제공하는 방법이 제공된다.According to another aspect of the present invention, in nucleic acids isolated from a biological sample of an individual, MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside Xylosyltransferase 1), ZNF879 (Zinc Finger Protein 879), EPS8L2 (EPS8 Like 2), TRPC6 (Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) genes. A method of providing information for diagnosing pancreatic cancer is provided, which includes measuring the methylation status of one or more genes or CpG regions thereof selected from the group consisting of.
일 구현예에서, 하나 이상의 유전자는 MIRLET7BHG를 포함할 수 있다.In one embodiment, one or more genes may include MIRLET7BHG.
다른 구현예에서, 하나 이상의 유전자는 TRPC6, AVPR1A, EPS8L2, TBCD 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함할 수 있다.In another embodiment, the one or more genes may further include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1.
또 다른 구현예에서, 하나 이상의 유전자는 EPS8L2 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함할 수 있다.In another embodiment, the one or more genes may further include one or more genes selected from the group consisting of EPS8L2 and BNC1.
또 다른 구현예에서, 하나 이상의 유전자는 TRPC6, AVPR1A 또는 TBCD를 추가로 포함할 수 있다.In another embodiment, the one or more genes may further include TRPC6, AVPR1A, or TBCD.
일 구현예에서, 메틸화 상태를 측정하는 단계는 하기 (i), (ii), (iii), (iv), (v) 또는 (vi) 조합의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 측정하는 것을 포함할 수 있다: (i) BNC1, TRPC6 및 MIRLET7BHG; (ii) BNC1, AVPR1A 및 MIRLET7BHG; (iii) BNC1 및 MIRLET7BHG; (iv) BNC1, MIRLET7BHG 및 EPS8L2; (v) MIRLET7BHG 및 EPS8L2; 또는 (vi) MIRLET7BHG, EPS8L2 및 TBCD.In one embodiment, the step of measuring the methylation status includes measuring the methylation status of a marker gene or a CpG region thereof of a combination of (i), (ii), (iii), (iv), (v), or (vi) below. It may include: (i) BNC1, TRPC6 and MIRLET7BHG; (ii) BNC1, AVPR1A, and MIRLET7BHG; (iii) BNC1 and MIRLET7BHG; (iv) BNC1, MIRLET7BHG, and EPS8L2; (v) MIRLET7BHG and EPS8L2; or (vi) MIRLET7BHG, EPS8L2, and TBCD.
일 구현예에서, 하나 이상의 유전자는 TBCD 및 EPS8L2로 이루어진 군에서 선택된 하나 이상의 유전자를 포함할 수 있다.In one embodiment, the one or more genes may include one or more genes selected from the group consisting of TBCD and EPS8L2.
다른 구현예에서, 하나 이상의 유전자는 BNC1을 추가로 포함할 수 있다.In another embodiment, the one or more genes may further comprise BNC1.
일부 구현예에서, 생물학적 시료는 혈액, 혈장 또는 혈청일 수 있다.In some embodiments, the biological sample can be blood, plasma, or serum.
다른 구현예에서, 생물학적 시료로부터 분리된 핵산은 cfDNA일 수 있다.In another embodiment, the nucleic acid isolated from a biological sample may be cfDNA.
일부 구현예에서, 메틸화 상태를 측정하는 단계는, i) 바이설파이트, 하이드로젠 설파이트, 다이설파이트, 이들의 염, 및 이들의 조합으로 이루어진 군에서 선택되는 상기 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제 및 ii) 상기 하나 이상의 유전자를 특이적으로 증폭하는 프라이머로 상기 유전자를 처리하는 단계를 포함할 수 있다.In some embodiments, measuring the methylation status includes i) methylation of the gene or CpG region thereof selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, salts thereof, and combinations thereof. It may include the step of treating the gene with an agent that detects the condition and ii) a primer that specifically amplifies the one or more genes.
일부 구현예에서, 메틸화 상태를 측정하는 단계는 메틸화 특이적 중합효소 연쇄 반응(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 중합효소 연쇄 반응(real time methylation-specific polymerase chain reaction), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, 파이로시퀀싱, 또는 바이설파이트 시퀀싱에 의해 수행될 수 있다.In some embodiments, the step of measuring methylation status includes methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, methylation DNA-specific polymerase chain reaction. It can be performed by PCR using a binding protein, quantitative PCR, pyrosequencing, or bisulfite sequencing.
일부 구현예에서, 메틸화 상태를 측정하는 단계는 대조군 시료의 해당 마커의 상응하는 메틸화 측정 결과로부터 생성된 대조 메틸화 프로파일과 비교하는 단계를 추가로 포함할 수 있다.In some embodiments, determining the methylation status may further include comparing a control methylation profile generated from the corresponding methylation measurement results of the corresponding marker in the control sample.
다른 구현예에서, 대조 메틸화 프로파일은 상기 획득된 하나 이상의 유전자의 메틸화 패턴을 기계학습시킴으로써 구축된 모델을 구성하며, 정상 대조군과 상이한 췌장암 환자군 특이적 메틸화 패턴을 식별하기 위한 것일 수 있다.In another embodiment, the control methylation profile constitutes a model constructed by machine learning the methylation patterns of one or more genes obtained above, and may be used to identify pancreatic cancer patient-specific methylation patterns that are different from normal controls.
또 다른 구현예에서, 기계학습은 랜덤 포레스트, 로지스틱 회귀 분석, 서포트 벡터 머신, 의사결정나무, 연관성 규칙 마이닝, 신경망 네트워크 및 딥러닝으로 이루어진 군에서 선택되는 하나 이상의 알고리즘에 의해 수행될 수 있다.In another implementation, machine learning may be performed by one or more algorithms selected from the group consisting of random forests, logistic regression, support vector machines, decision trees, association rule mining, neural networks, and deep learning.
일 구현예에서, 대조군 시료는 정상 개체의 시료일 수 있다.In one embodiment, the control sample may be a sample from a normal individual.
일부 구현예에서, 췌장암은 조기 췌장암, 전이성 췌장암, 또는 재발성 또는 난치성 췌장암일 수 있다.In some embodiments, the pancreatic cancer may be early pancreatic cancer, metastatic pancreatic cancer, or recurrent or refractory pancreatic cancer.
또 다른 태양에 따르면, MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2)로 이루어진 군에서 선택된 하나 이상의 마커 유전자 또는 이의 CpG 영역을 특이적으로 증폭하는 제제 및 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제로부터 선택된 하나 이상의 제제를 포함하는 생물학적 시료에서 분리된 핵산을 이용하는 췌장암 진단을 위한 조성물이 제공된다.According to another embodiment, MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside One or more marker genes or their CpG region selected from the group consisting of Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) A composition for diagnosing pancreatic cancer using nucleic acids isolated from a biological sample containing one or more agents selected from a specifically amplifying agent and an agent for detecting the methylation status of the one or more marker genes or CpG regions thereof is provided.
일 구현예에서, 하나 이상의 마커 유전자는 MIRLET7BHG를 포함할 수 있다.In one embodiment, the one or more marker genes may include MIRLET7BHG.
다른 구현예에서, 하나 이상의 마커 유전자는 TRPC6, AVPR1A, EPS8L2, TBCD 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함할 수 있다.In another embodiment, the one or more marker genes may further include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1.
또 다른 구현예에서, 하나 이상의 마커 유전자는 EPS8L2 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함할 수 있다.In another embodiment, the one or more marker genes may further include one or more genes selected from the group consisting of EPS8L2 and BNC1.
또 다른 구현예에서, 하나 이상의 마커 유전자는 TRPC6, TBCD 또는 AVPR1A을 추가로 포함할 수 있다.In another embodiment, the one or more marker genes may further include TRPC6, TBCD, or AVPR1A.
일 구현예에서, 조성물은 하기 (i), (ii), (iii), (iv), (v) 또는 (vi) 조합의 마커 유전자 또는 이의 CpG 영역을 증폭하는 제제 및 상기 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제로부터 선택된 하나 이상의 제제를 포함할 수 있다: (i) BNC1, TRPC6 및 MIRLET7BHG; (ii) BNC1, AVPR1A 및 MIRLET7BHG; (iii) BNC1 및 MIRLET7BHG; (iv) BNC1, MIRLET7BHG 및 EPS8L2; (v) MIRLET7BHG 및 EPS8L2; 또는 (vi) MIRLET7BHG, EPS8L2 및 TBCD.In one embodiment, the composition comprises an agent that amplifies a marker gene or a CpG region thereof of a combination of (i), (ii), (iii), (iv), (v) or (vi) and the marker gene or CpG region thereof. It may include one or more agents selected from agents that detect the methylation status of a region: (i) BNC1, TRPC6, and MIRLET7BHG; (ii) BNC1, AVPR1A, and MIRLET7BHG; (iii) BNC1 and MIRLET7BHG; (iv) BNC1, MIRLET7BHG, and EPS8L2; (v) MIRLET7BHG and EPS8L2; or (vi) MIRLET7BHG, EPS8L2, and TBCD.
일 구현예에서, 하나 이상의 마커 유전자는 TBCD 및 EPS8L2로 이루어진 군에서 선택된 하나 이상의 유전자를 포함할 수 있다.In one embodiment, the one or more marker genes may include one or more genes selected from the group consisting of TBCD and EPS8L2.
다른 구현예에서, 하나 이상의 마커 유전자는 BNC1을 추가로 포함할 수 있다.In another embodiment, the one or more marker genes may further include BNC1.
또 다른 구현예에서, 유전자의 CpG 영역은 다음의 특징을 갖는 것일 수 있다:In another embodiment, the CpG region of a gene may have the following characteristics:
일부 구현예에서, 증폭하는 제제는 상기 유전자에 상보적으로 결합하는 프라이머, 프로브 또는 안티센스 뉴클레오티드를 포함할 수 있다.In some embodiments, the amplifying agent may include a primer, probe, or antisense nucleotide that binds complementary to the gene.
일부 구현예에서, 조성물은 하나 이상의 마커 유전자 또는 이의 CpG 영역을 특이적으로 증폭하는 제제 및 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제를 포함할 수 있다.In some embodiments, the composition may include an agent that specifically amplifies one or more marker genes or CpG regions thereof and an agent that detects the methylation status of the one or more marker genes or CpG regions thereof.
다른 구현예에서, 메틸화 상태를 측정하는 제제는 바이설파이트, 하이드로겐 설파이트, 다이설파이트 또는 이들의 조합일 수 있다.In other embodiments, the agent that measures methylation status can be a bisulfite, hydrogen sulfite, disulfite, or a combination thereof.
또 다른 태양에 따르면, 췌장암 진단을 위한 조성물을 포함하는, 개체의 생물학적 시료로부터 분리된 핵산을 이용하는 췌장암 진단을 위한 키트가 제공될 수 있다.According to another aspect, a kit for diagnosing pancreatic cancer using nucleic acids isolated from a biological sample of an individual may be provided, including a composition for diagnosing pancreatic cancer.
일 구현예에서, 키트는 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역을 포함하는 검출 대상 유전자 또는 이의 CpG 영역을 포함하는 단편과 혼성화할 수 있는 프로브가 고정되어 있는 진단용 핵산 칩(Chip)을 포함할 수 있다. In one embodiment, the kit may include a diagnostic nucleic acid chip on which a probe capable of hybridizing with a gene to be detected including the one or more marker genes or a CpG region thereof or a fragment including a CpG region thereof is immobilized. there is.
본 발명의 또 다른 태양에 따르면, 췌장암의 치료 방법으로서, (i) 개체의 생물학적 시료로부터 분리된 핵산에서 상술한 메틸화 마커 유전자 또는 이의 CpG 영역이 정상 대조군에 비해 저메틸화된 상태 또는 과메틸화 상태를 검출하는 단계; 및 (ii) 상기 개체에서 췌장암에 대한 치료요법을 시행하는 단계를 포함하는 방법이 제공된다. According to another aspect of the present invention, there is a method for treating pancreatic cancer, wherein (i) the above-described methylation marker gene or its CpG region in nucleic acid isolated from a biological sample of an individual is hypomethylated or hypermethylated compared to the normal control group. detecting; and (ii) administering therapy for pancreatic cancer in said individual.
본 발명의 또 다른 태양에 따르면, 췌장암을 가진 것으로 의심되거나 췌장암 발병 위험이 있는 개체에서 메틸화 마커를 검출하는 방법이 제공된다. 구체적으로, 상기 방법은 상기 개체로부터 수득된 생물학적 시료에서 하나 이상의 메틸화 마커 유전자의 메틸화 상태를 측정하는 단계를 포함하고, 상기 하나 이상의 메틸화 마커 유전자는 MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2)로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함할 수 있다. According to another aspect of the invention, a method is provided for detecting methylation markers in an individual suspected of having or at risk of developing pancreatic cancer. Specifically, the method includes measuring the methylation status of one or more methylation marker genes in a biological sample obtained from the individual, wherein the one or more methylation marker genes include MIRLET7B Host Gene (MIRLET7BHG) and Xyloside Xylosyltransferase 1 (XXYLT1). , ZNF879 (Zinc Finger Protein 879), EPS8L2 (EPS8 Like 2), TRPC6 (Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D) , may include one or more genes or a CpG region thereof selected from the group consisting of MAFB (MAF BZIP Transcription Factor B) and TCEA2 (Transcription Elongation Factor A2).
본 발명에 따르면, 췌장암 환자군에서 차별적으로 메틸화 수준이 변화되어 있는 새로운 메틸화 마커 유전자가 발굴되었다. 상기 마커 유전자를 사용하면 우수한 민감도, 특이도 및/또는 정확도로 췌장암을 진단할 수 있다. 특히, 상기 메틸화 마커 유전자는 혈액에 기반한 생물학적 시료를 이용하여 조기에 췌장암 진단이 가능하다는 점에서 유용하다.According to the present invention, a new methylation marker gene whose methylation level was differentially changed in a group of pancreatic cancer patients was discovered. Using the marker gene, pancreatic cancer can be diagnosed with excellent sensitivity, specificity and/or accuracy. In particular, the methylation marker gene is useful in that it enables early diagnosis of pancreatic cancer using blood-based biological samples.
도 1은 본 발명의 일 구현예에 따른 췌장암 식별 모델을 수립하기 위한 과정을 나타내는 도면이다.Figure 1 is a diagram showing a process for establishing a pancreatic cancer identification model according to an embodiment of the present invention.
도 2는 신규 메틸화 마커 유전자의 DMR에서 췌장암 환자군(33례) 및 정상 대조군(42례) 샘플 간의 메틸화 수준 차이를 컬러로 표시한 차트를 나타내는 도면이다.Figure 2 is a diagram showing a color chart showing the difference in methylation levels between pancreatic cancer patient group (33 cases) and normal control (42 cases) samples in the DMR of a new methylation marker gene.
