WO2014046199A1 - Method for obtaining information about brain tumor and marker and kit for obtaining information about brain tumor - Google Patents
Method for obtaining information about brain tumor and marker and kit for obtaining information about brain tumor Download PDFInfo
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- WO2014046199A1 WO2014046199A1 PCT/JP2013/075336 JP2013075336W WO2014046199A1 WO 2014046199 A1 WO2014046199 A1 WO 2014046199A1 JP 2013075336 W JP2013075336 W JP 2013075336W WO 2014046199 A1 WO2014046199 A1 WO 2014046199A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Definitions
- the present invention relates to a method for acquiring information related to a brain tumor of a subject.
- the present invention also relates to markers and kits used in the method.
- Brain tumor is a general term for tumors that develop in the cranium, and is classified into primary brain tumors that arise from the brain and its surrounding tissues, and metastatic brain tumors that have metastasized to cancer in another organ.
- primary brain tumors including malignant tumors and benign tumors.
- gliomas also called gliomas
- gliomas have a high incidence of about 30% of all brain tumors. .
- AFP ⁇ -fetoprotein
- HCG human chorionic ggonadotropin
- a method based on information on DNA methylation examples include a method based on information on DNA methylation.
- a CpG site (5 ′-(CG) -3 ′) in the base sequence of a predetermined gene is used as a marker.
- information such as the presence or absence of cancer cells is obtained, and cancer is diagnosed using this information as an index.
- the information obtained from the analysis results includes not only information on brain tumors but also other types. Information about other tumors may also be included. For example, even if information that tumor cells are present in a sample is obtained based on the analysis results, it is difficult to determine whether the tumor cells are derived from brain tumors or other types of tumors. It is.
- the specificity of the markers to brain tumors is a very important factor. Therefore, it is desired to develop a novel marker specific to a brain tumor that makes it possible to specifically detect tumor cells derived from the brain tumor.
- an object of the present invention is to provide a method for acquiring information related to a brain tumor by identifying such a novel marker and analyzing the methylation state of the marker. Moreover, an object of this invention is to provide the kit used suitably for the method.
- the present inventors have identified, as a novel marker, a gene region in which methylation is specifically observed in DNA obtained from a cancerous tissue of a brain tumor. Based on the results obtained by analyzing the methylation status of these markers, the present inventors differed from tumor cells derived from brain tumors and other cells (normal tissue cells and brain tumors). The present invention was completed by discovering that tumor cells derived from different types of tumors can be clearly distinguished.
- a step of preparing a DNA sample from a biological sample collected from a subject At least one gene selected from CREM (cAMP responsive element modulator), VRK2 (Vaccinia related kinase 2) and MYL12A (Myosin, light chain 12A, regulatory, non-sarcomeric) contained in the DNA sample obtained in the preparation process Analyzing the methylation status of the CpG site present in the promoter region;
- a method for acquiring information related to a brain tumor is provided, including the step of acquiring information related to the brain tumor of the subject based on the analysis result obtained in the analysis step.
- a marker for obtaining information on brain tumors by methylation analysis which is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
- information on a brain tumor including a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A is obtained. Kits are provided for doing so.
- the present invention can provide a novel marker that makes it possible to obtain information on brain tumors. Moreover, this invention can provide the method and kit for acquiring the information regarding a test subject's brain tumor by analyzing the methylation state about such a marker.
- a DNA sample is prepared from a biological sample collected from a subject.
- the brain tumor targeted by the present invention is not particularly limited as long as it is a primary brain tumor, and examples thereof include glioma, meningioma, pituitary adenoma, and schwannoma. Among them, glioma is particularly suitable as the object of the method of the present invention. Glioma is classified into glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, etc., but is not particularly limited in the present invention.
- the biological sample is not particularly limited as long as it is a biological sample containing DNA of a subject, but is preferably a sample containing genomic DNA, for example, a clinical specimen.
- clinical specimens include body fluids, urine, tissues collected by surgery or biopsy, and the like.
- body fluid include blood, serum, plasma, lymph, ascites, cerebrospinal fluid, bone marrow fluid, and nipple secretion.
- a culture obtained by culturing cells or tissues collected from a subject can also be used as a biological sample.
- Preparation of a DNA sample can be performed by extracting DNA from a biological sample.
- Methods for extracting DNA from biological samples are known in the art.
- a biological sample and a treatment solution containing a surfactant eg, sodium cholate, sodium dodecyl sulfate, etc.
- a surfactant eg, sodium cholate, sodium dodecyl sulfate, etc.
- physical treatment eg.g, sodium cholate, sodium dodecyl sulfate, etc.
- DNA can be extracted by performing ultrasonic crushing or the like to release DNA contained in the biological sample into the mixed solution.
- the obtained supernatant may be purified by a method known in the art.
- the extraction and purification of DNA from a biological sample can also be performed using a commercially available kit.
- the preparation step preferably further includes a step of fragmenting the extracted DNA.
- the methylated DNA immunoprecipitation (MeDIP) method and the unmethylated cytosine conversion treatment described later can be performed efficiently.
- DNA fragmentation can be performed by ultrasonic treatment, alkali treatment, restriction enzyme treatment, or the like.
- alkali treatment a DNA solution is fragmented by adding a sodium hydroxide solution to the DNA solution to a final concentration of 0.1 to 1.0 N and incubating at 10 to 40 ° C. for 5 to 15 minutes. It becomes.
- restriction enzyme treatment the restriction enzyme to be used is appropriately selected based on the base sequence of the DNA, such as MseI or BamHI.
- the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the DNA obtained in the above preparation step is analyzed.
- the “CpG site” means a site of a sequence in which cytosine (C) and guanine (G) in the base sequence are adjacent in this order in the 5 ′ to 3 ′ direction.
- the letter “p” in CpG represents a phosphodiester bond between cytosine and guanine.
- analyzing the methylation state means analyzing the presence or absence of methylation of a CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A, or It means analyzing the frequency of methylation of the promoter region.
- the base sequences of promoter regions of CREM, VRK2 and MYL12A genes are known. These nucleotide sequences are publicly known such as a database (http://www.ncbi.nlm.nih.gov/) provided by the National Center for Biological Information (NCBI) of the US National Library of Medicine. You can know from the database. Various ID numbers of the above genes are shown in Table 1. In addition, the base sequence of the promoter region of each gene is shown as the sequence of SEQ ID NO: 1, 2 and 3, respectively (note that the base sequence of SEQ ID NO: 1 is a minus-strand sequence, and the base sequences of SEQ ID NO: 2 and 3 are Plus strand sequence).
- the analyzing step is a step of analyzing the presence or absence of methylation of at least one CpG site among CpG sites existing in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A.
- Presence / absence of methylation means whether cytosine at the CpG site present in the promoter region is methylated.
- the number of CpG sites to be analyzed may be one, but it is preferable to analyze the presence or absence of methylation of a plurality of CpG sites.
- the plurality of CpG sites may be selected from a promoter region of one gene, or may be selected from each of the promoter regions of a plurality of genes.
- the analyzing step may be a step of analyzing the frequency of methylation in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A.
- the “frequency of methylation” means the ratio of the number of methylated CpG sites to the number of CpG sites present in the promoter region.
- the analysis target may be the entire promoter region described above or a part including at least one CpG site. Note that the analysis target may include only one CpG site, but preferably includes a plurality of CpG sites.
- the analysis target may be determined from any one promoter region of the above genes, or may be determined from promoter regions of a plurality of genes.
- the number of methylated CpG sites in the promoter region itself is It can be used as the frequency of methylation.
- the above methylation frequency may be a “methylation score” obtained by analyzing the methylation state of a CpG site in DNA by mass spectrometry such as MassARRAY (registered trademark) described later.
- MassARRAY registered trademark
- a DNA fragment is measured, and a methylation score is calculated from an area ratio between a peak derived from a methylated DNA fragment and a peak derived from an unmethylated DNA fragment.
- CpG site or a predetermined range including the CpG site in the promoter region of each gene of CREM, VRK2 and MYL12A is to be analyzed.
- a method for analyzing the methylation state various methods are known in the art.
- which analysis method is used is not particularly limited, but preferably, a step of distinguishing methylated DNA from unmethylated DNA, a step of amplifying DNA, methylated DNA and / or Detecting unmethylated DNA.
- Examples of the step of distinguishing methylated DNA from unmethylated DNA include a step of performing methylation sensitive restriction enzyme treatment, MeDIP method, unmethylated cytosine conversion treatment, and the like.
- Examples of the step of amplifying DNA include a step of performing PCR method, quantitative PCR method, IVT (in vitro transcription) amplification method, SPIA (trademark) amplification method and the like.
- Examples of the process for detecting methylated DNA and / or unmethylated DNA include a process for performing electrophoresis, sequence analysis, microarray analysis, mass spectrometry, Southern hybridization, and the like.
- the MeDIP method is a method of concentrating methylated DNA contained in a biological sample by immunoprecipitation using an anti-methylated cytosine antibody, an anti-methylated cytidine antibody, or an antibody that specifically recognizes a methylated DNA-binding protein.
- the analysis step may be a step of concentrating methylated DNA contained in the DNA obtained in the extraction step by the MeDIP method and analyzing the methylation state of the obtained methylated DNA.
- methylated DNA concentrated by the MeDIP method can be amplified by the IVT amplification method or the like, and the methylated state of the obtained amplification product can be analyzed using a microarray. Such an analysis method is called the MeDIP on chip method.
- Unmethylated cytosine conversion treatment is a reaction between a DNA extracted from a biological sample and an unmethylated cytosine converting agent to convert the unmethylated cytosine in the DNA to another base (uracil, thymine, adenine or guanine). It is processing to convert to.
- the unmethylated cytosine converting agent is a substance that can react with DNA to convert unmethylated cytosine in the DNA into another base (uracil, thymine, adenine or guanine).
- an unmethylated cytosine converting agent for example, bisulfite such as sodium sulfite, potassium salt, calcium salt, magnesium salt of bisulfite is preferably used.
- the addition amount (concentration) of bisulfite is not particularly limited as long as it can sufficiently convert unmethylated cytosine in DNA.
- the final concentration in a solution containing DNA is 1M.
- the above is preferably 1 to 15 ⁇ M, more preferably 3 to 10 ⁇ M.
- the incubation conditions (temperature and time) after the addition of bisulfite can be appropriately set according to the amount of bisulfite added. For example, when bisulfite is added at a final concentration of 6M, the incubation conditions are 50 to 80 ° C. Incubate for 10-90 minutes.
- the methylation state of the CpG site contained in DNA can be analyzed by sequence analysis of bisulfite-treated DNA and detecting differences from the original base sequence. This method is called the bisulfite sequence method.
- the methylation state of the CpG site can be analyzed by mass spectrometry. Specifically, using a bisulfite-treated DNA as a template, PCR amplification was performed using a primer set specific for the base sequence to be analyzed, and then the resulting PCR product was further IVT amplified to obtain methylated cytosine. And uracil become guanine (G) and adenine (A), respectively.
- the obtained IVT amplification product is cleaved with RNase A, and the mass difference between G and A (16Da) between the obtained nucleic acid fragments is calculated using a MALDI-TOF (Matrix Assisted Laser Desorption / Ionization-Time of Flight) mass spectrometer.
- MALDI-TOF Mass Assisted Laser Desorption / Ionization-Time of Flight
- the site cleaved by RNase A in the IVT product is between any base and uracil (U) or thymine (T) adjacent to the base. Therefore, the base sequence and mass of the IVT product cleaved by RNase A can be predicted from the base sequence of the DNA used as a template. Therefore, for each peak obtained by MassARRAY (registered trademark), it can be identified from which part of the base sequence of DNA used as a template. For example, when one CpG site in the DNA fragment is methylated, the peak obtained with MassARRAY (registered trademark) is shifted 16 ⁇ Da toward the high mass side.
- MassARRAY registered trademark
- the methylation state of the CpG site can be analyzed by a methylation specific PCR (MSP) method.
- MSP methylation specific PCR
- the MSP method is a method of analyzing the methylation status (presence / absence of methylation) of the CpG site by performing PCR amplification on the bisulfite-treated DNA using the primer set described below and confirming the presence or absence of the PCR product. It is.
- a base sequence in which the CpG site to be analyzed is methylated (ie, cytosine is not converted to uracil) can be amplified, but the CpG site is not methylated (ie, the cytosine is converted to uracil).
- the CpG site to be analyzed is methylated when a PCR product is present.
- the MSP method cannot be used to amplify a base sequence in which cytosine at the CpG site to be analyzed is not converted to uracil, but a primer set that can amplify a base sequence in which cytosine at the CpG site is converted to uracil should be used You can also. In this case, it can be seen that the CpG site to be analyzed is methylated when no PCR product is present.
- Each primer included in the primer set used in the MSP method can be appropriately designed by those skilled in the art according to the base sequence including the CpG site to be analyzed, but the CpG site to be analyzed is located at or near the 3 ′ end of the primer. It is preferably designed to include cytosine.
- the methylation state of the CpG site can also be analyzed using a microarray.
- the analysis microarray can be prepared by immobilizing on the substrate a nucleic acid probe complementary to the nucleotide sequence of the promoter region of each gene of CREM, VRK2 and MYL12A.
- Such a microarray can be produced by a method known in the art.
- DNA extracted from a biological sample is preferably labeled with a labeling substance known in the art. Therefore, it is preferable that the determination method of the present invention further includes a step of labeling the extracted DNA. Since this labeling step can label all DNA in the biological sample, it is advantageous to be performed after the DNA amplification step.
- labeling substances include fluorescent substances, haptens such as biotin, radioactive substances, and the like.
- the fluorescent substance include Cy3, Cy5, FITC, Alexa Fluor (trademark) and the like.
- the above signal may be an appropriate signal depending on the type of microarray.
- the signal may be an electrical signal generated when a DNA fragment hybridized with each probe of the microarray is present, or when the DNA to be analyzed is labeled as described above, a labeling substance Signals such as fluorescence and luminescence generated from The signal can be detected by a scanner provided in a normal microarray measurement apparatus. Examples of the scanner include GeneChip (registered trademark) Scanner 3000-7G (Affymetrix), Illumina (registered trademark) BeadArray Reader (Illumina).
- information on the brain tumor of the subject is acquired based on the analysis result obtained in the above analysis step.
- the information regarding the brain tumor is not particularly limited as long as it is information that can serve as an index for diagnosis of the brain tumor, but is preferably information indicating the occurrence and / or state of the brain tumor in the subject.
- Such information includes, for example, the presence or absence of brain tumor-derived tumor cells in a biological sample collected from a subject, the possibility that a brain tumor has occurred in the subject, and the risk that a brain tumor will occur in the subject.
- information on the brain tumor includes the prognosis of the subject, the degree of progression of the tumor (stage), and the like.
- the information regarding the brain tumor acquired based on the analysis result obtained by said analysis process does not contain the information regarding the kind of tumor different from a brain tumor substantially. This is because the promoter region of the above gene used as a marker in the method of the present invention is specifically methylated in tumor cells derived from a brain tumor.
- the analysis step when an analysis result indicating that there is a methylated CpG site is obtained, information indicating that a brain tumor has occurred, or the state of the brain tumor is poor (or , Bad) information can be acquired.
- the methylation frequency obtained in the analysis step is higher than a predetermined threshold or the same result as the threshold is obtained, such information may be acquired. it can. More specifically, information that tumor cells derived from brain tumors are present in the biological sample can be acquired. Alternatively, information that the subject has a high risk of developing a brain tumor or information that a brain tumor has already occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is poor (or bad), or information that the tumor state is a more advanced stage can be acquired.
- the analysis process when the result that there is no methylated CpG site is obtained, information indicating that no brain tumor has occurred or information indicating that the brain tumor is in good condition can be obtained. .
- information can be acquired when a result that the frequency of methylation obtained in the analysis step is lower than a predetermined threshold is obtained. More specifically, information that tumor cells derived from brain tumors are not present in the biological sample can be acquired. Alternatively, information that the subject has a low risk of developing a brain tumor or information that a brain tumor has not yet occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is good or information that the tumor state is not so advanced can be acquired.
- the predetermined threshold is not particularly limited, and can be set empirically by accumulating data on various biological samples.
- a predetermined threshold value may be set as follows. First, for DNA extracted from each of biological samples (normal brain tissue or normal brain cells) that have been confirmed in advance not to contain tumor cells derived from brain tumors, and biological samples containing tumor cells derived from brain tumors, Analyze methylation frequency. Next, based on the obtained analysis result, a threshold value is set from a range that is higher than the methylation frequency of the biological sample that does not include tumor cells and lower than the methylation frequency of the biological sample that includes tumor cells. Preferably, a threshold value is set as a value capable of accurately distinguishing between a biological sample not containing tumor cells and a biological sample containing tumor cells.
- the scope of the present invention includes a marker (hereinafter, also simply referred to as “marker”) for acquiring information related to a brain tumor by methylation analysis.
- the marker of the present invention is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
- the methylation state of a marker in a DNA sample prepared from a biological sample collected from a subject can be analyzed, and information related to the brain tumor of the subject can be acquired based on the obtained analysis result.
- the analysis of methylation status and the acquisition of brain tumor information are the same as described above.
- kits for acquiring information on brain tumors comprising a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
- the primer set included in the kit is used to determine the methylation state of the CpG site by an analysis method involving PCR amplification such as mass spectrometry such as MassARRAY (registered trademark), MSP method, bisulfite sequencing method, etc.
- mass spectrometry such as MassARRAY (registered trademark), MSP method, bisulfite sequencing method, etc.
- a primer set for analysis may be used, but a primer set used in mass spectrometry such as MassARRAY (registered trademark) or MSP method is preferable.
- the base sequence of each primer included in the primer set can be appropriately set by those skilled in the art according to the base sequence of the promoter region.
- a primer set that is the base sequence of SEQ ID NO: 4 and SEQ ID NO: 5 a primer set that is the base sequence of SEQ ID NO: 6 and SEQ ID NO: 7, a primer set that is the base sequence of SEQ ID NO: 8 and SEQ ID NO: 9, and SEQ ID NO: 12
- a primer set that is the base sequence of SEQ ID NO: 13 a primer set that is the base sequence of SEQ ID NO: 14 and SEQ ID NO: 15, and a primer set that is the base sequence of SEQ ID NO: 16 and SEQ ID NO: 17.
- the present invention also includes a system suitable for providing information on brain tumors in a subject.
- a system suitable for providing information on brain tumors in a subject is, for example, as follows.
- a computer comprising a processor and memory under control of the processor;
- the memory has the following steps: Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample; Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
- the present invention also includes a computer program product for causing a computer to provide information related to a brain tumor in a subject.
- a computer program product for example, as follows.
- a computer-readable medium includes the following steps: Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample; Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
- a computer program product that causes a computer to provide information about brain tumors in a subject.
- FIG. 7 is a schematic diagram illustrating an example of a determination device for providing information related to a brain tumor of a subject.
- the determination apparatus 1 shown in FIG. 7 includes a measurement apparatus 2 and a computer system 3 connected to the measurement apparatus 2.
- the measuring device 2 is a MALDI-TOF type mass spectrometer. This measuring device 2 acquires mass spectrometry information such as the flight time and mass-to-charge ratio (m / z value) of the test substance.
- mass spectrometry information such as the flight time and mass-to-charge ratio (m / z value) of the test substance.
- the measurement device 2 acquires mass spectrometry information of the nucleic acid contained in the measurement sample, and the obtained mass spectrometry information is stored in the computer system 3. Send to.
- the measuring device 2 may be a gel imaging device such as a fluorescence image scanner.
- the measuring device 2 detects the amplification product. Then, the measuring device 2 acquires the band intensity data of the amplification product and transmits the obtained data to the computer system 3.
- the computer system 3 includes a computer main body 3a, an input device 3b, and a display unit 3c that displays sample information, determination results, and the like.
- the computer system 3 receives mass spectrometry information from the measuring device 2. Then, the processor of the computer system 3 executes a program that provides information related to the brain tumor in the subject based on the mass spectrometry information.
- FIG. 8 is a block diagram showing a functional configuration of the determination apparatus shown in FIG.
- the computer system 3 includes an acquisition unit 301, a storage unit 302, a calculation unit 303, a determination unit 304, and an output unit 305.
- the acquisition unit 301 is connected to the measurement apparatus 2 via a network so that communication is possible.
- the calculation unit 303 and the determination unit 304 constitute a control unit 306.
- the acquisition unit 301 acquires information transmitted from the measurement device 2.
- the storage unit 302 stores a threshold and a formula for calculating a methylation score necessary for determination.
- the calculation unit 303 calculates the methylation score according to the formula stored in the storage unit 302 using the information acquired by the acquisition unit 301.
- the determination unit 304 determines whether the methylation score calculated by the calculation unit 303 is lower than the threshold value stored in the storage unit 302.
- the output unit 305 outputs the determination result by the determination unit 304 as information on the brain tumor of the subject (for example, the presence or absence of cancer cells derived from the brain tumor in the biological sample collected from the subject).
- FIG. 9 is a block diagram showing a hardware configuration of the determination apparatus shown in FIG.
- the computer main body 3 a includes a CPU (Central Processing Unit) 30, a ROM (Read Only Memory) 121, a ROM 32, a hard disk 33, an input / output interface 34, a reading device 35, and a communication.
- An interface 36 and an image output interface 37 are provided.
- a CPU 30, a ROM 31, a RAM (Random Access Memory) 32, a hard disk 33, an input / output interface 34, a reading device 35, a communication interface 36 and an image output interface 37 are connected by a bus 38 so that data communication is possible.
- the CPU 30 can execute a computer program stored in the ROM 31 and a computer program loaded in the ROM 32. Each functional block described above is realized by the CPU 30 executing the application program. Thereby, a computer system functions as a terminal as a determination apparatus for providing the information regarding a test subject's brain tumor.
