JP7142872B2 - Method for assisting diagnosis of gastric cancer risk, artificial DNA used in the method, and kit for diagnosing gastric cancer risk - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for assisting diagnosis of gastric cancer development risk of a subject. The present invention also relates to an artificial DNA and a kit for diagnosing the risk of developing gastric cancer that are used in the method.

The main cause of gastric cancer is Helicobacter pylori infection. When gastric mucosal changes occur due to Helicobacter pylori infection, regular follow-up is recommended for screening of gastric cancer even after eradication. Gastric cancer examinations include gastric endoscopy and gastric X-ray examination. However, gastroscopy may be accompanied by a vomiting reflex when a gastroscope is inserted, and gastric X-rays require swallowing barium. heavy burden on

In recent years, cancer diagnostic methods based on genetic abnormalities accumulated in normal gastric mucosa have been studied. Such methods include, for example, methods based on information about abnormal DNA methylation. In this method, a CpG site in the nucleotide sequence of a given gene is used as a marker. Then, information such as the presence or absence of cancer cells and the degree of accumulation of genetic abnormality is obtained based on the analysis results of the methylation state of the marker using gastric mucosal DNA collected by endoscopy or the like, and this information is used as an index. Then, the presence of cancer, the pathology of cancer, the risk of carcinogenesis, etc. are diagnosed.

For example, Patent Document 1 discloses that gastric cancer can be diagnosed at an early stage by measuring the methylation level of the CpG island of the SDC2 gene. In addition, p53 gene, ADCY3 gene, BARHL2 gene, ACGM1 gene, VLDLR gene and the like have been reported as highly methylated genes in gastric cancer (Patent Documents 2 to 6).

U.S. Patent Application Publication No. 2016/040244 U.S. Patent Application Publication No. 2015/254400 U.S. Patent Application Publication No. 2015/240313 JP 2014-161308 Japanese Patent Application Laid-Open No. 2007-54059 U.S. Patent Application Publication No. 2009/054245

In order to reduce the burden on subjects who wish to be tested regularly, it is desirable to develop a methylation marker that can further evaluate cancer risk as one of cancer risk diagnoses using DNA methylation. was rare.

The present inventors focused on the fact that among those who have been infected with Helicobacter pylori, there are those who develop gastric cancer and those who do not. The present inventors verified genomic DNA obtained from non-cancerous tissues of gastric cancer patients who had experience of H. pylori infection and those who had experience of H. pylori infection but did not develop gastric cancer. We identified a gene region in which methylation was observed at the site as a novel marker. Based on the results obtained by analyzing the methylation status of these markers, the present inventors have found that the risk of developing gastric cancer in persons who have been infected with Helicobacter pylori can be evaluated, and have completed the present invention.

That is, according to the present invention, based on the results obtained in the step of analyzing the methylation state of the CpG site present in the gene region of the gene contained in the DNA sample of the subject and the analysis step, including a step of obtaining information on the risk of developing gastric cancer, wherein the DNA sample is prepared from a non-cancerous tissue sample collected from a subject with a history of H. pylori infection, and the genes are BHLHE22 gene, GUSBP5 gene, RIMS1 gene, LINC00643 gene, Provided is at least one selected from the group consisting of FGF12 gene, FLT3 gene, RPRM gene, JAM2 gene, SEPT9 gene and STX16 gene, and a method for assisting the diagnosis of gastric cancer risk.

Further, according to the present invention, the continuous bases of all or part of the genetic region of a gene, which is prepared from a non-cancerous sample collected from a subject who has a history of infection with H. pylori, is used in the diagnostic aid method described above. It has a sequence obtained by bisulfite treatment of the sequence, the gene region contains a CpG site and a cytosine base that does not constitute the CpG site, is used as a marker for diagnosing the risk of developing gastric cancer, and the gene is BHLHE22 An artificial DNA is provided which is at least one selected from the group consisting of genes, GUSBP5 gene, RIMS1 gene, LINC00643 gene, FGF12 gene, FLT3 gene, RPRM gene, JAM2 gene, SEPT9 gene and STX16 gene.

Furthermore, according to the present invention, a forward primer and a reverse primer, which are used in the diagnostic aid method described above, are included, and the forward primer and the reverse primer are present in a nucleotide sequence having a CpG site in the gene region other than the CpG site. It hybridizes to a nucleic acid consisting of a base sequence in which cytosine is converted to another base, or hybridizes to a region containing a methylated CpG site, and the gene is BHLHE22 gene, GUSBP5 gene, RIMS1 gene, LINC00643 gene, FGF12 gene, A kit comprising at least one selected from the group consisting of FLT3 gene, RPRM gene, JAM2 gene, SEPT9 gene and STX16 gene is provided.

INDUSTRIAL APPLICABILITY The present invention can provide a method capable of assisting diagnosis of the risk of developing gastric cancer. In addition, the present invention can provide an artificial DNA and a kit for diagnosing the risk of developing gastric cancer that are used in the method.

FIG. 10 is a diagram showing an ROC curve in Example 3; FIG. 2 is a schematic diagram showing an example of a diagnostic assisting device for providing information on a subject's risk of developing stomach cancer. FIG. 3 is a block diagram showing the functional configuration of the diagnosis assisting device of FIG. 2; FIG. FIG. 3 is a block diagram showing the hardware configuration of the diagnosis assisting device shown in FIG. 2; FIG. FIG. 3 is a flow chart of determination for providing information on the risk of developing gastric cancer of a subject using the diagnosis assisting device shown in FIG. 2. FIG.

In the method for assisting the diagnosis of the risk of developing gastric cancer (hereinafter also simply referred to as the "method") of the present embodiment, first, a DNA sample is prepared from a non-cancerous tissue sample collected from a subject who has been infected with Helicobacter pylori. Prepare. Subjects with a history of H. pylori infection include H. pylori infected carriers and previously infected individuals who have been eradicated. The presence or absence of a history of infection with Helicobacter pylori can be determined by, for example, whether or not atrophic gastritis in the pyloric region is observed by endoscopic observation. Whether or not a person is a carrier infected with Helicobacter pylori can be determined by, for example, a Helicobacter pylori IgG antibody test, a urea respiration test method, or the like.