도 3은 본 발명의 일 구현예에 따른 MIRLET7BHG와 BNC1의 조합을 기반으로 환자군(33례) 및 정상 대조군(42례) 샘플에 대해 leave-one-out 테스트를 수행하여 얻은 민감도 수치를 암 기수별로 정리한 결과를 나타내는 도면이다.Figure 3 shows the sensitivity values obtained by performing a leave-one-out test on samples of the patient group (33 cases) and the normal control group (42 cases) based on the combination of MIRLET7BHG and BNC1 according to an embodiment of the present invention, by cancer stage. This is a drawing showing the organized results.
후술하는 본 발명에 대한 상세한 설명은, 본 발명이 실시될 수 있는 특정 구현예에 관하여 특정 도면을 참조하여 기술될 것이지만, 본 발명은 이에 한정되지 않고, 적절하게 설명된다면, 그 청구항들이 주장하는 것과 균등한 모든 범위와 더불어 첨부된 청구항에 의해서만 한정된다. 본 발명의 다양한 구현예/실시예는 서로 다르지만 상호 배타적일 필요는 없음이 이해되어야 한다. 예를 들어, 본 명세서에 기재되어 있는 특정 형상, 구조 및 특성은 본 발명의 정신과 범위를 벗어나지 않으면서 일 구현예/실시예에서 다른 구현예/실시예로 변경되거나 구현예/실시예들이 조합되어 구현될 수 있다. 본 발명을 설명하면서 제시된 용어는 달리 나타내지 않는 한, 일반적으로 그의 통상적인 의미로 이해되어야 하며, 그 용어가 정의된 발명의 태양 또는 구현예뿐만 아니라 본 명세서에 사용된 동일한 용어에 모두 적용된다. 본 명세서를 해석할 목적으로 하기 정의들이 적용될 것이고, 단수로 사용된 용어는 적절한 경우에는 복수형을 포함할 것이며 그 반대도 마찬가지이다. The detailed description of the invention set forth below will be described with reference to specific drawings with respect to specific embodiments in which the invention may be practiced, but the invention is not limited thereto and, if properly described, is as claimed in the claims. It is limited only by the appended claims, along with all equivalents. It should be understood that the various implementations/embodiments of the present invention are different from one another but are not necessarily mutually exclusive. For example, specific shapes, structures and characteristics described herein may be changed from one embodiment/embodiment to another embodiment/embodiment or combinations of embodiments/embodiments without departing from the spirit and scope of the invention. It can be implemented. Terms presented in describing the present invention, unless otherwise indicated, are generally to be understood in their ordinary meaning and apply both to the same term used herein as well as to the aspect or embodiment of the invention for which it is defined. For purposes of interpreting this specification, the following definitions will apply and terms used in the singular will include the plural where appropriate and vice versa.
췌장암 진단을 위한 마커 유전자Marker genes for pancreatic cancer diagnosis
본 발명자들은 췌장암 진단을 위한 혈액 기반 메틸화 마커 유전자를 확보하기 위해 연구한 결과, 췌장암 환자군에서 차별적으로 메틸화 수준이 변화되어 있는 신규 유전자 또는 유전자 조합을 발굴하였고 이들 마커 유전자 또는 마커 유전자 조합을 사용하는 경우에 우수한 민감도, 특이도 및/또는 정확도로 췌장암을 진단할 수 있음을 확인하여 본 발명을 완성하였다.As a result of research to secure blood-based methylation marker genes for diagnosing pancreatic cancer, the present inventors discovered new genes or gene combinations whose methylation levels were differentially changed in pancreatic cancer patient groups, and when using these marker genes or combinations of marker genes The present invention was completed by confirming that pancreatic cancer can be diagnosed with excellent sensitivity, specificity, and/or accuracy.
본 발명의 일 태양에 따르면, MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2) 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함하는 췌장암 진단을 위한 마커 유전자가 제공된다. 구체적으로 상기 췌장암 진단을 위한 마커 유전자는 상기 각 유전자 또는 이의 CpG 영역을 단독으로 포함할 수 있거나 상기 유전자들로부터 선택된 둘 이상의 유전자 또는 이의 CpG 영역의 조합을 포함할 수 있다. 또한, 상기 췌장암 진단을 위한 마커 유전자는 당업계에 췌장암 진단을 위해 알려진 다른 마커 유전자, 예컨대 BNC1과 같은 췌장암 진단 마커 유전자와 조합하여 사용할 수 있다. According to one aspect of the present invention, MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside Xylosyltransferase 1), ZNF879 (Zinc Finger Protein 879), EPS8L2 (EPS8 Like 2), TRPC6 (Transient Receptor Potential Cation Channel Subfamily C Member 6), One or more genes or CpGs thereof selected from the group consisting of AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) genes A marker gene for pancreatic cancer diagnosis containing a region is provided. Specifically, the marker gene for diagnosing pancreatic cancer may include each of the above genes or their CpG regions alone, or may include a combination of two or more genes or their CpG regions selected from the above genes. Additionally, the marker gene for diagnosing pancreatic cancer can be used in combination with other marker genes known in the art for diagnosing pancreatic cancer, such as BNC1.
유전자 MIRLET7BHG는 lncRNA 클래스에 속하는 RNA로서, 이와 관련된 질병으로는 단지증 타입 B2 질환이 있다.The gene MIRLET7BHG is an RNA belonging to the lncRNA class, and diseases related to it include brachydactyly type B2 disease.
유전자 XXYLT1에 의해 암호화된 단백질은 표적 단백질의 세포외 도메인에서 EGF 유사 반복부에 부착된 O-연결된 자일로스-글루코스 이당류를 연장하는 알파-1,3-자일로실트랜스퍼라제 효소이다.The protein encoded by the gene XXYLT1 is an alpha-1,3-xylosyltransferase enzyme that extends O-linked xylose-glucose disaccharides attached to EGF-like repeats in the extracellular domain of target proteins.
유전자 ZNF879에 의해 암호화된 단백질은 N-말단 kruppel-associated box(KRAB) 도메인과 13개의 C-말단 C2H2-유형 징크 핑거 도메인을 포함하는 전사억제인자이다.The protein encoded by gene ZNF879 is a transcriptional repressor containing an N-terminal kruppel-associated box (KRAB) domain and 13 C-terminal C2H2-type zinc finger domains.
유전자 EPS8L2는 표피 성장 인자 수용체에 대한 기질인 표피 성장 인자 수용체 경로 기질 8(EPS8)과 관련된 단백질을 인코딩하며, EPS8L2 단백질은 액틴 세포골격 리모델링에 연관된 기능을 하는 것으로 알려져 있다.The gene EPS8L2 encodes a protein related to epidermal growth factor receptor pathway substrate 8 (EPS8), a substrate for the epidermal growth factor receptor, and the EPS8L2 protein is known to function involved in actin cytoskeleton remodeling.
유전자 TRPC6에 의해 암호화된 단백질은 TRPC 서브패밀리의 일시적인 수용체 전위 채널로 작용하며, 이와 관련된 질병으로는 우울증, 불안 및 국소 분절 사구체 경화 등이 있다. The protein encoded by the gene TRPC6 acts as a transient receptor potential channel of the TRPC subfamily, and diseases associated with it include depression, anxiety, and focal segmental glomerulosclerosis.
유전자 AVPR1A에 의해 암호화된 단백질은 아르기닌 바소프레신에 대한 수용체로 작용한다. 이 수용체는 세포 수축과 증식, 혈소판 응집, 응고 인자의 방출 및 글리코겐 분해를 매개한다.The protein encoded by the gene AVPR1A acts as a receptor for arginine vasopressin. This receptor mediates cell contraction and proliferation, platelet aggregation, release of clotting factors, and glycogenolysis.
유전자 GSC는 짝지어진(paired) homeobox 단백질 패밀리의 바이코이드(bicoid) 서브패밀리의 구성원을 암호화하며, GSC 단백질은 전사 인자로 작용하며 자가 조절될 수 있다. The gene GSC encodes a member of the bicoid subfamily of the paired homeobox protein family, and the GSC protein acts as a transcription factor and can be self-regulated.
유전자 TBCD는 베타 튜불린의 중간체를 포착하고 안정화하는 역할을 하며, TBCD와 관련된 질병에는 뇌병증 및 지루성 피부염이 포함된다.The gene TBCD is responsible for capturing and stabilizing intermediates of beta tubulin, and diseases associated with TBCD include encephalopathy and seborrheic dermatitis.
유전자 MAFB는 계통 특이적 조혈 조절에 중요한 역할을 하는 기본 류신 지퍼(bZIP) 전사 인자이며, 암호화된 핵 단백질은 골수 세포에서 적혈구 특이적 유전자의 ETS1 매개 전사를 억제한다.The gene MAFB is a basic leucine zipper (bZIP) transcription factor that plays an important role in the regulation of lineage-specific hematopoiesis, and the encoded nuclear protein represses ETS1-mediated transcription of erythroid-specific genes in myeloid cells.
유전자 TCEA2에 의해 암호화된 단백질은 핵에서 SII 클래스 전사 연장 인자로 기능하며, 기본 전사 인자인 일반 전사 인자 IIB와 상호작용한다. The protein encoded by the gene TCEA2 functions as a SII class transcription elongation factor in the nucleus and interacts with the basic transcription factor, general transcription factor IIB.
유전자 BNC1에 의해 암호화된 단백질은 표피의 기저 세포층과 모낭에 존재하는 징크 핑거 단백질로 생식세포에서 풍부하게 발현되며, 각질세포의 증식을 조절하는 역할을 하는 것으로 알려져 있다.The protein encoded by the gene BNC1 is a zinc finger protein present in the basal cell layer of the epidermis and hair follicles, is abundantly expressed in germ cells, and is known to play a role in regulating the proliferation of keratinocytes.
상기 유전자들의 뉴클레오티드 서열 등 구체적인 정보는 미국 국립보건원에 의해 유지되는 NCBI(미국 국립 생물공학 정보센터)에 의해 제공되는 데이터베이스, 스위스 생물 정보학 연구소에 의해 제공되는 UniProt 지식베이스(UniProtKB) 및 Swiss-Prot 데이터베이스를 포함한 다양한 DB 엔진에 의하여 입수할 수 있으며, 당업자에 의하여 적절하게 선택될 수 있다. Specific information, such as the nucleotide sequences of the genes, can be found in the database provided by the National Center for Biotechnology Information (NCBI) maintained by the National Institutes of Health, the UniProt Knowledge Base (UniProtKB) and the Swiss-Prot database provided by the Swiss Bioinformatics Institute. It can be obtained from various DB engines, including , and can be appropriately selected by those skilled in the art.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG, EPS8L2, TRPC6, AVPR1A 및 TBCD로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함할 수 있다. 구체적으로 췌장암 진단을 위한 마커 유전자는 상기 각 유전자 또는 이의 CpG 영역을 단독으로 포함할 수 있거나 상기 유전자들로부터 선택된 둘 이상의 마커 유전자 또는 이의 CpG 영역의 조합을 포함할 수 있다. 또한, 췌장암 진단을 위한 마커 유전자는 당업계에 췌장암 진단을 위해 알려진 다른 마커 유전자, 예컨대 BNC1과 같은 췌장암 진단 마커 유전자와 조합하여 사용할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer may include one or more genes selected from the group consisting of MIRLET7BHG, EPS8L2, TRPC6, AVPR1A, and TBCD, or a CpG region thereof. Specifically, a marker gene for diagnosing pancreatic cancer may include each of the above genes or their CpG regions alone, or may include a combination of two or more marker genes or their CpG regions selected from the above genes. Additionally, the marker gene for diagnosing pancreatic cancer can be used in combination with other marker genes known in the art for diagnosing pancreatic cancer, such as a pancreatic cancer diagnostic marker gene such as BNC1.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역이거나 이를 포함할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include MIRLET7BHG or its CpG region.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 EPS8L2 또는 이의 CpG 영역이거나 이를 포함할 수 있다. In one embodiment, the marker gene for pancreatic cancer diagnosis may be or include EPS8L2 or its CpG region.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 TRPC6 또는 이의 CpG 영역이거나 이를 포함할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include TRPC6 or its CpG region.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 AVPR1A 또는 이의 CpG 영역이거나 이를 포함할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include AVPR1A or its CpG region.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 TBCD 또는 이의 CpG 영역이거나 이를 포함할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include TBCD or its CpG region.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역을 포함하고, TRPC6, AVPR1A, EPS8L2, TBCD 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함할 수 있다. 바람직하게는, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역을 포함하고, EPS8L2 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함할 수 있다. 또한, 췌장암 진단을 위한 마커 유전자는 TRPC6 또는 이의 CpG 영역, TBCD 또는 이의 CpG 영역, 또는 AVPR1A 또는 이의 CpG 영역을 추가로 포함할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer includes MIRLET7BHG or a CpG region thereof, and may include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1, or a CpG region thereof. Preferably, the marker gene for diagnosing pancreatic cancer includes MIRLET7BHG or a CpG region thereof, and may include one or more genes selected from the group consisting of EPS8L2 and BNC1 or a CpG region thereof. Additionally, the marker gene for diagnosing pancreatic cancer may further include TRPC6 or its CpG region, TBCD or its CpG region, or AVPR1A or its CpG region.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역 및 EPS8L2 또는 이의 CpG 영역의 조합이거나 이를 포함할 수 있다. In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include a combination of MIRLET7BHG or a CpG region thereof and EPS8L2 or a CpG region thereof.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역 및 TRPC6 또는 이의 CpG 영역의 조합이거나 이를 포함할 수 있다.In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include a combination of MIRLET7BHG or a CpG region thereof and TRPC6 or a CpG region thereof.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역 및 AVPR1A 또는 이의 CpG 영역의 조합이거나 이를 포함할 수 있다.In one embodiment, the marker gene for diagnosing pancreatic cancer may be or include a combination of MIRLET7BHG or a CpG region thereof and AVPR1A or a CpG region thereof.
일 구현예에서, 췌장암 진단을 위한 마커 유전자는 MIRLET7BHG 또는 이의 CpG 영역, EPS8L2 또는 이의 CpG 영역 및 TBCD 또는 이의 CpG 영역의 조합이거나 이를 포함할 수 있다.In one embodiment, the marker gene for pancreatic cancer diagnosis may be or include a combination of MIRLET7BHG or a CpG region thereof, EPS8L2 or a CpG region thereof, and TBCD or a CpG region thereof.
다른 구현예에서, 췌장암 진단을 위한 마커 유전자는 BNC1 또는 이의 CpG 영역과 조합하여 췌장암 진단에 사용될 수 있다. In another embodiment, a marker gene for diagnosing pancreatic cancer can be used for diagnosing pancreatic cancer in combination with BNC1 or its CpG region.