- the ROM 31 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like.
- the ROM 31 stores a computer program executed by the CPU 30 and data used therefor.
- the ROM 32 is configured by SRAM, DRAM or the like.
- the ROM 32 is used for reading computer programs recorded in the ROM 31 and the hard disk 33.
- the ROM 32 is also used as a work area for the CPU 30 when executing these computer programs.
- the hard disk 33 is installed with an operating system to be executed by the CPU 30, a computer program such as an application program (a computer program for providing information on a brain tumor of a subject), and data used for executing the computer program.
- a computer program such as an application program (a computer program for providing information on a brain tumor of a subject), and data used for executing the computer program.
- the reading device 35 includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, and the like.
- the reading device 35 can read the computer program or data recorded on the portable recording medium 40.
- the input / output interface 34 includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. It is configured.
- An input device 3 b such as a keyboard and a mouse is connected to the input / output interface 34. The operator can input data to the computer main body 3a by using the input device 3b.
- the communication interface 36 is, for example, an Ethernet (registered trademark) interface.
- the computer system 3 can transmit print data to the printer via the communication interface 36.
- the image output interface 37 is connected to a display unit 3c composed of an LCD, a CRT, or the like. Thereby, the display part 3c can output the video signal according to the image data given from CPU30.
- the display unit 3c displays an image (screen) according to the input video signal.
- FIG. 10 is a flowchart for providing information related to a brain tumor using the determination apparatus shown in FIG.
- the peak area is calculated from the mass spectrometry information of the nucleic acid contained in the measurement sample prepared from the subject-derived DNA sample
- the methylation score is calculated from the obtained peak area
- the obtained methylation score is the threshold value.
- step S1-1 the acquisition unit 301 of the determination apparatus 1 acquires mass spectrometry information from the measurement apparatus 2.
- step S ⁇ b> 1-2 the calculation unit 303 calculates a peak area from the mass spectrometry information acquired by the acquisition unit 301 and transmits it to the storage unit 302.
- step S ⁇ b> 1-3 the calculation unit 303 calculates a methylation score according to the formula stored in the storage unit 302 based on the peak area stored in the storage unit 302.
- step S1-4 the determination unit 304 determines whether or not the methylation score calculated by the calculation unit 303 is lower than the threshold value stored in the storage unit 302.
- the process proceeds to step S1-5, and the determination unit 304 displays a determination result indicating that the biological sample collected from the subject has no brain tumor-derived cancer cells.
- the data is transmitted to the output unit 305.
- the output unit 305 outputs a determination result indicating that the biological sample collected from the subject has brain tumor-derived cancer cells. Send to.
- step S1-7 the output unit 305 outputs the determination result as information on the brain tumor of the subject, and displays it on the display unit 3c or prints it on a printer. Thereby, it is possible to provide a doctor or the like with information that assists in determining whether the subject has a brain tumor or not.
- Reference Example 1 Identification of a new marker using methylation data of cancerous tissue and normal brain tissue of brain tumor (1) Acquisition of methylation data
- cancerous tissue (283 samples) of brain tumor and normal brain tissue (2 specimens)
- Infinium HumanMethylation27 BeadChip (Illumina) methylation data published on TCGA (The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) I got it.
- the gene methylation positive rate is calculated by “(number of methylation positive samples / total number of cancer tissue samples among cancer tissue samples) ⁇ 100”.
- the gene was used as a marker for methylation observed in the cancer tissue.
- the promoter region of each gene of CREM, VRK2 and MYL12A was identified as a marker with high methylation observed in the cancerous tissue of brain tumor (FIG. 1A- C).
- these markers are also called markers of the present invention.
- Reference Example 2 Comparison of methylation data in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples and normal tissue samples (1) Acquisition of methylation data
- reference example 1 In addition to the indicated cancerous tissue (283 samples) and normal brain tissue (2 samples) of the brain tumor, methylation of 9 types of cancer / tumor tissue samples, 7 types of non-cancerous tissue samples and 12 types of normal tissue samples The data were compared. The number of specimens in each tissue is shown in the following table.
- cancer tissue specimens derived from gastric cancer cancer tissue specimens derived from hepatocellular carcinoma, non-cancerous tissue specimens derived from hepatocellular carcinoma, normal tissue specimens derived from normal liver, 11 types of normal tissue specimens.
- methylation data measured by Infinium Methylation Assay using Infinium HumanMethylation27 BeadChip (Illumina) was obtained.
- TCGA The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp
- lung cancer prostate cancer
- 93 healthy human peripheral blood specimens 93 healthy human peripheral blood specimens below.
- the methylation data referred to here is the methylation rate (mCpG) of the CpG region of CREM, VRK2 and MYL12A obtained as described in (2) of Reference Example 1 above.
- all the markers of the present invention are not highly methylated in other types of cancer, and normal human tissues and normal human blood (healthy human peripheral blood, Salhia B et al.). It was found to be specifically and highly methylated.
- Comparative Example 1 Comparison of methylation positive rates in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples, and normal tissue samples It is already known that the tumor cells derived from brain tumors are methylated For each of the MGMT and CDKN2A genes (hereinafter referred to as “known markers”), the methylation positive rate in each tissue was calculated in the same manner as in Reference Example 2. The results are shown in FIGS. 3A and B.
- both MGMT and CDKN2A have a high positive rate of cases in brain tumors, but methylation is detected in other types of cancer and other non-cancerous tissues.
- the known marker has high sensitivity as a brain tumor marker, but has low specificity. Therefore, known markers have many problems in performance as diagnostic markers for brain tumors. That is, it was shown that it is necessary to conduct a marker search in consideration of specificity in other types of cancer and other non-cancerous tissues.
- Example 1 Comparison of methylation data (MassARRAY) in normal brain and brain tumor (1) Biological sample
- normal brain tissue (1 sample) and cancerous tissue of brain tumor (8 samples) are used as the biological sample. It was.
- Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO). Further, in order to prepare a calibration curve in mass spectrometry, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA.
- the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA).
- a portion of the solution of this unmethylated DNA fragment is taken and reacted with SssI methylase (New England Biolab) to methylate all cytosines in the CG sequence to obtain a methylated DNA fragment (100% methyl). Solution).
- SssI methylase New England Biolab
- a 0% methylated DNA solution and a 100% methylated DNA solution were mixed at a predetermined ratio to obtain 25%, 50% and 75% methylated DNA solutions.
- Table 5 shows the primer set sequences for the markers of the present invention. Further, regarding the promoter regions of the CREM, VRK2 and MYL12A genes, the nucleotide sequences of the regions analyzed with the primer sets shown in Table 5 are shown in SEQ ID NOs: 18 to 20, respectively (the nucleotide sequence of SEQ ID NO: 18 is a minus strand) And the base sequences of SEQ ID NOs: 19 and 20 are positive strand sequences).
- Tag array AGGAAGAGAG (SEQ ID NO: 10) -T7 promoter sequence: CAGTAATACGACTCACTATAGGGAGAAGGCT (SEQ ID NO: 11)
- a PCR reaction solution was prepared by mixing the following reagents. 10x Hot Star buffer (QIAGEN) 0.5 ⁇ L 25 mM dNTP mix 0.04 ⁇ L Hot Star Taq (5U / ⁇ l) (QIAGEN) 0.04 ⁇ L Primer mix 2.0 ⁇ L DNA solution 1.0 ⁇ L 1.42 ⁇ L water Total 5 ⁇ L
- PCR reaction was performed on condition of the following. 1 cycle at 94 ° C for 15 minutes, 45 cycles of 94 ° C for 20 seconds, 52 ° C for 30 seconds and 72 ° C for 1 minute, 3 minutes at 72 ° C.
- each reaction product obtained was purified by Clean Resin (SEQUENOM) to obtain a sample for mass spectrometry.
- SEQUENOM Clean Resin
- the methylation score was calculated from the area ratio of the peak of the fragment containing the methylated CpG site and the peak of the fragment not containing the methylated CpG site. The results are shown in FIGS.
- FIG. 4 shows that in the analysis of the methylation frequency using the marker of the present invention, it is possible to set a threshold value capable of distinguishing between a brain tumor specimen and a normal brain tissue specimen.
- Example 2 Comparison of methylation data (MSP) in normal brain and brain tumor (1) Biological sample
- MSP methylation data
- Biological sample as a biological sample, the same normal brain tissue (one specimen) and brain tumor cancer as in Example 1 above. Tissue (8 specimens) was used.
- Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO). Further, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA. Subsequently, the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA).
- Bioruptor manufactured by COSMO BIO
- MSP Methylation specific PCR
- Table 6 shows primer sets used in the above MSP. These are primer sets that can obtain an amplification product when the DNA of the region to be amplified is methylated.
- a primer set for determining whether or not the bisulfite treatment was appropriately performed was also used as a quality control primer set (see Table 7).
- the base sequences of the regions analyzed with the primer sets shown in Table 6 are shown in SEQ ID NOs: 23 to 25, respectively (the base sequence of SEQ ID NO: 23 is a minus strand) And the base sequences of SEQ ID NOs: 24 and 25 are positive strand sequences).
- ⁇ PCR reaction conditions > 6 minutes at 95 ° C Y cycle for 30 seconds at 95 °C, 30 seconds at X °C, 30 seconds at 72 °C, 7 minutes at 72 ° C Leave at 16 ° C.
- “X” and “Y” represent the annealing temperature and the number of cycles shown in Tables 6 and 7, respectively.
- MSP methylation specific PCR
- Example 3 Methylation Analysis by MSP Method Using Blood Sample methylation analysis was performed on CpG sites in CREM and VRK2 promoter regions by MSP using peripheral blood as a biological sample.
- peripheral blood 8 samples collected from a brain tumor patient was used as the biological sample.
- healthy human peripheral blood (3 samples) was used as a control sample.
- MSP Qualitative MSP was performed using the measurement sample obtained in (2) above.
- the composition of PCR reagents used and the PCR conditions are shown below.
- the base sequences of the primer sets (CREM, VRK2, and accuracy control) used in this example are shown in Table 6 and Table 7.
- ⁇ PCR reaction conditions > 6 minutes at 95 ° C Y cycle for 30 seconds at 95 °C, 30 seconds at X °C, 30 seconds at 72 °C, 7 minutes at 72 ° C Leave at 16 ° C.
- “X” and “Y” represent the annealing temperature and the number of cycles shown in Table 8, respectively.
- FIG. 6C shows that bands were detected in all samples by PCR using the primer set for quality control. This indicates that the bisulfite treatment of the sample was properly performed.
- the marker of the present invention showed a high methylation tendency in brain tumor patients even when peripheral blood was used as a biological sample, and methylation was not detected in healthy individuals. Therefore, it was suggested that the marker of the present invention is a marker highly specific for brain tumors, and is useful for determining the presence or absence of cancer cells derived from brain tumors in blood samples.
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Abstract
The present invention provides a method in which the methylation state of a CpG site in a promoter region of at least one gene selected from among CREM, VRK2, and MYL12A in DNA extracted from a biosample from a subject is analyzed and information about a brain tumor in said subject is obtained on the basis of the result of that analysis.
Description
本発明は、被験者の脳腫瘍に関する情報の取得方法に関する。また、本発明は、その方法に用いられるマーカーおよびキットに関する。
The present invention relates to a method for acquiring information related to a brain tumor of a subject. The present invention also relates to markers and kits used in the method.
脳腫瘍は、頭蓋内に発生する腫瘍の総称であり、脳およびその周辺組織から発生する原発性脳腫瘍と、別の臓器の癌が転移してきた転移性脳腫瘍とに分類される。原発性脳腫瘍には、悪性腫瘍および良性腫瘍を含めて様々な種類があるが、それらの中でも、悪性腫瘍である神経膠腫(グリオーマともいう)は脳腫瘍全体の約30%と高い発症頻度を示す。
Brain tumor is a general term for tumors that develop in the cranium, and is classified into primary brain tumors that arise from the brain and its surrounding tissues, and metastatic brain tumors that have metastasized to cancer in another organ. There are various types of primary brain tumors, including malignant tumors and benign tumors. Among them, gliomas (also called gliomas), which are malignant tumors, have a high incidence of about 30% of all brain tumors. .
脳腫瘍の検査では、主にCTやMRIによる画像検査が行われている。また、血液中の腫瘍マーカーの検査としてAFP(α-fetoprotein)およびHCG(human chorionic gonadotropin)の測定も行われている。しかし、これらの腫瘍マーカーの検査は、脳腫瘍が胚細胞腫である場合にしか適用されない。現時点では、胚細胞腫以外の脳腫瘍に有用な腫瘍マーカーは見出されておらず、脳腫瘍の簡便な検査法は確立されていない。
In brain tumor testing, CT and MRI image tests are mainly performed. In addition, AFP (α-fetoprotein) and HCG (human chorionic ggonadotropin) are also measured as a test for tumor markers in blood. However, these tumor marker tests are only applicable when the brain tumor is a germinoma. At present, no useful tumor marker has been found for brain tumors other than germinoma, and a simple test method for brain tumors has not been established.
他方で、近年、新たな癌の診断方法として、遺伝子情報に基づく方法が研究されている。そのような方法としては、例えば、DNAのメチル化に関する情報に基づく方法が挙げられる。この方法では、所定の遺伝子の塩基配列中のCpG部位(5'-(CG)-3')をマーカーとして用いる。そして、該マーカーのメチル化状態の解析結果に基づいて癌細胞の存否などの情報を取得し、これを指標にして癌の診断が行われる。
On the other hand, in recent years, methods based on genetic information have been studied as new cancer diagnosis methods. Examples of such a method include a method based on information on DNA methylation. In this method, a CpG site (5 ′-(CG) -3 ′) in the base sequence of a predetermined gene is used as a marker. Based on the analysis result of the methylation state of the marker, information such as the presence or absence of cancer cells is obtained, and cancer is diagnosed using this information as an index.
脳腫瘍においても、DNAのメチル化解析を利用した腫瘍の判定方法の研究・開発が行なわれている。例えば、Bello M.J.らの文献には、p16(「CDKN2A」とも呼ばれる)などの遺伝子が正常な脳の試料ではメチル化されていないが、グリオーマの生検試料では高度にメチル化されていることが開示されている(非特許文献1参照)。また、Gomori E.らの文献には、DNMT1、MGMTおよびEGFRの3つの遺伝子がグリオーマにおいて高度にメチル化されていることが開示されている(非特許文献2参照)。
Also in brain tumors, research and development of tumor determination methods using DNA methylation analysis is being conducted. For example, Bello 文献 MJ et al. Found that genes such as p16 (also called “CDKN2A”) are not methylated in normal brain samples, but are highly methylated in glioma biopsy samples. It is disclosed (see Non-Patent Document 1). Further, Gomori E. et al. Discloses that three genes, DNMT1, MGMT and EGFR, are highly methylated in glioma (see Non-Patent Document 2).
上述のように、脳腫瘍(特にグリオーマ)において異常メチル化を示す遺伝子がいくつか報告されているが、それらの遺伝子の中には、脳腫瘍とは異なる種類の腫瘍においてもある程度メチル化されている遺伝子が含まれている。しかし、脳腫瘍を含む複数の種類の腫瘍においてメチル化されている遺伝子をマーカーとして用いてメチル化解析を行った場合、解析結果から得られた情報には、脳腫瘍に関する情報のみならず、他の種類の腫瘍に関する情報も含まれる可能性がある。例えば、解析結果に基づいて試料中に腫瘍細胞が存在するという情報を得たとしても、その腫瘍細胞が脳腫瘍に由来するのか、または、他の種類の腫瘍に由来するのかを判別することは困難である。
As described above, several genes that exhibit abnormal methylation in brain tumors (particularly glioma) have been reported, but some of these genes are also methylated to some extent even in different types of tumors from brain tumors. It is included. However, when methylation analysis is performed using genes methylated in multiple types of tumors including brain tumors as markers, the information obtained from the analysis results includes not only information on brain tumors but also other types. Information about other tumors may also be included. For example, even if information that tumor cells are present in a sample is obtained based on the analysis results, it is difficult to determine whether the tumor cells are derived from brain tumors or other types of tumors. It is.
このように、マーカーのメチル化解析により脳腫瘍に関する情報を取得するためには、該マーカーの脳腫瘍への特異性が非常に重要な要素となる。それゆえ、脳腫瘍に由来する腫瘍細胞を特異的に検出することを可能にするような、脳腫瘍に特異的な新規マーカーの開発が望まれている。
Thus, in order to obtain information on brain tumors by methylation analysis of markers, the specificity of the markers to brain tumors is a very important factor. Therefore, it is desired to develop a novel marker specific to a brain tumor that makes it possible to specifically detect tumor cells derived from the brain tumor.
そこで、本発明は、そのような新規マーカーを同定し、そのマーカーについてメチル化の状態を解析することにより、脳腫瘍に関する情報の取得方法を提供することを目的とする。また、本発明は、その方法に好適に用いられるキットを提供することを目的とする。
Therefore, an object of the present invention is to provide a method for acquiring information related to a brain tumor by identifying such a novel marker and analyzing the methylation state of the marker. Moreover, an object of this invention is to provide the kit used suitably for the method.
本発明者らは、脳腫瘍の癌部組織から得たDNAに特異的にメチル化が認められる遺伝子領域を、新規マーカーとして同定した。そして、本発明者らは、これらのマーカーについてメチル化状態を解析して得られた結果に基づいて、脳腫瘍に由来する腫瘍細胞と、その他の細胞(正常組織の細胞、および、脳腫瘍とは異なる種類の腫瘍に由来する腫瘍細胞)とを明確に区別できることを見出して、本発明を完成した。
The present inventors have identified, as a novel marker, a gene region in which methylation is specifically observed in DNA obtained from a cancerous tissue of a brain tumor. Based on the results obtained by analyzing the methylation status of these markers, the present inventors differed from tumor cells derived from brain tumors and other cells (normal tissue cells and brain tumors). The present invention was completed by discovering that tumor cells derived from different types of tumors can be clearly distinguished.
すなわち、本発明によれば、
被験者から採取した生体試料からDNA試料を調製する工程と、
調製工程で得られたDNA試料に含まれるCREM(cAMP responsive element modulator)、VRK2(Vaccinia related kinase 2)およびMYL12A(Myosin, light chain 12A, regulatory, non-sarcomeric)から選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態を解析する工程と、
解析工程で得られた解析結果に基づいて、上記の被験者の脳腫瘍に関する情報を取得する工程と
を含む、脳腫瘍に関する情報の取得方法が提供される。 That is, according to the present invention,
A step of preparing a DNA sample from a biological sample collected from a subject;
At least one gene selected from CREM (cAMP responsive element modulator), VRK2 (Vaccinia related kinase 2) and MYL12A (Myosin,light chain 12A, regulatory, non-sarcomeric) contained in the DNA sample obtained in the preparation process Analyzing the methylation status of the CpG site present in the promoter region;
A method for acquiring information related to a brain tumor is provided, including the step of acquiring information related to the brain tumor of the subject based on the analysis result obtained in the analysis step.
被験者から採取した生体試料からDNA試料を調製する工程と、
調製工程で得られたDNA試料に含まれるCREM(cAMP responsive element modulator)、VRK2(Vaccinia related kinase 2)およびMYL12A(Myosin, light chain 12A, regulatory, non-sarcomeric)から選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態を解析する工程と、
解析工程で得られた解析結果に基づいて、上記の被験者の脳腫瘍に関する情報を取得する工程と
を含む、脳腫瘍に関する情報の取得方法が提供される。 That is, according to the present invention,
A step of preparing a DNA sample from a biological sample collected from a subject;
At least one gene selected from CREM (cAMP responsive element modulator), VRK2 (Vaccinia related kinase 2) and MYL12A (Myosin,
A method for acquiring information related to a brain tumor is provided, including the step of acquiring information related to the brain tumor of the subject based on the analysis result obtained in the analysis step.
また、本発明によれば、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つである、メチル化解析により脳腫瘍に関する情報を取得するためのマーカーが提供される。
さらに、本発明によれば、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つのメチル化の状態を解析するためのプライマーセットを含む、脳腫瘍に関する情報を取得するためのキットが提供される。 In addition, according to the present invention, there is provided a marker for obtaining information on brain tumors by methylation analysis, which is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A. The
Furthermore, according to the present invention, information on a brain tumor including a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A is obtained. Kits are provided for doing so.
さらに、本発明によれば、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つのメチル化の状態を解析するためのプライマーセットを含む、脳腫瘍に関する情報を取得するためのキットが提供される。 In addition, according to the present invention, there is provided a marker for obtaining information on brain tumors by methylation analysis, which is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A. The
Furthermore, according to the present invention, information on a brain tumor including a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A is obtained. Kits are provided for doing so.
本発明は、脳腫瘍に関する情報の取得を可能にする新規マーカーを提供することができる。また、本発明は、そのようなマーカーについてメチル化の状態を解析することにより、被験者の脳腫瘍に関する情報を取得するための方法およびキットを提供することができる。
The present invention can provide a novel marker that makes it possible to obtain information on brain tumors. Moreover, this invention can provide the method and kit for acquiring the information regarding a test subject's brain tumor by analyzing the methylation state about such a marker.
本発明の脳腫瘍に関する情報の取得方法(以下、単に「方法」ともいう)では、まず、被験者から採取した生体試料からDNA試料を調製する。
In the method for acquiring information related to brain tumor of the present invention (hereinafter also simply referred to as “method”), first, a DNA sample is prepared from a biological sample collected from a subject.