In this embodiment, the non-cancerous tissue sample is not limited as long as it is prepared from the non-cancerous tissue sample collected from the subject. A non-cancerous part refers to a part that does not substantially contain cancer cells. Preferred are samples containing genomic DNA, such as clinical specimens. Clinical specimens include, for example, body fluids, urine, tissues obtained by surgery or biopsy, and the like. Body fluids include blood, serum, plasma, lymph, ascites, bone marrow fluid, nipple discharge, and the like. A culture obtained by culturing cells or tissues collected from a subject can also be used as a non-cancerous tissue sample. Furthermore, a formalin-fixed, paraffin-embedded (FFPE) sample of tissue taken from a subject may be used as a non-cancerous sample. Gastric mucosa substantially free of gastric cancer cells is particularly preferred as the non-cancerous sample.

A DNA sample can be prepared by extracting DNA from a non-cancerous tissue sample. Methods for extracting DNA from non-cancerous samples are known in the art. For example, a non-cancerous tissue sample is mixed with a treatment solution containing a surfactant that solubilizes cells or tissues (for example, sodium cholate, sodium dodecyl sulfate, etc.), and the resulting mixture is subjected to physical treatment (stirring, DNA can be extracted by subjecting the sample to homogenization, sonication, etc. to liberate the DNA contained in the non-cancerous tissue sample into the mixture. In this case, the mixture is preferably centrifuged to precipitate cell debris, and the supernatant containing the released DNA is preferably used in the analysis step described below. Also, the resulting supernatant may be purified by methods known in the art. The extraction and purification of DNA from non-cancerous tissue samples can also be performed using commercially available kits.

The above preparation step preferably further includes a step of fragmenting the extracted DNA. By fragmenting DNA into appropriate lengths, the methylated DNA immunoprecipitation (MeDIP) method and unmethylated cytosine conversion treatment, which will be described later, can be performed efficiently.
Fragmentation of DNA can be performed by sonication, alkali treatment, restriction enzyme treatment, or the like. For example, when DNA is fragmented by alkali treatment, a sodium hydroxide solution is added to the DNA solution to a final concentration of 0.1 to 1.0N, and the DNA is fragmented by incubating at 10 to 40°C for 5 to 15 minutes. become. When DNA is fragmented by restriction enzyme treatment, the restriction enzyme to be used is appropriately selected based on the base sequence of DNA, and for example, MseI or BamHI is used.

In the method of the present embodiment, the BHLHE22 gene, FGF12 gene, FLT3 gene, RPRM gene, RIMS1 gene, JAM2 gene, LINC00643 gene, SEPT9 gene, STX16 gene and GUSBP5 gene contained in the DNA obtained in the above preparation step. The methylation state of CpG sites present in the gene region of at least one gene selected from the group is analyzed. As used herein, the term "gene region" may include not only transcription regions but also regions involved in expression control of the gene, such as promoter regions upstream of transcription initiation sites. A transcribed region can include a 5' untranslated region (5'UTR), a gene body (body), a 3' untranslated region (3'UTR), and the like. A gene body may also contain exons and introns. As used herein, the term "CpG site" means a sequence site in which cytosine (C) and guanine (G) are adjacent in this order from 5' to 3' in the nucleotide sequence. The "p" letter in CpG represents the phosphodiester bond between cytosine and guanine.

The nucleotide sequences of the BHLHE22 gene, FGF12 gene, FLT3 gene, RPRM gene, RIMS1 gene, JAM2 gene, LINC00643 gene, SEPT9 gene, STX16 gene and GUSBP5 gene are known per se, and are available from known databases such as GeneBank and Ensembl. be. Table 1 shows the Gene bank or Ensembl accession numbers of the above genes.

The gene sequence of each gene described above may include, for example, the base sequences of SEQ ID NOs: 1 to 9 or 62, or their reverse complementary strand sequences. SEQ ID NO: 1 is BHLHE22, SEQ ID NO: 2 is FGF12, SEQ ID NO: 3 is FLT3, SEQ ID NO: 4 is RPRM, SEQ ID NO: 5 is RIMS1, SEQ ID NO: 6 is JAM2, SEQ ID NO: 7 is LONC00643, SEQ ID NO: 8 is SEPT9, SEQ ID NO: 8 9 is the region of STX16, and SEQ ID NO: 62 is the region of GUSBP5.

In this embodiment, the analysis of the methylation state of CpG sites is performed, for example, by examining whether cytosine in at least one CpG site among the CpG sites present in each gene region is methylated. can be done. The CpG site to be analyzed may be on the Top strand side or on the Bottom strand side. Also, one or more CpG sites may be analyzed. A plurality of CpG sites may be selected from the gene region of one gene, or may be selected from each of the gene regions of a plurality of genes. Among the CpG sites present in each gene region, the following CpG sites were analyzed because they are CpG sites with specific methylation in non-cancerous tissues of gastric cancer patients who have been infected with Helicobacter pylori. preferably included. A plurality of CpG sites contained within the same base sequence shown below are sites that have been found to be either mostly methylated or mostly unmethylated. Therefore, it is not necessary to detect the methylation of all the CpG sites in the same base sequence, and by detecting the presence or absence of methylation of at least one CpG site, it is possible to detect the methylation of the entire CpG site. Presence or absence can be determined.
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 1, the 1st to 5th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 2, the 1st, 2nd, 26th, 30th, and 32nd CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 3, the 1, 18, 19, 31, and 72 CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 4, the 1st, 6th, 9th, 11th, and 25th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 5, the 1st, 29th, 32nd, 34th, and 47th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 6, the 1st, 2nd, 13th, 18th, and 29th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 7, the 1, 13, 17, 18, 20, and 23 CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 8, the 1st, 16th, 17th, 18th, and 42nd CpG sites counted from the 5' end side,
Among the CpG sites contained in the base sequence of SEQ ID NO: 9, the 1, 34, and 116th CpG sites counted from the 5' end side and Of the CpG sites contained in the base sequence of SEQ ID NO: 62, from the 5' end side 7th, 8th, 10th, 11th, 12th CpG sites.

In another embodiment, methylation analysis may be performed by examining the frequency of methylation in each gene region. Here, the "methylation frequency" can be, for example, the ratio of the number of methylated CpG sites to the number of CpG sites present in each gene region. In this embodiment, the analysis target may be the entire gene region of each gene described above, or a portion containing at least one CpG site. The analysis target may be determined from the gene region of any one of the above genes, or may be determined from the gene regions of a plurality of genes. Also in this embodiment, it is preferable to analyze the portion containing the CpG site described above.

The above methylation frequency may be a "methylation score" obtained by analyzing the methylation state of CpG sites in DNA by mass spectrometry such as MassARRAY (registered trademark) described below. With MassARRAY (registered trademark), DNA fragments can be measured and a methylation score can be calculated from the area ratio of the peak derived from the methylated DNA fragment and the peak derived from the unmethylated DNA fragment.

