JP6583817B2 - Diagnostic markers for tumors in uterine smooth muscle - Google Patents

Description

  The present invention relates to a method for collecting data for diagnosing a tumor in uterine smooth muscle and a diagnostic kit for a tumor in uterine smooth muscle.

  Uterine sarcoma is a malignant tumor that arises from uterine smooth muscle, and is an intractable tumor with extremely high malignancy that causes metastasis to the lungs and liver even in relatively early cases. Tumors derived from uterine smooth muscle include benign uterine leiomyoma (uterine fibroids) and malignant uterine leiomyosarcoma (uterine sarcomas). Uterine sarcoma is a tumor with an extremely poor prognosis, although its onset frequency is 1% or less compared with uterine fibroids, and the 5-year survival rate is about 37%. With regard to uterine sarcoma, existing chemotherapy is hardly expected to have a life-prolonging effect, and no effective treatment is currently established other than surgical operation.

  Uterine fibroids, on the other hand, are benign tumors and are the most frequently occurring tumors observed in more than 30% of sexually mature women. In recent years, due to changes in women's lifestyles such as late marriage and aging of pregnancy, there is an increased chance of selecting hysteromyctomy surgery (uterine enucleation) that preserves the uterus as a method of uterine fibroid treatment. ing.

Uterine sarcoma is less frequent than uterine fibroids, but both are similar in location and shape, so it is very important to diagnose benign or malignant tumors when determining patient uterine preservation . However, the current diagnosis relies on morphological criteria that comprehensively judge based on three factors: the number of dividing cells in the pathological specimen, cell atypia, and the presence or absence of necrosis.
Since morphological criteria may differ depending on the facility or the diagnosed individual, an objective and reliable diagnostic method is desired. However, there is currently no useful biomarker that distinguishes between uterine leiomyoma and uterine leiomyosarcoma.

  Currently, the most reliable biomarker for uterine fibroids is a mutation in the MED12 gene, and it is reported that gene mutations are detected in about 70% of uterine fibroids (Non-patent Document 1: Science. 2011 Oct 14; 334). (6053): 252-5.). In addition, a diagnosis by immunostaining of LMP2 protein, which has been found to decrease in expression due to the development of uterine sarcoma, has been reported (Non-patent Document 2: Sci Rep. 2011; 1: 180.). However, both of these determination methods are extremely lacking in objectivity and reliability for use in actual diagnosis.

  On the other hand, in recent years, there has been a growing interest in epigenetics (genetics or molecular biology research fields resulting from the regulation of gene expression without alteration of gene sequence due to acquired modifications to chromatin). Research on the relationship with diseases such as cancer is underway. Among them, methylation of genes on genomic DNA has attracted attention. Phenomenon in which cytosine (C) located on the 5 ′ side of guanine (G) is methylated at a 5′-CG-3 ′ DNA (hereinafter, CpG) site present in the genomic DNA sequence of higher eukaryotes And this CpG methylation modification is thought to affect gene expression. In particular, when a region rich in CpG sites (CpG island) is present in the promoter region of a gene, it has an important influence on gene expression. Normally, many genes on the genome are protected from these methylation modifications, but if for some reason, CpG islands in the promoter region of the gene are methylated, transcription of the gene is suppressed. Become. For this reason, for example, when transcription of a tumor suppressor gene in the human body is inactivated due to abnormal methylation of CpG island, the control of cell proliferation becomes ineffective, and cell proliferative diseases such as cancer progress. It will end up. In fact, in some cancers, it has been reported that the methylation frequency of a specific gene is increased, and recently, the methylation frequency of a CpG island of a specific gene is used for diagnosis of a specific cancer. Several attempts have been made to use it. For example, a method for diagnosing liver cancer by detecting the degree of methylation of a CpG island of a gene such as BASP1 is known (Patent Document 1: Japanese Patent Application Laid-Open No. 2008-245635). Moreover, in gynecological cancers such as uterine cancer, it has been reported that the methylation frequency of each gene of hMLH-1 and CDKN2A / p16 is increased (Non-Patent Document 3: Eur J Gynaecol Oncol). 2009; 30: 267-270.). Further, one or more genes selected from DCC, GHSR, AJAP1, ZNF560, FERD3L, FLJ23514, BXDC1, HKR1, DRD4, PENK, FAM12B, HIST1H4F, NEF3, SKIP, CX36, EOMES, and SOLCS3. A method of using the frequency of methylation at the CpG site of a gene for determination of endometrial cancer has been reported (Patent Document 2: JP 2011-160711 A). However, the degree of increase in the methylation frequency of genes in cancer varies depending on the gene, etc., and genes that cause an increase in the methylation frequency enough to withstand the diagnosis of practical gynecological cancer have been known so far. Absent. In addition, the present inventors have conducted genome-wide DNA methylation analysis (DNA methylome) of uterine fibroids and uterine smooth muscle (normal muscle layer), and DNA methylation is more clearly normal than mRNA expression. It has been found that muscle layers and uterine fibroids can be distinguished (Non-patent Document 4: PLoS One, 8 (6): e66632, 2013). There is no distinction between myoma and uterine leiomyosarcoma.