本発明が対象とする脳腫瘍は原発性脳腫瘍であれば特に限定されず、例えば、グリオーマ、髄膜腫、下垂体腺腫、神経鞘腫などが挙げられる。それらの中でもグリオーマが本発明の方法の対象として特に好適である。なお、グリオーマは、膠芽腫、星細胞腫、乏突起膠腫、上衣腫、髄芽腫などに分類されるが、本発明では特に限定されない。
The brain tumor targeted by the present invention is not particularly limited as long as it is a primary brain tumor, and examples thereof include glioma, meningioma, pituitary adenoma, and schwannoma. Among them, glioma is particularly suitable as the object of the method of the present invention. Glioma is classified into glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, etc., but is not particularly limited in the present invention.
本実施形態において、生体試料は、被験者のDNAを含む生体由来の試料であれば特に制限されないが、好ましくはゲノムDNAを含む試料、例えば臨床検体である。臨床検体としては、例えば、体液、尿、手術または生検により採取した組織などが挙げられる。体液としては、血液、血清、血漿、リンパ液、腹水、脳脊髄液、骨髄液、乳頭分泌液などが挙げられる。また、被検者から採取した細胞または組織を培養して得られた培養物を生体試料として用いることもできる。
In the present embodiment, the biological sample is not particularly limited as long as it is a biological sample containing DNA of a subject, but is preferably a sample containing genomic DNA, for example, a clinical specimen. Examples of clinical specimens include body fluids, urine, tissues collected by surgery or biopsy, and the like. Examples of the body fluid include blood, serum, plasma, lymph, ascites, cerebrospinal fluid, bone marrow fluid, and nipple secretion. In addition, a culture obtained by culturing cells or tissues collected from a subject can also be used as a biological sample.
DNA試料の調製は、生体試料からDNAを抽出することにより行うことができる。生体試料からのDNAの抽出方法は当該技術において公知である。例えば、生体試料と、細胞または組織を可溶化する界面活性剤(例えばコール酸ナトリウム、ドデシル硫酸ナトリウムなど)を含む処理液とを混合し、得られた混合液に物理的処理(撹拌、ホモジナイズ、超音波破砕など)を施して、生体試料に含まれるDNAを該混合液中に遊離させることによって、DNAを抽出することができる。この場合、混合液を遠心分離して細胞破片を沈殿させ、遊離したDNAを含む上清を後述の解析工程に用いることが好ましい。また、得られた上清を当該技術において公知の方法により精製してもよい。なお、生体試料からのDNAの抽出および精製は、市販のキットを用いて行うこともできる。
Preparation of a DNA sample can be performed by extracting DNA from a biological sample. Methods for extracting DNA from biological samples are known in the art. For example, a biological sample and a treatment solution containing a surfactant (eg, sodium cholate, sodium dodecyl sulfate, etc.) that solubilizes cells or tissues are mixed, and physical treatment (stirring, homogenization, DNA can be extracted by performing ultrasonic crushing or the like to release DNA contained in the biological sample into the mixed solution. In this case, it is preferable to centrifuge the mixed solution to precipitate cell debris and use the supernatant containing the released DNA in the analysis step described later. The obtained supernatant may be purified by a method known in the art. The extraction and purification of DNA from a biological sample can also be performed using a commercially available kit.
上記の調製工程は、抽出したDNAを断片化する工程をさらに含むことが好ましい。DNAを適当な長さに断片化することにより、後述するメチル化DNA免疫沈降(MeDIP)法および非メチル化シトシン変換処理を効率よく行うことができる。
DNAの断片化は、超音波処理、アルカリ処理、制限酵素処理などにより行うことができる。例えば、アルカリ処理によりDNAの断片化を行なう場合は、DNA溶液に水酸化ナトリウム溶液を終濃度0.1~1.0 Nとなるよう添加し、10~40℃で5~15分間インキュベーションすることによりDNAが断片化される。また、制限酵素処理によりDNAの断片化を行なう場合、用いる制限酵素はDNAの塩基配列に基づいて適宜選択され、例えばMseIやBamHIなどが用いられる。 The preparation step preferably further includes a step of fragmenting the extracted DNA. By fragmenting DNA into an appropriate length, the methylated DNA immunoprecipitation (MeDIP) method and the unmethylated cytosine conversion treatment described later can be performed efficiently.
DNA fragmentation can be performed by ultrasonic treatment, alkali treatment, restriction enzyme treatment, or the like. For example, when DNA fragmentation is performed by alkali treatment, a DNA solution is fragmented by adding a sodium hydroxide solution to the DNA solution to a final concentration of 0.1 to 1.0 N and incubating at 10 to 40 ° C. for 5 to 15 minutes. It becomes. When DNA is fragmented by restriction enzyme treatment, the restriction enzyme to be used is appropriately selected based on the base sequence of the DNA, such as MseI or BamHI.
DNAの断片化は、超音波処理、アルカリ処理、制限酵素処理などにより行うことができる。例えば、アルカリ処理によりDNAの断片化を行なう場合は、DNA溶液に水酸化ナトリウム溶液を終濃度0.1~1.0 Nとなるよう添加し、10~40℃で5~15分間インキュベーションすることによりDNAが断片化される。また、制限酵素処理によりDNAの断片化を行なう場合、用いる制限酵素はDNAの塩基配列に基づいて適宜選択され、例えばMseIやBamHIなどが用いられる。 The preparation step preferably further includes a step of fragmenting the extracted DNA. By fragmenting DNA into an appropriate length, the methylated DNA immunoprecipitation (MeDIP) method and the unmethylated cytosine conversion treatment described later can be performed efficiently.
DNA fragmentation can be performed by ultrasonic treatment, alkali treatment, restriction enzyme treatment, or the like. For example, when DNA fragmentation is performed by alkali treatment, a DNA solution is fragmented by adding a sodium hydroxide solution to the DNA solution to a final concentration of 0.1 to 1.0 N and incubating at 10 to 40 ° C. for 5 to 15 minutes. It becomes. When DNA is fragmented by restriction enzyme treatment, the restriction enzyme to be used is appropriately selected based on the base sequence of the DNA, such as MseI or BamHI.
本発明の方法では、上記の調製工程で得られたDNAに含まれるCREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態を解析する。
なお、本明細書において、「CpG部位」とは、塩基配列中のシトシン(C)とグアニン(G)とが5'から3'への方向にこの順序で隣り合った配列の部位を意味する。なお、CpGの「p」の文字は、シトシンとグアニンとの間のホスホジエステル結合を表わす。
また、本明細書において、「メチル化の状態を解析する」とは、CREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の有無解析すること、または該プロモータ領域のメチル化の頻度を解析することを意味する。 In the method of the present invention, the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the DNA obtained in the above preparation step is analyzed.
In the present specification, the “CpG site” means a site of a sequence in which cytosine (C) and guanine (G) in the base sequence are adjacent in this order in the 5 ′ to 3 ′ direction. . The letter “p” in CpG represents a phosphodiester bond between cytosine and guanine.
Further, in the present specification, “analyzing the methylation state” means analyzing the presence or absence of methylation of a CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A, or It means analyzing the frequency of methylation of the promoter region.
なお、本明細書において、「CpG部位」とは、塩基配列中のシトシン(C)とグアニン(G)とが5'から3'への方向にこの順序で隣り合った配列の部位を意味する。なお、CpGの「p」の文字は、シトシンとグアニンとの間のホスホジエステル結合を表わす。
また、本明細書において、「メチル化の状態を解析する」とは、CREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の有無解析すること、または該プロモータ領域のメチル化の頻度を解析することを意味する。 In the method of the present invention, the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the DNA obtained in the above preparation step is analyzed.
In the present specification, the “CpG site” means a site of a sequence in which cytosine (C) and guanine (G) in the base sequence are adjacent in this order in the 5 ′ to 3 ′ direction. . The letter “p” in CpG represents a phosphodiester bond between cytosine and guanine.
Further, in the present specification, “analyzing the methylation state” means analyzing the presence or absence of methylation of a CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A, or It means analyzing the frequency of methylation of the promoter region.
当該技術において、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域の塩基配列自体は公知である。これらの塩基配列は、例えば米国国立医学図書館の国立生物情報センター(National Center for Biotechnology Information:NCBI)により提供されるデータベース(http://www.ncbi.nlm.nih.gov/)などの公知のデータベースから知ることができる。上記の遺伝子の各種ID番号を、表1に示す。また、各遺伝子のプロモータ領域の塩基配列を、それぞれ配列番号1、2および3の配列として示す(なお、配列番号1の塩基配列はマイナス鎖の配列であり、配列番号2および3の塩基配列はプラス鎖の配列である)。
In this technology, the base sequences of promoter regions of CREM, VRK2 and MYL12A genes are known. These nucleotide sequences are publicly known such as a database (http://www.ncbi.nlm.nih.gov/) provided by the National Center for Biological Information (NCBI) of the US National Library of Medicine. You can know from the database. Various ID numbers of the above genes are shown in Table 1. In addition, the base sequence of the promoter region of each gene is shown as the sequence of SEQ ID NO: 1, 2 and 3, respectively (note that the base sequence of SEQ ID NO: 1 is a minus-strand sequence, and the base sequences of SEQ ID NO: 2 and 3 are Plus strand sequence).
本発明の実施形態において、解析工程は、CREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のうち、少なくとも1つのCpG部位のメチル化の有無を解析する工程であってもよい。ここで、「メチル化の有無」とは、上記のプロモータ領域に存在するCpG部位のシトシンがメチル化されているか否かを意味する。この実施形態では、解析するCpG部位は1つであってもよいが、複数のCpG部位のメチル化の有無を解析することが好ましい。なお、複数のCpG部位は、1つの遺伝子のプロモータ領域から選択してもよいし、複数の遺伝子のプロモータ領域のそれぞれの中から選択してもよい。
In the embodiment of the present invention, the analyzing step is a step of analyzing the presence or absence of methylation of at least one CpG site among CpG sites existing in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A. There may be. Here, “presence / absence of methylation” means whether cytosine at the CpG site present in the promoter region is methylated. In this embodiment, the number of CpG sites to be analyzed may be one, but it is preferable to analyze the presence or absence of methylation of a plurality of CpG sites. The plurality of CpG sites may be selected from a promoter region of one gene, or may be selected from each of the promoter regions of a plurality of genes.
本発明の別の実施形態において、解析工程は、CREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域におけるメチル化の頻度を解析する工程であってもよい。ここで、「メチル化の頻度」とは、上記のプロモータ領域に存在するCpG部位の数に対する、メチル化されたCpG部位の数の割合を意味する。この実施形態では、解析対象は、上記のプロモータ領域の全部であってもよいし、少なくとも1つのCpG部位を含む一部分であってもよい。なお、解析対象は、CpG部位を1つしか含んでいなくてもよいが、複数のCpG部位を含んでいることが好ましい。また、解析対象は、上記の遺伝子のうちのいずれか1つのプロモータ領域から決定しもよいし、複数の遺伝子のプロモータ領域から決定してもよい。
なお、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位の位置および数は既知であるので、本発明の実施形態においては、該プロモータ領域におけるメチル化されたCpG部位の数自体をメチル化の頻度として利用できる。 In another embodiment of the present invention, the analyzing step may be a step of analyzing the frequency of methylation in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A. Here, the “frequency of methylation” means the ratio of the number of methylated CpG sites to the number of CpG sites present in the promoter region. In this embodiment, the analysis target may be the entire promoter region described above or a part including at least one CpG site. Note that the analysis target may include only one CpG site, but preferably includes a plurality of CpG sites. The analysis target may be determined from any one promoter region of the above genes, or may be determined from promoter regions of a plurality of genes.
Since the position and number of CpG sites existing in the promoter region of each gene of CREM, VRK2 and MYL12A are known, in the embodiment of the present invention, the number of methylated CpG sites in the promoter region itself is It can be used as the frequency of methylation.
なお、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位の位置および数は既知であるので、本発明の実施形態においては、該プロモータ領域におけるメチル化されたCpG部位の数自体をメチル化の頻度として利用できる。 In another embodiment of the present invention, the analyzing step may be a step of analyzing the frequency of methylation in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A. Here, the “frequency of methylation” means the ratio of the number of methylated CpG sites to the number of CpG sites present in the promoter region. In this embodiment, the analysis target may be the entire promoter region described above or a part including at least one CpG site. Note that the analysis target may include only one CpG site, but preferably includes a plurality of CpG sites. The analysis target may be determined from any one promoter region of the above genes, or may be determined from promoter regions of a plurality of genes.
Since the position and number of CpG sites existing in the promoter region of each gene of CREM, VRK2 and MYL12A are known, in the embodiment of the present invention, the number of methylated CpG sites in the promoter region itself is It can be used as the frequency of methylation.
上記のメチル化の頻度は、DNA中のCpG部位のメチル化状態を、後述するMassARRAY(登録商標)などの質量分析法で解析することにより得られる「メチル化スコア」であってもよい。MassARRAY(登録商標)では、DNA断片を測定して、メチル化DNA断片に由来するピークと非メチル化DNA断片に由来するピークとの面積比からメチル化スコアが算出される。
The above methylation frequency may be a “methylation score” obtained by analyzing the methylation state of a CpG site in DNA by mass spectrometry such as MassARRAY (registered trademark) described later. In MassARRAY (registered trademark), a DNA fragment is measured, and a methylation score is calculated from an area ratio between a peak derived from a methylated DNA fragment and a peak derived from an unmethylated DNA fragment.
本発明の実施形態では、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域中のいずれのCpG部位(あるいは該CpG部位を含む所定の範囲)を解析対象とするかについては特に限定されず、当業者が適宜決定することができる。なお、これらの遺伝子のプロモータ領域に存在するCpG部位の位置および数は既知であるので、解析対象とするCpG部位または範囲は、後述の公知の解析方法を用いたルーチンの実験により決定することができる。
In the embodiment of the present invention, there is no particular limitation on which CpG site (or a predetermined range including the CpG site) in the promoter region of each gene of CREM, VRK2 and MYL12A is to be analyzed. Can be determined as appropriate. Since the position and number of CpG sites existing in the promoter region of these genes are known, the CpG site or range to be analyzed can be determined by routine experiments using known analysis methods described below. it can.
メチル化の状態を解析する方法は、当該技術において種々の方法が公知である。本発明の方法では、いずれの解析方法を用いるかは特に限定されないが、好ましくは、メチル化DNAと非メチル化DNAとを区別する工程と、DNAを増幅する工程と、メチル化DNAおよび/または非メチル化DNAを検出する工程とを含む方法である。
As a method for analyzing the methylation state, various methods are known in the art. In the method of the present invention, which analysis method is used is not particularly limited, but preferably, a step of distinguishing methylated DNA from unmethylated DNA, a step of amplifying DNA, methylated DNA and / or Detecting unmethylated DNA.
メチル化DNAと非メチル化DNAとを区別する工程としては、メチル化感受性制限酵素処理、MeDIP法、非メチル化シトシン変換処理などを行う工程が挙げられる。
DNAを増幅する工程としては、PCR法、定量的PCR法、IVT(in vitro transcription)増幅法、SPIA(商標)増幅法などを行う工程が挙げられる。
メチル化DNAおよび/または非メチル化DNAを検出する工程としては、電気泳動法、シークエンス解析法、マイクロアレイ解析法、質量分析法、サザンハイブリダイゼーションなどを行う工程が挙げられる。 Examples of the step of distinguishing methylated DNA from unmethylated DNA include a step of performing methylation sensitive restriction enzyme treatment, MeDIP method, unmethylated cytosine conversion treatment, and the like.
Examples of the step of amplifying DNA include a step of performing PCR method, quantitative PCR method, IVT (in vitro transcription) amplification method, SPIA (trademark) amplification method and the like.
Examples of the process for detecting methylated DNA and / or unmethylated DNA include a process for performing electrophoresis, sequence analysis, microarray analysis, mass spectrometry, Southern hybridization, and the like.
DNAを増幅する工程としては、PCR法、定量的PCR法、IVT(in vitro transcription)増幅法、SPIA(商標)増幅法などを行う工程が挙げられる。
メチル化DNAおよび/または非メチル化DNAを検出する工程としては、電気泳動法、シークエンス解析法、マイクロアレイ解析法、質量分析法、サザンハイブリダイゼーションなどを行う工程が挙げられる。 Examples of the step of distinguishing methylated DNA from unmethylated DNA include a step of performing methylation sensitive restriction enzyme treatment, MeDIP method, unmethylated cytosine conversion treatment, and the like.
Examples of the step of amplifying DNA include a step of performing PCR method, quantitative PCR method, IVT (in vitro transcription) amplification method, SPIA (trademark) amplification method and the like.
Examples of the process for detecting methylated DNA and / or unmethylated DNA include a process for performing electrophoresis, sequence analysis, microarray analysis, mass spectrometry, Southern hybridization, and the like.
MeDIP法とは、抗メチル化シトシン抗体もしくは抗メチル化シチジン抗体、またはメチル化DNA結合タンパク質を特異的に認識する抗体を用いる免疫沈降により、生体試料に含まれるメチル化DNAを濃縮する方法である。本発明の実施形態において、解析工程は、抽出工程で得られたDNAに含まれるメチル化DNAをMeDIP法により濃縮し、得られたメチル化DNAのメチル化状態を解析する工程であってもよい。また、MeDIP法により濃縮したメチル化DNAを、IVT増幅法などにより増幅し、得られた増幅産物について、マイクロアレイを用いてメチル化状態を解析することもできる。このような解析方法は、MeDIP on chip法と呼ばれる。
The MeDIP method is a method of concentrating methylated DNA contained in a biological sample by immunoprecipitation using an anti-methylated cytosine antibody, an anti-methylated cytidine antibody, or an antibody that specifically recognizes a methylated DNA-binding protein. . In the embodiment of the present invention, the analysis step may be a step of concentrating methylated DNA contained in the DNA obtained in the extraction step by the MeDIP method and analyzing the methylation state of the obtained methylated DNA. . In addition, methylated DNA concentrated by the MeDIP method can be amplified by the IVT amplification method or the like, and the methylated state of the obtained amplification product can be analyzed using a microarray. Such an analysis method is called the MeDIP on chip method.
非メチル化シトシン変換処理とは、生体試料から抽出したDNAと非メチル化シトシン変換剤とを反応させることにより、該DNA中の非メチル化シトシンを他の塩基(ウラシル、チミン、アデニンまたはグアニン)に変換する処理である。ここで、非メチル化シトシン変換剤とは、DNAと反応して該DNA中の非メチル化シトシンを他の塩基(ウラシル、チミン、アデニンまたはグアニン)に変換できる物質である。このような非メチル化シトシン変換剤としては、例えば亜硫酸水素のナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩などの亜硫酸水素塩(バイサルファイト)が好適に用いられる。
バイサルファイトを用いる処理では、DNA中の非メチル化シトシンは、脱アミノ化反応によりウラシルに変換されるが、メチル化シトシンには、このような塩基の変換が起こらない。
したがって、DNA中のCpG部位のメチル化状態の違いは、バイサルファイトを用いる非メチル化シトシン変換処理によって、塩基配列の違い(CおよびU)に変換される。なお、バイサルファイトによる非メチル化シトシン変換処理は、バイサルファイト処理と呼ばれる。 Unmethylated cytosine conversion treatment is a reaction between a DNA extracted from a biological sample and an unmethylated cytosine converting agent to convert the unmethylated cytosine in the DNA to another base (uracil, thymine, adenine or guanine). It is processing to convert to. Here, the unmethylated cytosine converting agent is a substance that can react with DNA to convert unmethylated cytosine in the DNA into another base (uracil, thymine, adenine or guanine). As such an unmethylated cytosine converting agent, for example, bisulfite such as sodium sulfite, potassium salt, calcium salt, magnesium salt of bisulfite is preferably used.
In the treatment using bisulfite, unmethylated cytosine in DNA is converted to uracil by deamination reaction, but such base conversion does not occur in methylated cytosine.
Therefore, the difference in the methylation state of the CpG site in DNA is converted into a difference in base sequence (C and U) by unmethylated cytosine conversion treatment using bisulfite. In addition, the unmethylated cytosine conversion process by bisulfite is called a bisulfite process.
バイサルファイトを用いる処理では、DNA中の非メチル化シトシンは、脱アミノ化反応によりウラシルに変換されるが、メチル化シトシンには、このような塩基の変換が起こらない。
したがって、DNA中のCpG部位のメチル化状態の違いは、バイサルファイトを用いる非メチル化シトシン変換処理によって、塩基配列の違い(CおよびU)に変換される。なお、バイサルファイトによる非メチル化シトシン変換処理は、バイサルファイト処理と呼ばれる。 Unmethylated cytosine conversion treatment is a reaction between a DNA extracted from a biological sample and an unmethylated cytosine converting agent to convert the unmethylated cytosine in the DNA to another base (uracil, thymine, adenine or guanine). It is processing to convert to. Here, the unmethylated cytosine converting agent is a substance that can react with DNA to convert unmethylated cytosine in the DNA into another base (uracil, thymine, adenine or guanine). As such an unmethylated cytosine converting agent, for example, bisulfite such as sodium sulfite, potassium salt, calcium salt, magnesium salt of bisulfite is preferably used.
In the treatment using bisulfite, unmethylated cytosine in DNA is converted to uracil by deamination reaction, but such base conversion does not occur in methylated cytosine.
Therefore, the difference in the methylation state of the CpG site in DNA is converted into a difference in base sequence (C and U) by unmethylated cytosine conversion treatment using bisulfite. In addition, the unmethylated cytosine conversion process by bisulfite is called a bisulfite process.