In this embodiment, the frequency of methylation in each gene region may be calculated manually or by a machine such as a computer.

As a means of methylation analysis, various analysis methods are known in the art. In the method of this embodiment, there is no particular limitation as to which analysis method is used. Preferably, the method comprises a step of distinguishing between methylated DNA and unmethylated DNA, a step of amplifying DNA, and a step of detecting methylated DNA and/or unmethylated DNA.

The step of distinguishing between methylated DNA and unmethylated DNA includes 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 steps of PCR, quantitative PCR, IVT (in vitro transcription) amplification, SPIA (trademark) amplification, and the like.
The step of detecting methylated DNA and/or unmethylated DNA includes steps of performing electrophoresis, sequence analysis, microarray analysis, mass spectrometry, Southern hybridization, and the like.

The MeDIP method is a method for 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 this embodiment, the methylated DNA contained in the DNA obtained in the extraction step may be concentrated by the MeDIP method, and the obtained methylated DNA may be subjected to methylation analysis. Alternatively, the methylated DNA enriched by the MeDIP method can be amplified by the IVT amplification method or the like, and the resulting amplification product can be subjected to methylation analysis using a microarray. Such an analysis method is called a MeDIP on chip method.

The unmethylated cytosine conversion treatment involves reacting DNA extracted from a biological sample with an unmethylated cytosine converting agent to convert unmethylated cytosines in the DNA to other bases (uracil, thymine, adenine or guanine). This is the process of converting to Here, the unmethylated cytosine-converting agent is a substance capable of reacting with DNA and converting unmethylated cytosine in the DNA into other bases (uracil, thymine, adenine or guanine). As such a non-methylated cytosine converting agent, for example, hydrogen sulfite salts (bisulfites) such as sodium, potassium, calcium, and magnesium hydrogen sulfites are preferably used.

In the treatment of DNA with bisulfite, unmethylated cytosines in the DNA are converted to uracil by deamination reaction, but methylated cytosines do not undergo such base conversion. Therefore, differences in the methylation status of CpG sites in DNA are converted into differences in base sequences (differences between C and U) by unmethylated cytosine conversion treatment using bisulfite. In addition, such conversion treatment of unmethylated cytosine by bisulfite is called bisulfite treatment.

When performing bisulfite treatment, the amount (concentration) of bisulfite to be added is not particularly limited as long as it is sufficient to convert unmethylated cytosines in DNA. For example, the final concentration in a solution containing DNA is 1M or more, preferably 1-15M, more preferably 3-10M. 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, incubation is carried out at 50-80°C for 10-90 minutes.

Methylation of CpG sites contained in DNA can be analyzed by sequence analysis of bisulfite-treated DNA and detection of differences from the original base sequence. This method is called a bisulfite sequencing method.

The pyrosequencing method is a method of amplifying bisulfite-treated DNA by PCR and analyzing the PCR product with a pyrosequencer. During PCR, a biotinylated primer (or a biotinylated universal primer) is used as a primer on one side to label only one strand of the PCR product with biotin. This is denatured to be single-stranded, adsorbed with beads and screened, then annealed with a sequencing primer and short-sequenced. DNA methylation levels can be analyzed by measuring the percentage of methylated CpG sites.

Also, methylation of CpG sites can be analyzed by mass spectrometry. Specifically, using bisulfite-treated DNA as a template, PCR amplification is performed using a primer set specific to the base sequence to be analyzed, and the resulting PCR product is further subjected to IVT amplification to obtain methylated cytosine. and uracil become guanine (G) and adenine (A), respectively. The resulting IVT amplification product was cleaved with RNase A, and the mass difference (16 Da) between G and A between the resulting nucleic acid fragments was measured using a MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometer. DNA methylation can be analyzed by detection using . This method is called MassARRAY® analysis.

The sites cleaved by RNase A in IVT products are known to be between any base and uracil (U) or thymine (T) adjacent to that base. Therefore, the base sequence and mass of the IVT product cleaved by RNase A can be predicted from the base sequence of DNA used as a template. Therefore, each peak obtained by MassARRAY (registered trademark) can be identified from which part of the nucleotide sequence of the template DNA is derived. For example, when one CpG site in the DNA fragment is methylated, the peak obtained with MassARRAY (registered trademark) shifts by 16 Da toward the high mass side. In the analysis of a DNA fragment having multiple CpG sites, for example, when the CpG sites in the DNA fragment are methylated at two sites, the shift is 32 Da, and when the CpG site is methylated at three sites, the shift is 48 Da.

A mass spectrometry method such as MassARRAY (registered trademark) can calculate the methylation score of the measured DNA fragment. For example, for a DNA fragment of a given sequence, if the area ratio of the peak of the unmethylated DNA fragment and 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. In theory, the methylation score is 1 when all CpG sites of the DNA fragment are methylated, and 0 when all CpG sites are unmethylated.

Methylation of CpG sites can be analyzed by methylation-specific PCR (MSP) and quantitative MSP (qMSP) methods. The MSP method is a method in which bisulfite-treated DNA is subjected to PCR amplification using a primer set described below, and the presence or absence of PCR products is confirmed to analyze the presence or absence of methylation at CpG sites. In the MSP method, a nucleotide sequence in which the CpG site to be analyzed is methylated (that is, cytosine is not converted to uracil) can be amplified, but the CpG site is not methylated (that is, the cytosine is converted to uracil). A primer set that cannot amplify the base sequence (converted) is used. In the MSP method using such a primer set, it is found that the CpG site to be analyzed is methylated when a PCR product is obtained. In addition, the MSP method cannot 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. can also In this case, if no PCR product is obtained, it is found that the CpG site to be analyzed is methylated.

The presence or absence of PCR products can be confirmed, for example, by gel electrophoresis. The reaction solution after amplification may be subjected to gel electrophoresis to visually confirm whether or not a band derived from the PCR product is present on the gel. Alternatively, an image of the gel after electrophoresis may be obtained and confirmed by image analysis. Image analysis can be performed, for example, by obtaining a value (e.g., band intensity) indicating the density of pixels in a region where a PCR product is expected to exist in a gel image, and comparing this value with a predetermined threshold. can. Specifically, it can be determined that a PCR product has been obtained when the value indicating the pixel density is equal to or greater than a predetermined threshold, and it can be determined that the PCR product has not been obtained when the value indicating the pixel density is smaller than the predetermined threshold. I can judge. Note that the predetermined threshold for pixel density is not particularly limited, and may be, for example, a value indicating the density of a background pixel, or a value twice or three times that value.