JP 2008-245635 A JP 2011-160711 A

Science. 2011 Oct 14; 334 (6053): 252-5. Sci Rep. 2011; 1: 180. Eur J Gynaecol Oncol. 2009; 30: 267-270. PLoS One, 8 (6): e66632, 2013

  An object of the present invention is to provide a diagnostic marker capable of accurately diagnosing whether a tumor generated in uterine smooth muscle is a benign tumor (uterine leiomyoma) or a malignant tumor (uterine leiomyosarcoma). .

  The inventors have so far performed genome-wide DNA methylation analysis (DNA methylome) of uterine fibroids and uterine smooth muscle (normal muscle layer), and DNA methylation is more clearly compared with normal muscle layer and compared with mRNA expression. It has been found that uterine fibroids can be distinguished. Based on the methylation analysis, we first examined whether the normal muscle layer and uterine fibroids can be distinguished from the genes in which these DNA methylation occurred. However, it is not possible to distinguish only by detecting the methylation level (degree) of one gene. It became clear that it was difficult. Therefore, when a combination of genes that can minimize the labor of methylation analysis while maintaining discrimination accuracy was examined, 10 genes of ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 (hereinafter referred to as “the genes”) CpG in a region that encodes these genes (sometimes referred to as “the present biomarker gene”) or a region that encodes the transcriptional control region of each gene (hereinafter also referred to as “the present biomarker gene region”) By measuring the frequency of methylation of the sequence, conducting multivariate analysis, and creating this biomarker gene panel for CpG methylation, not only can the normal muscle layer and uterine fibroids be clearly distinguished, but also the tumor is uterine smooth Can determine if it is myoma or uterine leiomyosarcoma They found the door, which resulted in the completion of the present invention.