バイサルファイト処理を行なう場合、バイサルファイトの添加量(濃度)は、DNA中の非メチル化シトシンを十分に変換できる程度であれば特に限定されないが、例えば、DNAを含む溶液中の終濃度として1M以上、好ましくは1~15 M、より好ましくは3~10 Mである。また、バイサルファイトを添加した後のインキュベーションの条件(温度および時間)は、バイサルファイトの添加量に応じて適宜設定できるが、例えば、バイサルファイトを終濃度6Mで添加した場合、50~80℃で10~90分間インキュベーションする。
When bisulfite treatment is performed, the addition amount (concentration) of bisulfite is not particularly limited as long as it can sufficiently convert unmethylated cytosine in DNA. For example, the final concentration in a solution containing DNA is 1M. The above is preferably 1 to 15 μM, more preferably 3 to 10 μM. The incubation conditions (temperature and time) after the addition of bisulfite can be appropriately set according to the amount of bisulfite added. For example, when bisulfite is added at a final concentration of 6M, the incubation conditions are 50 to 80 ° C. Incubate for 10-90 minutes.
DNAに含まれるCpG部位のメチル化状態は、バイサルファイト処理したDNAをシークエンス解析して、本来の塩基配列との違いを検出することにより解析できる。この方法は、バイサルファイトシークエンス法と呼ばれている。
The methylation state of the CpG site contained in DNA can be analyzed by sequence analysis of bisulfite-treated DNA and detecting differences from the original base sequence. This method is called the bisulfite sequence method.
また、CpG部位のメチル化状態は、質量分析法によって解析することができる。具体的には、バイサルファイト処理したDNAを鋳型として、解析対象とする塩基配列に特異的なプライマーセットを用いてPCR増幅した後、得られたPCR産物をさらにIVT増幅することにより、メチル化シトシンおよびウラシルはそれぞれグアニン(G)およびアデニン(A)となる。得られたIVT増幅産物をRNase Aで切断し、得られた核酸断片間におけるGとAとの質量差(16 Da)をMALDI-TOF(マトリックス支援レーザ脱離イオン化-飛行時間)型質量分析装置を用いて検出することにより、DNAのメチル化状態を解析できる。この方法はMassARRAY(登録商標)解析と呼ばれる。
Also, the methylation state of the CpG site can be analyzed by mass spectrometry. Specifically, using a bisulfite-treated DNA as a template, PCR amplification was performed using a primer set specific for the base sequence to be analyzed, and then the resulting PCR product was further IVT amplified to obtain methylated cytosine. And uracil become guanine (G) and adenine (A), respectively. The obtained IVT amplification product is cleaved with RNase A, and the mass difference between G and A (16Da) between the obtained nucleic acid fragments is calculated using a MALDI-TOF (Matrix Assisted Laser Desorption / Ionization-Time of Flight) mass spectrometer. By detecting using the DNA, the methylation state of DNA can be analyzed. This method is called MassARRAY® analysis.
IVT産物においてRNase Aによって切断される部位は、任意の塩基と該塩基に隣接するウラシル(U)またはチミン(T)との間であることが知られている。したがって、RNase Aによって切断されたIVT産物の塩基配列および質量は、鋳型として用いたDNAの塩基配列から予測することが可能である。したがって、MassARRAY(登録商標)で得られた各ピークについて、鋳型としたDNAの塩基配列のどの部分に由来するかを同定できる。例えば、DNA断片中の1か所のCpG部位がメチル化していた場合、MassARRAY(登録商標)で得られるピークは、高質量側へ16 Daシフトする。複数のCpG部位を有するDNA断片の解析では、例えば、該DNA断片中のCpG部位が2か所メチル化していた場合は32 Daシフトし、3か所メチル化していた場合は48 Daシフトする。
It is known that the site cleaved by RNase A in the IVT product is between any base and uracil (U) or thymine (T) adjacent to the base. Therefore, the base sequence and mass of the IVT product cleaved by RNase A can be predicted from the base sequence of the DNA used as a template. Therefore, for each peak obtained by MassARRAY (registered trademark), it can be identified from which part of the base sequence of DNA used as a template. For example, when one CpG site in the DNA fragment is methylated, the peak obtained with MassARRAY (registered trademark) is shifted 16 高 Da toward the high mass side. In the analysis of a DNA fragment having a plurality of CpG sites, for example, if the CpG site in the DNA fragment is methylated at 2 sites, it is shifted 32 Da, and if it is methylated at 3 sites, it is shifted 48 Da.
MassARRAY(登録商標)などの質量分析法では、測定したDNA断片のメチル化スコアを算出することができる。例えば、所定の配列のDNA断片について、分析で得られたチャートにおける非メチル化DNA断片のピークとメチル化DNA断片のピークとの面積比が1:3であった場合、このDNA断片のメチル化スコアは、3/(1+3)=0.75より、0.75となる。なお、メチル化スコアは、理論上、DNA断片が有する全てのCpG部位がメチル化している場合は1であり、全てのCpG部位がメチル化していない場合は0である。
In mass spectrometry such as MassARRAY (registered trademark), the methylation score of the measured DNA fragment can be calculated. For example, for a DNA fragment of a predetermined sequence, when the area ratio of the peak of the unmethylated DNA fragment to the peak of the methylated DNA fragment in the chart obtained by analysis is 1: 3, methylation of this DNA fragment The score is 0.75 from 3 / (1 + 3) = 0.75. Theoretically, the methylation score is 1 when all the CpG sites of the DNA fragment are methylated, and 0 when all the CpG sites are not methylated.
CpG部位のメチル化状態は、メチル化特異的PCR(MSP)法によって解析することができる。MSP法とは、バイサルファイト処理したDNAを、後述するプライマーセットを用いてPCR増幅を行い、PCR産物の有無を確認することによって、CpG部位のメチル化状態(メチル化の有無)を解析する方法である。
MSP法では、解析対象のCpG部位がメチル化されている(すなわち、シトシンがウラシルに変換されていない)塩基配列は増幅できるが、CpG部位がメチル化されていない(すなわち、該シトシンがウラシルに変換されている)塩基配列は増幅できないプライマーセットを用いる。このようなプライマーセットを用いるMSP法では、PCR産物が存在する場合に、解析対象のCpG部位がメチル化されていることがわかる。
また、MSP法は、解析対象のCpG部位のシトシンがウラシルに変換されていない塩基配列は増幅できないが、CpG部位のシトシンがウラシルに変換されている塩基配列は増幅できるプライマーセットを用いて行なうこともできる。この場合、PCR産物が存在しない場合に、解析対象のCpG部位がメチル化されていることがわかる。 The methylation state of the CpG site can be analyzed by a methylation specific PCR (MSP) method. The MSP method is a method of analyzing the methylation status (presence / absence of methylation) of the CpG site by performing PCR amplification on the bisulfite-treated DNA using the primer set described below and confirming the presence or absence of the PCR product. It is.
In the MSP method, a base sequence in which the CpG site to be analyzed is methylated (ie, cytosine is not converted to uracil) can be amplified, but the CpG site is not methylated (ie, the cytosine is converted to uracil). Use a primer set that cannot amplify the converted base sequence. In the MSP method using such a primer set, it can be seen that the CpG site to be analyzed is methylated when a PCR product is present.
In addition, the MSP method cannot be used to amplify a base sequence in which cytosine at the CpG site to be analyzed is not converted to uracil, but a primer set that can amplify a base sequence in which cytosine at the CpG site is converted to uracil should be used You can also. In this case, it can be seen that the CpG site to be analyzed is methylated when no PCR product is present.
MSP法では、解析対象のCpG部位がメチル化されている(すなわち、シトシンがウラシルに変換されていない)塩基配列は増幅できるが、CpG部位がメチル化されていない(すなわち、該シトシンがウラシルに変換されている)塩基配列は増幅できないプライマーセットを用いる。このようなプライマーセットを用いるMSP法では、PCR産物が存在する場合に、解析対象のCpG部位がメチル化されていることがわかる。
また、MSP法は、解析対象のCpG部位のシトシンがウラシルに変換されていない塩基配列は増幅できないが、CpG部位のシトシンがウラシルに変換されている塩基配列は増幅できるプライマーセットを用いて行なうこともできる。この場合、PCR産物が存在しない場合に、解析対象のCpG部位がメチル化されていることがわかる。 The methylation state of the CpG site can be analyzed by a methylation specific PCR (MSP) method. The MSP method is a method of analyzing the methylation status (presence / absence of methylation) of the CpG site by performing PCR amplification on the bisulfite-treated DNA using the primer set described below and confirming the presence or absence of the PCR product. It is.
In the MSP method, a base sequence in which the CpG site to be analyzed is methylated (ie, cytosine is not converted to uracil) can be amplified, but the CpG site is not methylated (ie, the cytosine is converted to uracil). Use a primer set that cannot amplify the converted base sequence. In the MSP method using such a primer set, it can be seen that the CpG site to be analyzed is methylated when a PCR product is present.
In addition, the MSP method cannot be used to amplify a base sequence in which cytosine at the CpG site to be analyzed is not converted to uracil, but a primer set that can amplify a base sequence in which cytosine at the CpG site is converted to uracil should be used You can also. In this case, it can be seen that the CpG site to be analyzed is methylated when no PCR product is present.
MSP法で用いられるプライマーセットに含まれる各プライマーは、解析対象のCpG部位を含む塩基配列に応じて当業者が適宜設計できるが、プライマーの3'末端またはその付近に、解析対象のCpG部位のシトシンを含むように設計することが好ましい。
Each primer included in the primer set used in the MSP method can be appropriately designed by those skilled in the art according to the base sequence including the CpG site to be analyzed, but the CpG site to be analyzed is located at or near the 3 ′ end of the primer. It is preferably designed to include cytosine.
CpG部位のメチル化状態は、マイクロアレイを用いて解析することもできる。この場合、解析用マイクロアレイは、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域の塩基配列に相補的な核酸プローブを、基板上に固定して作製できる。なお、このようなマイクロアレイは、当該技術において公知の方法により作製できる。
The methylation state of the CpG site can also be analyzed using a microarray. In this case, the analysis microarray can be prepared by immobilizing on the substrate a nucleic acid probe complementary to the nucleotide sequence of the promoter region of each gene of CREM, VRK2 and MYL12A. Such a microarray can be produced by a method known in the art.
マイクロアレイによる解析では、生体試料から抽出したDNAは、当該技術において公知の標識物質により標識されていることが好ましい。よって、本発明の判定方法は、抽出したDNAを標識する工程をさらに含むことが好ましい。この標識工程は、生体試料中の全てのDNAを標識できるので、上記のDNA増幅工程の後に行われるのが有利である。なお、標識物質としては、蛍光物質、ビオチンなどのハプテン、放射性物質などが挙げられる。また、蛍光物質としては、Cy3、Cy5、FITC、Alexa Fluor(商標)などが挙げられる。このようにDNAを標識することにより、マイクロアレイ上のプローブからのシグナルの測定が容易になる。なお、DNAをこれらの標識物質で標識する方法は、当該技術において公知である。
In analysis using a microarray, DNA extracted from a biological sample is preferably labeled with a labeling substance known in the art. Therefore, it is preferable that the determination method of the present invention further includes a step of labeling the extracted DNA. Since this labeling step can label all DNA in the biological sample, it is advantageous to be performed after the DNA amplification step. Examples of labeling substances include fluorescent substances, haptens such as biotin, radioactive substances, and the like. Examples of the fluorescent substance include Cy3, Cy5, FITC, Alexa Fluor (trademark) and the like. By labeling the DNA in this way, the signal from the probe on the microarray can be easily measured. A method for labeling DNA with these labeling substances is known in the art.
上記のシグナルは、マイクロアレイの種類に応じて適切なシグナルであり得る。例えば、シグナルは、マイクロアレイの各プローブとハイブリダイズしたDNA断片が存在する場合に発生する電気的シグナルであってもよいし、上記のように解析対象のDNAが標識されている場合は、標識物質から生じる蛍光、発光などのシグナルであってもよい。シグナルの検出は、通常のマイクロアレイ測定装置に備えられたスキャナーにより行うことができる。スキャナーとしては、例えば、GeneChip(登録商標)Scanner3000 7G(Affymetrix社)、Illumina(登録商標) BeadArray Reader (Illumina社)などが挙げられる。
The above signal may be an appropriate signal depending on the type of microarray. For example, the signal may be an electrical signal generated when a DNA fragment hybridized with each probe of the microarray is present, or when the DNA to be analyzed is labeled as described above, a labeling substance Signals such as fluorescence and luminescence generated from The signal can be detected by a scanner provided in a normal microarray measurement apparatus. Examples of the scanner include GeneChip (registered trademark) Scanner 3000-7G (Affymetrix), Illumina (registered trademark) BeadArray Reader (Illumina).
本発明の方法では、上記の解析工程で得られた解析結果に基づいて、被験者の脳腫瘍に関する情報を取得する。ここで、脳腫瘍に関する情報とは、脳腫瘍の診断の指標となり得る情報であれば特に限定されないが、好ましくは、被験者における脳腫瘍の発生もしくは状態またはその両方を示す情報である。そのような情報としては、例えば、被験者から採取した生体試料における脳腫瘍由来の腫瘍細胞の存否、脳腫瘍が被験者に発生している可能性、脳腫瘍がこれから被験者に発生する危険性などが挙げられる。また、被験者に脳腫瘍が発生している場合は、脳腫瘍に関する情報として、被験者の予後、腫瘍の進行度(ステージ)などが挙げられる。なお、上記の解析工程で得られた解析結果に基づいて取得した脳腫瘍に関する情報は、脳腫瘍とは異なる種類の腫瘍に関する情報を実質的に含まない。これは、本発明の方法でマーカーとして用いる上記の遺伝子のプロモータ領域が、脳腫瘍に由来する腫瘍細胞に特異的にメチル化していることによる。
In the method of the present invention, information on the brain tumor of the subject is acquired based on the analysis result obtained in the above analysis step. Here, the information regarding the brain tumor is not particularly limited as long as it is information that can serve as an index for diagnosis of the brain tumor, but is preferably information indicating the occurrence and / or state of the brain tumor in the subject. Such information includes, for example, the presence or absence of brain tumor-derived tumor cells in a biological sample collected from a subject, the possibility that a brain tumor has occurred in the subject, and the risk that a brain tumor will occur in the subject. In addition, when a brain tumor has occurred in the subject, information on the brain tumor includes the prognosis of the subject, the degree of progression of the tumor (stage), and the like. In addition, the information regarding the brain tumor acquired based on the analysis result obtained by said analysis process does not contain the information regarding the kind of tumor different from a brain tumor substantially. This is because the promoter region of the above gene used as a marker in the method of the present invention is specifically methylated in tumor cells derived from a brain tumor.
本発明の実施形態では、解析工程において、メチル化されたCpG部位が有るという解析結果が得られた場合、脳腫瘍が発生していることを示唆する情報、または脳腫瘍の状態が不良である(もしくは、悪い)という情報を取得することができる。
また、本発明の別の実施形態では、解析工程で得られたメチル化の頻度が所定の閾値より高いか、または閾値と同じという結果が得られた場合、そのような情報を取得することができる。
より具体的には、生体試料中に脳腫瘍由来の腫瘍細胞が存在するという情報を取得することができる。あるいは、被験者が脳腫瘍になる危険性が高いという情報、または脳腫瘍がすでに発生しているという情報を取得することができる。また、被験者に脳腫瘍が発生しているならば、被験者の予後が不良である(もしくは、悪い)という情報、または腫瘍の状態がより進行したステージであるという情報を取得することができる。 In an embodiment of the present invention, in the analysis step, when an analysis result indicating that there is a methylated CpG site is obtained, information indicating that a brain tumor has occurred, or the state of the brain tumor is poor (or , Bad) information can be acquired.
In another embodiment of the present invention, when the methylation frequency obtained in the analysis step is higher than a predetermined threshold or the same result as the threshold is obtained, such information may be acquired. it can.
More specifically, information that tumor cells derived from brain tumors are present in the biological sample can be acquired. Alternatively, information that the subject has a high risk of developing a brain tumor or information that a brain tumor has already occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is poor (or bad), or information that the tumor state is a more advanced stage can be acquired.
また、本発明の別の実施形態では、解析工程で得られたメチル化の頻度が所定の閾値より高いか、または閾値と同じという結果が得られた場合、そのような情報を取得することができる。
より具体的には、生体試料中に脳腫瘍由来の腫瘍細胞が存在するという情報を取得することができる。あるいは、被験者が脳腫瘍になる危険性が高いという情報、または脳腫瘍がすでに発生しているという情報を取得することができる。また、被験者に脳腫瘍が発生しているならば、被験者の予後が不良である(もしくは、悪い)という情報、または腫瘍の状態がより進行したステージであるという情報を取得することができる。 In an embodiment of the present invention, in the analysis step, when an analysis result indicating that there is a methylated CpG site is obtained, information indicating that a brain tumor has occurred, or the state of the brain tumor is poor (or , Bad) information can be acquired.
In another embodiment of the present invention, when the methylation frequency obtained in the analysis step is higher than a predetermined threshold or the same result as the threshold is obtained, such information may be acquired. it can.
More specifically, information that tumor cells derived from brain tumors are present in the biological sample can be acquired. Alternatively, information that the subject has a high risk of developing a brain tumor or information that a brain tumor has already occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is poor (or bad), or information that the tumor state is a more advanced stage can be acquired.
反対に、解析工程において、メチル化されたCpG部位が無いという結果が得られた場合、脳腫瘍が発生していないことを示唆する情報または脳腫瘍の状態が良好であるという情報を取得することができる。あるいは、解析工程で得られたメチル化の頻度が所定の閾値より低いという結果が得られた場合に、そのような情報を取得することができる。より具体的には、生体試料中に脳腫瘍に由来する腫瘍細胞が存在しないという情報を取得することができる。あるいは、被験者が脳腫瘍になる危険性が低いという情報、または脳腫瘍はまだ発生していないという情報を取得することができる。また、被験者に脳腫瘍が発生しているならば、被験者の予後が良好であるという情報、または腫瘍の状態がそれほど進行したステージではないという情報を取得することができる。
On the other hand, in the analysis process, when the result that there is no methylated CpG site is obtained, information indicating that no brain tumor has occurred or information indicating that the brain tumor is in good condition can be obtained. . Alternatively, such information can be acquired when a result that the frequency of methylation obtained in the analysis step is lower than a predetermined threshold is obtained. More specifically, information that tumor cells derived from brain tumors are not present in the biological sample can be acquired. Alternatively, information that the subject has a low risk of developing a brain tumor or information that a brain tumor has not yet occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is good or information that the tumor state is not so advanced can be acquired.
なお、所定の閾値は特に限定されず、種々の生体試料についてのデータの蓄積により経験的に設定することができる。あるいは、次のようにして所定の閾値を設定してもよい。まず、脳腫瘍に由来する腫瘍細胞を含まないことが予め確認されている生体試料(正常脳組織または正常脳細胞)、および、脳腫瘍に由来する腫瘍細胞を含む生体試料のそれぞれから抽出したDNAについて、メチル化の頻度を解析する。次いで、得られた解析結果に基づいて、腫瘍細胞を含まない生体試料のメチル化の頻度よりも高く、且つ、腫瘍細胞を含む生体試料のメチル化の頻度よりも低い範囲から閾値を設定する。好ましくは、腫瘍細胞を含まない生体試料と腫瘍細胞を含む生体試料とを高精度に区別し得る値を、閾値として設定する。
The predetermined threshold is not particularly limited, and can be set empirically by accumulating data on various biological samples. Alternatively, a predetermined threshold value may be set as follows. First, for DNA extracted from each of biological samples (normal brain tissue or normal brain cells) that have been confirmed in advance not to contain tumor cells derived from brain tumors, and biological samples containing tumor cells derived from brain tumors, Analyze methylation frequency. Next, based on the obtained analysis result, a threshold value is set from a range that is higher than the methylation frequency of the biological sample that does not include tumor cells and lower than the methylation frequency of the biological sample that includes tumor cells. Preferably, a threshold value is set as a value capable of accurately distinguishing between a biological sample not containing tumor cells and a biological sample containing tumor cells.
本発明の範囲には、メチル化解析により脳腫瘍に関する情報を取得するためのマーカー(以下、単に「マーカー」ともいう)も含まれる。本発明のマーカーは、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つである。
本発明の実施形態では、被験者から採取した生体試料から調製したDNA試料におけるマーカーのメチル化状態を解析し、得られた解析結果に基づいて該被験者の脳腫瘍に関する情報を取得することができる。なお、メチル化状態の解析および脳腫瘍の情報の取得については、これまでに述べたことと同様である。 The scope of the present invention includes a marker (hereinafter, also simply referred to as “marker”) for acquiring information related to a brain tumor by methylation analysis. The marker of the present invention is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
In the embodiment of the present invention, the methylation state of a marker in a DNA sample prepared from a biological sample collected from a subject can be analyzed, and information related to the brain tumor of the subject can be acquired based on the obtained analysis result. The analysis of methylation status and the acquisition of brain tumor information are the same as described above.
本発明の実施形態では、被験者から採取した生体試料から調製したDNA試料におけるマーカーのメチル化状態を解析し、得られた解析結果に基づいて該被験者の脳腫瘍に関する情報を取得することができる。なお、メチル化状態の解析および脳腫瘍の情報の取得については、これまでに述べたことと同様である。 The scope of the present invention includes a marker (hereinafter, also simply referred to as “marker”) for acquiring information related to a brain tumor by methylation analysis. The marker of the present invention is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
In the embodiment of the present invention, the methylation state of a marker in a DNA sample prepared from a biological sample collected from a subject can be analyzed, and information related to the brain tumor of the subject can be acquired based on the obtained analysis result. The analysis of methylation status and the acquisition of brain tumor information are the same as described above.