바람직하게는, 췌장암 진단을 위한 마커 유전자는 하기 조합의 마커 유전자 또는 이의 CpG 영역을 포함할 수 있다: Preferably, the marker gene for diagnosing pancreatic cancer may include the following combination of marker genes or CpG regions thereof:
(i) BNC1, TRPC6 및 MIRLET7BHG;(i) BNC1, TRPC6, and MIRLET7BHG;
(ii) BNC1, AVPR1A 및 MIRLET7BHG;(ii) BNC1, AVPR1A, and MIRLET7BHG;
(iii) BNC1 및 MIRLET7BHG; (iii) BNC1 and MIRLET7BHG;
(iv) BNC1, MIRLET7BHG 및 EPS8L2; (iv) BNC1, MIRLET7BHG, and EPS8L2;
(v) MIRLET7BHG 및 EPS8L2; 또는 (v) MIRLET7BHG and EPS8L2; or
(vi) MIRLET7BHG, EPS8L2 및 TBCD. (vi) MIRLET7BHG, EPS8L2, and TBCD.
또 다른 구현예에서, 췌장암 진단을 위한 마커 유전자는 TBCD 또는 이의 CpG 영역, EPS8L2 또는 이의 CpG 영역, 또는 이들 둘 모두를 포함할 수 있다. 이들 마커 유전자는 당업계에 췌장암 진단을 위해 알려진 다른 마커 유전자, 예컨대 BNC1과 같은 췌장암 진단 마커 유전자와 조합하여 사용할 수 있다. 바람직하게는 췌장암 진단을 위한 마커 유전자는 하기 조합의 마커 유전자 또는 이의 CpG 영역을 포함할 수 있다:In another embodiment, the marker gene for diagnosing pancreatic cancer may include TBCD or a CpG region thereof, EPS8L2 or a CpG region thereof, or both. These marker genes can be used in combination with other marker genes known in the art for diagnosing pancreatic cancer, such as BNC1. Preferably, the marker gene for diagnosing pancreatic cancer may include the following combination of marker genes or CpG regions thereof:
(i) BNC1 및 TBCD; 또는 (i) BNC1 and TBCD; or
(ii) BNC1 및 EPS8L2.(ii) BNC1 and EPS8L2.
상기 메틸화 마커 유전자는 개체로부터 분리된 생물학적 시료, 예컨대 액체 시료에 기반하여 우수한 민감도, 특이도 및/또는 정확도로 췌장암을 진단하거나 췌장암 진단에 관한 정보를 제공할 수 있다. 상기 메틸화 마커 유전자는 혈액, 혈장, 혈청과 같은 액체 시료를 이용한 경우, 우수한 진단 정확도를 나타낸다는 점에서 비침습적 진단을 가능하게 한다.The methylation marker gene can diagnose pancreatic cancer with excellent sensitivity, specificity, and/or accuracy based on a biological sample isolated from an individual, such as a liquid sample, or provide information regarding the diagnosis of pancreatic cancer. The methylation marker gene enables non-invasive diagnosis in that it shows excellent diagnostic accuracy when using liquid samples such as blood, plasma, and serum.
용어 "메틸화"는 DNA를 구성하는 염기에 메틸기가 부착되는 것을 의미한다. 일 구현예에서, 메틸화 상태는 특정 유전자의 특정 CpG 영역의 사이토신에서 일어나는 메틸화 여부 또는 메틸화 정도를 의미할 수 있다. 메틸화가 일어난 경우 그로 인하여 전사인자의 결합이 방해를 받아 해당 유전자의 발현이 억제될 수 있다. 발현 억제는 메틸화 정도에 따라 상대적으로 일어날 수 있다. 반대로, 비메틸화 또는 저메틸화가 일어나는 경우 특정 유전자의 발현이 증가할 수 있다. 이러한 이유로 DNA 메틸화는 유전자 발현 조절에 중추적인 역할을 하는 후성유전적 변형으로서, 사이토신의 5번 탄소에 메틸기(-CH3)가 붙어 5-메틸사이토신 형태로 변형된다. DNA 메틸화는 주로 CpG 디뉴클레오티드의 사이토신 기에서 발생하며, CpG 섬 또는 CpG 영역이라고 불리는 CpG가 집중적으로 몰려 있는 DNA 영역에서 많이 나타난다.The term “methylation” refers to the attachment of a methyl group to the bases that make up DNA. In one embodiment, the methylation status may mean whether or not methylation occurs at a cytosine in a specific CpG region of a specific gene or the degree of methylation. If methylation occurs, it may interfere with the binding of transcription factors and suppress the expression of the gene in question. Expression suppression may occur relative to the degree of methylation. Conversely, when unmethylation or hypomethylation occurs, the expression of certain genes may increase. For this reason, DNA methylation is an epigenetic modification that plays a central role in regulating gene expression. A methyl group (-CH3) is attached to the 5th carbon of cytosine, transforming it into 5-methylcytosine. DNA methylation mainly occurs at the cytosine group of CpG dinucleotides and occurs in DNA regions where CpGs are concentrated, called CpG islands or CpG regions.
용어 "CpG 영역"은 CpG 섬이라고 불리는 CpG가 집중적으로 몰려있는 유전자의 DNA 영역 또는 그의 일부를 지칭한다. 예컨대, 상기 CpG 영역은 프로모터 영역 등 전사 조절 영역, 단백질 코딩 영역(open reading frame, ORF) 또는 터미네이터 영역 등에 존재할 수 있다. 암이 진행되는 과정 동안 게놈 전반적으로는 DNA 저메틸화가 나타나고, CpG 섬의 일부 영역에서 국소적인 과메틸화가 빈번하게 일어나는 것으로 알려져 있다. 예를 들어, 암세포에서는 암 억제 유전자의 프로모터 영역의 CpG 부위가 비정상적으로 과메틸화되면 이 유전자의 발현이 억제되어 암을 일으킬 수 있다. CpG 부위의 메틸화는 암 발생의 초기에 일어나기 때문에 암의 조기 진단에 유용하다. The term “CpG region” refers to a region of the DNA of a gene or a portion thereof where CpGs are concentrated, called a CpG island. For example, the CpG region may exist in a transcriptional control region such as a promoter region, a protein coding region (open reading frame, ORF), or a terminator region. It is known that during the course of cancer, DNA hypomethylation occurs throughout the genome, and local hypermethylation occurs frequently in some regions of CpG islands. For example, in cancer cells, if the CpG region of the promoter region of a cancer suppressor gene is abnormally hypermethylated, the expression of this gene may be suppressed, causing cancer. Methylation of CpG sites occurs early in cancer development, so it is useful for early diagnosis of cancer.
일 구현예에서, 상기 유전자의 CpG 부위는 C와 G가 포스페이트로 연결된 서열이 200bp 이상 연속된 부분일 수 있고, 1kb에서 9kb 정도의 범위에서 임의의 크기를 가질 수 있다.In one embodiment, the CpG region of the gene may be a continuous sequence of 200 bp or more in which C and G are linked by phosphate, and may have any size ranging from 1 kb to 9 kb.
다른 구현예에서, 상기 유전자의 CpG 부위는 유전자의 전사 시작 부위(transcription start site, TSS)로부터 +/- 2000 염기(2kb) 사이에 위치할 수 있다.In another embodiment, the CpG region of the gene may be located between +/- 2000 bases (2 kb) from the transcription start site (TSS) of the gene.
본 발명의 일 실시예에서, 상기 유전자의 CpG 부위는 하기 표 1에 기재된 유전자의 CpG 부위 또는 그에 포함된 일부의 사이토신의 메틸화 상태일 수 있다. 하기 표 1에서 CpG 부위는 DMR(Differentially Methylated Region)이라고 표시한다.In one embodiment of the present invention, the CpG region of the gene may be the CpG region of the gene listed in Table 1 below or the methylation state of a portion of the cytosine contained therein. In Table 1 below, the CpG region is indicated as DMR (Differentially Methylated Region).
| 유전자 명칭gene name | 염색체 번호chromosome number | DMR 위치 (GRCh37)DMR location (GRCh37) | DMR 크기DMR size | |
| 시작start | 끝end | |||
| XXYLT1XXYLT1 | chr3chr3 | 194,800,905194,800,905 | 194,802,072194,802,072 | 1,1671,167 |
| ZNF879ZNF879 | chr5chr5 | 178,450,154178,450,154 | 178,451,476178,451,476 | 1,3221,322 |
| EPS8L2EPS8L2 | chr11chr11 | 704,570704,570 | 713,015713,015 | 8,4458,445 |
| TRPC6TRPC6 | chr11chr11 | 101,453,938101,453,938 | 101,455,234101,455,234 | 1,2961,296 |
| AVPR1AAVPR1A | chr12chr12 | 63,543,59863,543,598 | 63,544,75663,544,756 | 1,1581,158 |
| GSCGSC | chr14chr14 | 95,237,29195,237,291 | 95,238,48995,238,489 | 1,1981,198 |
| TBCDTBCD | chr17chr17 | 80,831,80880,831,808 | 80,833,01280,833,012 | 1,2041,204 |
| MAFBMAFB | chr20chr20 | 39,318,91539,318,915 | 39,320,06839,320,068 | 1,1531,153 |
| TCEA2TCEA2 | chr20chr20 | 62,696,70162,696,701 | 62,697,89662,697,896 | 1,1951,195 |
| MIRLET7BHGMIRLET7BHG | chr22chr22 | 46,479,06546,479,065 | 46,481,48046,481,480 | 2,4152,415 |
| BNC1BNC1 | chr15chr15 | 83,951,73083,951,730 | 83,954,13383,954,133 | 2,4032,403 |
본 발명에서 인간 게놈 염색체 부위의 염기서열은 The February 2009 Human reference sequence(GRCh37)에 따라 표현하였지만, 상기 인간 게놈 염색체 부위의 구체적 서열은 게놈 서열 연구 결과가 업데이트됨에 따라서 그 표현이 다소 변경될 수 있으며, 이러한 변경에 따라 본 발명의 상기 인간 게놈 염색체 부위의 표현이 상이해질 수 있다. 따라서, 본 발명의 The February 2009 Human reference sequence(GRCh37)에 따라 표현된 인간 게놈 염색체 부위는 본 발명의 출원일 이후 인간 표준 염기서열(human reference sequence)이 업데이트되어 상기 인간 게놈 염색체 부위의 표현이 지금과 다르게 변경된다고 하여도, 본 발명의 범위가 상기 변경된 인간 게놈 염색체 부위에 미치게 됨은 자명하다고 할 것이다. 이러한 변경 내용은 본 발명이 속하는 기술분야의 통상의 지식을 가진 자라면 누구라도 용이하게 알 수 있는 사항이다. In the present invention, the base sequence of the human genome chromosome region is expressed according to The February 2009 Human reference sequence (GRCh37), but the specific sequence of the human genome chromosome region may change somewhat as the results of genome sequence research are updated. , depending on these changes, the expression of the human genomic chromosomal region of the present invention may be different. Accordingly, the human genome chromosomal region expressed according to The February 2009 Human reference sequence (GRCh37) of the present invention has been updated as a human reference sequence after the filing date of the present invention, so that the expression of the human genomic chromosomal region is the same as now. Even if it is changed differently, it will be obvious that the scope of the present invention extends to the changed human genome chromosomal region. These changes can be easily known by anyone with ordinary knowledge in the technical field to which the present invention pertains.
췌장암 진단을 위한 정보 제공 방법How to provide information for pancreatic cancer diagnosis
본 발명의 다른 태양에 따르면, 개체의 생물학적 시료로부터 분리된 핵산에서 상술한 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 측정하는 단계를 포함하는 췌장암 진단을 위한 정보를 제공하는 방법 또는 췌장암 진단 방법이 제공된다. 다른 측면에서, 췌장암을 가진 것으로 의심되거나 췌장암 발병 위험이 있는 개체로부터 수득한 시료에서 상술한 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 결정하는 단계를 포함하는 상기 개체에서 메틸화 마커를 검출하는 방법이 제공된다. According to another aspect of the present invention, a method for providing information for diagnosing pancreatic cancer or a method for diagnosing pancreatic cancer is provided, comprising measuring the methylation status of the above-described marker gene or its CpG region in nucleic acid isolated from a biological sample of an individual. do. In another aspect, a method is provided for detecting a methylation marker in an individual suspected of having or at risk of developing pancreatic cancer, comprising determining the methylation status of the marker gene or CpG region thereof in a sample obtained from the individual. do.
용어 "개체"는 췌장암의 진단을 필요로 하는 포유동물, 예를 들어, 영장류(예: 인간), 반려 동물(예: 개, 고양이 등), 가축 동물(예: 소, 돼지, 말, 양, 염소 등) 및 실험실 동물(예: 랫트, 마우스, 기니피그 등)일 수 있다. 본 발명의 일 구현예에서, 개체는 인간이다. The term “subject” refers to mammals in need of a diagnosis of pancreatic cancer, such as primates (e.g. humans), companion animals (e.g. dogs, cats, etc.), domestic animals (e.g. cattle, pigs, horses, sheep, etc.) goats, etc.) and laboratory animals (e.g. rats, mice, guinea pigs, etc.). In one embodiment of the invention, the individual is a human.
상기 생물학적 시료는 혈액, 혈장 또는 혈청일 수 있지만, 이로 한정되지 않는다. 구체적으로, 상기 생물학적 시료로부터 분리된 핵산은 cfDNA일 수 있다. "cfDNA"는 게놈 DNA 중 세포 유리(cell free) DNA를 지칭한다.The biological sample may be, but is not limited to, blood, plasma, or serum. Specifically, the nucleic acid isolated from the biological sample may be cfDNA. “cfDNA” refers to cell free DNA among genomic DNA.
상기 생물학적 시료에서 게놈 DNA 또는 그의 단편을 분리 또는 단리하는 방법은 당업계에서 통상적으로 사용되는 페놀/클로로포름 추출법, SDS 추출법(Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB 분리법(Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) 또는 상업적으로 판매되는 DNA 추출 키트를 이용하여 수행할 수 있다. 일 구현예에서, 해당 DNA가 세포막으로 싸여 있는 경우, 생물학적 샘플은 효소적, 화학적 또는 기계적 수단에 의해 파괴하고 용해하는 과정을 거칠 수 있다. 이어서, DNA 용액에서 단백질 및 기타 오염물을, 예를 들면, 프로테이나제 K로 분해함으로써 제거하고, 게놈 DNA를 용액으로부터 회수한다. DNA의 정제 방법은 염석(salting out), 유기 추출, 또는 고체상 지지체에 대한 DNA의 결합을 포함한 다양한 방법에 의해 수행될 수 있다. DNA 분리 방법 및 정제 방법은 시간, 비용 및 DNA의 필요량을 포함한 여러 요인들을 고려하여 당업자에게 맞게 선택될 수 있다.Methods for separating or isolating genomic DNA or fragments thereof from the biological sample include phenol/chloroform extraction and SDS extraction (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990) commonly used in the art. ), CTAB separation method (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980), or a commercially available DNA extraction kit. In one embodiment, if the DNA of interest is surrounded by a cell membrane, the biological sample may be subjected to a process of destruction and dissolution by enzymatic, chemical, or mechanical means. Proteins and other contaminants are then removed from the DNA solution, for example by digestion with proteinase K, and genomic DNA is recovered from the solution. Purification of DNA can be performed by a variety of methods, including salting out, organic extraction, or binding of DNA to a solid phase support. DNA isolation and purification methods can be selected by those skilled in the art taking into account several factors including time, cost, and required amount of DNA.