Each primer contained in the primer set used in the MSP method can be appropriately designed by those skilled in the art according to the nucleotide sequence containing the CpG site to be analyzed. It is preferred to design to include cytosine.

Quantitative MSP methods are carried out using real-time PCR techniques, such as real-time MSP using SYBR Green I and MethyLight using TaqMan probes.

Methylation of CpG sites can also be analyzed using microarrays. In this case, the analysis microarray can be produced by immobilizing nucleic acid probes complementary to the base sequences of the regions of each gene on a substrate. In addition, such a microarray can be produced by a method known in the art.

In microarray analysis, DNA extracted from a biological sample is preferably labeled with a labeling substance known in the art. Therefore, the determination method of this embodiment preferably further includes a step of labeling the extracted DNA. Since this labeling step can label all the DNA in the biological sample, it is advantageously performed after the DNA amplification step described above. Examples of labeling substances include fluorescent substances, haptens such as biotin, and radioactive substances. Fluorescent substances include Cy3, Cy5, FITC, and Alexa Fluor (trademark). Labeling the DNA in this way facilitates the measurement of signals from the probes on the microarray. Methods for labeling DNA with these labeling substances are known in the art.

The above signal may be a suitable signal depending on the type of microarray. For example, the signal may be an electrical signal generated when there is a DNA fragment hybridized with each probe of the microarray. It may be a signal such as fluorescence or luminescence generated from. Signal detection can be performed by a scanner provided in a normal microarray measurement device. Scanners include, for example, GeneChip (registered trademark) Scanner3000 7G (Affymetrix) and Illumina (registered trademark) BeadArray Reader (Illumina).

In the present embodiment, when an analysis result indicating the presence of a methylated CpG site is obtained in the analysis step, information indicating that the subject is in a high-risk group for developing gastric cancer (high risk of developing gastric cancer). can be obtained.
In another embodiment, when the frequency of methylation obtained by methylation analysis is higher than a predetermined threshold or the same as the threshold, information that the subject is in a high risk group for developing gastric cancer can be obtained.
The predetermined threshold value is not particularly limited, and can be empirically set by accumulating data on various biological samples. Alternatively, a predetermined threshold may be set as follows. First, the frequency of methylation is analyzed for DNA extracted from a biological sample of a healthy subject who has ever been infected with H. pylori and a non-cancerous tissue sample from a patient with gastric cancer who has ever been infected with H. pylori. Next, based on the obtained analysis results, the methylation frequency is higher than the methylation frequency of biological samples from healthy subjects who have been infected with Helicobacter pylori and the methylation frequency of non-cancerous tissue samples from gastric cancer patients who have been infected with Helicobacter pylori. Set the threshold from the lowest range. Preferably, the threshold value is set to a value that allows highly accurate discrimination between a biological sample from a healthy subject who has been infected with H. pylori and a non-cancerous tissue sample from a patient with gastric cancer who has been infected with H. pylori.

On the other hand, in the present embodiment, if the analysis result indicates that there is no methylated CpG site in the analysis step, the subject is in the low-risk group for developing gastric cancer (low risk of developing gastric cancer). You can get information about
In another embodiment, when the methylation frequency obtained by methylation analysis is lower than a predetermined threshold, information can be obtained that the subject is in the low risk group for developing gastric cancer.

Within the scope of the present invention, the continuous base sequences of all or part of the gene regions of the above genes, which are used as markers for obtaining information on the risk of developing gastric cancer by methylation analysis, are obtained by bisulfite treatment. An artificial DNA having a sequence that is In such an artificial DNA, the gene region of each gene may contain CpG sites and cytosines that do not constitute CpG sites. Here, the cytosine that does not constitute the CpG site may be other than the cytosine contained in the CpG site, for example, cytosine (C) and adenine (A), thymine (T) or cytosine (C) Cytosines in adjacent base sequences (ie, CA, CT or CC) in this order in the 3′ direction. Alternatively, such artificial DNA can be derived from DNA isolated from a subject.
In this embodiment, methylation analysis is performed on the above-mentioned markers in a DNA sample prepared from a non-cancerous tissue sample collected from a subject who has a history of H. pylori infection, and the subject is tested based on the obtained analysis results. It is possible to obtain information on the risk of developing gastric cancer in the elderly. The methylation analysis and acquisition of information on the risk of developing gastric cancer are the same as those described above.

In the artificial DNA used as a marker in the present embodiment, unmethylated cytosine in the isolated DNA can be converted to uracil by the bisulfite treatment of the isolated DNA, but methylated cytosine can remain intact. . In this embodiment, the presence or absence of methylation and the frequency of methylation can be detected by analyzing the nucleotide sequence of the artificial DNA used as such a marker. The above isolated DNA can be obtained in the same manner as the DNA sample preparation described above. Bisulfite treatment, methylation analysis, and acquisition of information on the risk of developing gastric cancer are also the same as described above.

In this embodiment, the size of the artificial DNA used as a marker is not particularly limited as long as it is a size that allows methylation analysis by MSP, qMSP, bisulfite sequencing, pyrosequencing or mass spectrometry, but is preferred. may be 80-500 bases, more preferably 100-400 bases.
In this embodiment, artificial DNAs used as markers include, for example, artificial DNAs consisting of the nucleotide sequences of SEQ ID NOS: 10-19 and 63. Among them, the artificial DNA used as a marker consisting of the base sequences of SEQ ID NOs: 11 and 12 is suitable for methylation analysis by the MSP method. In addition, artificial DNAs consisting of nucleotide sequences of SEQ ID NOS: 10, 13 to 19, and 63 used as markers are suitable for methylation analysis by the pyrosequencing method.
SEQ. SEQ ID NO: 19 corresponds to STX16 and SEQ ID NO: 63 to GUSBP5.

The scope of the present invention also includes a reagent kit for diagnosing the risk of developing gastric cancer (hereinafter also simply referred to as "kit"). The kit of this embodiment may contain primers for amplifying bisulfite-treated DNA. Such a primer hybridizes to a nucleic acid consisting of a nucleotide sequence in which cytosines present outside the CpG site in the nucleotide sequence having the CpG site in the gene region of each gene are converted to other nucleotides, and methylated CpG It can hybridize to a region containing the site.