また、本発明の範囲には、脳腫瘍に関する情報を取得するためのキット(以下、単に「キット」ともいう)も含まれる。本発明のキットは、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つのメチル化の状態を解析するためのプライマーセットを含む。
Also, the scope of the present invention includes a kit for acquiring information on brain tumors (hereinafter also simply referred to as “kit”). The kit of the present invention comprises a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
本発明の実施形態において、キットに含まれるプライマーセットは、MassARRAY(登録商標)などの質量分析法、MSP法、バイサルファイトシーケンス法などのPCR増幅を伴う解析方法によりCpG部位のメチル化の状態を解析するためのプライマーセットであればよいが、好ましくはMassARRAY(登録商標)などの質量分析法またはMSP法で用いられるプライマーセットである。なお、プライマーセットに含まれる各プライマーの塩基配列は、上記のプロモータ領域の塩基配列に応じて当業者が適宜設定できる。例えば、配列番号4および配列番号5の塩基配列であるプライマーセット、配列番号6および配列番号7の塩基配列であるプライマーセット、配列番号8および配列番号9の塩基配列であるプライマーセット、配列番号12および配列番号13の塩基配列であるプライマーセット、配列番号14および配列番号15の塩基配列であるプライマーセット、ならびに配列番号16および配列番号17の塩基配列であるプライマーセットが挙げられる。
In the embodiment of the present invention, the primer set included in the kit is used to determine the methylation state of the CpG site by an analysis method involving PCR amplification such as mass spectrometry such as MassARRAY (registered trademark), MSP method, bisulfite sequencing method, etc. A primer set for analysis may be used, but a primer set used in mass spectrometry such as MassARRAY (registered trademark) or MSP method is preferable. The base sequence of each primer included in the primer set can be appropriately set by those skilled in the art according to the base sequence of the promoter region. For example, a primer set that is the base sequence of SEQ ID NO: 4 and SEQ ID NO: 5, a primer set that is the base sequence of SEQ ID NO: 6 and SEQ ID NO: 7, a primer set that is the base sequence of SEQ ID NO: 8 and SEQ ID NO: 9, and SEQ ID NO: 12 And a primer set that is the base sequence of SEQ ID NO: 13, a primer set that is the base sequence of SEQ ID NO: 14 and SEQ ID NO: 15, and a primer set that is the base sequence of SEQ ID NO: 16 and SEQ ID NO: 17.
本発明には、被験者における脳腫瘍に関する情報の提供に適するシステムも含まれる。そのようなシステムは、例えば、次のとおりである。
The present invention also includes a system suitable for providing information on brain tumors in a subject. Such a system is, for example, as follows.
プロセッサおよび該プロセッサの制御下にあるメモリを含むコンピュータを備え、
該メモリには、下記のステップ:
被験者由来のDNA試料に含まれるCREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態の解析結果を取得するステップと、
得られた解析結果に基づいて、上記の被験者の脳腫瘍に関する情報を提供するステップと
を該コンピュータに実行させるためのコンピュータプログラムが記録されている、
被験者における脳腫瘍に関する情報の提供に適するシステム。 A computer comprising a processor and memory under control of the processor;
The memory has the following steps:
Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample;
Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
A system suitable for providing information about brain tumors in subjects.
該メモリには、下記のステップ:
被験者由来のDNA試料に含まれるCREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態の解析結果を取得するステップと、
得られた解析結果に基づいて、上記の被験者の脳腫瘍に関する情報を提供するステップと
を該コンピュータに実行させるためのコンピュータプログラムが記録されている、
被験者における脳腫瘍に関する情報の提供に適するシステム。 A computer comprising a processor and memory under control of the processor;
The memory has the following steps:
Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample;
Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
A system suitable for providing information about brain tumors in subjects.
また、本発明には、被験者における脳腫瘍に関する情報の提供をコンピュータに実行させるためのコンピュータプログラム製品も含まれる。そのようなコンピュータプログラム製品は、例えば、次のとおりである。
The present invention also includes a computer program product for causing a computer to provide information related to a brain tumor in a subject. Such a computer program product is, for example, as follows.
コンピュータに読み取り可能な媒体を備え、
該媒体には、下記のステップ:
被験者由来のDNA試料に含まれるCREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態の解析結果を取得するステップと、
得られた解析結果に基づいて、上記の被験者の脳腫瘍に関する情報を提供するステップと
を該コンピュータに実行させるためのコンピュータプログラムが記録されている、
被験者における脳腫瘍に関する情報の提供をコンピュータに実行させるためのコンピュータプログラム製品。 A computer-readable medium,
The medium includes the following steps:
Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample;
Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
A computer program product that causes a computer to provide information about brain tumors in a subject.
該媒体には、下記のステップ:
被験者由来のDNA試料に含まれるCREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態の解析結果を取得するステップと、
得られた解析結果に基づいて、上記の被験者の脳腫瘍に関する情報を提供するステップと
を該コンピュータに実行させるためのコンピュータプログラムが記録されている、
被験者における脳腫瘍に関する情報の提供をコンピュータに実行させるためのコンピュータプログラム製品。 A computer-readable medium,
The medium includes the following steps:
Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample;
Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
A computer program product that causes a computer to provide information about brain tumors in a subject.
以下に、本発明の方法を実施するのに好適な装置の一形態を、図面を参照して説明する。しかし、本発明はこの実施形態のみに限定されるものではない。図7は、被験者の脳腫瘍に関する情報を提供するための判定装置の一例を示した概略図である。図7に示された判定装置1は、測定装置2と、該測定装置2と接続されたコンピュータシステム3とを含んでいる。
Hereinafter, an embodiment of an apparatus suitable for carrying out the method of the present invention will be described with reference to the drawings. However, the present invention is not limited only to this embodiment. FIG. 7 is a schematic diagram illustrating an example of a determination device for providing information related to a brain tumor of a subject. The determination apparatus 1 shown in FIG. 7 includes a measurement apparatus 2 and a computer system 3 connected to the measurement apparatus 2.
本実施形態においては、測定装置2は、MALDI-TOF型質量分析装置である。この測定装置2は、被験物質の飛行時間や質量電荷比(m/z値)などの質量分析情報を取得する。被験者由来のDNA試料から調製した測定用試料を測定装置2にセットすると、測定装置2は、該測定用試料に含まれる核酸の質量分析情報を取得し、得られた質量分析情報をコンピュータシステム3に送信する。
In the present embodiment, the measuring device 2 is a MALDI-TOF type mass spectrometer. This measuring device 2 acquires mass spectrometry information such as the flight time and mass-to-charge ratio (m / z value) of the test substance. When a measurement sample prepared from a DNA sample derived from a subject is set in the measurement device 2, the measurement device 2 acquires mass spectrometry information of the nucleic acid contained in the measurement sample, and the obtained mass spectrometry information is stored in the computer system 3. Send to.
なお、メチル化の状態をMSP法により解析する場合、測定装置2は、蛍光イメージスキャナなどのゲルイメージング装置であってもよい。この場合、MSP法による核酸増幅を行った反応液をゲル電気泳動し、泳動後のゲルを測定装置2にセットすると、測定装置2は増幅産物を検出する。そして、測定装置2は、増幅産物のバンド強度データを取得し、得られたデータをコンピュータシステム3に送信する。
When the methylation state is analyzed by the MSP method, the measuring device 2 may be a gel imaging device such as a fluorescence image scanner. In this case, when the reaction solution subjected to nucleic acid amplification by the MSP method is subjected to gel electrophoresis and the gel after electrophoresis is set in the measuring device 2, the measuring device 2 detects the amplification product. Then, the measuring device 2 acquires the band intensity data of the amplification product and transmits the obtained data to the computer system 3.
コンピュータシステム3は、コンピュータ本体3aと、入力デバイス3bと、検体情報、判定結果などを表示する表示部3cとを含む。コンピュータシステム3は、測定装置2から質量分析情報を受信する。そして、コンピュータシステム3のプロセッサは、質量分析情報に基づいて、被験者における脳腫瘍に関する情報を提供するプログラムを実行する。
The computer system 3 includes a computer main body 3a, an input device 3b, and a display unit 3c that displays sample information, determination results, and the like. The computer system 3 receives mass spectrometry information from the measuring device 2. Then, the processor of the computer system 3 executes a program that provides information related to the brain tumor in the subject based on the mass spectrometry information.
図8は、図7に示された判定装置の機能構成を示すブロック図である。図8に示されるように、コンピュータシステム3は、取得部301と、記憶部302と、算出部303と、判定部304と、出力部305とを備える。取得部301は、測定装置2と、ネットワークを介して通信可能に接続されている。なお、算出部303と判定部304とは、制御部306を構成している。
FIG. 8 is a block diagram showing a functional configuration of the determination apparatus shown in FIG. As illustrated in FIG. 8, the computer system 3 includes an acquisition unit 301, a storage unit 302, a calculation unit 303, a determination unit 304, and an output unit 305. The acquisition unit 301 is connected to the measurement apparatus 2 via a network so that communication is possible. The calculation unit 303 and the determination unit 304 constitute a control unit 306.
取得部301は、測定装置2から送信された情報を取得する。記憶部302は、判定に必要な閾値およびメチル化スコアを算出するための式を記憶する。算出部303は、取得部301で取得された情報を用い、記憶部302に記憶された式にしたがって、メチル化スコアを算出する。判定部304は、算出部303によって算出されたメチル化スコアが、記憶部302に記憶された閾値よりも低いか否かを判定する。出力部305は、判定部304による判定結果を、被験者の脳腫瘍に関する情報(例えば、該被験者から採取した生体試料中の脳腫瘍由来の癌細胞の有無)として出力する。
The acquisition unit 301 acquires information transmitted from the measurement device 2. The storage unit 302 stores a threshold and a formula for calculating a methylation score necessary for determination. The calculation unit 303 calculates the methylation score according to the formula stored in the storage unit 302 using the information acquired by the acquisition unit 301. The determination unit 304 determines whether the methylation score calculated by the calculation unit 303 is lower than the threshold value stored in the storage unit 302. The output unit 305 outputs the determination result by the determination unit 304 as information on the brain tumor of the subject (for example, the presence or absence of cancer cells derived from the brain tumor in the biological sample collected from the subject).
図9は、図7に示された判定装置のハードウェア構成を示すブロック図である。図9に示されるように、コンピュータ本体3aは、CPU(Central Processing Unit)30と、ROM(Read Only Memory)121と、ROM32と、ハードディスク33と、入出力インターフェイス34と、読出装置35と、通信インターフェイス36と、画像出力インターフェイス37とを備えている。CPU30、ROM31、RAM(Random Access Memory)32、ハードディスク33、入出力インターフェイス34、読出装置35、通信インターフェイス36および画像出力インターフェイス37は、バス38によってデータ通信可能に接続されている。
FIG. 9 is a block diagram showing a hardware configuration of the determination apparatus shown in FIG. As shown in FIG. 9, the computer main body 3 a includes a CPU (Central Processing Unit) 30, a ROM (Read Only Memory) 121, a ROM 32, a hard disk 33, an input / output interface 34, a reading device 35, and a communication. An interface 36 and an image output interface 37 are provided. A CPU 30, a ROM 31, a RAM (Random Access Memory) 32, a hard disk 33, an input / output interface 34, a reading device 35, a communication interface 36 and an image output interface 37 are connected by a bus 38 so that data communication is possible.
CPU30は、ROM31に記憶されているコンピュータプログラムおよびROM32にロードされたコンピュータプログラムを実行することが可能である。CPU30がアプリケーションプログラムを実行することにより、上述した各機能ブロックが実現される。これにより、コンピュータシステムが、被験者の脳腫瘍に関する情報を提供するための判定装置としての端末として機能する。
The CPU 30 can execute a computer program stored in the ROM 31 and a computer program loaded in the ROM 32. Each functional block described above is realized by the CPU 30 executing the application program. Thereby, a computer system functions as a terminal as a determination apparatus for providing the information regarding a test subject's brain tumor.
ROM31は、マスクROM、PROM、EPROM、EEPROMなどによって構成されている。ROM31には、CPU30によって実行されるコンピュータプログラムおよびこれに用いるデータが記録されている。
The ROM 31 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like. The ROM 31 stores a computer program executed by the CPU 30 and data used therefor.
ROM32は、SRAM、DRAMなどによって構成されている。ROM32は、ROM31およびハードディスク33に記録されているコンピュータプログラムの読み出しに用いられる。ROM32はまた、これらのコンピュータプログラムを実行するときに、CPU30の作業領域として利用される。
The ROM 32 is configured by SRAM, DRAM or the like. The ROM 32 is used for reading computer programs recorded in the ROM 31 and the hard disk 33. The ROM 32 is also used as a work area for the CPU 30 when executing these computer programs.
ハードディスク33は、CPU30に実行させるためのオペレーティングシステム、アプリケーションプログラム(被験者の脳腫瘍に関する情報を提供するためのコンピュータプログラム)などのコンピュータプログラムおよび当該コンピュータプログラムの実行に用いるデータがインストールされている。
The hard disk 33 is installed with an operating system to be executed by the CPU 30, a computer program such as an application program (a computer program for providing information on a brain tumor of a subject), and data used for executing the computer program.
読出装置35は、フレキシブルディスクドライブ、CD-ROMドライブ、DVD-ROMドライブなどによって構成されている。読出装置35は、可搬型記録媒体40に記録されたコンピュータプログラムまたはデータを読み出すことができる。
The reading device 35 includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, and the like. The reading device 35 can read the computer program or data recorded on the portable recording medium 40.
入出力インターフェイス34は、例えば、USB、IEEE1394、RS-232Cなどのシリアルインターフェイスと、SCSI、IDE、IEEE1284などのパラレルインターフェイスと、D/A変換器、A/D変換器などからなるアナログインターフェイスとから構成されている。入出力インターフェイス34には、キーボード、マウスなどの入力デバイス3bが接続されている。操作者は、当該入力デバイス3bを使用することにより、コンピュータ本体3aにデータを入力することが可能である。
The input / output interface 34 includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. It is configured. An input device 3 b such as a keyboard and a mouse is connected to the input / output interface 34. The operator can input data to the computer main body 3a by using the input device 3b.
通信インターフェイス36は、例えば、Ethernet(登録商標)インターフェイスなどである。コンピュータシステム3は、通信インターフェイス36により、プリンタへの印刷データの送信が可能である。
The communication interface 36 is, for example, an Ethernet (registered trademark) interface. The computer system 3 can transmit print data to the printer via the communication interface 36.
画像出力インターフェイス37は、LCD、CRTなどで構成される表示部3cに接続されている。これにより、表示部3cは、CPU30から与えられた画像データに応じた映像信号を出力することができる。表示部3cは、入力された映像信号にしたがって画像(画面)を表示する。
The image output interface 37 is connected to a display unit 3c composed of an LCD, a CRT, or the like. Thereby, the display part 3c can output the video signal according to the image data given from CPU30. The display unit 3c displays an image (screen) according to the input video signal.
次に、判定装置1による、被験者の脳腫瘍に関する情報の提供の処理手順を説明する。図10は、図7に示された判定装置を用いた脳腫瘍に関する情報の提供のフローチャートである。ここでは、被験者由来のDNA試料から調製した測定用試料に含まれる核酸の質量分析情報からピーク面積を算出し、得られたピーク面積からメチル化スコアを算出し、得られたメチル化スコアが閾値よりも低いか否かの判定を行なう場合を例として挙げて説明する。しかし、本発明は、この実施形態のみに限定されるものではない。
Next, a processing procedure for providing information related to the brain tumor of the subject by the determination apparatus 1 will be described. FIG. 10 is a flowchart for providing information related to a brain tumor using the determination apparatus shown in FIG. Here, the peak area is calculated from the mass spectrometry information of the nucleic acid contained in the measurement sample prepared from the subject-derived DNA sample, the methylation score is calculated from the obtained peak area, and the obtained methylation score is the threshold value. The case where it is determined whether it is lower than the above will be described as an example. However, the present invention is not limited only to this embodiment.
まず、ステップS1-1において、判定装置1の取得部301は、測定装置2から質量分析情報を取得する。次に、ステップS1-2において、算出部303は、取得部301が取得した質量分析情報からピーク面積を算出し、記憶部302に送信する。そして、ステップS1-3において、算出部303は、記憶部302に記憶された該ピーク面積に基づき、記憶部302に記憶された式にしたがって、メチル化スコアを算出する。
First, in step S1-1, the acquisition unit 301 of the determination apparatus 1 acquires mass spectrometry information from the measurement apparatus 2. Next, in step S <b> 1-2, the calculation unit 303 calculates a peak area from the mass spectrometry information acquired by the acquisition unit 301 and transmits it to the storage unit 302. In step S <b> 1-3, the calculation unit 303 calculates a methylation score according to the formula stored in the storage unit 302 based on the peak area stored in the storage unit 302.
その後、ステップS1-4において、判定部304は、算出部303で算出されたメチル化スコアが、記憶部302に記憶された閾値よりも低いか否かの判定を行う。ここで、メチル化スコアが閾値よりも低いとき、処理は、ステップS1-5に進行し、判定部304は、被験者から採取した生体試料には脳腫瘍由来の癌細胞がないことを示す判定結果を出力部305に送信する。また、メチル化スコアが閾値よりも低くないとき(すなわち、メチル化スコアが閾値以上であるとき)、被験者から採取した生体試料には脳腫瘍由来の癌細胞があることを示す判定結果を出力部305に送信する。
Thereafter, in step S1-4, the determination unit 304 determines whether or not the methylation score calculated by the calculation unit 303 is lower than the threshold value stored in the storage unit 302. Here, when the methylation score is lower than the threshold value, the process proceeds to step S1-5, and the determination unit 304 displays a determination result indicating that the biological sample collected from the subject has no brain tumor-derived cancer cells. The data is transmitted to the output unit 305. In addition, when the methylation score is not lower than the threshold value (that is, when the methylation score is equal to or higher than the threshold value), the output unit 305 outputs a determination result indicating that the biological sample collected from the subject has brain tumor-derived cancer cells. Send to.
そして、ステップS1-7において、出力部305は、判定結果を、被験者の脳腫瘍に関する情報として出力し、表示部3cに表示させたり、プリンタに印刷させたりする。これにより、被験者において脳腫瘍があるのか、またはないのかについて判定することを補助する情報を医師などに提供することができる。
In step S1-7, the output unit 305 outputs the determination result as information on the brain tumor of the subject, and displays it on the display unit 3c or prints it on a printer. Thereby, it is possible to provide a doctor or the like with information that assists in determining whether the subject has a brain tumor or not.
以下に、本発明を実施例によって詳細に説明するが、本発明はこれらの実施例に限定されない。
Hereinafter, the present invention will be described in detail by examples, but the present invention is not limited to these examples.
参考例1: 脳腫瘍の癌部組織および正常脳組織のメチル化データを利用した新規マーカーの同定
(1)メチル化データの取得
本参考例では、脳腫瘍の癌部組織(283検体)および正常脳組織(2検体)について、TCGA(The Cancer Genome Atlas:http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp)において公開されているInfinium HumanMethylation27 BeadChip(Illumina社)のメチル化データを取得した。 Reference Example 1: Identification of a new marker using methylation data of cancerous tissue and normal brain tissue of brain tumor (1) Acquisition of methylation data In this reference example, cancerous tissue (283 samples) of brain tumor and normal brain tissue (2 specimens) Infinium HumanMethylation27 BeadChip (Illumina) methylation data published on TCGA (The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) I got it.
(1)メチル化データの取得
本参考例では、脳腫瘍の癌部組織(283検体)および正常脳組織(2検体)について、TCGA(The Cancer Genome Atlas:http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp)において公開されているInfinium HumanMethylation27 BeadChip(Illumina社)のメチル化データを取得した。 Reference Example 1: Identification of a new marker using methylation data of cancerous tissue and normal brain tissue of brain tumor (1) Acquisition of methylation data In this reference example, cancerous tissue (283 samples) of brain tumor and normal brain tissue (2 specimens) Infinium HumanMethylation27 BeadChip (Illumina) methylation data published on TCGA (The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) I got it.
(2)新規マーカーの同定
Infinium HumanMethylation27 BeadChipには、ヒトのゲノム上にあるCpG部位のうち、27,578ヶ所のCpG部位ごとにメチル化用プローブと非メチル化用プローブを設けている。本参考例では、CpG部位のメチル化用プローブのシグナル強度(シグナルM)と非メチル化用プローブのシグナル強度(シグナルU)とをBeadArray Readerで検出し、以下の計算式により各遺伝子のCpG領域のメチル化率(mCpG)を算出した。
(mCpG)=(シグナルM)/(シグナルM)+(シグナルU) (2) Identification of novel markers Infinium HumanMethylation27 BeadChip has a methylation probe and an unmethylation probe for each of 27,578 CpG sites in the CpG site on the human genome. In this reference example, the signal intensity (signal M) of the methylation probe at the CpG site and the signal intensity (signal U) of the non-methylation probe are detected by the BeadArray Reader, and the CpG region of each gene is calculated using the following formula: The methylation rate (mCpG) was calculated.
(MCpG) = (Signal M) / (Signal M) + (Signal U)
Infinium HumanMethylation27 BeadChipには、ヒトのゲノム上にあるCpG部位のうち、27,578ヶ所のCpG部位ごとにメチル化用プローブと非メチル化用プローブを設けている。本参考例では、CpG部位のメチル化用プローブのシグナル強度(シグナルM)と非メチル化用プローブのシグナル強度(シグナルU)とをBeadArray Readerで検出し、以下の計算式により各遺伝子のCpG領域のメチル化率(mCpG)を算出した。
(mCpG)=(シグナルM)/(シグナルM)+(シグナルU) (2) Identification of novel markers Infinium HumanMethylation27 BeadChip has a methylation probe and an unmethylation probe for each of 27,578 CpG sites in the CpG site on the human genome. In this reference example, the signal intensity (signal M) of the methylation probe at the CpG site and the signal intensity (signal U) of the non-methylation probe are detected by the BeadArray Reader, and the CpG region of each gene is calculated using the following formula: The methylation rate (mCpG) was calculated.