다른 구현예에서, 샘플 DNA가 막에 둘러싸여 있지 않은 경우(예를 들면, 혈액 샘플로부터의 순환 또는 유리 DNA), DNA의 분리 및/또는 정제를 위한 당해 분야에서의 표준 방법을 사용할 수 있다. 상기 방법은 단백질 변성 시약, 예를 들면, 카오트로픽(chaotropic) 염, 예를 들어, 구아니딘 하이드로클로라이드 또는 우레아; 또는 세제, 예를 들어, 나트륨 도데실설페이트(SDS), 시아노겐 브로마이드의 사용을 포함한다. 대안적 방법으로는 에탄올 침전 또는 프로판올 침전, 특히 원심분리에 의한 진공 농축이 포함되나, 이로 한정되지는 않는다. 당해 분야에 숙련된 자라면 또한 필터 장치와 같은 장치, 예를 들면, 한외여과, 실리카 표면 또는 막, 자기 입자, 폴리스티롤 입자, 폴리스티롤 표면, 양으로 하전된 표면 및 양으로 하전된 막, 하전된 막, 하전된 표면, 하전된 전환 막, 하전된 전환 표면을 사용할 수 있다.In other embodiments, when the sample DNA is not surrounded by a membrane (e.g., circulating or free DNA from a blood sample), standard methods in the art for isolation and/or purification of DNA can be used. The method includes protein denaturing reagents, such as chaotropic salts such as guanidine hydrochloride or urea; or the use of detergents such as sodium dodecyl sulfate (SDS), cyanogen bromide. Alternative methods include, but are not limited to, ethanol precipitation or propanol precipitation, especially vacuum concentration by centrifugation. Those skilled in the art will also understand devices such as filter devices, e.g., ultrafiltration, silica surfaces or membranes, magnetic particles, polystyrol particles, polystyrol surfaces, positively charged surfaces and positively charged membranes, charged A charged film, a charged surface, a charged conversion film, or a charged conversion surface can be used.
상기 생물학적 시료에서 분리 또는 단리된 DNA는 상술한 마커 유전자의 메틸화 상태를 측정하기 위한 방법으로 사용될 수 있다. DNA separated or isolated from the biological sample can be used as a method to measure the methylation status of the marker gene described above.
또 다른 구현예에서, 상기 메틸화 상태를 측정하는 단계는, i) 상술한 하나 이상의 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제 및/또는 ii) 상술한 하나 이상의 유전자 또는 이의 CpG 영역을 특이적으로 증폭하는 프라이머로 상기 DNA를 처리하는 단계를 포함할 수 있다.In another embodiment, the step of measuring the methylation status comprises i) an agent that detects the methylation status of one or more genes or CpG regions thereof described above and/or ii) an agent that specifically detects the methylation state of one or more genes or CpG regions thereof described above. It may include the step of treating the DNA with a primer to amplify.
상기 메틸화 상태를 검출(또는 측정)하는 제제는 사이토신 염기를 변형시키는 화합물 또는 메틸화 민감성 제한 효소일 수 있다. 상기 사이토신 염기를 변형시키는 화합물은 비메틸화 사이토신 또는 메틸화 사이토신을 변형시키는 화합물일 수 있다. 구체적으로, 바이설파이트, 하이드로젠 설파이트, 다이설파이트, 이들의 염, 및 이들의 조합으로 이루어진 군에서 선택되는 어느 하나일 수 있다. 또한, 메틸화 사이토신을 변형시키는 TET 단백질일 수 있으나 이에 제한되지 않는다. 사이토신 염기를 변형시켜 CpG 영역의 메틸화 여부를 검출하는 방법은 당업계에 널리 공지되어 있다.The agent that detects (or measures) the methylation status may be a compound that modifies a cytosine base or a methylation-sensitive restriction enzyme. The compound that modifies the cytosine base may be a compound that modifies unmethylated cytosine or methylated cytosine. Specifically, it may be any one selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, salts thereof, and combinations thereof. Additionally, it may be a TET protein that modifies methylated cytosine, but is not limited thereto. Methods for detecting methylation of a CpG region by modifying cytosine bases are well known in the art.
또한, 상기 메틸화 민감성 제한효소는 CpG 영역의 메틸화를 특이적으로 검출할 수 있는 제한효소로서 제한효소의 인식부위로 CG를 함유하는 제한효소일 수 있다. 예를 들면, SmaI, SacII, EagI, HpaII, MspI, BssII,BstUI, NotI 등이 있으며 이에 제한되지 않는다. 상기 제한효소 인식부위의 C에서의 메틸화 또는 비메틸화에 따라 제한효소에 의한 절단 여부가 달라지고 이를 PCR 또는 서던블롯(Southern Blot) 분석을 통해 검출할 수 있게 된다. 상기 제한효소 이외의 다른 메틸화 민감성 제한효소는 당업계에 잘 알려져 있다. Additionally, the methylation-sensitive restriction enzyme is a restriction enzyme that can specifically detect methylation of the CpG region and may be a restriction enzyme that contains CG as a recognition site. For example, SmaI, SacII, EagI, HpaII, MspI, BssII, BstUI, NotI, etc., but are not limited thereto. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, cleavage by restriction enzymes varies and 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.
상기 프라이머는 상기 하나 이상의 상술한 마커 유전자의 메틸화된 대립형질 서열에 특이적인 프라이머 및/또는 비메틸화된 대립형질 서열에 특이적인 프라이머를 포함할 수 있다.The primers may include primers specific to the methylated allele sequence and/or primers specific to the unmethylated allele sequence of the one or more aforementioned marker genes.
또 다른 구현예에서, 상기 메틸화 상태를 측정하는 단계는 메틸화 특이적 중합효소 연쇄 반응(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 중합효소 연쇄 반응(real time methylation-specific polymerase chain reaction), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, 파이로시퀀싱, 또는 바이설파이트 시퀀싱에 의해 수행될 수 있지만, 이에 제한되지 않는다. 상기에 나열된 PCR 또는 시퀀싱의 다른 예로는 MethyLight PCR, MehtyLight 디지털 PCR, EpiTYPER, CpG 섬 마이크로어레이 등이 포함된다. 또한, TET 단백질(ten-eleven translocation protein)을 이용한 검출법을 이용하여 측정할 수 있다. 상기 TET 단백질은 DNA에 작용하는 효소로 염기의 화학적 변화에 관여하며, 바이설파이트를 처리할 경우 메틸화된 C를 제외한 모든 C가 T 염기로 바뀌는 것과 달리 TET 단백질은 메틸화된 C만이 T로 바뀌어 효율적인 검출이 가능하다.In another embodiment, the step of measuring the methylation status includes methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, methylation It may be performed by, but is not limited to, PCR using a DNA-specific binding protein, quantitative PCR, pyrosequencing, or bisulfite sequencing. Other examples of PCR or sequencing listed above include MethyLight PCR, MehtyLight digital PCR, EpiTYPER, CpG island microarray, etc. Additionally, it can be measured using a detection method using TET protein (ten-eleven translocation protein). The TET protein is an enzyme that acts on DNA and is involved in the chemical change of bases. When treated with bisulfite, all Cs except methylated C are changed to T bases, but in TET protein, only methylated C is changed to T, making it efficient. Detection is possible.
또 다른 구현예에서, 상기 방법은 상기 측정된 메틸화 수준을 대조군 시료의 상기 동일한 마커 유전자 또는 이의 CpG 부위의 메틸화 수준과 비교하는 단계를 추가로 포함할 수 있다. In another embodiment, the method may further include comparing the measured methylation level with the methylation level of the same marker gene or CpG region thereof in a control sample.
또 다른 구현예에서, 상기 메틸화 상태를 측정하는 단계는 대조군 시료의 해당 마커 유전자의 상응하는 메틸화 측정 결과로부터 생성된 대조 메틸화 프로파일과 비교하는 단계를 추가로 포함할 수 있다.In another embodiment, the step of measuring the methylation status may further include comparing the methylation profile with a control methylation profile generated from the corresponding methylation measurement result of the corresponding marker gene in the control sample.
또 다른 구현예에서, 상기 대조 메틸화 프로파일은, 상기 획득된 하나 이상의 유전자의 메틸화 패턴을 기계학습 시킴으로써 구축된 모델을 구성하며, 정상 대조군과 상이한 췌장암 환자군 특이적 메틸화 패턴을 식별하기 위한 것일 수 있다. In another embodiment, the control methylation profile constitutes a model constructed by machine learning the methylation patterns of one or more genes obtained, and may be used to identify pancreatic cancer patient-specific methylation patterns that are different from normal controls.
또 다른 구현예에서, 상기 기계학습은 랜덤 포레스트, 로지스틱 회귀 분석, 서포트 벡터 머신, 의사결정나무, 연관성 규칙 마이닝, 신경망 네트워크 및 딥러닝으로 이루어진 군에서 선택되는 하나 이상의 알고리즘에 의해 수행될 수 있다. In another implementation, the machine learning may be performed by one or more algorithms selected from the group consisting of random forest, logistic regression, support vector machine, decision tree, association rule mining, neural network, and deep learning.
또 다른 구현예에서, 상기 대조군 시료는 정상 개체의 시료일 수 있다. 또한, 상기 대조군 시료는 췌장암 환자의 시료일 수 있으며, 상기 췌장암 환자는 다양한 췌장암 병기를 갖는 환자일 수 있다.In another embodiment, the control sample may be a sample from a normal individual. Additionally, the control sample may be a sample from a pancreatic cancer patient, and the pancreatic cancer patient may be a patient with various pancreatic cancer stages.
또 다른 구현예에서, 상기 췌장암은 조기 췌장암, 전이성 췌장암, 또는 재발성 또는 난치성 췌장암일 수 있다. 이때, 각각의 췌장암은 다양한 병기를 가질 수 있다.In another embodiment, the pancreatic cancer may be early pancreatic cancer, metastatic pancreatic cancer, or recurrent or refractory pancreatic cancer. At this time, each pancreatic cancer may have various stages.
상술한 복수의 구현예는 본 발명의 목적을 달성할 수 있는 한, 제한 없이 상호 조합되어 다양한 형태로 구현될 수 있다.The plurality of embodiments described above can be implemented in various forms by combining them without limitation, as long as the purpose of the present invention can be achieved.
예를 들어, 본 발명에 따른 췌장암 진단 방법 또는 췌장암 진단을 위한 정보를 제공하는 방법은, (i) 개체의 생물학적 시료로부터 핵산을 분리하는 단계, (ii) 상기 분리된 핵산에서, 바이설파이트, 하이드로젠 설파이트, 다이설파이트, 이들의 염, 및 이들의 조합으로 이루어진 군에서 선택되는 어느 하나를 처리하는 단계, (iii) 상술한 마커 유전자에 특이적인 프라이머를 사용하여 상기 핵산을 증폭하는 단계, 및 (iv) 메틸화 특이적 중합효소 연쇄 반응, 실시간 메틸화 특이적 중합효소 연쇄 반응, 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, 파이로시퀀싱, 또는 바이설파이트 시퀀싱에 의해 상술한 마커 유전자에 대한 하나 이상의 CpG 영역의 메틸화 수준을 측정하는 단계를 포함하여 구현될 수 있다.For example, the method for diagnosing pancreatic cancer or providing information for diagnosing pancreatic cancer according to the present invention includes (i) isolating nucleic acid from a biological sample of an individual, (ii) from the isolated nucleic acid, bisulfite, Processing any one selected from the group consisting of hydrogen sulfite, disulfite, salts thereof, and combinations thereof, (iii) amplifying the nucleic acid using primers specific for the marker gene described above. , and (iv) the marker genes described above by methylation-specific polymerase chain reaction, real-time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, or bisulfite sequencing. It may be implemented including measuring the methylation level of one or more CpG regions.
한편, 본 발명의 또 다른 태양에 따르면, 췌장암의 치료 방법으로서, (i) 개체의 생물학적 시료로부터 분리된 핵산에서 상술한 마커 유전자 또는 이의 CpG 영역이 정상 대조군에 비해 저메틸화된 상태 또는 과메틸화 상태를 검출하는 단계; 및 (ii) 상기 개체에서 췌장암에 대한 치료요법을 시행하는 단계를 포함하는 방법이 제공된다.Meanwhile, according to another aspect of the present invention, there is a method of treating pancreatic cancer, wherein (i) the above-described marker gene or its CpG region in the nucleic acid isolated from the biological sample of the individual is in a hypomethylated or hypermethylated state compared to the normal control group. detecting; and (ii) administering therapy for pancreatic cancer in said individual.
일 구현예에서, 치료 방법은 저메틸화된 상태 또는 과메틸화된 상태가 검출되는 경우에, (ii)의 단계 전에 조직 생검 등의 추가 분석 또는 진단 방법을 통해 개체에 췌장암이 있음을 확인하는 단계를 추가로 포함할 수 있다. In one embodiment, the method of treatment includes confirming that the subject has pancreatic cancer through additional analysis or diagnostic methods, such as tissue biopsy, before step (ii) when a hypomethylated state or a hypermethylated state is detected. Additional items may be included.
일 구현예에서, 상기 췌장암에 대한 치료요법은 병기에 따라, 외과적 수술, 방사선 요법, 화학 요법, 표적 요법 또는 이들의 조합을 포함할 수 있다. In one embodiment, the treatment for pancreatic cancer may include surgery, radiation therapy, chemotherapy, targeted therapy, or a combination thereof, depending on the stage.
상기 각 마커 유전자, "생물학적 시료", "CpG 영역", "메틸화", 메틸화 상태를 검출하는 방법 등 동일 구성에 대한 구체적인 설명은 상기를 참조한다.For detailed descriptions of the same components, such as each marker gene, “biological sample,” “CpG region,” “methylation,” and method for detecting methylation status, please refer to the above.
췌장암 진단을 위한 조성물Composition for diagnosing pancreatic cancer
본 발명의 또 다른 태양에 따르면, 상술한 마커 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역에서 메틸화 상태를 검출 또는 측정하기 위한 제제를 포함하는 조성물이 제공된다. 구체적으로, 생물학적 시료에서 분리된 핵산을 이용하는 췌장암 진단을 위한 조성물로서, 상술한 마커 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 특이적으로 증폭하거나 이와 혼성화(hybridization)할 수 있는 제제 및 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제로부터 선택된 하나 이상의 제제를 포함하는 조성물이 제공된다. According to another aspect of the present invention, a composition comprising an agent for detecting or measuring the methylation status of one or more genes selected from the group consisting of the above-described marker genes or CpG regions thereof is provided. Specifically, it is a composition for diagnosing pancreatic cancer using nucleic acids isolated from biological samples, and is an agent capable of specifically amplifying or hybridizing one or more genes or their CpG regions selected from the group consisting of the above-mentioned marker genes, and A composition comprising one or more agents selected from agents that detect the methylation status of the one or more marker genes or CpG regions thereof is provided.