In this embodiment, the primers included in the kit are used for analysis methods involving PCR amplification such as the MSP method, bisulfite sequencing method and pyrosequencing method, and mass spectrometry methods such as MassARRAY (registered trademark) for methylation analysis of CpG sites. can be a primer to perform Preferably, they are forward and reverse primers used in the MSP method, bisulfite sequencing method, pyrosequencing method, or mass spectrometry such as MassARRAY (registered trademark). The nucleotide sequences of the primers can be appropriately determined by those skilled in the art according to the nucleotide sequences of the gene regions of the above genes. For example, any of the following primer sets can be used.
When the analysis target is the BHLHE22 gene, the primer set of SEQ ID NOs: 20 and 21, or SEQ ID NOs: 64 and 65,
When the subject of analysis is the FGF12 gene, the primer set of SEQ ID NOs: 22 and 23,
When the subject of analysis is the FLT3 gene, the primer set of SEQ ID NOs: 24 and 25, or SEQ ID NOs: 38 and 39,
When the analysis target is the RPRM gene, the primer set of SEQ ID NOs: 26 and 27,
When the analysis target is the RIMS1 gene, the primer set of SEQ ID NOs: 28 and 29,
when the target of analysis is the JAM2 gene, the primer set of SEQ ID NOs: 30 and 31;
When the analysis target is the LINC00643 gene, the primer set of SEQ ID NOs: 32 and 33,
When the subject of analysis is the SEPT9 gene, the primer set of SEQ ID NOs: 34 and 35,
When the target of analysis is the STX16 gene, the primer set of SEQ ID NOs: 36 and 37, or
A primer set of SEQ ID NOs: 66 and 67 when the target of analysis is the GUSBP5 gene.

The above kit may contain a second primer. Such a second primer can hybridize to a nucleic acid consisting of a nucleotide sequence in which cytosine in the nucleotide sequence having the CpG site in the gene region of each gene is replaced with another nucleotide. Also, the second primer can hybridize to a region containing a sequence in which cytosines at unmethylated CpG sites have been converted to other bases by conversion treatment. For example, when the subject of analysis is the FGF12 gene, the primer set of SEQ ID NOs: 40 and 41 can be used. Also, when the FLT3 gene is to be analyzed, the primer set of SEQ ID NOS: 42 and 43 can be used.

In another embodiment, the kit described above may contain sequencing primers for use in pyrosequencing. The nucleotide sequences of the sequencing primers can be appropriately determined by those skilled in the art according to the nucleotide sequences of the gene regions of the above genes. For example, any of the following primers can be used.
When the analysis target is the BHLHE22 gene, the primer of SEQ ID NO: 44 or 68,
When the analysis target is the RPRM gene, the primer of SEQ ID NO: 45,
When the analysis target is the RIMS1 gene, the primer of SEQ ID NO: 46,
When the analysis target is the JAM2 gene, the primer of SEQ ID NO: 47,
When the analysis target is the LINC00643 gene, the primer of SEQ ID NO: 48,
When the subject of analysis is the SEPT9 gene, the primer of SEQ ID NO: 49,
When the target of analysis is the STX16 gene, the primer of SEQ ID NO: 50,
The primer of SEQ ID NO: 51 when the target of analysis is the FLT3 gene, or the primer of SEQ ID NO: 69 when the target of analysis is the GUSBP5 gene.

The present invention also includes a program product for causing a computer to obtain information on the patient's gastric cancer risk. Examples of such program products include a program that can be downloaded via the Internet or the like, a computer-readable recording medium that records the program, and the like.

For example, a program for causing a computer to execute the following steps is exemplified.
Group consisting of BHLHE22 gene, GUSBP5 gene, RIMS1 gene, LINC00643 gene, FGF12 gene, FLT3 gene, RPRM gene, JAM2 gene, SEPT9 gene and STX16 gene contained in a non-cancerous tissue sample derived from a subject who has been infected with Helicobacter pylori Acquiring analysis results of methylation of CpG sites present in the gene region of at least one gene selected from the measuring device;
A step of determining the risk of developing gastric cancer in the subject based on the acquired analysis results.

One form of a suitable apparatus for carrying out the method of the present embodiment will be described below with reference to the drawings. However, the invention is not limited to this embodiment only. FIG. 2 is a schematic diagram showing an example of a diagnosis assisting device used to acquire information on the risk of developing gastric cancer of a subject. The diagnostic assistance device 10 shown in FIG. 2 includes a measuring device 20 and a determination device 30 connected to the measuring device 20 .

In this embodiment, when performing methylation analysis by the MSP method, the measuring device 20 can be a gel imaging device such as a fluorescence image scanner. In this case, the reaction solution subjected to nucleic acid amplification by the MSP method is subjected to gel electrophoresis, and when the electrophoresed gel is set in the measurement device 20, the measurement device 20 detects the amplified product. Then, the measurement device 20 acquires gel image information of the amplified product and provides the obtained information to the determination device 30 .

Note that the measuring device 20 may be a MALDI-TOF mass spectrometer. In this case, the measuring device 20 acquires mass spectrometric 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 20, the measurement device 20 acquires mass spectrometry information of nucleic acids contained in the measurement sample, and determines the obtained mass spectrometry information. provided to device 30;

The determination device 30 includes a computer main body 300, an input section 301 including a keyboard and a mouse, and a display section 302 including an LCD and a CRT for displaying sample information, determination results, and the like. The determination device 30 acquires gel image information of the amplification product from the measurement device 20 . Then, based on this information, the determination device 30 executes a program that provides information on the risk of developing gastric cancer in the subject.
Note that the determination device 30 may be a device separate from the measurement device 20 as shown in FIG. 2, or may be a device including the measurement device 20 . In the latter case, the determination device 30 may itself be the diagnostic aid device 10 .

FIG. 3 is a block diagram showing the software of the computer main body 300 of the determination device 30 in functional blocks. As shown in FIG. 3 , the computer includes an acquisition section 321 , a storage section 322 , a calculation section 323 , a determination section 324 and an output section 325 . The acquisition unit 321 is communicably connected to the measuring device 20 via a network.

The acquisition unit 321 acquires information provided from the measurement device 20 . The storage unit 322 stores an equation, a processing program, and the like for obtaining the threshold value and band intensity necessary for determination. The calculation unit 323 uses the information acquired by the acquisition unit 321 and calculates the band intensity according to the stored processing program. The determination unit 324 determines whether the band intensity acquired by the acquisition unit 321 or calculated by the calculation unit 323 is equal to or greater than the threshold value stored in the storage unit 322 . The output unit 325 outputs the determination result by the determination unit 324 to the display unit 302 as information on the gastric cancer risk of the subject.