(MCpG) = (Signal M) / (Signal M) + (Signal U)
得られた各遺伝子のメチル化率(mCpG)の値について、閾値を「0.4」に設定し、メチル化率の値がこの閾値と同じか、またはこの閾値よりも高い検体を「メチル化陽性検体」とした。そして、メチル化陽性検体の数に基づいて、各種の組織における遺伝子のメチル化陽性率(%)を下記の式により算出した。
メチル化陽性率(%)=(メチル化陽性検体数/総検体数)×100 For the methylation rate (mCpG) value of each gene obtained, set the threshold value to “0.4” and select a sample with a methylation rate value equal to or higher than this threshold value as “methylation positive sample. " Based on the number of methylation positive specimens, gene methylation positive rates (%) in various tissues were calculated by the following formula.
Methylation positive rate (%) = (number of methylation positive samples / total number of samples) x 100
メチル化陽性率(%)=(メチル化陽性検体数/総検体数)×100 For the methylation rate (mCpG) value of each gene obtained, set the threshold value to “0.4” and select a sample with a methylation rate value equal to or higher than this threshold value as “methylation positive sample. " Based on the number of methylation positive specimens, gene methylation positive rates (%) in various tissues were calculated by the following formula.
Methylation positive rate (%) = (number of methylation positive samples / total number of samples) x 100
例えば、癌部組織検体の場合、遺伝子のメチル化陽性率は「(癌部組織検体のうちメチル化陽性検体数/癌部組織の総検体数)×100」によって算出される。そして、癌部組織検体と正常脳組織検体との間で統計学的に有意な差を認められる場合に、その遺伝子を癌部組織でメチル化が認められるマーカーとした。
Infinium HumanMethylation27 BeadChip(Illumina社)を用いたデータマイニングの結果、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域が、脳腫瘍の癌部組織に高度にメチル化が認められるマーカーとして同定された(図1A~C参照)。以降、これらのマーカーを本発明のマーカーとも呼ぶ。 For example, in the case of cancer tissue samples, the gene methylation positive rate is calculated by “(number of methylation positive samples / total number of cancer tissue samples among cancer tissue samples) × 100”. When a statistically significant difference was observed between the cancer tissue sample and the normal brain tissue sample, the gene was used as a marker for methylation observed in the cancer tissue.
As a result of data mining using Infinium HumanMethylation27 BeadChip (Illumina), the promoter region of each gene of CREM, VRK2 and MYL12A was identified as a marker with high methylation observed in the cancerous tissue of brain tumor (FIG. 1A- C). Hereinafter, these markers are also called markers of the present invention.
Infinium HumanMethylation27 BeadChip(Illumina社)を用いたデータマイニングの結果、CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域が、脳腫瘍の癌部組織に高度にメチル化が認められるマーカーとして同定された(図1A~C参照)。以降、これらのマーカーを本発明のマーカーとも呼ぶ。 For example, in the case of cancer tissue samples, the gene methylation positive rate is calculated by “(number of methylation positive samples / total number of cancer tissue samples among cancer tissue samples) × 100”. When a statistically significant difference was observed between the cancer tissue sample and the normal brain tissue sample, the gene was used as a marker for methylation observed in the cancer tissue.
As a result of data mining using Infinium HumanMethylation27 BeadChip (Illumina), the promoter region of each gene of CREM, VRK2 and MYL12A was identified as a marker with high methylation observed in the cancerous tissue of brain tumor (FIG. 1A- C). Hereinafter, these markers are also called markers of the present invention.
参考例2: 複数種類の癌/腫瘍由来の癌/腫瘍組織検体、非癌部組織検体および正常組織検体におけるメチル化データの比較
(1)メチル化データの取得
本参考例では、参考例1で示される脳腫瘍の癌部組織(283検体)および正常脳組織(2検体)に加えて、9種類の癌/腫瘍組織検体、7種類の非癌部組織検体および12種類の正常組織検体のメチル化データを比較した。なお、各組織の検体数を以下の表に示す。 Reference Example 2: Comparison of methylation data in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples and normal tissue samples (1) Acquisition of methylation data In this reference example, reference example 1 In addition to the indicated cancerous tissue (283 samples) and normal brain tissue (2 samples) of the brain tumor, methylation of 9 types of cancer / tumor tissue samples, 7 types of non-cancerous tissue samples and 12 types of normal tissue samples The data were compared. The number of specimens in each tissue is shown in the following table.
(1)メチル化データの取得
本参考例では、参考例1で示される脳腫瘍の癌部組織(283検体)および正常脳組織(2検体)に加えて、9種類の癌/腫瘍組織検体、7種類の非癌部組織検体および12種類の正常組織検体のメチル化データを比較した。なお、各組織の検体数を以下の表に示す。 Reference Example 2: Comparison of methylation data in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples and normal tissue samples (1) Acquisition of methylation data In this reference example, reference example 1 In addition to the indicated cancerous tissue (283 samples) and normal brain tissue (2 samples) of the brain tumor, methylation of 9 types of cancer / tumor tissue samples, 7 types of non-cancerous tissue samples and 12 types of normal tissue samples The data were compared. The number of specimens in each tissue is shown in the following table.
上記の検体のうち、胃癌由来の癌組織検体、肝細胞癌由来の癌組織検体、肝細胞癌由来の非癌部組織検体、正常肝臓由来の正常組織検体、11種類の正常組織検体については、上記参考例1と同様にして、Infinium HumanMethylation27 BeadChip(Illumina社)を用いたInfinium Methylation Assayにより測定されたメチル化データを取得した。それ以外の検体については、TCGA(The Cancer Genome Atlas:http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp)、または肺癌、前立腺癌、健常人末梢血93検体については下記の文献において公開されているInfinium HumanMethylation27 BeadChip(Illumina社)のメチル化データを取得した。なお、ここでいうメチル化データとは、上記参考例1の(2)の記載のようにして得られた、CREM、VRK2およびMYL12AのCpG領域のメチル化率(mCpG)である。
・肺癌:Selamat SAら、Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression. Genome Res. Jul;22(7):1197-211(2012).
・前立腺癌:Kobayashi Yら、 DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer. Genome Res. Jul 21(7):1017-27(2011)
・健常人末梢血93献体のデータ:Salhia Bら、DNA methylation analysis determines the high frequency of genic hypomethylation and low frequency of hypermethylation events in plasma cell tumors. Cancer Res. Sep 1;70(17):6934-44(2010). Among the above specimens, cancer tissue specimens derived from gastric cancer, cancer tissue specimens derived from hepatocellular carcinoma, non-cancerous tissue specimens derived from hepatocellular carcinoma, normal tissue specimens derived from normal liver, 11 types of normal tissue specimens, In the same manner as in Reference Example 1, methylation data measured by Infinium Methylation Assay using Infinium HumanMethylation27 BeadChip (Illumina) was obtained. For other specimens, see TCGA (The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp), or lung cancer, prostate cancer, and 93 healthy human peripheral blood specimens below. The methylation data of Infinium HumanMethylation27 BeadChip (Illumina) published in the above literature was obtained. The methylation data referred to here is the methylation rate (mCpG) of the CpG region of CREM, VRK2 and MYL12A obtained as described in (2) of Reference Example 1 above.
・ Lung cancer: Selamat SA et al., Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression. Genome Res. Jul; 22 (7): 1197-211 (2012).
・ Prostate cancer: Kobayashi Y et al., DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer. Genome Res. Jul 21 (7): 1017-27 (2011)
・ Data of healthy human peripheral blood 93 donation: Salhia B et al., DNA methylation analysis determines the high frequency of genic hypomethylation and low frequency of hypermethylation events in plasma cell tumors. Cancer Res.Sep 1; 70 (17): 6934-44 ( 2010).
・肺癌:Selamat SAら、Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression. Genome Res. Jul;22(7):1197-211(2012).
・前立腺癌:Kobayashi Yら、 DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer. Genome Res. Jul 21(7):1017-27(2011)
・健常人末梢血93献体のデータ:Salhia Bら、DNA methylation analysis determines the high frequency of genic hypomethylation and low frequency of hypermethylation events in plasma cell tumors. Cancer Res. Sep 1;70(17):6934-44(2010). Among the above specimens, cancer tissue specimens derived from gastric cancer, cancer tissue specimens derived from hepatocellular carcinoma, non-cancerous tissue specimens derived from hepatocellular carcinoma, normal tissue specimens derived from normal liver, 11 types of normal tissue specimens, In the same manner as in Reference Example 1, methylation data measured by Infinium Methylation Assay using Infinium HumanMethylation27 BeadChip (Illumina) was obtained. For other specimens, see TCGA (The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp), or lung cancer, prostate cancer, and 93 healthy human peripheral blood specimens below. The methylation data of Infinium HumanMethylation27 BeadChip (Illumina) published in the above literature was obtained. The methylation data referred to here is the methylation rate (mCpG) of the CpG region of CREM, VRK2 and MYL12A obtained as described in (2) of Reference Example 1 above.
・ Lung cancer: Selamat SA et al., Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression. Genome Res. Jul; 22 (7): 1197-211 (2012).
・ Prostate cancer: Kobayashi Y et al., DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer. Genome Res. Jul 21 (7): 1017-27 (2011)
・ Data of healthy human peripheral blood 93 donation: Salhia B et al., DNA methylation analysis determines the high frequency of genic hypomethylation and low frequency of hypermethylation events in plasma cell tumors. Cancer Res.
(2)複数種類の癌/腫瘍由来の癌/腫瘍組織検体、非癌部組織検体および正常組織検体におけるメチル化陽性率の比較
各遺伝子のメチル化率(mCpG)の値について、閾値を「0.4」に設定し、メチル化率の値がこの閾値と同じか、またはこの閾値よりも高い検体を「メチル化陽性検体」とした。メチル化陽性検体の数に基づいて、各種の組織における本発明のマーカーのメチル化陽性率(%)を上記の式により算出した。結果を図2A~Cに示す。なお、図2において、「正常組織」とは、表4に示される組織のうち、健常人末梢血93検体(Salhia Bら)を除いた正常組織を表し、「正常血液」とは健常人末梢血93検体(Salhia Bら)を表す。 (2) Comparison of methylation positive rate in multiple types of cancer / tumor-derived cancer / tumor tissue specimens, non-cancerous tissue specimens and normal tissue specimens For the methylation rate (mCpG) value of each gene, A sample having a methylation rate value equal to or higher than this threshold value was defined as a “methylation positive sample”. Based on the number of methylation positive specimens, the methylation positive rate (%) of the marker of the present invention in various tissues was calculated by the above formula. The results are shown in FIGS. In FIG. 2, “normal tissue” refers to normal tissue excluding 93 healthy human peripheral blood samples (Salhia B et al.) Among the tissues shown in Table 4, and “normal blood” refers to normal human peripheral blood. Represents 93 blood samples (Salhia B et al.).
各遺伝子のメチル化率(mCpG)の値について、閾値を「0.4」に設定し、メチル化率の値がこの閾値と同じか、またはこの閾値よりも高い検体を「メチル化陽性検体」とした。メチル化陽性検体の数に基づいて、各種の組織における本発明のマーカーのメチル化陽性率(%)を上記の式により算出した。結果を図2A~Cに示す。なお、図2において、「正常組織」とは、表4に示される組織のうち、健常人末梢血93検体(Salhia Bら)を除いた正常組織を表し、「正常血液」とは健常人末梢血93検体(Salhia Bら)を表す。 (2) Comparison of methylation positive rate in multiple types of cancer / tumor-derived cancer / tumor tissue specimens, non-cancerous tissue specimens and normal tissue specimens For the methylation rate (mCpG) value of each gene, A sample having a methylation rate value equal to or higher than this threshold value was defined as a “methylation positive sample”. Based on the number of methylation positive specimens, the methylation positive rate (%) of the marker of the present invention in various tissues was calculated by the above formula. The results are shown in FIGS. In FIG. 2, “normal tissue” refers to normal tissue excluding 93 healthy human peripheral blood samples (Salhia B et al.) Among the tissues shown in Table 4, and “normal blood” refers to normal human peripheral blood. Represents 93 blood samples (Salhia B et al.).
図2A~Cより、本発明のマーカーはいずれも、他の種類の癌、ならびにヒト正常組織およびヒト正常血液(健常人末梢血、Salhia Bら)では高度なメチル化は認められず、脳腫瘍において特異的に、且つ高度にメチル化していることがわかった。
From FIGS. 2A to C, all the markers of the present invention are not highly methylated in other types of cancer, and normal human tissues and normal human blood (healthy human peripheral blood, Salhia B et al.). It was found to be specifically and highly methylated.
比較例1: 複数種類の癌/腫瘍由来の癌/腫瘍組織検体、非癌部組織検体および正常組織検体におけるメチル化陽性率の比較
脳腫瘍由来の腫瘍細胞においてメチル化されていることが既に知られているMGMTおよびCDKN2Aの各遺伝子(以下、「既知マーカー」という)について、参考例2と同様にして各組織におけるメチル化陽性率を算出した。結果を図3AおよびBに示す。 Comparative Example 1: Comparison of methylation positive rates in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples, and normal tissue samples It is already known that the tumor cells derived from brain tumors are methylated For each of the MGMT and CDKN2A genes (hereinafter referred to as “known markers”), the methylation positive rate in each tissue was calculated in the same manner as in Reference Example 2. The results are shown in FIGS. 3A and B.
脳腫瘍由来の腫瘍細胞においてメチル化されていることが既に知られているMGMTおよびCDKN2Aの各遺伝子(以下、「既知マーカー」という)について、参考例2と同様にして各組織におけるメチル化陽性率を算出した。結果を図3AおよびBに示す。 Comparative Example 1: Comparison of methylation positive rates in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples, and normal tissue samples It is already known that the tumor cells derived from brain tumors are methylated For each of the MGMT and CDKN2A genes (hereinafter referred to as “known markers”), the methylation positive rate in each tissue was calculated in the same manner as in Reference Example 2. The results are shown in FIGS. 3A and B.
図3AおよびBより、MGMTおよびCDKN2Aはいずれも脳腫瘍で症例の陽性率が高いが、他の種類の癌および他の非癌部組織でもメチル化を検出しているので、脳腫瘍への特異性は低い。したがって、既知マーカーは、脳腫瘍マーカーとして感度は高いが、特異性の面では低い。よって、既知マーカーは脳腫瘍の診断マーカーとしての性能において課題が多い。すなわち、他の種類の癌および他の非癌部組織における特異性を考慮したマーカー探索を実施する必要あることが示された。
From FIGS. 3A and B, both MGMT and CDKN2A have a high positive rate of cases in brain tumors, but methylation is detected in other types of cancer and other non-cancerous tissues. Low. Therefore, the known marker has high sensitivity as a brain tumor marker, but has low specificity. Therefore, known markers have many problems in performance as diagnostic markers for brain tumors. That is, it was shown that it is necessary to conduct a marker search in consideration of specificity in other types of cancer and other non-cancerous tissues.
実施例1: 正常脳および脳腫瘍におけるメチル化データ(MassARRAY)の比較
(1)生体試料
本実施例では、生体試料として、正常脳組織(1検体)および脳腫瘍の癌部組織(8検体)を用いた。 Example 1: Comparison of methylation data (MassARRAY) in normal brain and brain tumor (1) Biological sample In this example, normal brain tissue (1 sample) and cancerous tissue of brain tumor (8 samples) are used as the biological sample. It was.
(1)生体試料
本実施例では、生体試料として、正常脳組織(1検体)および脳腫瘍の癌部組織(8検体)を用いた。 Example 1: Comparison of methylation data (MassARRAY) in normal brain and brain tumor (1) Biological sample In this example, normal brain tissue (1 sample) and cancerous tissue of brain tumor (8 samples) are used as the biological sample. It was.
(2)測定用試料および対照試料の作製
(i)ゲノムDNAの抽出
上記の各組織からゲノムDNAを、QIAamp DNA Mini Kit(QIAGEN社)を用いて抽出した。得られたDNA溶液に含まれるゲノムDNAを、Bioruptor(COSMO BIO社製)によって断片化した。また、質量分析法における検量線を作成するため、対照用ゲノムDNAとして、ヒト末梢血リンパ球のゲノムDNAを用いた。このヒト末梢血リンパ球のゲノムDNAを、GenomiPhi v2DNA amplificationキット(GEヘルスケアライフサイエンス社)を用いて増幅した。得られた増幅産物は非メチル化DNAからなる。次いで、該増幅産物をBioruptor(COSMO BIO社製)により断片化して、非メチル化DNA断片(0%メチル化DNA)の溶液を得た。この非メチル化DNA断片の溶液の一部を取り、これにSssIメチラーゼ(New England Biolab社)を反応させることによりCG配列にある全てのシトシンをメチル化させて、メチル化DNA断片(100%メチル化DNA)の溶液を得た。そして、0%メチル化DNAの溶液と100%メチル化DNAの溶液とを所定の割合で混合して、25%、50%および75%メチル化DNAの溶液を得た。
(ii)バイサルファイト処理
上記で得られた各DNA断片(500 ng)を、EZ DNA Methylation Kit(Zymo Research社)を用いてバイサルファイト処理を行い、処理後のゲノムDNAを滅菌蒸留水(80μl)に溶出した。
(iii)PCRおよびIVTによる増幅
バイサルファイト処理した各DNA断片に含まれるメチル化シトシンおよびウラシルを、PCR法およびIVT増幅法により、それぞれグアニンおよびアデニンに変換した。
なお、PCR法に用いたプライマーセットがメチル化DNAおよび非メチル化DNAのいずれも偏りなく増幅できることは、後述する対照試料を用いたMassARRAY(登録商標)解析によって確認されている。本発明のマーカーに対するプライマーセットの配列を、表5に示した。また、CREM、VRK2およびMYL12A遺伝子のプロモータ領域について、表5に示されるプライマーセットで解析される領域の塩基配列を、それぞれ配列番号18~20で示した(配列番号18の塩基配列はマイナス鎖の配列であり、配列番号19および20の塩基配列はプラス鎖の配列である)。 (2) Preparation of measurement sample and control sample (i) Extraction of genomic DNA Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO). Further, in order to prepare a calibration curve in mass spectrometry, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA. Subsequently, the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA). A portion of the solution of this unmethylated DNA fragment is taken and reacted with SssI methylase (New England Biolab) to methylate all cytosines in the CG sequence to obtain a methylated DNA fragment (100% methyl). Solution). Then, a 0% methylated DNA solution and a 100% methylated DNA solution were mixed at a predetermined ratio to obtain 25%, 50% and 75% methylated DNA solutions.
(Ii) Bisulfite treatment Each DNA fragment (500 ng) obtained above was bisulfite treated using EZ DNA Methylation Kit (Zymo Research), and the treated genomic DNA was sterilized distilled water (80 μl). Eluted.
(Iii) Amplification by PCR and IVT Methylated cytosine and uracil contained in each bisulfite-treated DNA fragment were converted to guanine and adenine, respectively, by the PCR method and the IVT amplification method.
It has been confirmed by MassARRAY (registered trademark) analysis using a control sample described later that the primer set used in the PCR method can amplify both methylated DNA and unmethylated DNA without bias. Table 5 shows the primer set sequences for the markers of the present invention. Further, regarding the promoter regions of the CREM, VRK2 and MYL12A genes, the nucleotide sequences of the regions analyzed with the primer sets shown in Table 5 are shown in SEQ ID NOs: 18 to 20, respectively (the nucleotide sequence of SEQ ID NO: 18 is a minus strand) And the base sequences of SEQ ID NOs: 19 and 20 are positive strand sequences).
(i)ゲノムDNAの抽出
上記の各組織からゲノムDNAを、QIAamp DNA Mini Kit(QIAGEN社)を用いて抽出した。得られたDNA溶液に含まれるゲノムDNAを、Bioruptor(COSMO BIO社製)によって断片化した。また、質量分析法における検量線を作成するため、対照用ゲノムDNAとして、ヒト末梢血リンパ球のゲノムDNAを用いた。このヒト末梢血リンパ球のゲノムDNAを、GenomiPhi v2DNA amplificationキット(GEヘルスケアライフサイエンス社)を用いて増幅した。得られた増幅産物は非メチル化DNAからなる。次いで、該増幅産物をBioruptor(COSMO BIO社製)により断片化して、非メチル化DNA断片(0%メチル化DNA)の溶液を得た。この非メチル化DNA断片の溶液の一部を取り、これにSssIメチラーゼ(New England Biolab社)を反応させることによりCG配列にある全てのシトシンをメチル化させて、メチル化DNA断片(100%メチル化DNA)の溶液を得た。そして、0%メチル化DNAの溶液と100%メチル化DNAの溶液とを所定の割合で混合して、25%、50%および75%メチル化DNAの溶液を得た。
(ii)バイサルファイト処理
上記で得られた各DNA断片(500 ng)を、EZ DNA Methylation Kit(Zymo Research社)を用いてバイサルファイト処理を行い、処理後のゲノムDNAを滅菌蒸留水(80μl)に溶出した。
(iii)PCRおよびIVTによる増幅
バイサルファイト処理した各DNA断片に含まれるメチル化シトシンおよびウラシルを、PCR法およびIVT増幅法により、それぞれグアニンおよびアデニンに変換した。
なお、PCR法に用いたプライマーセットがメチル化DNAおよび非メチル化DNAのいずれも偏りなく増幅できることは、後述する対照試料を用いたMassARRAY(登録商標)解析によって確認されている。本発明のマーカーに対するプライマーセットの配列を、表5に示した。また、CREM、VRK2およびMYL12A遺伝子のプロモータ領域について、表5に示されるプライマーセットで解析される領域の塩基配列を、それぞれ配列番号18~20で示した(配列番号18の塩基配列はマイナス鎖の配列であり、配列番号19および20の塩基配列はプラス鎖の配列である)。 (2) Preparation of measurement sample and control sample (i) Extraction of genomic DNA Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO). Further, in order to prepare a calibration curve in mass spectrometry, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA. Subsequently, the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA). A portion of the solution of this unmethylated DNA fragment is taken and reacted with SssI methylase (New England Biolab) to methylate all cytosines in the CG sequence to obtain a methylated DNA fragment (100% methyl). Solution). Then, a 0% methylated DNA solution and a 100% methylated DNA solution were mixed at a predetermined ratio to obtain 25%, 50% and 75% methylated DNA solutions.