용어 "혼성화"는 이중체(duplex) 구조를 형성하는, 샘플 DNA 중 왓슨-크릭(Watson-Crick) 염기쌍과 유사한 상보성 서열에 대한 올리고뉴클레오티드의 결합으로 이해될 수 있다.The term “hybridization” can be understood as the binding of an oligonucleotide to a complementary sequence similar to Watson-Crick base pairs in sample DNA, forming a duplex structure.
상기 생물학적 시료는 혈액, 혈장 또는 혈청일 수 있지만, 이로 제한되지 않는다. 일 구현예에서, 상기 생물학적 시료로부터 분리된 핵산은 cfDNA일 수 있다.The biological sample may be, but is not limited to, blood, plasma, or serum. In one embodiment, the nucleic acid isolated from the biological sample may be cfDNA.
다른 구현예에서, 상기 제제는 상기 유전자에 상보적으로 결합하는 하나 이상의 올리고뉴클레오티드, 예컨대, 프라이머, 프로브 또는 안티센스 뉴클레오티드를 포함할 수 있다.In other embodiments, the agent may include one or more oligonucleotides, such as primers, probes, or antisense nucleotides, that bind complementary to the gene.
본 발명에서 사용되는 용어 "프라이머"는 시료 내의 유전자의 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고, 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 핵산 서열을 의미한다. 프라이머의 서열은 반드시 주형의 서열과 정확히 같을 필요는 없으며, 충분히 상보적이어서 주형과 혼성화될 수 있으면 된다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 제제 및 상이한 4가지 뉴클레오사이드 3인산 (nucleoside triphosphate)의 존재하에 DNA 합성을 개시할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 것을 기초로 변형할 수 있다.The term “primer” used in the present invention refers to a nucleic acid sequence that can form a base pair with a complementary template of a gene in a sample and serves as a starting point for copying the template strand. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but just needs to be sufficiently complementary to hybridize with the template. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization at appropriate buffer solutions and temperatures. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.
본 발명에서 사용되는 용어 "프로브"는 시료 내의 검출하고자 하는 유전자와 특이적으로 결합할 수 있는 물질로서, 상기 결합을 통하여 특이적으로 시료 내의 유전자의 존재를 확인할 수 있는 물질을 의미한다. 프로브는 올리고뉴클레오티드 프로브, 단쇄 DNA 프로브, 이중쇄 DNA 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당업계에 공지된 것을 기초로 변형할 수 있다.The term “probe” used in the present invention refers to a substance that can specifically bind to a gene to be detected in a sample, and can specifically confirm the presence of the gene in the sample through this binding. Probes may be manufactured in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
본 발명에서 사용되는 용어 "안티센스 뉴클레오티드"는 타겟으로 하는 유전자에 대한 상보적인 서열을 가지고 있어 타겟 유전자와 이합체를 형성할 수 있는 핵산 기반의 분자를 의미하며, 타겟 유전자를 검출하는 데 사용될 수 있다. 상기 안티센스 뉴클레오티드는 검출 특이성을 증가시키기 위하여 적절한 길이를 선택할 수 있다.The term “antisense nucleotide” used in the present invention refers to a nucleic acid-based molecule that has a complementary sequence to the target gene and can form a dimer with the target gene, and can be used to detect the target gene. The antisense nucleotide may be of an appropriate length to increase detection specificity.
본 발명에서, 프라이머, 프로브 또는 안티센스 뉴클레오티드와 같은 올리고뉴클레오티드는 메틸화 상태를 분석하는 대상이 되는 특정 CpG 영역의 서열에 따라 당업계에 알려진 공지된 지식 및 기술을 사용하여 바람직하게 디자인될 수 있다. In the present invention, oligonucleotides such as primers, probes or antisense nucleotides can be preferably designed using known knowledge and techniques known in the art according to the sequence of a specific CpG region whose methylation status is to be analyzed.
일 구현예에서, 상기 조성물은 상기 유전자의 CpG 영역의 메틸화 상태를 검출하기 위한 제제를 포함할 수 있다. 상기 메틸화 상태를 검출하기 위한 제제는 앞서 설명한 바를 참조한다. 예컨대, 상기 제제는 바이설파이트, 하이드로겐 설파이트, 다이설파이트, 이들의 염, 또는 이들의 조합일 수 있다. 그러나, 상기 제제는 이로 한정되지 않는다. 바이설파이트, 하이드로겐 설파이트, 다이설파이트와 같은 비메틸화 사이토신 염기를 변형시키는 화합물 및 이를 이용하여 비메틸화 사이토신 잔기를 변형시켜 유전자의 메틸화 여부를 검출하는 방법은 당업계에 잘 알려져 있다(WO 01/26536; US 2003/0148326 A1).In one embodiment, the composition may include an agent for detecting the methylation status of the CpG region of the gene. For the agent for detecting the methylation status, please refer to the previous description. For example, the agent may be bisulfite, hydrogen sulfite, disulfite, a salt thereof, or a combination thereof. However, the agent is not limited thereto. Compounds that modify unmethylated cytosine bases, such as bisulfite, hydrogen sulfite, and disulfite, and methods for detecting methylation of genes by modifying unmethylated cytosine residues using the same are well known in the art. (WO 01/26536; US 2003/0148326 A1).
또한, 타겟 유전자 부위의 염기서열의 변환된 정도로 확인하여 메틸화 여부 및 정도를 알아보기 위해 NGS가 이용될 수 있다. NGS에 의한 서열 분석은 Illumina사의 MiSeq, NextSeq500, NextSeq550, Hiseq 2500, Hiseq 4000, Hiseq X Ten, NovaSeq 6000, Thermo Fisher Scientific사의 Ion PGM, Ion Proton, Ion S5, BGI 사의 BGI-500, MGISEQ-2000, MGISEQ-T7 등을 이용하여 수행될 수 있지만 이에 제한되지 않는다.In addition, NGS can be used to determine the presence and extent of methylation by confirming the degree of conversion of the base sequence of the target gene region. Sequence analysis by NGS was performed using Illumina's MiSeq, NextSeq500, NextSeq550, Hiseq 2500, Hiseq 4000, Hiseq It can be performed using MGISEQ-T7, etc., but is not limited to this.
다른 구현예에서, 생물학적 시료에서 분리된 핵산을 이용하는 췌장암 진단을 위한 조성물로서, 상술한 마커 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제를 포함하는 조성물이 제공된다.In another embodiment, a composition for diagnosing pancreatic cancer using nucleic acids isolated from biological samples is provided, including an agent for detecting the methylation status of one or more genes selected from the group consisting of the above-described marker genes or a CpG region thereof. .
상기 구현예에서, 생물학적 시료, 핵산, 유전자, CpG 영역, 메틸화 상태를 검출하는 제제 등 동일한 구성에 대해서는 상술한 바를 참조한다.In the above embodiments, refer to the above description for the same components, such as biological samples, nucleic acids, genes, CpG regions, and agents for detecting methylation status.
키트kit
본 발명의 또 다른 태양에 따르면, 상기 조성물을 포함하는, 개체의 생물학적 시료로부터 분리된 핵산을 이용하여 췌장암 진단을 위한 키트를 제공한다. 구체적으로, 상술한 마커 유전자로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역에서 메틸화 상태를 검출 또는 측정하기 위한 제제를 포함하는 키트가 제공된다.According to another aspect of the present invention, a kit for diagnosing pancreatic cancer using nucleic acid isolated from a biological sample of an individual including the composition is provided. Specifically, a kit containing an agent for detecting or measuring the methylation status of one or more genes selected from the group consisting of the above-mentioned marker genes or their CpG region is provided.
상기 키트는 (i) 엄격하거나 또는 적당히 엄격한 조건하에서, 암에서는 메틸화되지만 비-암성 조직에서는 메틸화되지 않는 상술한 하나 이상의 췌장암 특이적 유전자에 혼성화될 수 있는 하나 이상 또는 다수의 올리고뉴클레오티드, 예컨대 프라이머, 프로브 또는 안티센스 뉴클레오티드; (ii) 상기 유전자에 혼성화될 수 있는 하나 이상 또는 다수의 올리고뉴클레오티드, 예컨대 프라이머, 프로브 또는 안티센스 뉴클레오티드 및 대상의 생물학적 샘플(예컨대, 생물학적 시료로부터 분리된 핵산)을 함유하기에 적합한 용기; (iii) (ii)의 혼성화를 검출하기 위한 수단; 및/또는 선택적으로 (iv) 사용 설명서 및 키트 결과의 해석을 포함할 수 있다. The kit includes (i) one or more oligonucleotides, such as primers, that can hybridize under stringent or moderately stringent conditions to one or more of the pancreatic cancer-specific genes described above that are methylated in cancer but not methylated in non-cancerous tissue; Probe or antisense nucleotide; (ii) a container suitable for containing one or more oligonucleotides, such as primers, probes or antisense nucleotides, capable of hybridizing to the gene and a biological sample of interest (e.g., a nucleic acid isolated from the biological sample); (iii) means for detecting hybridization of (ii); and/or optionally (iv) instructions for use and interpretation of kit results.
상기 키트는 또한 별도의 용기에 패키징된 혼성화 용액을 포함한 다른 성분들을 함유할 수 있다. 상기 혼성화 용액에서 상기 유전자의 핵산과 다수의 프라이머, 프로브 또는 안티센스 뉴클레오티드가 혼성화될 수 있다. The kit may also contain other components, including hybridization solution, packaged in separate containers. In the hybridization solution, the nucleic acid of the gene and a plurality of primers, probes, or antisense nucleotides may hybridize.
구체적으로, 상기 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치로 구성된다. 예를 들어, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(Enzyme-linked immunosorbent assay) 키트, 단백질 칩 키트 또는 래피드(rapid) 키트일 수 있다. 상기 키트는 상기 제제 이외에도 중합효소 아가로스, 전기영동에 필요한 완충용액 등이 추가로 포함될 수 있다. Specifically, the kit consists of one or more different component compositions, solutions, or devices suitable for the analysis method. For example, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, or a rapid kit. In addition to the above agents, the kit may additionally include polymerase agarose, a buffer solution required for electrophoresis, etc.
일 구현예에 따르면, 상기 키트는, 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역을 포함하는 검출 대상 유전자 또는 이의 CpG 영역을 포함하는 단편과 혼성화할 수 있는 프로브가 고정되어 있는 진단용 핵산 칩(CHIP) 또는 유전자 패널을 포함할 수 있다. According to one embodiment, the kit is a diagnostic nucleic acid chip (CHIP) on which a probe capable of hybridizing with a target gene to be detected including the one or more marker genes or a CpG region thereof or a fragment including a CpG region thereof is immobilized, or May include a genetic panel.
또한, 본 발명의 또 다른 태양에 따르면, 상술한 하나 이상의 마커 유전자 또는 이의 CpG 영역을 포함하는 검출 대상 유전자 또는 이의 CpG 영역을 포함하는 단편과 혼성화할 수 있는 프로브가 고정되어 있는 진단용 핵산 칩 또는 유전자 패널이 제공된다. In addition, according to another aspect of the present invention, a diagnostic nucleic acid chip or gene on which a probe capable of hybridizing with a detection target gene containing the above-described one or more marker genes or a CpG region thereof or a fragment containing a CpG region thereof is immobilized. A panel is provided.
구체적으로, 상기 핵산 칩은 올리고뉴클레오티드 및/또는 PNA(Peptide Nucleic Acid)-올리고머의 배열이 고체상에 결합된 형태 예를 들면, 직사각형 또는 육각형 격자의 형태로 고체상 위에 배열되는 것을 특징으로 할 수 있다. 고체상 표면은 실리콘, 유리, 폴리스티렌, 알루미늄, 스틸, 철, 구리, 니켈, 은 또는 금으로 이루어질 수 있다. 추가적으로, 니트로셀룰로스, 및 펠릿의 형태로 존재하거나 또는 또한 수지 매트릭스로서 존재할 수 있는 나일론과 같은 플라스틱도 또한 사용될 수 있다. 핵산 칩의 제조는 선행 문헌인 네이처 제네틱스(Nature Genetics)의 특별판 [Nature Genetics Supplement, Volume 21, January 1999] 및 그에 인용된 문헌들을 참조한다. 상기 올리고뉴클레오티드는 프라이머, 프로브 또는 안티센스 뉴클레오티드를 모두 포함하는 개념으로 해석될 수 있으며, 상기 "PNA"는 인공적으로 합성된 올리고뉴클레오티드를 의미한다. 형광 표지된 프로브도 고정화된 DNA 칩의 스캐닝에 사용될 수 있으며, Cy3 및 Cy5 염료의 특정 프로브의 5'-OH에 대한 단순한 결합이 형광성 표지에 특히 적합하다. 혼성화된 프로브의 형광 검출은, 예를 들면, 공초점 현미경에 의해 수행될 수 있다.Specifically, the nucleic acid chip may be characterized in that an array of oligonucleotides and/or PNA (Peptide Nucleic Acid)-oligomers are bound to a solid phase and arranged on a solid phase, for example, in the form of a rectangular or hexagonal lattice. The solid surface may be made of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold. Additionally, plastics such as nitrocellulose and nylon, which may exist in the form of pellets or also as a resin matrix, may also be used. For the production of nucleic acid chips, refer to the preceding literature, Nature Genetics' special edition [Nature Genetics Supplement, Volume 21, January 1999] and the literature cited therein. The oligonucleotide can be interpreted as a concept that includes all primers, probes, or antisense nucleotides, and the “PNA” refers to an artificially synthesized oligonucleotide. Fluorescently labeled probes can also be used for scanning immobilized DNA chips, and the simple binding of Cy3 and Cy5 dyes to the 5'-OH of specific probes is particularly suitable for fluorescent labeling. Fluorescence detection of hybridized probes can be performed, for example, by confocal microscopy.
이하, 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위해 제시된 것으로, 어떠한 방식으로든 이의 범위를 제한하려는 의도가 아니며, 제한하려는 것으로 해석되어서는 안 된다. Hereinafter, the present invention will be explained in more detail by the following examples. The following examples are presented to aid understanding of the present invention and are not intended to limit its scope in any way and should not be construed as limiting.
실시예Example
실시예 1. 임상 연구 개요Example 1. Clinical study overview
2020년 3월부터 2021년 12월까지 강북삼성병원 예방건진센터에서 국민건강보험공단 일반검진과 암검진을 받은 수검자들의 혈장 시료를 제공받았다. 이때, 수검자들의 개인 정보는 일체 수집되지 않았다.From March 2020 to December 2021, plasma samples were provided from examinees who underwent National Health Insurance Corporation general examination and cancer screening at the Kangbuk Samsung Hospital Preventive Health Checkup Center. At this time, no personal information of the examinees was collected.