FIG. 4 is a block diagram showing the hardware configuration of computer main body 300 shown in FIG. As shown in FIG. 4, the computer main body 300 includes a CPU (Central Processing Unit) 310, a ROM (Read Only Memory) 311, a RAM (Random Access Memory) 312, a hard disk 313, an input/output interface 314, A reading device 315 , a communication interface 316 and an image output interface 317 are provided. CPU 310, ROM 311, RAM 312, hard disk 313, input/output interface 314, reading device 315, communication interface 316 and image output interface 317 are connected by bus 318 so as to be capable of data communication.

CPU 310 can execute programs stored in ROM 311 and programs loaded in RAM 312 . Each function shown in FIG. 3 is executed by CPU 310 executing the program. As a result, the determination device 30 functions as a determination device for providing information on the subject's gastric cancer risk.

The ROM 311 is composed of mask ROM, PROM, EPROM, EEPROM, and the like. The ROM 311 stores programs executed by the CPU 310 and data used for the programs as described above.

The RAM 312 is composed of an SRAM, a DRAM, or the like. The RAM 312 is used for reading programs recorded in the ROM 311 and hard disk 313 . RAM 312 is also used as a work area for CPU 310 when executing these programs.

The hard disk 313 is installed with programs such as an operating system and an application program (computer program for providing information on the risk of developing gastric cancer of a subject) to be executed by the CPU 310, and data used for executing the programs.

The reading device 315 is configured by a flexible disk drive, CD-ROM drive, DVD-ROM drive, or the like. The reading device 315 can read programs or data recorded on the portable recording medium 40 .

The input/output interface 314 includes serial interfaces such as USB, IEEE1394 and RS-232C, parallel interfaces such as SCSI, IDE and IEEE1284, and analog interfaces such as D/A converters and A/D converters. It is configured. An input unit 301 such as a keyboard and a mouse is connected to the input/output interface 314 . The operator can input various commands to the computer main body 300 through the input unit 301 .

The communication interface 316 is, for example, an Ethernet (registered trademark) interface. The computer main body 300 can also transmit print data to a printer or the like through the communication interface 316 .

The image output interface 317 is connected to the display section 302 configured by an LCD, CRT, or the like. Thereby, the display unit 302 can output a video signal corresponding to the image data given from the CPU 310 . The display unit 302 displays an image (screen) according to the input video signal.

Next, a processing procedure for obtaining information on the gastric cancer development risk of the subject by the diagnosis assisting device 10 will be described.
FIG. 5 is an example of a flow chart for acquiring information on the risk of developing stomach cancer. Here, the band intensity is obtained from gel image information obtained using a non-cancerous tissue sample derived from a subject who has a history of H. pylori infection, and whether or not the obtained band intensity is equal to or greater than a threshold value is determined. A case in which it is performed will be described as an example. However, the invention is not limited to this embodiment only.

First, in step S1-1, the acquiring unit 321 of the diagnosis assisting device 10 acquires gel image information about each gene region from the measuring device 20. FIG. Next, in step S1-2, the calculation unit 323 acquires the band intensity from the obtained gel image information and transmits it to the storage unit 322. FIG.

After that, in step S1-3, the determination unit 324 determines whether or not the band intensity acquired in step S1-2 is equal to or greater than the threshold value stored in the storage unit 322. Here, when the band intensity is less than the threshold, the routine proceeds to step S1-4. Then, the determination unit 324 transmits to the output unit 325 the determination result indicating that the subject is in the low-risk group for developing gastric cancer. On the other hand, if the band intensity is greater than or equal to the threshold, the routine proceeds to step S1-5. Then, the determination unit 324 transmits to the output unit 325 the determination result indicating that the subject is in the high-risk group for developing stomach cancer.

Finally, in step S1-6, the output unit 325 outputs the determination result as information on the gastric cancer risk of the subject, and causes the display unit 302 to display the information. As a result, the diagnosis assisting device 10 can provide a doctor or the like with information that assists in diagnosing whether the subject has a high risk of developing stomach cancer.

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these Examples.

Example 1: Identification of novel markers based on normal gastric mucosa of healthy individuals who have been infected with H. pylori and non-cancerous tissues after endoscopic resection of gastric cancer patients who have been infected with H. pylori
(1) Collection of Biological Samples Mucous membranes at one site of the lesser curvature of the antrum were collected from healthy subjects who had been infected with Helicobacter pylori (58 specimens; group 2 (G2)). Non-cancerous tissues were similarly collected from gastric cancer patients (96 specimens; group 3 (G3)) after endoscopic resection and who had a history of Helicobacter pylori infection. As controls, mucous membranes were similarly collected from healthy volunteers (8 specimens; group 1 (G1)) who had never been infected with Helicobacter pylori.
(2) Preparation of measurement samples
(i) Genomic DNA extraction
For 12 samples of G2 and 12 samples of G3, the tissue preservation solution such as RNAlater in which gastric mucosal tissue is stored was rinsed and replaced with an aqueous solution consisting of EDTA and salts with nucleic acid protection, SDS (final concentration 1%) and A protease (Protease K) was added to perform proteolytic treatment overnight at 55°C.
Next, 1 μl of RNase (20 mg/ml) was added and RNA degradation treatment was performed at 37° C. for 1 hour, followed by protein removal by phenol/chloroform treatment. Further, DNA was purified by ethanol precipitation and dissolved in 50 μl of 1×TE solution to prepare genomic DNA.
(ii) genomic DNA fragmentation
5 μg of the genomic DNA obtained in (i) was treated with 100 units of BamHI (HC: high concentration, TKR1010AH 50 units/μl) at 30° C. for 15 to 16 hours. After removing protein by phenol/chloroform treatment, the DNA was purified by ethanol precipitation and dissolved in 20 μl of 1×TE solution to obtain a DNA fragment.
(iii) Bisulfite treatment
1 μg of the DNA fragment obtained in (ii) was treated with bisulfite using innuCONVERT Bisulfite Basic Kit (Analytik jena AG), and the treated DNA fragment was added to 40 μl of 1xTE buffer (10 mM Tris-HCl, 1 mM EDTA). eluted.
(3) Infinium Analysis The DNA fragments after bisulfite conversion were subjected to methylation analysis using Infinium HumanMethylation450 BeadChip (Illumina). The Infinium HumanMethylation450 BeadChip has methylation probes and non-methylation probes for each of the 482,421 CpG sites on the human genome. The signal intensity of the methylation probe (signal M) and the signal intensity of the non-methylation probe (signal U) of the CpG site of the target gene are detected with the iScan system (ilumina), and the target is determined by the following calculation formula. The methylation rate (mCpG) of the CpG site of each gene was calculated.
(mCpG) = (Signal M) / {(Signal M) + (Signal U)}

The methylation rates of G2 and G3 were compared, and the following two methods (conventional method and iEVORA method (Teschendorff, AE, et al. (2016). "DNA methylation outliers in normal breast tissue identify field defects that are enriched in cancer ." Nat Commun 7: 10478.)), gene regions of 10 genes in which specific methylation was observed in non-cancerous tissues of gastric cancer patients who had been infected with Helicobacter pylori were selected as markers.