(Ii) Bisulfite treatment Each DNA fragment (500 ng) obtained above was bisulfite treated using EZ DNA Methylation Kit (Zymo Research), and the treated genomic DNA was sterilized distilled water (80 μl). Eluted.
(Iii) Amplification by PCR and IVT Methylated cytosine and uracil contained in each bisulfite-treated DNA fragment were converted to guanine and adenine, respectively, by the PCR method and the IVT amplification method.
It has been confirmed by MassARRAY (registered trademark) analysis using a control sample described later that the primer set used in the PCR method can amplify both methylated DNA and unmethylated DNA without bias. Table 5 shows the primer set sequences for the markers of the present invention. Further, regarding the promoter regions of the CREM, VRK2 and MYL12A genes, the nucleotide sequences of the regions analyzed with the primer sets shown in Table 5 are shown in SEQ ID NOs: 18 to 20, respectively (the nucleotide sequence of SEQ ID NO: 18 is a minus strand) And the base sequences of SEQ ID NOs: 19 and 20 are positive strand sequences).
なお、上記のプライマーセットのフォワードプライマーおよびリバースプライマーの5’末端には、IVT反応のために、それぞれ以下のタグ配列およびT7プロモータ配列が付加されている。
・タグ配列:AGGAAGAGAG (配列番号10)
・T7プロモータ配列:CAGTAATACGACTCACTATAGGGAGAAGGCT (配列番号11) The following tag sequence and T7 promoter sequence are added to the 5 ′ ends of the forward primer and reverse primer of the above primer set, respectively, for the IVT reaction.
Tag array: AGGAAGAGAG (SEQ ID NO: 10)
-T7 promoter sequence: CAGTAATACGACTCACTATAGGGAGAAGGCT (SEQ ID NO: 11)
・タグ配列:AGGAAGAGAG (配列番号10)
・T7プロモータ配列:CAGTAATACGACTCACTATAGGGAGAAGGCT (配列番号11) The following tag sequence and T7 promoter sequence are added to the 5 ′ ends of the forward primer and reverse primer of the above primer set, respectively, for the IVT reaction.
Tag array: AGGAAGAGAG (SEQ ID NO: 10)
-T7 promoter sequence: CAGTAATACGACTCACTATAGGGAGAAGGCT (SEQ ID NO: 11)
PCR反応液は、下記の試薬類を混合して調製した。
10x Hot Starバッファー(QIAGEN社) 0.5μL
25 mM dNTPミックス 0.04μL
Hot Star Taq(5U/μl)(QIAGEN社) 0.04μL
プライマーミックス 2.0μL
DNA溶液 1.0μL
水 1.42μL
トータル 5μL A PCR reaction solution was prepared by mixing the following reagents.
10x Hot Star buffer (QIAGEN) 0.5μL
25 mM dNTP mix 0.04 μL
Hot Star Taq (5U / μl) (QIAGEN) 0.04μL
Primer mix 2.0μL
DNA solution 1.0 μL
1.42μL water
Total 5μL
10x Hot Starバッファー(QIAGEN社) 0.5μL
25 mM dNTPミックス 0.04μL
Hot Star Taq(5U/μl)(QIAGEN社) 0.04μL
プライマーミックス 2.0μL
DNA溶液 1.0μL
水 1.42μL
トータル 5μL A PCR reaction solution was prepared by mixing the following reagents.
10x Hot Star buffer (QIAGEN) 0.5μL
25 mM dNTP mix 0.04 μL
Hot Star Taq (5U / μl) (QIAGEN) 0.04μL
Primer mix 2.0μL
DNA solution 1.0 μL
1.42μL water
Total 5μL
上記の反応液について、下記の条件でPCR反応を行った。
94℃で15分を1サイクル、
94℃で20秒、52℃で30秒および72℃で1分を45サイクル、
72℃で3分。 About said reaction liquid, PCR reaction was performed on condition of the following.
1 cycle at 94 ° C for 15 minutes,
45 cycles of 94 ° C for 20 seconds, 52 ° C for 30 seconds and 72 ° C for 1 minute,
3 minutes at 72 ° C.
94℃で15分を1サイクル、
94℃で20秒、52℃で30秒および72℃で1分を45サイクル、
72℃で3分。 About said reaction liquid, PCR reaction was performed on condition of the following.
1 cycle at 94 ° C for 15 minutes,
45 cycles of 94 ° C for 20 seconds, 52 ° C for 30 seconds and 72 ° C for 1 minute,
3 minutes at 72 ° C.
得られた各PCR産物を、MassCLEAVE(商標)Reagentキット(SEQUENOM社)に含まれるSAP(Shrimp Alkaline Phosphatase)を用いて脱リン酸化処理した。次いで、これらに、上記のキットを用いて調製した下記の反応液を添加した。
5x T7 R&DNAポリメラーゼバッファー 0.89μL
T Cleavageミックス 0.24μL
100 mM DTT 0.22μL
T7 R&DNAポリメラーゼ 0.44μL
RNase A 0.06μL
RNaseフリーの水 3.15μL
トータル 5μL Each obtained PCR product was dephosphorylated using SAP (Shrimp Alkaline Phosphatase) contained in the MassCLEAVE ™ Reagent kit (SEQUENOM). Subsequently, the following reaction liquid prepared using said kit was added to these.
5x T7 R & DNA polymerase buffer 0.89μL
T Cleavage mix 0.24μL
100 mM DTT 0.22 μL
T7 R & DNA polymerase 0.44μL
RNase A 0.06μL
RNase-free water 3.15 μL
Total 5μL
5x T7 R&DNAポリメラーゼバッファー 0.89μL
T Cleavageミックス 0.24μL
100 mM DTT 0.22μL
T7 R&DNAポリメラーゼ 0.44μL
RNase A 0.06μL
RNaseフリーの水 3.15μL
トータル 5μL Each obtained PCR product was dephosphorylated using SAP (Shrimp Alkaline Phosphatase) contained in the MassCLEAVE ™ Reagent kit (SEQUENOM). Subsequently, the following reaction liquid prepared using said kit was added to these.
5x T7 R & DNA polymerase buffer 0.89μL
T Cleavage mix 0.24μL
100 mM DTT 0.22 μL
T7 R & DNA polymerase 0.44μL
RNase A 0.06μL
RNase-free water 3.15 μL
Total 5μL
得られた混合物を37℃で3時間インキュベーションして、IVT反応と、ウラシルまたはチミン特異的切断反応とを行った。そして、得られた各反応物をClean Resin(SEQUENOM社)により精製して、質量分析法のための試料を得た。これらの試料について、上記の臨床検体から抽出したゲノムDNAに由来する試料を測定用試料とし、対照ゲノムDNAに由来する試料を対照試料として、後述する質量分析法に用いた。
The obtained mixture was incubated at 37 ° C. for 3 hours to perform IVT reaction and uracil or thymine-specific cleavage reaction. Then, each reaction product obtained was purified by Clean Resin (SEQUENOM) to obtain a sample for mass spectrometry. About these samples, the sample derived from the genomic DNA extracted from the above-mentioned clinical specimen was used as a measurement sample, and the sample derived from the control genomic DNA was used as a control sample for mass spectrometry described later.
(3)MassARRAY(登録商標)を用いた質量分析法によるメチル化状態の解析
(i)検量線の作成
上記で得られた対照試料を2回ずつ独立に質量分析を行った。得られた解析結果より、各プライマーセットについての検量線を作成し、相関係数を算出した。これにより、用いたプライマーセットは、メチル化DNAおよび非メチル化DNAのいずれにも偏りなく増幅できることが確認された。
(ii)測定用試料の解析
上記で得られた測定用試料の質量分析を行い、測定用試料に含まれるDNA断片のピークを得た。次いで、得られた各ピークが、CREM、VRK2およびMYL12Aの塩基配列のどの部分に由来するかを同定した。そして、同一の塩基配列に由来する断片について、メチル化CpG部位を含む断片のピークとメチル化CpG部位を含まない断片のピークとの面積比から、メチル化スコアを算出した。結果を図4A~Cに示す。 (3) Analysis of methylation state by mass spectrometry using MassARRAY (registered trademark) (i) Preparation of calibration curve Mass spectrometry of the control sample obtained above was carried out independently twice. From the obtained analysis results, a calibration curve for each primer set was created, and a correlation coefficient was calculated. Thereby, it was confirmed that the used primer set can be amplified to both methylated DNA and unmethylated DNA without bias.
(Ii) Analysis of measurement sample Mass analysis of the measurement sample obtained above was performed to obtain a peak of a DNA fragment contained in the measurement sample. Next, it was identified which part of the base sequences of CREM, VRK2 and MYL12A each peak obtained. For the fragments derived from the same base sequence, the methylation score was calculated from the area ratio of the peak of the fragment containing the methylated CpG site and the peak of the fragment not containing the methylated CpG site. The results are shown in FIGS.
(i)検量線の作成
上記で得られた対照試料を2回ずつ独立に質量分析を行った。得られた解析結果より、各プライマーセットについての検量線を作成し、相関係数を算出した。これにより、用いたプライマーセットは、メチル化DNAおよび非メチル化DNAのいずれにも偏りなく増幅できることが確認された。
(ii)測定用試料の解析
上記で得られた測定用試料の質量分析を行い、測定用試料に含まれるDNA断片のピークを得た。次いで、得られた各ピークが、CREM、VRK2およびMYL12Aの塩基配列のどの部分に由来するかを同定した。そして、同一の塩基配列に由来する断片について、メチル化CpG部位を含む断片のピークとメチル化CpG部位を含まない断片のピークとの面積比から、メチル化スコアを算出した。結果を図4A~Cに示す。 (3) Analysis of methylation state by mass spectrometry using MassARRAY (registered trademark) (i) Preparation of calibration curve Mass spectrometry of the control sample obtained above was carried out independently twice. From the obtained analysis results, a calibration curve for each primer set was created, and a correlation coefficient was calculated. Thereby, it was confirmed that the used primer set can be amplified to both methylated DNA and unmethylated DNA without bias.
(Ii) Analysis of measurement sample Mass analysis of the measurement sample obtained above was performed to obtain a peak of a DNA fragment contained in the measurement sample. Next, it was identified which part of the base sequences of CREM, VRK2 and MYL12A each peak obtained. For the fragments derived from the same base sequence, the methylation score was calculated from the area ratio of the peak of the fragment containing the methylated CpG site and the peak of the fragment not containing the methylated CpG site. The results are shown in FIGS.
図4A~Cより、CREM、VRK2およびMYL12Aのメチル化スコアは、正常脳組織で低く、脳腫瘍で顕著に高いことを確認した。参考例1のInfinium法の結果と同様に、本発明のマーカーのメチル化スコアと脳腫瘍とが相関することが示された。また、図4より、本発明のマーカーを用いるメチル化の頻度の解析では、脳腫瘍の検体と正常脳組織の検体とを区別可能な閾値を設定できることがわかった。
4A to 4C, it was confirmed that the methylation scores of CREM, VRK2 and MYL12A were low in normal brain tissues and remarkably high in brain tumors. Similar to the result of the Infinium method of Reference Example 1, it was shown that the methylation score of the marker of the present invention correlates with the brain tumor. Further, FIG. 4 shows that in the analysis of the methylation frequency using the marker of the present invention, it is possible to set a threshold value capable of distinguishing between a brain tumor specimen and a normal brain tissue specimen.
実施例2: 正常脳および脳腫瘍におけるメチル化データ(MSP)の比較
(1)生体試料
本実施例では、生体試料として、上記の実施例1と同じ、正常脳組織(1検体)および脳腫瘍の癌部組織(8検体)を用いた。 Example 2: Comparison of methylation data (MSP) in normal brain and brain tumor (1) Biological sample In this example, as a biological sample, the same normal brain tissue (one specimen) and brain tumor cancer as in Example 1 above. Tissue (8 specimens) was used.
(1)生体試料
本実施例では、生体試料として、上記の実施例1と同じ、正常脳組織(1検体)および脳腫瘍の癌部組織(8検体)を用いた。 Example 2: Comparison of methylation data (MSP) in normal brain and brain tumor (1) Biological sample In this example, as a biological sample, the same normal brain tissue (one specimen) and brain tumor cancer as in Example 1 above. Tissue (8 specimens) was used.
(2)測定用試料および対照試料の作製
(i)ゲノムDNAの抽出
上記の各組織からゲノムDNAを、QIAamp DNA Mini Kit(QIAGEN社)を用いて抽出した。得られたDNA溶液に含まれるゲノムDNAを、Bioruptor(COSMO BIO社製)によって断片化した。
また、対照用ゲノムDNAとして、ヒト末梢血リンパ球のゲノムDNAを用いた。このヒト末梢血リンパ球のゲノムDNAを、GenomiPhi v2DNA amplificationキット(GEヘルスケアライフサイエンス社)を用いて増幅した。得られた増幅産物は非メチル化DNAからなる。次いで、該増幅産物をBioruptor(COSMO BIO社製)により断片化して、非メチル化DNA断片(0%メチル化DNA)の溶液を得た。この非メチル化DNA断片の溶液の一部を取り、これにSssIメチラーゼ(New England Biolab社)を反応させることによりCG配列にある全てのシトシンをメチル化させて、メチル化DNA断片(100%メチル化DNA)の溶液を得た。
(ii)バイサルファイト処理
上記で得られた各DNA断片(500 ng)を、EZ DNA Methylation Kit(Zymo Research社)を用いてバイサルファイト処理を行い、処理後のゲノムDNAを滅菌蒸留水(80μl)に溶出した。 (2) Preparation of measurement sample and control sample (i) Extraction of genomic DNA Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO).
Further, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA. Subsequently, the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA). A portion of the solution of this unmethylated DNA fragment is taken and reacted with SssI methylase (New England Biolab) to methylate all cytosines in the CG sequence to obtain a methylated DNA fragment (100% methyl). Solution).
(Ii) Bisulfite treatment Each DNA fragment (500 ng) obtained above was subjected to bisulfite treatment using EZ DNA Methylation Kit (Zymo Research), and the treated genomic DNA was sterilized distilled water (80 μl) Eluted.
(i)ゲノムDNAの抽出
上記の各組織からゲノムDNAを、QIAamp DNA Mini Kit(QIAGEN社)を用いて抽出した。得られたDNA溶液に含まれるゲノムDNAを、Bioruptor(COSMO BIO社製)によって断片化した。
また、対照用ゲノムDNAとして、ヒト末梢血リンパ球のゲノムDNAを用いた。このヒト末梢血リンパ球のゲノムDNAを、GenomiPhi v2DNA amplificationキット(GEヘルスケアライフサイエンス社)を用いて増幅した。得られた増幅産物は非メチル化DNAからなる。次いで、該増幅産物をBioruptor(COSMO BIO社製)により断片化して、非メチル化DNA断片(0%メチル化DNA)の溶液を得た。この非メチル化DNA断片の溶液の一部を取り、これにSssIメチラーゼ(New England Biolab社)を反応させることによりCG配列にある全てのシトシンをメチル化させて、メチル化DNA断片(100%メチル化DNA)の溶液を得た。
(ii)バイサルファイト処理
上記で得られた各DNA断片(500 ng)を、EZ DNA Methylation Kit(Zymo Research社)を用いてバイサルファイト処理を行い、処理後のゲノムDNAを滅菌蒸留水(80μl)に溶出した。 (2) Preparation of measurement sample and control sample (i) Extraction of genomic DNA Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO).
Further, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA. Subsequently, the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA). A portion of the solution of this unmethylated DNA fragment is taken and reacted with SssI methylase (New England Biolab) to methylate all cytosines in the CG sequence to obtain a methylated DNA fragment (100% methyl). Solution).
(Ii) Bisulfite treatment Each DNA fragment (500 ng) obtained above was subjected to bisulfite treatment using EZ DNA Methylation Kit (Zymo Research), and the treated genomic DNA was sterilized distilled water (80 μl) Eluted.
(3)メチル化特異的PCR(MSP)
上記(2)で得られた測定用試料および対照試料(バイサルファイト処理後のDNA)を用いて、MSPを行った。MSPで用いたPCR試薬の組成、プライマーセットおよびPCRの反応条件を以下に示す。
<PCR試薬>
DDW(滅菌水) 16.8μL
10×PCR buffer with MgCl2(Roche社) 2.5μL
2mM dNTP mix 2.5μL
10μMセンスプライマー 1.0μL
10μMアンチセンスプライマー 1.0μL
Faststart Taq polymerase(Roche社) 0.2μL
測定用試料または対照試料 1.0μL
トータル 25μL (3) Methylation specific PCR (MSP)
MSP was performed using the measurement sample and control sample (DNA after bisulfite treatment) obtained in (2) above. The PCR reagent composition, primer set and PCR reaction conditions used in MSP are shown below.
<PCR reagents>
DDW (sterile water) 16.8μL
10 × PCR buffer with MgCl 2 (Roche) 2.5μL
2mM dNTP mix 2.5μL
10 μM sense primer 1.0 μL
10 μM antisense primer 1.0 μL
Faststart Taq polymerase (Roche) 0.2μL
Sample for measurement or control sample 1.0 μL
Total 25μL
上記(2)で得られた測定用試料および対照試料(バイサルファイト処理後のDNA)を用いて、MSPを行った。MSPで用いたPCR試薬の組成、プライマーセットおよびPCRの反応条件を以下に示す。
<PCR試薬>
DDW(滅菌水) 16.8μL
10×PCR buffer with MgCl2(Roche社) 2.5μL
2mM dNTP mix 2.5μL
10μMセンスプライマー 1.0μL
10μMアンチセンスプライマー 1.0μL
Faststart Taq polymerase(Roche社) 0.2μL
測定用試料または対照試料 1.0μL
トータル 25μL (3) Methylation specific PCR (MSP)
MSP was performed using the measurement sample and control sample (DNA after bisulfite treatment) obtained in (2) above. The PCR reagent composition, primer set and PCR reaction conditions used in MSP are shown below.
<PCR reagents>
DDW (sterile water) 16.8μL
10 × PCR buffer with MgCl 2 (Roche) 2.5μL
2mM dNTP mix 2.5μL
10 μM sense primer 1.0 μL
10 μM antisense primer 1.0 μL
Faststart Taq polymerase (Roche) 0.2μL
Sample for measurement or control sample 1.0 μL
Total 25μL
<プライマーセット>
上記のMSPで用いたプライマーセットを表6に示す。これらは、増幅される領域のDNAがメチル化している場合に増幅産物を得ることができるプライマーセットである。また、精度管理用プライマーセットとして、バイサルファイト処理が適切に行なわれたか否かを判定するためのプライマーセットも用いた(表7参照)。なお、CREM、VRK2およびMYL12A遺伝子のプロモータ領域について、表6に示されるプライマーセットで解析される領域の塩基配列を、それぞれ配列番号23~25で示した(配列番号23の塩基配列はマイナス鎖の配列であり、配列番号24および25の塩基配列はプラス鎖の配列である)。 <Primer set>
Table 6 shows primer sets used in the above MSP. These are primer sets that can obtain an amplification product when the DNA of the region to be amplified is methylated. In addition, a primer set for determining whether or not the bisulfite treatment was appropriately performed was also used as a quality control primer set (see Table 7). Regarding the promoter regions of the CREM, VRK2 and MYL12A genes, the base sequences of the regions analyzed with the primer sets shown in Table 6 are shown in SEQ ID NOs: 23 to 25, respectively (the base sequence of SEQ ID NO: 23 is a minus strand) And the base sequences of SEQ ID NOs: 24 and 25 are positive strand sequences).
上記のMSPで用いたプライマーセットを表6に示す。これらは、増幅される領域のDNAがメチル化している場合に増幅産物を得ることができるプライマーセットである。また、精度管理用プライマーセットとして、バイサルファイト処理が適切に行なわれたか否かを判定するためのプライマーセットも用いた(表7参照)。なお、CREM、VRK2およびMYL12A遺伝子のプロモータ領域について、表6に示されるプライマーセットで解析される領域の塩基配列を、それぞれ配列番号23~25で示した(配列番号23の塩基配列はマイナス鎖の配列であり、配列番号24および25の塩基配列はプラス鎖の配列である)。 <Primer set>
Table 6 shows primer sets used in the above MSP. These are primer sets that can obtain an amplification product when the DNA of the region to be amplified is methylated. In addition, a primer set for determining whether or not the bisulfite treatment was appropriately performed was also used as a quality control primer set (see Table 7). Regarding the promoter regions of the CREM, VRK2 and MYL12A genes, the base sequences of the regions analyzed with the primer sets shown in Table 6 are shown in SEQ ID NOs: 23 to 25, respectively (the base sequence of SEQ ID NO: 23 is a minus strand) And the base sequences of SEQ ID NOs: 24 and 25 are positive strand sequences).
<PCR反応条件>
95℃で6分、
95℃で30秒、X℃で30秒、72℃で30秒をYサイクル、
72℃で7分、
16℃で放置。
なお、上記の反応条件において「X」および「Y」はそれぞれ、表6および7に示されるアニーリング温度およびサイクル数を表す。 <PCR reaction conditions>
6 minutes at 95 ° C
Y cycle for 30 seconds at 95 ℃, 30 seconds at X ℃, 30 seconds at 72 ℃,
7 minutes at 72 ° C
Leave at 16 ° C.