상기 검체를 췌장암 환자군과 정상 대조군으로 분류하였다. 환자군의 경우, 췌장암은 소화기 전문의에 의해 TNM 병기 체계 및 AJCC(American Joint Committee on Cancer, 미국암연합회) 병기 분류법에 따라 0기부터 4기까지 분류되었다.The samples were classified into pancreatic cancer patient group and normal control group. For the patient group, pancreatic cancer was classified from stage 0 to stage 4 by gastroenterologists according to the TNM staging system and the American Joint Committee on Cancer (AJCC) staging system.
이렇게 분류된 검체를 이용하여 도 1에 제시된 과정을 수행하였다. 그 결과, 췌장암 환자군에서 차별적으로 메틸화 수준이 변화되어 있는 11종의 메틸화 마커 유전자 XXYLT1, ZNF879, EPS8L2, TRPC6, AVPR1A, GSC, TBCD, MAFB, TCEA2, MIRLET7BHG 및 BNC1를 발굴하였다. 또한, 이렇게 발굴된 11종의 메틸화 마커 유전자와 관련된 정보에 기반하여 기계학습 알고리즘에 의해 췌장암 식별 모델을 수립하고, 이러한 모델을 검증하였다.The process shown in Figure 1 was performed using the samples classified in this way. As a result, 11 methylation marker genes XXYLT1, ZNF879, EPS8L2, TRPC6, AVPR1A, GSC, TBCD, MAFB, TCEA2, MIRLET7BHG, and BNC1, whose methylation levels were differentially changed in the pancreatic cancer patient group, were discovered. In addition, based on the information related to the 11 methylation marker genes discovered in this way, a pancreatic cancer identification model was established using a machine learning algorithm, and this model was verified.
이하의 실시예에서는 이러한 과정을 상세히 설명한다.The following examples describe this process in detail.
실시예 2. 무세포 DNA 추출Example 2. Cell-free DNA extraction
차세대 염기서열 분석을 위한 준비 과정으로서, 각 검체를 3000 rpm에서 15분간 원심분리하여 혈장을 분리하였다. 분리된 혈장으로부터 무세포 DNA의 추출은 MagMAX™ Cell-Free DNA Isolation Kit(Thermo fisher scientific)를 이용하여 수행하였다. 추출 방법은 제조사의 매뉴얼에 따라 진행하였다. 추출된 무세포 DNA는 형광 강도 측정기(Fluorometer)(Qubit Flex, Invitrogen)를 이용하여 정량하였으며, DNA 상태는 자동화된 전기영동 장비(4150 Tapestation system, Agilent)를 이용하여 DNA degradation 여부 및 크기를 확인하였다.In preparation for next-generation sequencing, each sample was centrifuged at 3000 rpm for 15 minutes to separate plasma. Extraction of cell-free DNA from separated plasma was performed using the MagMAX™ Cell-Free DNA Isolation Kit (Thermo fisher scientific). The extraction method was performed according to the manufacturer's manual. Extracted cell-free DNA was quantified using a fluorescence intensity meter (Qubit Flex, Invitrogen), and DNA status was checked for DNA degradation and size using automated electrophoresis equipment (4150 Tapestation system, Agilent). .
실시예 3. 라이브러리 제작 및 정도관리(QC)Example 3. Library production and quality control (QC)
실시예 2에서 추출된 무세포 DNA에 EZ DNA methylation-Lightning™ kit(Zymo Research, USA)를 이용하여 바이설파이트를 처리하였다.The cell-free DNA extracted in Example 2 was treated with bisulfite using the EZ DNA methylation-Lightning™ kit (Zymo Research, USA).
무세포 DNA에 바이설파이트를 처리하면, 비메틸화된 사이토신은 우라실로 변형되고, 메틸화된 사이토신은 변화없이 남게 된다. 상기 바이설파이트가 처리된 DNA를 멸균 증류수 20㎕로 용출시켜 차세대 염기서열 분석을 위한 라이브러리를 제작하였다.When cell-free DNA is treated with bisulfite, unmethylated cytosine is modified to uracil, and methylated cytosine remains unchanged. The bisulfite-treated DNA was eluted with 20 μl of sterilized distilled water to prepare a library for next-generation sequencing.
라이브러리 제작용 준비 시약으로서 Accel-NGS® Methyl-Seq DNA Library Kit(Swift Biosciences, USA)를 사용하였다. 이 과정은 메틸화 어댑터 라이게이션, 라이브러리의 인덱싱, AMPure XP 비드를 이용한 라이게이션된 DNA의 순수 분리 과정을 포함한다.Accel-NGS® Methyl-Seq DNA Library Kit (Swift Biosciences, USA) was used as a preparation reagent for library production. This process includes methylation adapter ligation, indexing of the library, and pure isolation of ligated DNA using AMPure XP beads.
바이설파이트-라이브러리는 제작된 패널을 사용하는 단일 혼합 반응으로 설계하였고, SureSelectXT Human Methyl Seq Kit(Agilent, USA)를 이용하여 라이브러리를 완성하였다. 이 과정은 라이브러리 혼성화, 스트렙트아비딘 비드를 이용한 하이브리드 캡쳐, 라이브러리 증폭, AMPure XP 비드를 이용한 순수 분리 과정을 포함한다.The bisulfite-library was designed as a single mixing reaction using the fabricated panel, and the library was completed using the SureSelectXT Human Methyl Seq Kit (Agilent, USA). This process includes library hybridization, hybrid capture using streptavidin beads, library amplification, and pure separation using AMPure XP beads.
제작된 라이브러리가 염기서열 분석에 적합한지 확인하기 위하여, Agilent Bioanalyzer 2200(Agilent, USA) 기기 및 High Sensitivity chip을 이용하여 라이브러리의 길이와 양을 측정하였고, 제조사가 제시하는 정도 관리(QC) 조건에 만족하는 라이브러리를 서열분석에 사용하였다.In order to confirm whether the produced library is suitable for base sequence analysis, the length and amount of the library were measured using an Agilent Bioanalyzer 2200 (Agilent, USA) instrument and a high sensitivity chip, and the quality control (QC) conditions suggested by the manufacturer were measured. Satisfactory libraries were used for sequence analysis.
실시예 4. 차세대 염기서열 분석 수행Example 4. Performing next-generation sequencing
실시예 3에서 제작된 라이브러리에 대해 차세대 염기서열 분석 장비(NovaSeq 6000, Illumina)를 이용하여 바이설파이트 염기서열 분석을 수행하였다. 서열분석 장비로부터 생산된 염기량, 퀄리티 스코어(Q30), 온-타겟(on-target) 비율, 타겟 영역별 분석에 필요한 평균 리드 개수, 고유한 분자 식별자(Unique Molecular Identifier, UMI) 중복 비율, 매핑된 데이터량 등을 확인하여 생성된 데이터의 퀄리티를 평가하였다. 이후, 무세포 DNA의 8,400만개 염기 가운데 총 370만개 CpG 부위를 타겟하여 그 메틸화 수준을 분석하였다. Bisulfite sequencing was performed on the library produced in Example 3 using next-generation sequencing equipment (NovaSeq 6000, Illumina). Quantity of bases produced from sequencing equipment, quality score (Q30), on-target ratio, average number of reads required for analysis by target region, Unique Molecular Identifier (UMI) duplication ratio, mapping The quality of the generated data was evaluated by checking the amount of data generated. Afterwards, a total of 3.7 million CpG sites out of 84 million bases of cell-free DNA were targeted and their methylation levels were analyzed.
실시예 5. DNA 메틸화 분석 및 메틸화 마커 유전자 발굴Example 5. DNA methylation analysis and methylation marker gene discovery
실시예 4에서 캡쳐된 타겟 영역인 총 370만개 CpG 좌위에 대하여 DNA 메틸화 정도를 측정하였다.The degree of DNA methylation was measured for a total of 3.7 million CpG loci, which were the target regions captured in Example 4.
DNA 메틸화 정도는 실시예 4에서 수행된 차세대 염기서열 분석 결과에 기반하여 측정하였다. 각각의 CpG 좌위에서 메틸화된 염기서열 단편(리드(read))과 메틸화되지 않은 리드를 각각 카운트하고 그 값을 아래의 식에 대입하여 계산하였다:The degree of DNA methylation was measured based on the results of next-generation sequencing performed in Example 4. At each CpG locus, methylated base sequence fragments (reads) and unmethylated reads were counted and calculated by substituting the values into the formula below:
상기 식에서,In the above equation,
계산된 메틸화 정도는 0 내지 1 범위의 값으로 표시되며, 값 0은 해당 CpG 좌위가 완전히 메틸화되지 않은 것을 의미하며, 1은 해당 CpG 좌위가 완전히 메틸화된 것을 의미한다.The calculated degree of methylation is expressed as a value ranging from 0 to 1, where a value of 0 means that the corresponding CpG locus is not completely methylated, and a value of 1 means that the corresponding CpG locus is completely methylated.
췌장암 환자군과 정상 대조군에서 차별적으로 메틸화되어 있는 유전자 영역(Differentially methylated region, DMR)을 발굴하기 위하여 우선 개개의 CpG 좌위에 대해 t-test, wald test, z-score 방법을 사용하여 유의성을 평가하였다. 이후, 개개의 CpG 좌위에서 유의하게 나타난(p 값<0.05) 영역들에 대해 다양한 인자의 여러 값에 대해 평가한 후에 최적의 조합을 선정하였다. 최적화 테스트에 포함된 상기 다양한 인자의 예로는 최소 CpG 수, DMR 구간의 최소 길이, p 값 한계치, 윈도우(window) 크기 등이 있다.To discover differentially methylated regions (DMRs) in pancreatic cancer patients and normal controls, the significance of each CpG locus was first evaluated using t-test, wald test, and z-score methods. Afterwards, the optimal combination was selected after evaluating various values of various factors for regions that appeared significantly (p value <0.05) in each CpG locus. Examples of the various factors included in the optimization test include minimum number of CpGs, minimum length of DMR interval, p value threshold, window size, etc.
평가는 유전자와 DMR 단위로 진행하였고, 여러 기계학습 알고리즘을 사용하여 leave-one-out 테스트로 진행하였다. 이때, 기계학습 알고리즘으로는 서포트 벡터 머신(Support Vector Machine), 랜덤 포레스트(Random Forest), 신경망 네트워크(Neural Network) 및 딥러닝(Deep Learning)을 사용하였다.The evaluation was conducted on a gene and DMR basis, and was conducted as a leave-one-out test using several machine learning algorithms. At this time, the machine learning algorithms used were Support Vector Machine, Random Forest, Neural Network, and Deep Learning.
그 결과, 췌장암 환자군에서 차별적으로 메틸화 수준이 변화되어 있는 11종의 유전자 및 각 유전자에 포함된 DMR을 선별하였다(표 2). 11종의 유전자는 기존 유전자(BNC1) 및 신규 유전자(XXXLT1, ZNF879, EPS8L2, TRPC6, AVPR1A, GSC, TBCD, MAFB, TCEA2 및 MIRLET7BHG)로 구성되었다. As a result, 11 genes whose methylation levels were differentially changed in the pancreatic cancer patient group and the DMRs contained in each gene were selected (Table 2). The 11 genes consisted of existing genes (BNC1) and novel genes (XXXLT1, ZNF879, EPS8L2, TRPC6, AVPR1A, GSC, TBCD, MAFB, TCEA2, and MIRLET7BHG).
| 유전자 명칭gene name | 염색체 번호chromosome number | DMR 위치 (GRCh37)DMR location (GRCh37) | DMR 크기DMR size |
메틸화 상태methylation status
+:과메틸화+: Hypermethylation -:저메틸화-:Hypomethylated |
|
| 시작start | 끝end | ||||
| XXYLT1XXYLT1 | chr3chr3 | 194,800,905194,800,905 | 194,802,072194,802,072 | 1,1671,167 | -- |
| ZNF879ZNF879 | chr5chr5 | 178,450,154178,450,154 | 178,451,476178,451,476 | 1,3221,322 | ++ |
| EPS8L2EPS8L2 | chr11chr11 | 704,570704,570 | 713,015713,015 | 8,4458,445 | -- |
| TRPC6TRPC6 | chr11chr11 | 101,453,938101,453,938 | 101,455,234101,455,234 | 1,2961,296 | ++ |
| AVPR1AAVPR1A | chr12chr12 | 63,543,59863,543,598 | 63,544,75663,544,756 | 1,1581,158 | ++ |
| GSCGSC | chr14chr14 | 95,237,29195,237,291 | 95,238,48995,238,489 | 1,1981,198 | ++ |
| TBCDTBCD | chr17chr17 | 80,831,80880,831,808 | 80,833,01280,833,012 | 1,2041,204 | -- |
| MAFBMAFB | chr20chr20 | 39,318,91539,318,915 | 39,320,06839,320,068 | 1,1531,153 | ++ |
| TCEA2TCEA2 | chr20chr20 | 62,696,70162,696,701 | 62,697,89662,697,896 | 1,1951,195 | -- |
| MIRLET7BHGMIRLET7BHG | chr22chr22 | 46,479,06546,479,065 | 46,481,48046,481,480 | 2,4152,415 | -- |
| BNC1BNC1 | chr15chr15 | 83,951,73083,951,730 | 83,954,13383,954,133 | 2,4032,403 | ++ |
상기 11종의 유전자의 DMR에서 췌장암 환자군 33례와 정상 대조군 42례의 샘플을 이용한 메틸화 수준을 타겟 영역 기반 바이설파이트 시퀀싱을 통해 분석하였고, 그 결과를 도 2에 도시하였다. Methylation levels in the DMRs of the above 11 genes using samples from 33 pancreatic cancer patients and 42 normal controls were analyzed through target region-based bisulfite sequencing, and the results are shown in Figure 2.
실시예 6. 기계학습 모델 수립 및 평가Example 6. Machine learning model establishment and evaluation
실시예 5에서 발굴된 마커 유전자 내에 존재하는 DMR 영역에서의 메틸화 수치를 기반으로 기계학습 알고리즘을 이용하여 췌장암 식별 모델을 수립하였다.A pancreatic cancer identification model was established using a machine learning algorithm based on the methylation level in the DMR region present in the marker gene discovered in Example 5.