(conventional method)
1. Remove probes on sex chromosomes and probes lacking methylation rate data from all probes.
2. Among the remaining probes, probes with a methylation rate of 0.2 or less in blood samples from healthy subjects (data from 3 subjects are averaged) are extracted.
3. Extract probes with a methylation rate of 0.2 or less in G1.
4. Extract probes in which the methylation rate of G3 is greater than the methylation rate of G2 by 0.2 or more.
Extract probes in which the methylation rate of G3 is greater than the methylation rate of G2 by 0.2 or more in two probes before and after the extracted probe (five consecutive probes).

(iEVORA method)
1. Remove probes on sex chromosomes and probes lacking methylation rate data from all probes.
2. Extract probes based on the iEVORA algorithm (set FDR < 0.001, P-value for methylation difference < 0.05 as significant).

In addition, GUSBP5 is the above iEVORA method 1. and 2. In addition to the following 3. and 4. was selected by adding the conditions of
3. Extract probes in which the methylation rate of G3 is greater than the methylation rate of G2 by 0.2 or more. Alternatively, one probe before and after (three consecutive probes) is 2. Extract the probes extracted by .
4. Among the remaining probes, probes with a methylation rate of 0.2 or less in blood samples from healthy subjects (data from 3 subjects are averaged) are extracted.

Table 2 shows the sequence number and gene name of each gene region. In addition, the CpG sites where the G3 methylation rate was found to be 0.2 or more higher than the G2 methylation rate by Infinium analysis are shown below.
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 1, the 1st to 5th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 2, the 1st, 2nd, 26th, 30th, and 32nd CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 3, the 1, 18, 19, 31, and 72 CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 4, the 1st, 6th, 9th, 11th, and 25th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 5, the 1st, 29th, 32nd, 34th, and 47th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 6, the 1st, 2nd, 13th, 18th, and 29th CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 7, the 1, 13, 17, 18, 20, and 23 CpG sites counted from the 5' end side,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 8, the 1st, 16th, 17th, 18th, and 42nd CpG sites counted from the 5' end side,
Among the CpG sites contained in the base sequence of SEQ ID NO: 9, the 1, 34, and 116th CpG sites counted from the 5' end side and Of the CpG sites contained in the base sequence of SEQ ID NO: 62, from the 5' end side 7th, 8th, 10th, 11th, 12th CpG sites.

Among the above CpG sites, the difference in methylation rate between G2 and G3 and the ratio of methylation rate between G2 and G3 were determined for the following CpG sites. The difference in methylation rate was obtained by subtracting the average G2 methylation rate from the average G3 methylation rate. The methylation rate ratio was obtained by dividing the average methylation rate of G3 by the average methylation rate of G2. Welch's t-test was also used to test the methylation rates of G3 and G2. Table 2 shows the results.
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 1, the third CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 2, the 26th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 3, the 19th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 4, the 9th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 5, the 32nd CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 6, the 13th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 7, the 17th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 8, the 17th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 9, the 34th CpG site counting from the 5' end,
The 10th CpG site counted from the 5' end side among the CpG sites contained in the base sequence of SEQ ID NO:62.

Example 2:
The gene regions of the 10 genes selected in Example 1 were verified using MSP or pyrosequencing.
(1) Measurement samples were prepared in the same manner as in Example 1 for 12 samples of G2 and 12 samples of G3, which were different samples collected from the same group as the samples used in Example 1.

(2) Quantitative methylation-specific PCR (qMSP)
Using the measurement samples obtained in (1) above, the FLT3 gene and FGF12 gene were subjected to methylation analysis by qMSP. The composition of PCR reagents used, primer set, PCR conditions and CpG sites to be analyzed are shown below. qMSP was performed by real-time PCR using SYBR Green I (BioWhittaker Molecular Applications) and CFX connect (Bio-Rad Laboratories). The number of methylated molecules (M) and the number of unmethylated molecules (U) were measured, and the methylation rate was calculated by the following formula. The difference in methylation rate was obtained by subtracting the average G2 methylation rate from the average G3 methylation rate. The methylation rate ratio was obtained by dividing the average methylation rate of G3 by the average methylation rate of G2. Welch's t-test was also used to test the methylation rates of G3 and G2. Table 4 shows the results.
Methylation rate = M/(M + U)

<PCR reagent>
DW (sterilized water) 14.3 μL
10X PCR buffer (MgCl2 15mM) _2.0μL
dNTPs (2mM) 2.0μL
10x SYBR Green I solution 0.1 μL
AmpliTaq Gold (5U/μl) 0.2μL
20μM forward primer 0.2μL
20 μM reverse primer 0.2 μL
Sample for measurement 1 μL
Total 20μL

<PCR reaction conditions>
Annealing temperature: FGF12 (M 62℃, U 62℃), FLT3 (M 62℃, U 64℃)
PCR program: 95℃(15min)-[94℃(30sec)-annealing temperature(30sec)-72℃(30sec)]x40 cycle-72℃(10min)-15℃(∞)

<Primer set>
Table 3 shows the primer set used in the above qMSP. In the table, "M" is a primer set (hereinafter also referred to as "methylation detection primer set") capable of obtaining an amplification product when the DNA in the region to be amplified is methylated, and "UM" is It is a primer set capable of obtaining an amplification product when the DNA in the region to be amplified is not methylated (hereinafter also referred to as "unmethylation detection primer set"). In each gene region, the nucleotide sequences of the regions analyzed with the methylation detection primer set are shown in SEQ ID NOs: 53 (FGF12) and 54 (FLT3).
The primer sets of SEQ ID NOs: 22 and 23 and SEQ ID NOs: 40 and 41 target the 24th to 28th CpG sites counted from the 5' end of the nucleotide sequence of SEQ ID NO:2. The primer sets of SEQ ID NOs: 24 and 25 and SEQ ID NOs: 42 and 43 target the 18th to 20th and 29th to 30th CpG sites counted from the 5' end of the nucleotide sequence of SEQ ID NO:3.