In the above reaction conditions, “X” and “Y” represent the annealing temperature and the number of cycles shown in Tables 6 and 7, respectively.
95℃で6分、
95℃で30秒、X℃で30秒、72℃で30秒をYサイクル、
72℃で7分、
16℃で放置。
なお、上記の反応条件において「X」および「Y」はそれぞれ、表6および7に示されるアニーリング温度およびサイクル数を表す。 <PCR reaction conditions>
6 minutes at 95 ° C
Y cycle for 30 seconds at 95 ℃, 30 seconds at X ℃, 30 seconds at 72 ℃,
7 minutes at 72 ° C
Leave at 16 ° C.
In the above reaction conditions, “X” and “Y” represent the annealing temperature and the number of cycles shown in Tables 6 and 7, respectively.
(4)メチル化特異的PCR(MSP)の結果解析
上述のMSPで得られた増幅産物を2%アガロースゲル電気泳動で確認した。結果を図5に示す。なお、図中で「control」として示されている「0」および「100」はそれぞれ0%メチル化対照試料および100%メチル化対照試料を表す。 (4) Result analysis of methylation specific PCR (MSP) The amplification product obtained by the above-mentioned MSP was confirmed by 2% agarose gel electrophoresis. The results are shown in FIG. In the figure, “0” and “100” shown as “control” represent a 0% methylated control sample and a 100% methylated control sample, respectively.
上述のMSPで得られた増幅産物を2%アガロースゲル電気泳動で確認した。結果を図5に示す。なお、図中で「control」として示されている「0」および「100」はそれぞれ0%メチル化対照試料および100%メチル化対照試料を表す。 (4) Result analysis of methylation specific PCR (MSP) The amplification product obtained by the above-mentioned MSP was confirmed by 2% agarose gel electrophoresis. The results are shown in FIG. In the figure, “0” and “100” shown as “control” represent a 0% methylated control sample and a 100% methylated control sample, respectively.
図5より、精度管理用プライマーセットを用いたPCRにより全てのレーンにバンドが現れていた。このことから、試料のバイサルファイト処理が適切に行なわれたことがわかる。本発明のマーカーについてのPCRでは、正常脳組織ではメチル化CpGに由来するバンドを検出しなかった。他方で、脳腫瘍組織では、CREM、VRK2およびMYL12Aはそれぞれ8症例中8症例、8症例中7症例、および8例中7症例でバンドを検出した。このように、MSP法による本発明のマーカーのメチル化解析では、参考例1のInfinium法の結果と同様に、本発明のマーカーのメチル化と脳腫瘍とが相関することが示された。すなわち、CREM、VRK2およびMYL12Aは高率に脳腫瘍でメチル化傾向を示すが、他の正常組織では検出されない特異性の高いマーカーであることが示唆された。
From FIG. 5, bands appeared in all lanes by PCR using the primer set for quality control. This shows that the bisulfite treatment of the sample was performed appropriately. PCR for the markers of the present invention did not detect bands derived from methylated CpG in normal brain tissue. On the other hand, in the brain tumor tissue, CREM, VRK2 and MYL12A detected bands in 8 out of 8 cases, 7 out of 8 cases, and 7 out of 8 cases, respectively. As described above, the methylation analysis of the marker of the present invention by the MSP method showed that the methylation of the marker of the present invention and the brain tumor correlated with the results of the Infinium method of Reference Example 1. That is, it was suggested that CREM, VRK2 and MYL12A are highly specific markers that are highly methylated in brain tumors but not detected in other normal tissues.
実施例3: 血液試料を用いたMSP法によるメチル化解析
本実施例では、生体試料として末梢血を用いて、MSPにより、CREMおよびVRK2のプロモータ領域中のCpG部位についてメチル化解析を行った。
(1)生体試料
本実施例では、生体試料として、脳腫瘍患者から採取した末梢血(8検体)を用いた。また、対照試料として、健常人末梢血(3検体)を用いた。 Example 3 Methylation Analysis by MSP Method Using Blood Sample In this example, methylation analysis was performed on CpG sites in CREM and VRK2 promoter regions by MSP using peripheral blood as a biological sample.
(1) Biological Sample In this example, peripheral blood (8 samples) collected from a brain tumor patient was used as the biological sample. Moreover, healthy human peripheral blood (3 samples) was used as a control sample.
本実施例では、生体試料として末梢血を用いて、MSPにより、CREMおよびVRK2のプロモータ領域中のCpG部位についてメチル化解析を行った。
(1)生体試料
本実施例では、生体試料として、脳腫瘍患者から採取した末梢血(8検体)を用いた。また、対照試料として、健常人末梢血(3検体)を用いた。 Example 3 Methylation Analysis by MSP Method Using Blood Sample In this example, methylation analysis was performed on CpG sites in CREM and VRK2 promoter regions by MSP using peripheral blood as a biological sample.
(1) Biological Sample In this example, peripheral blood (8 samples) collected from a brain tumor patient was used as the biological sample. Moreover, healthy human peripheral blood (3 samples) was used as a control sample.
(2)測定用試料の作製
(i)ゲノムDNAの抽出
上記の各末梢血(2ml)から常法により血漿を得た。得られた各血漿からゲノムDNAを、QIAamp Circulating Nucleic Acid Kit(QIAGEN社)を用いて抽出した。
(ii)バイサルファイト処理
上記で得られた各DNA断片(500 ng)を、EZ DNA Methylation Kit(Zymo Research社)を用いてバイサルファイト処理を行い、処理後のゲノムDNAを滅菌蒸留水(80μl)に溶出した。 (2) Preparation of measurement sample (i) Extraction of genomic DNA Plasma was obtained from each of the above peripheral blood (2 ml) by a conventional method. Genomic DNA was extracted from each plasma obtained using QIAamp Circulating Nucleic Acid Kit (QIAGEN).
(Ii) Bisulfite treatment Each DNA fragment (500 ng) obtained above was subjected to bisulfite treatment using EZ DNA Methylation Kit (Zymo Research), and the treated genomic DNA was sterilized distilled water (80 μl) Eluted.
(i)ゲノムDNAの抽出
上記の各末梢血(2ml)から常法により血漿を得た。得られた各血漿からゲノムDNAを、QIAamp Circulating Nucleic Acid Kit(QIAGEN社)を用いて抽出した。
(ii)バイサルファイト処理
上記で得られた各DNA断片(500 ng)を、EZ DNA Methylation Kit(Zymo Research社)を用いてバイサルファイト処理を行い、処理後のゲノムDNAを滅菌蒸留水(80μl)に溶出した。 (2) Preparation of measurement sample (i) Extraction of genomic DNA Plasma was obtained from each of the above peripheral blood (2 ml) by a conventional method. Genomic DNA was extracted from each plasma obtained using QIAamp Circulating Nucleic Acid Kit (QIAGEN).
(Ii) Bisulfite treatment Each DNA fragment (500 ng) obtained above was subjected to bisulfite treatment using EZ DNA Methylation Kit (Zymo Research), and the treated genomic DNA was sterilized distilled water (80 μl) Eluted.
(3)MSP
上記(2)で得られた測定用試料を用いて、定性MSPを行った。用いたPCR試薬の組成およびPCR条件を以下に示す。なお、本実施例に用いたプライマーセット(CREM、VRK2および精度管理)の塩基配列は、表6および表7に示されている。
<PCR試薬>
DW(滅菌水) 10.88μL
10×PCR buffer with MgCl2(Roche社) 1.5μL
10mM dNTP mix 0.3μL
10μMセンスプライマー 0.6μL
10μMアンチセンスプライマー 0.6μL
Faststart Taq polymerase(Roche社) 0.12μL
測定用試料 1.0μL
トータル 15μL (3) MSP
Qualitative MSP was performed using the measurement sample obtained in (2) above. The composition of PCR reagents used and the PCR conditions are shown below. The base sequences of the primer sets (CREM, VRK2, and accuracy control) used in this example are shown in Table 6 and Table 7.
<PCR reagents>
DW (sterile water) 10.88μL
10 × PCR buffer with MgCl 2 (Roche) 1.5μL
10mM dNTP mix 0.3μL
10μM sense primer 0.6μL
10μM antisense primer 0.6μL
Faststart Taq polymerase (Roche) 0.12μL
Sample for measurement 1.0μL
Total 15μL
上記(2)で得られた測定用試料を用いて、定性MSPを行った。用いたPCR試薬の組成およびPCR条件を以下に示す。なお、本実施例に用いたプライマーセット(CREM、VRK2および精度管理)の塩基配列は、表6および表7に示されている。
<PCR試薬>
DW(滅菌水) 10.88μL
10×PCR buffer with MgCl2(Roche社) 1.5μL
10mM dNTP mix 0.3μL
10μMセンスプライマー 0.6μL
10μMアンチセンスプライマー 0.6μL
Faststart Taq polymerase(Roche社) 0.12μL
測定用試料 1.0μL
トータル 15μL (3) MSP
Qualitative MSP was performed using the measurement sample obtained in (2) above. The composition of PCR reagents used and the PCR conditions are shown below. The base sequences of the primer sets (CREM, VRK2, and accuracy control) used in this example are shown in Table 6 and Table 7.
<PCR reagents>
DW (sterile water) 10.88μL
10 × PCR buffer with MgCl 2 (Roche) 1.5μL
10mM dNTP mix 0.3μL
10μM sense primer 0.6μL
10μM antisense primer 0.6μL
Faststart Taq polymerase (Roche) 0.12μL
Sample for measurement 1.0μL
Total 15μL
<PCR反応条件>
95℃で6分、
95℃で30秒、X℃で30秒、72℃で30秒をYサイクル、
72℃で7分、
16℃で放置。
なお、上記の反応条件において「X」および「Y」はそれぞれ、表8に示されるアニーリング温度およびサイクル数を表す。 <PCR reaction conditions>
6 minutes at 95 ° C
Y cycle for 30 seconds at 95 ℃, 30 seconds at X ℃, 30 seconds at 72 ℃,
7 minutes at 72 ° C
Leave at 16 ° C.
In the above reaction conditions, “X” and “Y” represent the annealing temperature and the number of cycles shown in Table 8, respectively.
95℃で6分、
95℃で30秒、X℃で30秒、72℃で30秒をYサイクル、
72℃で7分、
16℃で放置。
なお、上記の反応条件において「X」および「Y」はそれぞれ、表8に示されるアニーリング温度およびサイクル数を表す。 <PCR reaction conditions>
6 minutes at 95 ° C
Y cycle for 30 seconds at 95 ℃, 30 seconds at X ℃, 30 seconds at 72 ℃,
7 minutes at 72 ° C
Leave at 16 ° C.
In the above reaction conditions, “X” and “Y” represent the annealing temperature and the number of cycles shown in Table 8, respectively.
(4)MSPの結果解析
上述のMSPで得られた増幅産物を2%アガロースゲル電気泳動で確認した。各増幅産物のバンド強度をグラフで表した(図6A~C参照)。図6AおよびBにおいて、健常人の末梢血として表示されている棒グラフはバックグラウンドに相当し、バンドを検出していないことを示す。図6AおよびBより、CREMおよびVRK2についてのPCRの結果、健常人の末梢血ではバンドを検出せず、脳腫瘍患者の末梢血では8症例中7症例でバンドを検出することができたことがわかる。また、CREMおよびVRK2を組み合わせることで、8症例の全てにおいてバンドを検出することができ、本発明のマーカーの高い性能を示された。なお、図6Cより、精度管理用プライマーセットを用いたPCRにより、全てのサンプルにおいてバンドが検出されたことがわかる。これは、試料のバイサルファイト処理が適切に行なわれたことを示している。このように、本発明のマーカーは、生体試料として末梢血を用いた場合でも、脳腫瘍患者において高率にメチル化傾向を示し、健常人ではメチル化が検出されないことがわかった。したがって、本発明のマーカーは、脳腫瘍に特異性の高いマーカーであり、血液試料中で脳腫瘍に由来する癌細胞の存否を判定するのに有用であることが示唆された。 (4) Result analysis of MSP The amplification product obtained by the above-mentioned MSP was confirmed by 2% agarose gel electrophoresis. The band intensity of each amplification product was represented graphically (see FIGS. 6A-C). In FIGS. 6A and B, the bar graph displayed as the peripheral blood of a healthy person corresponds to the background and indicates that no band is detected. 6A and 6B, as a result of PCR for CREM and VRK2, it was found that no band was detected in the peripheral blood of healthy individuals, and a band could be detected in 7 out of 8 cases in the peripheral blood of brain tumor patients. . In addition, by combining CREM and VRK2, bands were detected in all 8 cases, and the high performance of the marker of the present invention was shown. FIG. 6C shows that bands were detected in all samples by PCR using the primer set for quality control. This indicates that the bisulfite treatment of the sample was properly performed. Thus, it was found that the marker of the present invention showed a high methylation tendency in brain tumor patients even when peripheral blood was used as a biological sample, and methylation was not detected in healthy individuals. Therefore, it was suggested that the marker of the present invention is a marker highly specific for brain tumors, and is useful for determining the presence or absence of cancer cells derived from brain tumors in blood samples.
上述のMSPで得られた増幅産物を2%アガロースゲル電気泳動で確認した。各増幅産物のバンド強度をグラフで表した(図6A~C参照)。図6AおよびBにおいて、健常人の末梢血として表示されている棒グラフはバックグラウンドに相当し、バンドを検出していないことを示す。図6AおよびBより、CREMおよびVRK2についてのPCRの結果、健常人の末梢血ではバンドを検出せず、脳腫瘍患者の末梢血では8症例中7症例でバンドを検出することができたことがわかる。また、CREMおよびVRK2を組み合わせることで、8症例の全てにおいてバンドを検出することができ、本発明のマーカーの高い性能を示された。なお、図6Cより、精度管理用プライマーセットを用いたPCRにより、全てのサンプルにおいてバンドが検出されたことがわかる。これは、試料のバイサルファイト処理が適切に行なわれたことを示している。このように、本発明のマーカーは、生体試料として末梢血を用いた場合でも、脳腫瘍患者において高率にメチル化傾向を示し、健常人ではメチル化が検出されないことがわかった。したがって、本発明のマーカーは、脳腫瘍に特異性の高いマーカーであり、血液試料中で脳腫瘍に由来する癌細胞の存否を判定するのに有用であることが示唆された。 (4) Result analysis of MSP The amplification product obtained by the above-mentioned MSP was confirmed by 2% agarose gel electrophoresis. The band intensity of each amplification product was represented graphically (see FIGS. 6A-C). In FIGS. 6A and B, the bar graph displayed as the peripheral blood of a healthy person corresponds to the background and indicates that no band is detected. 6A and 6B, as a result of PCR for CREM and VRK2, it was found that no band was detected in the peripheral blood of healthy individuals, and a band could be detected in 7 out of 8 cases in the peripheral blood of brain tumor patients. . In addition, by combining CREM and VRK2, bands were detected in all 8 cases, and the high performance of the marker of the present invention was shown. FIG. 6C shows that bands were detected in all samples by PCR using the primer set for quality control. This indicates that the bisulfite treatment of the sample was properly performed. Thus, it was found that the marker of the present invention showed a high methylation tendency in brain tumor patients even when peripheral blood was used as a biological sample, and methylation was not detected in healthy individuals. Therefore, it was suggested that the marker of the present invention is a marker highly specific for brain tumors, and is useful for determining the presence or absence of cancer cells derived from brain tumors in blood samples.
1 判定装置
2 測定装置
3 コンピュータシステム
3a コンピュータ本体
3b 入力デバイス
3c 表示部 DESCRIPTION OFSYMBOLS 1 Determination apparatus 2 Measuring apparatus 3 Computer system 3a Computer main body 3b Input device 3c Display part
2 測定装置
3 コンピュータシステム
3a コンピュータ本体
3b 入力デバイス
3c 表示部 DESCRIPTION OF
Claims (10)
- 被験者から採取した生体試料からDNA試料を調製する工程と、
調製工程で得られたDNA試料に含まれるCREM、VRK2およびMYL12Aから選択される少なくとも1つの遺伝子のプロモータ領域に存在するCpG部位のメチル化の状態を解析する工程と、
解析工程で得られた解析結果に基づいて、前記被験者の脳腫瘍に関する情報を取得する工程と
を含む、脳腫瘍に関する情報の取得方法。 A step of preparing a DNA sample from a biological sample collected from a subject;
Analyzing the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the DNA sample obtained in the preparation step;
A method of acquiring information related to a brain tumor, comprising: acquiring information related to a brain tumor of the subject based on an analysis result obtained in the analysis step. - 脳腫瘍がグリオーマである請求項1に記載の方法。 The method according to claim 1, wherein the brain tumor is glioma.
- 解析工程が、少なくとも1つのCpG部位のメチル化の有無を解析する工程である請求項1または2に記載の方法。 The method according to claim 1 or 2, wherein the analyzing step is a step of analyzing the presence or absence of methylation of at least one CpG site.
- 被験者の脳腫瘍に関する情報が、被験者から採取した生体試料における脳腫瘍に由来する腫瘍細胞の存否であり、
情報取得工程が、メチル化されたCpG部位が有るという解析結果が得られた場合に、生体試料中に脳腫瘍由来の腫瘍細胞が存在するという情報を取得する工程である請求項3に記載の方法。 Information regarding the brain tumor of the subject is the presence or absence of tumor cells derived from the brain tumor in a biological sample collected from the subject,
The method according to claim 3, wherein the information acquisition step is a step of acquiring information that a tumor cell derived from a brain tumor is present in a biological sample when an analysis result indicating that there is a methylated CpG site is obtained. . - 解析工程が、メチル化の頻度を解析する工程である請求項1または2に記載の方法。 The method according to claim 1 or 2, wherein the analysis step is a step of analyzing the frequency of methylation.
- 被験者の脳腫瘍に関する情報が、被験者から採取した生体試料における脳腫瘍に由来する腫瘍細胞の存否であり、
情報取得工程が、解析工程で得られたメチル化の頻度が所定の閾値より高い場合に、生体試料中に脳腫瘍由来の腫瘍細胞が存在するという情報を取得する工程である、
請求項5に記載の方法。 Information regarding the brain tumor of the subject is the presence or absence of tumor cells derived from the brain tumor in a biological sample collected from the subject,
The information acquisition step is a step of acquiring information that a tumor cell derived from a brain tumor is present in a biological sample when the methylation frequency obtained in the analysis step is higher than a predetermined threshold.
The method of claim 5. - CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つである、メチル化解析により脳腫瘍に関する情報を取得するためのマーカー。 A marker for acquiring information on brain tumors by methylation analysis, which is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
- CREM、VRK2およびMYL12Aの各遺伝子のプロモータ領域に存在するCpG部位から選択される少なくとも1つのメチル化の状態を解析するためのプライマーセットを含む、脳腫瘍に関する情報を取得するためのキット。 A kit for obtaining information on brain tumors, including a primer set for analyzing at least one methylation state selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
- プライマーセットが、質量分析法およびメチル化特異的PCR法から選択される少なくとも1つの方法によりCpG部位のメチル化の状態を解析するためのプライマーセットである請求項8に記載のキット。 The kit according to claim 8, wherein the primer set is a primer set for analyzing the methylation state of the CpG site by at least one method selected from mass spectrometry and methylation-specific PCR.
- プライマーセットが、
配列番号4および配列番号5の塩基配列であるプライマーセット、
配列番号6および配列番号7の塩基配列であるプライマーセット、
配列番号8および配列番号9の塩基配列であるプライマーセット、
配列番号12および配列番号13の塩基配列であるプライマーセット、
配列番号14および配列番号15の塩基配列であるプライマーセット、ならびに
配列番号16および配列番号17の塩基配列であるプライマーセット
から選択される少なくとも1つである請求項9に記載のキット。 Primer set
A primer set which is the base sequence of SEQ ID NO: 4 and SEQ ID NO: 5,
A primer set which is the base sequence of SEQ ID NO: 6 and SEQ ID NO: 7,
A primer set which is the base sequence of SEQ ID NO: 8 and SEQ ID NO: 9,
A primer set which is the base sequence of SEQ ID NO: 12 and SEQ ID NO: 13,
The kit according to claim 9, wherein the kit is at least one selected from a primer set that is the base sequence of SEQ ID NO: 14 and SEQ ID NO: 15 and a primer set that is the base sequence of SEQ ID NO: 16 and SEQ ID NO: 17.
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CN112626184A (en) * | 2020-12-31 | 2021-04-09 | 深圳市第二人民医院(深圳市转化医学研究院) | Method for detecting methylation state of MGMT gene promoter |
JP2022078120A (en) * | 2015-01-18 | 2022-05-24 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Method and system for determining state of cancer |
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JP2002504308A (en) * | 1997-11-22 | 2002-02-12 | ハイセック インコーポレイテッド | Novel EGF motif protein obtained from fetal liver-spleen cDNA library |
WO2011112845A2 (en) * | 2010-03-11 | 2011-09-15 | Board Of Regents, The University Of Texas System | Methods and compositions related to a multi-methylation assay to predict patient outcome |
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JP2002504308A (en) * | 1997-11-22 | 2002-02-12 | ハイセック インコーポレイテッド | Novel EGF motif protein obtained from fetal liver-spleen cDNA library |
WO2011112845A2 (en) * | 2010-03-11 | 2011-09-15 | Board Of Regents, The University Of Texas System | Methods and compositions related to a multi-methylation assay to predict patient outcome |
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JP2022078120A (en) * | 2015-01-18 | 2022-05-24 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Method and system for determining state of cancer |
CN112626184A (en) * | 2020-12-31 | 2021-04-09 | 深圳市第二人民医院(深圳市转化医学研究院) | Method for detecting methylation state of MGMT gene promoter |
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