일반적인 모델링 프로그램은 초기 입력값에 어떤 조건이 만족되면 최종 결과를 내보내도록 개발자가 미리 프로그래밍하는 명시적 프로그래밍에 의해 만들어진다. 반면, 기계학습에서는 초기 입력값이 들어왔을 때 결과가 특정 값이 되는 조건을 찾도록 컴퓨터를 학습시킨다. 이러한 학습 과정은 입력값에 대해 결과값이 제대로 도출되도록 최적의 매개변수를 찾는 과정이며, 학습의 결과물은 최적의 매개변수 혹은 가중치 값이다. A typical modeling program is created through explicit programming in which the developer pre-programs it to output the final result when certain conditions are met for the initial input values. On the other hand, machine learning trains the computer to find conditions under which the result will be a specific value when the initial input value is received. This learning process is a process of finding optimal parameters so that the result value can be properly derived for the input value, and the result of learning is the optimal parameter or weight value.
딥러닝 혹은 신경망 네트워크와 같은 기계학습 알고리즘을 이용하여 수립된 모델은 다수의 정상 대조군과 췌장암 환자군 샘플에서 얻은 마커 유전자 영역의 메틸화 패턴을 학습시킴으로써 정상 대조군과 상이한 췌장암 환자군 특이적 메틸화 패턴을 식별할 수 있도록 만들어진 수식 모델이라고 할 수 있다. 이렇게 만들어진 모델을 활용하면 새로운 패턴의 데이터에 대해 추론하고 예측할 수 있게 된다. 따라서, 상기 수립된 모델은 주어진 테스트 샘플이 췌장암 환자군 샘플에서 나타나는 메틸화 패턴을 보이는지 여부를 평가하여, 그러한 테스트 샘플이 유래된 대상체가 정상인지 췌장암이 있는지에 대한 판정 결과를 도출해 낸다.A model established using machine learning algorithms such as deep learning or neural networks can identify pancreatic cancer patient-specific methylation patterns that are different from normal controls by learning the methylation patterns of marker gene regions obtained from multiple normal control and pancreatic cancer patient samples. It can be said to be a mathematical model created for this purpose. Using the model created in this way, it is possible to infer and predict new patterns of data. Accordingly, the established model evaluates whether a given test sample shows a methylation pattern that appears in a pancreatic cancer patient sample, and derives a determination result as to whether the subject from which the test sample is derived is normal or has pancreatic cancer.
이렇게 11종의 마커 유전자를 기반으로 수립된 모델을 평가하기 위한 일례로, 딥러닝 알고리즘을 이용하여 leave-one-out 테스트를 실시하였다. 딥러닝 알고리즘은 여러 층의 인공신경망(Artificial Neural Network)을 이용하여 학습을 수행하는 기계학습의 한 종류이다. 기존 기계학습 알고리즘의 경우 데이터 분석자가 학습 데이터에서 어떤 특징을 추출해야 하는지 직접 관여해야 하는 반면, 딥러닝의 경우 컴퓨터가 자동으로 학습 데이터에서 특징을 추출하여 자가 학습을 한다. 이러한 딥러닝 테스트 결과를 표 3에 나타낸다.As an example to evaluate the model established based on 11 types of marker genes, a leave-one-out test was performed using a deep learning algorithm. Deep learning algorithms are a type of machine learning that performs learning using multiple layers of artificial neural networks. In the case of existing machine learning algorithms, data analysts must be directly involved in determining which features should be extracted from the training data, while in the case of deep learning, the computer automatically extracts features from the training data and performs self-learning. The results of these deep learning tests are shown in Table 3.
표 3에는, 본 발명에서 발굴된 11종 마커 유전자 중 2종 내지 3종을 기반으로 수립된 모델에 대해 계산된 민감도, 특이도 및 정확도 수치와 함께, 이들 유전자를 단일 마커로 적용시 계산된 해당 수치가 함께 제시되어 있다.Table 3 shows the sensitivity, specificity, and accuracy values calculated for the model established based on 2 to 3 of the 11 marker genes discovered in the present invention, as well as the corresponding values calculated when these genes are applied as single markers. The figures are presented together.
| 유전자 타겟 조합/단일 마커Gene target combination/single marker | 민감도 (%)Sensitivity (%) | 특이도 (%)Specificity (%) | 정확도 (%)accuracy (%) | 신규마커/기존마커New marker/Existing marker |
| BNC1, TRPC6, MIRLET7BHGBNC1, TRPC6, MIRLET7BHG | 90.91%90.91% | 95.24%95.24% | 93.33%93.33% | 기존마커 및 신규마커의 조합Combination of existing and new markers |
| BNC1, AVPR1A, MIRLET7BHGBNC1, AVPR1A, MIRLET7BHG | 90.91%90.91% | 95.24%95.24% | 93.33%93.33% | 기존마커 및 신규마커의 조합Combination of existing and new markers |
| BNC1, MIRLET7BHGBNC1, MIRLET7BHG | 93.94%93.94% | 90.48%90.48% | 92.00%92.00% | 기존마커 및 신규마커의 조합Combination of existing and new markers |
| BNC1, TBCDBNC1, TBCD | 96.97%96.97% | 83.33%83.33% | 89.33%89.33% | 기존마커 및 신규마커의 조합Combination of existing and new markers |
| BNC1, EPS8L2BNC1, EPS8L2 | 96.97%96.97% | 71.43%71.43% | 82.67%82.67% | 기존마커 및 신규마커의 조합Combination of existing and new markers |
| BNC1, MIRLET7BHG, EPS8L2BNC1, MIRLET7BHG, EPS8L2 | 96.97%96.97% | 78.57%78.57% | 86.67%86.67% | 기존마커 및 신규마커의 조합Combination of existing and new markers |
| MIRLET7BHG, EPS8L2MIRLET7BHG, EPS8L2 | 72.73%72.73% | 95.24%95.24% | 85.33%85.33% | 신규마커 조합New marker combination |
| MIRLET7BHG, EPS8L2, TBCDMIRLET7BHG, EPS8L2, TBCD | 90.91%90.91% | 80.95%80.95% | 85.33%85.33% | 신규마커 조합New marker combination |
| AVPR1AAVPR1A | 63.64%63.64% | 90.48%90.48% | 78.67%78.67% | 신규new |
| GSCGSC | 87.88%87.88% | 78.57%78.57% | 82.67%82.67% | 신규new |
| MAFBMAFB | 84.85%84.85% | 69.05%69.05% | 76.00%76.00% | 신규new |
| TRPC6TRPC6 | 69.70%69.70% | 97.62%97.62% | 85.33%85.33% | 신규new |
| ZNF879ZNF879 | 63.64%63.64% | 80.95%80.95% | 73.33%73.33% | 신규new |
| EPS8L2EPS8L2 | 87.88%87.88% | 76.19%76.19% | 81.33%81.33% | 신규new |
| TBCDTBCD | 81.82%81.82% | 90.48%90.48% | 86.67%86.67% | 신규new |
| MIRLET7BHGMIRLET7BHG | 72.73%72.73% | 97.62%97.62% | 86.67%86.67% | 신규new |
| TCEA2TCEA2 | 72.73%72.73% | 83.33%83.33% | 78.67%78.67% | 신규new |
| XXYLT1XXYLT1 | 90.91%90.91% | 64.29%64.29% | 76.00%76.00% | 신규new |
| BNC1BNC1 | 78.79%78.79% | 95.24%95.24% | 88.00%88.00% | 기존existing |
또한, 상기 메틸화 마커 유전자 BNC1 및 MIRLET7BHG의 조합모델에 대해 환자군 33례와 정상 대조군 42례의 샘플을 이용하여 leave-one-out 테스트를 수행하여 얻은 민감도 수치를 췌장암 기수별로 정리한 결과를 도 3에 도시하였다. 도 3으로부터 알 수 있는 바와 같이, 상기 모델은 1기의 경우에는 50%, 그리고 2기 내지 4기의 경우에는 100%의 민감도를 나타내었다.In addition, the sensitivity values obtained by performing a leave-one-out test using samples from 33 patients and 42 normal controls for the combination model of the methylation marker genes BNC1 and MIRLET7BHG are summarized by pancreatic cancer stage in Figure 3. Shown. As can be seen from Figure 3, the model showed a sensitivity of 50% for stage 1 and 100% for stages 2 to 4.
비교예 1. BNC1 유전자 및 ADAMTS1 유전자의 마커 조합Comparative Example 1. Marker combination of BNC1 gene and ADAMTS1 gene
췌장암 진단 마커로 알려져 있는 BNC1 유전자는 민감도 및 특이도 측면에서 개별 유전자보다 ADAMTS1(ADAM Metallopeptidase With Thrombospondin Type 1 Motif 1) 유전자와 조합하여 사용되는 경우 췌장암의 조기 진단 마커로 유용하게 사용될 수 있음이 선행 문헌에서 밝혀진 바 있으며, 그 결과를 표 4에 정리하였다(비특허문헌 1: Eissa, Maryam AL, et al. Clinical epigenetics 11.1 (2019): 1-10; 및 비특허문헌 2: Yi, Joo Mi, et al. Clinical Cancer Research 19.23 (2013): 6544-6555.). 상기 두 개의 비특허문헌은 정량적 메틸화 특이적 PCR(Quantitative Methylation Specific PCR)을 수행하여 췌장암의 진단 마커를 측정하였다. 구체적으로, 비특허문헌 1의 경우 TaqMan 프로브를 사용하였으며 비특허문헌 2의 경우 SYBR을 사용하였다.Previous literature has shown that the BNC1 gene, known as a diagnostic marker for pancreatic cancer, can be more useful as an early diagnostic marker for pancreatic cancer when used in combination with the ADAMTS1 (ADAM Metallopeptidase With Thrombospondin Type 1 Motif 1) gene than with individual genes in terms of sensitivity and specificity. It has been revealed in, and the results are summarized in Table 4 (Non-patent Document 1: Eissa, Maryam AL, et al. Clinical epigenetics 11.1 (2019): 1-10; and Non-patent Document 2: Yi, Joo Mi, et al. al. Clinical Cancer Research 19.23 (2013): 6544-6555.) The above two non-patent documents measured diagnostic markers for pancreatic cancer by performing quantitative methylation specific PCR (Quantitative Methylation Specific PCR). Specifically, in the case of non-patent document 1, a TaqMan probe was used, and in the case of non-patent document 2, SYBR was used.
| 비특허문헌 1Non-patent Document 1 | 비특허문헌 2Non-patent Document 2 | |||||
| 유전자 조합genetic combination | BNC1BNC1 | ADAMTS1ADAMTS1 | BNC1+ ADAMTS1BNC1+ ADAMTS1 | BNC1BNC1 | ADAMTS1ADAMTS1 | BNC1+ ADAMTS1BNC1+ ADAMTS1 |
| 민감도(%)responsiveness(%) | 64.1%64.1% | 87.2%87.2% | 97.3%97.3% | 79.0%79.0% | 48.0%48.0% | 81.0%81.0% |
| 특이도(%)Specificity (%) | 93.7%93.7% | 95.8%95.8% | 91.6%91.6% | 89.0%89.0% | 92.0%92.0% | 85.0%85.0% |
표 4의 결과에서, BNC1 및 ADAMTS1 유전자는 개별적 사용보다 조합해서 사용시 민감도 및 특이도가 함께 증가함을 확인할 수 있었지만, 측정 방법에 따라 결과값에 차이가 존재함을 확인할 수 있었다. 추가적으로, NGS 방법을 이용하여 상기 두 비특허문헌의 결과를 검증한 결과, 민감도 측면에서는 개별 유전자보다 유전자 조합이 우수한 진단 마커로 확인되었으나, 특이도 측면에서는 개선된 바가 없었다(표 5). From the results in Table 4, it was confirmed that the sensitivity and specificity of the BNC1 and ADAMTS1 genes increased when used in combination rather than individually, but it was confirmed that there was a difference in the result depending on the measurement method. Additionally, as a result of verifying the results of the two non-patent documents above using the NGS method, the gene combination was confirmed to be a better diagnostic marker than individual genes in terms of sensitivity, but there was no improvement in specificity (Table 5).
| NGS를 이용한 검증Verification using NGS | |||
| 유전자 조합genetic combination | BNC1BNC1 | ADAMTS1ADAMTS1 | BNC1+ ADAMTS1BNC1+ ADAMTS1 |
| 민감도(%)responsiveness(%) | 75.8%75.8% | 87.9%87.9% | 96.9%96.9% |
| 특이도(%)Specificity (%) | 95.2%95.2% | 69.0%69.0% | 69.0%69.0% |
이와 비교하여, 본 발명의 실시예 1 내지 6에서 확인한 신규 발굴된 췌장암 마커 및 그의 조합은 민감도, 특이도 및 정확도 측면에서 우수함이 증명되었다(표 3). 특히, 기존 마커 유전자인 BNC1 및 신규 마커인 MIRLET7BHG를 포함한 마커 유전자 조합은 췌장암 2기 내지 4기에서 우수한 마커로 확인되었으며, 췌장암 1기에서도 유효한 결과를 보였으므로(도 3), 췌장암의 조기 진단에 유용하게 사용될 수 있다.In comparison, the newly discovered pancreatic cancer markers and their combinations identified in Examples 1 to 6 of the present invention were proven to be superior in terms of sensitivity, specificity, and accuracy (Table 3). In particular, the combination of marker genes including BNC1, an existing marker gene, and MIRLET7BHG, a new marker, was confirmed as an excellent marker in stages 2 to 4 pancreatic cancer, and also showed effective results in stage 1 pancreatic cancer (Figure 3), making it suitable for early diagnosis of pancreatic cancer. It can be useful.
Claims (33)
- 췌장암 진단을 위한 정보를 제공하는 방법으로서, As a method of providing information for diagnosing pancreatic cancer,개체의 생물학적 시료로부터 분리된 핵산에서, MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2)로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역의 메틸화 상태를 측정하는 단계를 포함하는 In nucleic acids isolated from biological samples of individuals, MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside Xylosyltransferase 1), ZNF879 (Zinc Finger Protein 879), EPS8L2 (EPS8 Like 2), TRPC6 (Transient Receptor Potential Cation Channel Subfamily C Member 6) ), one or more genes selected from the group consisting of AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) Comprising the step of measuring the methylation status of the CpG region방법.method.
- 제1항에 있어서, According to paragraph 1,상기 하나 이상의 유전자는 MIRLET7BHG를 포함하는 The one or more genes include MIRLET7BHG방법.method.
- 제2항에 있어서, According to paragraph 2,상기 하나 이상의 유전자는 TRPC6, AVPR1A, EPS8L2, TBCD 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함하는 The one or more genes further include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1.방법.method.
- 제2항에 있어서, According to paragraph 2,상기 하나 이상의 유전자는 EPS8L2 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함하는The one or more genes further include one or more genes selected from the group consisting of EPS8L2 and BNC1.방법.method.
- 제4항에 있어서, According to paragraph 4,상기 하나 이상의 유전자는 TRPC6, AVPR1A 또는 TBCD를 추가로 포함하는 The one or more genes further include TRPC6, AVPR1A or TBCD방법.method.