<CpG site to be analyzed>
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 2, the 26th CpG site counting from the 5' end,
The 19th CpG site counted from the 5' end among the CpG sites contained in the base sequence of SEQ ID NO:3.

(3) pyrosequencing
The DNA of the measurement sample was amplified using the primer set described in Table 5. The composition of the PCR reagents used, the primer set, the PCR conditions, and the CpG sites to be analyzed are shown below. Pyrosequencing was performed using PSQ 96 Pyrosequencing System (Qiagen). The methylation rate was calculated using PSQ Assay Design software (Qiagen). The difference in methylation rate was obtained by subtracting the average G2 methylation rate from the average G3 methylation rate. The methylation rate ratio was obtained by dividing the average methylation rate of G3 by the average methylation rate of G2. Welch's t-test was also used to test the methylation rates of G3 and G2. Table 6 shows the results.

<PCR reagent>
DW (sterilized water) 15 μL
22 μL of 2x PyroMark PCR Mix
10x Coral Lord Conc. 4.4 μL
0.4 μL of ₂₅ mM MgCl2
5 μM forward primer 1.1 μL
5 μM reverse primer 1.1 μL
Sample for measurement 1 μL
Total 45μL

<PCR reaction conditions>
Annealing temperature: BHLHE22(1): 55℃, BHLHE22(2): 51℃, RPRM 50℃, RIMS1: 57℃, JAM2: 58, LINC00643: 52℃, STX16: 56℃, FLT3: 58℃, GUSBP5: 56 ℃
PCR program: 95℃(15min)-[94℃(30sec)-annealing temperature(30sec)-72℃(30sec)]x40 cycle-72℃(10min)-15℃(∞)

<Primer>
Table 5 shows the primer set for PCR amplification and the sequence primer for pyrosequencing used in the pyrosequencing. Nucleotide sequences of regions analyzed with each primer set are shown in SEQ ID NOs: 52, 55 to 61, 70.

<CpG site to be analyzed>
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 1, the third CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 3, the 19th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 4, the 9th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 5, the 32nd CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 6, the 13th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 7, the 17th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 8, the 17th CpG site counting from the 5' end,
Among the CpG sites contained in the nucleotide sequence of SEQ ID NO: 9, the 34th CpG site counting from the 5' end,
The 10th CpG site counted from the 5' end among the CpG sites contained in the base sequence of SEQ ID NO:62.

Example 3: Validation
(1) For 22 G2 specimens and 41 G3 specimens different from the specimens used in Examples 1 and 2, after preparing measurement samples in the same manner as in Example 1, qMSP and Pyrosequencing was performed. Table 7 shows the results.

(2) ROC analysis was performed for each gene region and miR124a-3. Table 8 shows the results. Also, the ROC curve is shown in FIG.

A statistically significant difference was observed in the methylation rate between G3 and G2 in each of the above gene regions, which was also reproduced in the validation cofort. In ROC analysis, these markers also showed sensitivity and specificity comparable to or higher than the conventionally used miR124a-3.

REFERENCE SIGNS LIST 10 diagnostic auxiliary device 20 measuring device 30 judging device 40 recording medium 300 computer body 301 input unit 302 display unit 310 CPU
311 ROMs
312 RAM
313 hard disk 314 input/output interface 315 reading device 316 communication interface 317 image output interface 318 bus 321 acquisition unit 322 storage unit 323 calculation unit 324 determination unit 325 output unit

Claims (12)

A method for assisting in diagnosing the risk of developing gastric cancer in a subject,
The step of analyzing the methylation state of the CpG site present in the gene region of the gene contained in the DNA sample of the subject, and based on the results obtained in the analysis step, the gastric cancer risk of the subject. obtaining information about
The DNA sample is prepared from a non-cancerous gastric mucosa collected from a subject who has a history of H. pylori infection,
wherein the gene is the BHLHE22 gene;
A diagnostic aid method for the risk of developing gastric cancer. 2. The method according to claim 1, wherein said analyzing step is a step of analyzing the presence or absence of methylation at CpG sites. 3. The method according to claim 1 or 2, wherein when an analysis result indicating the presence of methylation is obtained in the analyzing step, information is obtained in the information obtaining step that the subject is in a high risk group for developing gastric cancer. described method. Claims 1 to 3, wherein when an analysis result indicating that methylation does not exist is obtained in the analyzing step, information is obtained in the information obtaining step that the subject is in a low risk group for developing gastric cancer. The method according to any one of 2. The method according to claim 1, wherein said analyzing step is a step of analyzing the frequency of methylation. 6. The method according to claim 5, wherein, when the frequency of methylation is higher than a predetermined threshold in the analysis step, information indicating that the patient is in a high risk group for developing gastric cancer is obtained in the information obtaining step. 7. The method according to claim 5 or 6, wherein, when the methylation frequency is lower than a predetermined threshold in the analyzing step, information is obtained in the information obtaining step that the patient belongs to a low risk group for developing gastric cancer. the genetic region of the BHLHE22 gene comprises the base sequence of SEQ ID NO: 1;
The method according to any one of claims 1-7. The method according to any one of claims 1 to 8, wherein in the analyzing step, the CpG site whose methylation state is to be analyzed is a CpG site included in i) below:
i) Of the CpG sites contained in the base sequence of SEQ ID NO: 1, the 1st to 5th CpG sites counted from the 5' end side. The method according to any one of claims 1 to 9, wherein the analysis of the methylation status is performed by at least one selected from microarray, sequencing, mass spectrometry and methylation-specific PCR. A kit for diagnosing the risk of developing gastric cancer used in the method according to any one of claims 1 to 10,
comprising a forward primer and a reverse primer;
The forward primer and the reverse primer hybridize to a nucleic acid consisting of a nucleotide sequence in which cytosines present outside the CpG site in the nucleotide sequence having the CpG site in the gene region of the gene are converted to other bases,
hybridizes to a region containing a methylated CpG site,
wherein the gene is the BHLHE22 gene;
Said kit. The kit according to claim 11, wherein the combination of the base sequence of the forward primer and the base sequence of the reverse primer is i) below:
i) SEQ ID NO:20 and SEQ ID NO:21 or SEQ ID NO:64 and SEQ ID NO:65.

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