- 제2항에 있어서, According to paragraph 2,상기 메틸화 상태를 측정하는 단계는 하기 (i), (ii), (iii), (iv), (v) 또는 (vi) 조합의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 측정하는 것을 포함하는The step of measuring the methylation status includes measuring the methylation status of the marker gene or its CpG region of the following combination (i), (ii), (iii), (iv), (v) or (vi)방법:method:(i) BNC1, TRPC6 및 MIRLET7BHG;(i) BNC1, TRPC6, and MIRLET7BHG;(ii) BNC1, AVPR1A 및 MIRLET7BHG;(ii) BNC1, AVPR1A, and MIRLET7BHG;(iii) BNC1 및 MIRLET7BHG; (iii) BNC1 and MIRLET7BHG;(iv) BNC1, MIRLET7BHG 및 EPS8L2; (iv) BNC1, MIRLET7BHG, and EPS8L2;(v) MIRLET7BHG 및 EPS8L2; 또는 (v) MIRLET7BHG and EPS8L2; or(vi) MIRLET7BHG, EPS8L2 및 TBCD.(vi) MIRLET7BHG, EPS8L2, and TBCD.
- 제1항에 있어서,According to paragraph 1,상기 하나 이상의 유전자는 TBCD 및 EPS8L2로 이루어진 군에서 선택된 하나 이상의 유전자를 포함하는 The one or more genes include one or more genes selected from the group consisting of TBCD and EPS8L2.방법.method.
- 제7항에 있어서,In clause 7,상기 하나 이상의 유전자는 BNC1을 추가로 포함하는 The one or more genes further include BNC1방법.method.
- 제1항 내지 제8항 중 어느 한 항에 있어서,According to any one of claims 1 to 8,상기 생물학적 시료는 혈액, 혈장 또는 혈청인The biological sample is blood, plasma or serum.방법.method.
- 제9항에 있어서, According to clause 9,상기 생물학적 시료로부터 분리된 핵산은 cfDNA인 The nucleic acid isolated from the biological sample is cfDNA.방법.method.
- 제1항 내지 제8항 중 어느 한 항에 있어서,According to any one of claims 1 to 8,상기 메틸화 상태를 측정하는 단계는, i) 바이설파이트, 하이드로젠 설파이트, 다이설파이트, 이들의 염, 및 이들의 조합으로 이루어진 군에서 선택되는 상기 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제 및 ii) 상기 하나 이상의 유전자를 특이적으로 증폭하는 프라이머로 상기 유전자를 처리하는 단계를 포함하는The step of measuring the methylation state includes i) detecting the methylation state of the gene or its CpG region selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, salts thereof, and combinations thereof. agent and ii) treating the gene with a primer that specifically amplifies the one or more genes.방법.method.
- 제1항 내지 제8항 중 어느 한 항에 있어서,According to any one of claims 1 to 8,상기 메틸화 상태를 측정하는 단계는 메틸화 특이적 중합효소 연쇄 반응(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 중합효소 연쇄 반응(real time methylation-specific polymerase chain reaction), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, 파이로시퀀싱, 또는 바이설파이트 시퀀싱에 의해 수행되는The step of measuring the methylation status includes methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, and methylated DNA-specific binding protein. performed by quantitative PCR, pyrosequencing, or bisulfite sequencing.방법.method.
- 제1항 내지 제8항 중 어느 한 항에 있어서,According to any one of claims 1 to 8,상기 메틸화 상태를 측정하는 단계는 대조군 시료의 해당 마커의 상응하는 메틸화 측정 결과로부터 생성된 대조 메틸화 프로파일과 비교하는 단계를 추가로 포함하는The step of measuring the methylation status further includes comparing with a control methylation profile generated from the corresponding methylation measurement results of the corresponding marker in the control sample.방법.method.
- 제13항에 있어서,According to clause 13,상기 대조 메틸화 프로파일은 상기 획득된 하나 이상의 유전자의 메틸화 패턴을 기계학습시킴으로써 구축된 모델을 구성하며, 정상 대조군과 상이한 췌장암 환자군 특이적 메틸화 패턴을 식별하기 위한 것인The control methylation profile constitutes a model constructed by machine learning the methylation patterns of one or more genes obtained above, and is intended to identify pancreatic cancer patient group-specific methylation patterns that are different from normal controls.방법.method.
- 제14항에 있어서, According to clause 14,상기 기계학습은 랜덤 포레스트, 로지스틱 회귀 분석, 서포트 벡터 머신, 의사결정나무, 연관성 규칙 마이닝, 신경망 네트워크 및 딥러닝으로 이루어진 군에서 선택되는 하나 이상의 알고리즘에 의해 수행되는The machine learning is performed by one or more algorithms selected from the group consisting of random forest, logistic regression analysis, support vector machine, decision tree, association rule mining, neural network, and deep learning.방법.method.
- 제13항에 있어서, According to clause 13,상기 대조군 시료는 정상 개체의 시료인The control sample is a sample from a normal individual.방법.method.
- 제1항 내지 제8항 중 어느 한 항에 있어서, According to any one of claims 1 to 8,상기 췌장암은 조기 췌장암, 전이성 췌장암, 또는 재발성 또는 난치성 췌장암인The pancreatic cancer is early pancreatic cancer, metastatic pancreatic cancer, or recurrent or incurable pancreatic cancer.방법.method.
- 생물학적 시료에서 분리된 핵산을 이용하는 췌장암 진단을 위한 조성물로서, A composition for diagnosing pancreatic cancer using nucleic acids isolated from biological samples,MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2)로 이루어진 군에서 선택된 하나 이상의 마커 유전자 또는 이의 CpG 영역을 특이적으로 증폭하는 제제 및 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제로부터 선택된 하나 이상의 제제를 포함하는MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside An agent that specifically amplifies one or more marker genes or their CpG region selected from the group consisting of GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) And one or more agents selected from agents that detect the methylation status of the one or more marker genes or CpG regions thereof.조성물.Composition.
- 제18항에 있어서, According to clause 18,상기 하나 이상의 마커 유전자는 MIRLET7BHG를 포함하는 The one or more marker genes include MIRLET7BHG.조성물.Composition.
- 제19항에 있어서, According to clause 19,상기 하나 이상의 마커 유전자는 TRPC6, AVPR1A, EPS8L2, TBCD 및 BNC1으로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함하는The one or more marker genes further include one or more genes selected from the group consisting of TRPC6, AVPR1A, EPS8L2, TBCD, and BNC1.조성물.Composition.
- 제19항에 있어서, According to clause 19,상기 하나 이상의 마커 유전자는 EPS8L2 및 BNC1로 이루어진 군에서 선택된 하나 이상의 유전자를 추가로 포함하는The one or more marker genes further include one or more genes selected from the group consisting of EPS8L2 and BNC1.조성물.Composition.
- 제21항에 있어서, According to clause 21,상기 하나 이상의 마커 유전자는 TRPC6, TBCD 또는 AVPR1A을 추가로 포함하는 The one or more marker genes further include TRPC6, TBCD or AVPR1A.조성물.Composition.
- 제19항에 있어서,According to clause 19,상기 조성물은 하기 (i), (ii), (iii), (iv), (v) 또는 (vi) 조합의 마커 유전자 또는 이의 CpG 영역을 증폭하는 제제 및 상기 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제로부터 선택된 하나 이상의 제제를 포함하는 The composition includes an agent that amplifies a marker gene or a CpG region thereof in the following combination (i), (ii), (iii), (iv), (v) or (vi), and the methylation status of the marker gene or CpG region thereof. Containing one or more agents selected from agents that detect조성물:Composition:(i) BNC1, TRPC6 및 MIRLET7BHG;(i) BNC1, TRPC6, and MIRLET7BHG;(ii) BNC1, AVPR1A 및 MIRLET7BHG;(ii) BNC1, AVPR1A, and MIRLET7BHG;(iii) BNC1 및 MIRLET7BHG; (iii) BNC1 and MIRLET7BHG;(iv) BNC1, MIRLET7BHG 및 EPS8L2; (iv) BNC1, MIRLET7BHG, and EPS8L2;(v) MIRLET7BHG 및 EPS8L2; 또는 (v) MIRLET7BHG and EPS8L2; or(vi) MIRLET7BHG, EPS8L2 및 TBCD.(vi) MIRLET7BHG, EPS8L2, and TBCD.
- 제18항에 있어서,According to clause 18,상기 하나 이상의 마커 유전자는 TBCD 및 EPS8L2로 이루어진 군에서 선택된 하나 이상의 유전자를 포함하는 The one or more marker genes include one or more genes selected from the group consisting of TBCD and EPS8L2.조성물.Composition.
- 제24항에 있어서,According to clause 24,상기 하나 이상의 마커 유전자는 BNC1을 추가로 포함하는 The one or more marker genes further include BNC1조성물.Composition.
- 제18항에 있어서,According to clause 18,상기 유전자의 CpG 영역은 다음의 특징을 갖는The CpG region of the gene has the following characteristics조성물:Composition:
- 제18항 내지 제26항 중 어느 한 항에 있어서,According to any one of claims 18 to 26,상기 증폭하는 제제는 상기 마커 유전자에 상보적으로 결합하는 프라이머, 프로브 또는 안티센스 뉴클레오티드를 포함하는The amplifying agent includes a primer, probe, or antisense nucleotide that binds complementary to the marker gene.조성물.Composition.
- 제18항 내지 제26항 중 어느 한 항에 있어서,According to any one of claims 18 to 26,상기 하나 이상의 마커 유전자 또는 이의 CpG 영역을 특이적으로 증폭하는 제제 및 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역의 메틸화 상태를 검출하는 제제를 포함하는 Comprising an agent that specifically amplifies the one or more marker genes or CpG regions thereof and an agent that detects the methylation status of the one or more marker genes or CpG regions thereof.조성물.Composition.
- 제28항에 있어서, According to clause 28,상기 메틸화 상태를 측정하는 제제는 바이설파이트, 하이드로겐 설파이트, 다이설파이트 또는 이들의 조합인 The agent for measuring the methylation state is bisulfite, hydrogen sulfite, disulfite, or a combination thereof.조성물.Composition.
- 제18항 내지 제29항 중 어느 한 항에 따른 조성물을 포함하는, 개체의 생물학적 시료로부터 분리된 핵산을 이용하는 췌장암 진단을 위한 키트.A kit for diagnosing pancreatic cancer using nucleic acid isolated from a biological sample of an individual, comprising the composition according to any one of claims 18 to 29.
- 제30항에 있어서,According to clause 30,상기 키트는 상기 하나 이상의 마커 유전자 또는 이의 CpG 영역을 포함하는 검출 대상 유전자 또는 이의 CpG 영역을 포함하는 단편과 혼성화할 수 있는 프로브가 고정되어 있는 진단용 핵산 칩(Chip)을 포함하는 The kit includes a diagnostic nucleic acid chip (Chip) on which a probe capable of hybridizing with a detection target gene containing the one or more marker genes or a CpG region thereof or a fragment containing a CpG region thereof is immobilized.키트. kit.
- 췌장암의 치료 방법으로서,As a treatment method for pancreatic cancer,(i) 개체의 생물학적 시료로부터 분리된 핵산에서 메틸화 마커 유전자 또는 이의 CpG 영역이 정상 대조군에 비해 저메틸화된 상태 또는 과메틸화 상태를 검출하는 단계; 및 (ii) 상기 개체에서 췌장암에 대한 치료요법을 시행하는 단계를 포함하고, 상기 메틸화 마커 유전자 또는 이의 CpG 영역은 MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2)로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함하는(i) detecting a methylation marker gene or a CpG region thereof in a nucleic acid isolated from a biological sample of an individual in a hypomethylated or hypermethylated state compared to a normal control; and (ii) administering treatment for pancreatic cancer in the subject, wherein the methylation marker gene or its CpG region is MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside Xylosyltransferase 1), and ZNF879 (Zinc Finger Protein 879). , EPS8L2 (EPS8 Like 2), TRPC6 (Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B) ) and TCEA2 (Transcription Elongation Factor A2) containing one or more genes or their CpG region selected from the group consisting of방법.method.
- 췌장암을 가진 것으로 의심되거나 췌장암 발병 위험이 있는 개체에서 메틸화 마커를 검출하는 방법으로서, A method for detecting methylation markers in an individual suspected of having pancreatic cancer or at risk of developing pancreatic cancer, comprising:상기 개체로부터 수득된 생물학적 시료에서 하나 이상의 메틸화 마커 유전자의 메틸화 상태를 측정하는 단계를 포함하고,Measuring the methylation status of one or more methylation marker genes in a biological sample obtained from the individual,상기 하나 이상의 메틸화 마커 유전자는 MIRLET7BHG(MIRLET7B Host Gene), XXYLT1(Xyloside Xylosyltransferase 1), ZNF879(Zinc Finger Protein 879), EPS8L2(EPS8 Like 2), TRPC6(Transient Receptor Potential Cation Channel Subfamily C Member 6), AVPR1A(Arginine Vasopressin Receptor 1A), GSC(Goosecoid Homeobox), TBCD(Tubulin Folding Cofactor D), MAFB(MAF BZIP Transcription Factor B) 및 TCEA2(Transcription Elongation Factor A2)로 이루어진 군에서 선택된 하나 이상의 유전자 또는 이의 CpG 영역을 포함하는 The one or more methylation marker genes include MIRLET7BHG (MIRLET7B Host Gene), XXYLT1 (Xyloside One or more genes or their CpG region selected from the group consisting of (Arginine Vasopressin Receptor 1A), GSC (Goosecoid Homeobox), TBCD (Tubulin Folding Cofactor D), MAFB (MAF BZIP Transcription Factor B), and TCEA2 (Transcription Elongation Factor A2) containing방법.method.
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| WO2015153679A1 (en) * | 2014-03-31 | 2015-10-08 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Microrna assay for detection and management of pancreatic cancer precursors |
| WO2017158158A1 (en) * | 2016-03-18 | 2017-09-21 | Region Nordjylland, Aalborg University Hospital | Methylation markers for pancreatic cancer |
| JP2021126107A (en) * | 2020-02-17 | 2021-09-02 | 国立大学法人金沢大学 | Effectiveness prediction marker of chemotherapeutic agent for pancreatic carcinoma/biliary cancer, and effectiveness prediction kit corresponding to the same |
| KR102549013B1 (en) * | 2022-10-11 | 2023-06-28 | 주식회사 엔도믹스 | Methylation marker genes for pancreatic cancer diagnosis and use thereof |
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| WO2015153679A1 (en) * | 2014-03-31 | 2015-10-08 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Microrna assay for detection and management of pancreatic cancer precursors |
| WO2017158158A1 (en) * | 2016-03-18 | 2017-09-21 | Region Nordjylland, Aalborg University Hospital | Methylation markers for pancreatic cancer |
| JP2021126107A (en) * | 2020-02-17 | 2021-09-02 | 国立大学法人金沢大学 | Effectiveness prediction marker of chemotherapeutic agent for pancreatic carcinoma/biliary cancer, and effectiveness prediction kit corresponding to the same |
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