That is, the present invention is as follows.
[1] A method for collecting data for diagnosing a tumor in uterine smooth muscle, comprising the following steps (A) to (E):
(A) extracting genomic DNA from the collected uterine leiomyoma, uterine leiomyosarcoma and normal uterine leiomyosarcoma tissue;
(B) Regions encoding ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 genes in the genomic DNA of each tissue extracted in step (A), or transcriptional control of the respective genes Measuring the methylation frequency of the CpG sequence in the region;
(C) a step of extracting genomic DNA from tumor tissue collected from a subject;
(D) A region encoding the ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 genes in the genomic DNA extracted in the step (C) or the transcriptional control region of each gene is encoded. Measuring the methylation frequency of the CpG sequence in the region to be treated;
(E) Multivariate analysis is performed based on the methylation frequency of the CpG sequence measured in step (B) and the methylation frequency of the CpG sequence measured in step (D), and uterine leiomyoma and uterine smooth muscle in the subject Collecting data for diagnosing a tumor;
[2] The method according to [1] above, wherein the multivariate analysis is a hierarchical clustering analysis.
[3] In steps (B) and (D),
The CpG sequence in the region encoding the ALX1 gene or the transcriptional regulatory region of the gene is the nucleotide sequence 144th to 145th of the nucleotide sequence shown in SEQ ID NO: 1,
The CpG sequence in the region encoding the CBLN1 gene or the transcription control region of the gene is the nucleotide sequence of positions 146 to 147 of the nucleotide sequence shown in SEQ ID NO: 2,
The CpG sequence in the region encoding the CORIN gene or the transcriptional regulatory region of the gene is the nucleotide sequence 181 to 182 of the nucleotide sequence shown in SEQ ID NO: 3,
The CpG sequence in the region encoding the FOXP1 gene or the transcriptional regulatory region of the gene is the nucleotide sequence at positions 67-68 of the nucleotide sequence shown in SEQ ID NO: 4,
The CpG sequence in the region encoding the GATA2 gene or the transcription control region of the gene is a nucleotide sequence from positions 171 to 172 of the nucleotide sequence represented by SEQ ID NO: 5,
The CpG sequence in the region encoding the IGLON5 gene or the transcription control region of the gene is the nucleotide sequence of positions 377 to 378 of the nucleotide sequence shown in SEQ ID NO: 6,
The CpG sequence in the region encoding the NPTX2 gene or the transcriptional regulatory region of the gene is the nucleotide sequence at positions 159 to 160 of the nucleotide sequence shown in SEQ ID NO: 7,
The CpG sequence in the region encoding the NTRK2 gene or the transcription control region of the gene is the nucleotide sequence at positions 65-66 of the nucleotide sequence shown in SEQ ID NO: 8,
The CpG sequence in the region encoding the PRL gene or the transcriptional regulatory region of the gene is the nucleotide sequence 259 to 260 of the nucleotide sequence shown in SEQ ID NO: 9,
[1] or [2] above, wherein the CpG sequence in the region encoding STEAP4 gene or the transcription control region of said gene is the nucleotide sequence at positions 102-103 of the nucleotide sequence shown in SEQ ID NO: 10 the method of.
[4] Primer for measuring the methylation frequency of the CpG sequence in the region encoding the ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 genes or the transcription control region of each gene A kit for diagnosing a tumor in uterine smooth muscle, comprising a probe or a label thereof.
[5] The CpG sequence in the region encoding the ALX1 gene or the transcription control region of the gene is the 144th to 145th nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 1,
The CpG sequence in the region encoding the CBLN1 gene or the transcription control region of the gene is the nucleotide sequence of positions 146 to 147 of the nucleotide sequence shown in SEQ ID NO: 2,
The CpG sequence in the region encoding the CORIN gene or the transcriptional regulatory region of the gene is the nucleotide sequence 181 to 182 of the nucleotide sequence shown in SEQ ID NO: 3,
The CpG sequence in the region encoding the FOXP1 gene or the transcriptional regulatory region of the gene is the nucleotide sequence at positions 67-68 of the nucleotide sequence shown in SEQ ID NO: 4,
The CpG sequence in the region encoding the GATA2 gene or the transcription control region of the gene is a nucleotide sequence from positions 171 to 172 of the nucleotide sequence represented by SEQ ID NO: 5,
The CpG sequence in the region encoding the IGLON5 gene or the transcription control region of the gene is the nucleotide sequence of positions 377 to 378 of the nucleotide sequence shown in SEQ ID NO: 6,
The CpG sequence in the region encoding the NPTX2 gene or the transcriptional regulatory region of the gene is the nucleotide sequence at positions 159 to 160 of the nucleotide sequence shown in SEQ ID NO: 7,
The CpG sequence in the region encoding the NTRK2 gene or the transcription control region of the gene is the nucleotide sequence at positions 65-66 of the nucleotide sequence shown in SEQ ID NO: 8,
The CpG sequence in the region encoding the PRL gene or the transcriptional regulatory region of the gene is the nucleotide sequence 259 to 260 of the nucleotide sequence shown in SEQ ID NO: 9,
The kit according to [4] above, wherein the CpG sequence in the region encoding the STEAP4 gene or the transcription control region of the gene is the nucleotide sequence at positions 102 to 103 of the nucleotide sequence shown in SEQ ID NO: 10.

  The method of the present invention is a method for assisting a doctor in diagnosing a tumor in uterine smooth muscle, and does not include a diagnostic action by a doctor.

  Another embodiment of the present invention is a method for diagnosing a tumor in uterine smooth muscle.

  While determining benign or malignant tumors is very important in determining patient uterine preservation, there was no useful biomarker to distinguish between uterine leiomyoma and uterine leiomyosarcoma. By using the present biomarker gene panel for CpG methylation, it is possible to accurately determine whether a patient with a smooth muscle-derived tumor suffers from uterine leiomyoma or uterine leiomyosarcoma.

It is the result of having analyzed the methylation frequency of ALX1 gene using the normal muscle layer sample of each 18 cases, and the uterine fibroid sample. It is the result of having acquired CpG methylation data of this biomarker gene region about the normal muscle layer specimen of each 18 cases, and the uterine fibroid specimen, and performing hierarchical clustering. CpG methylation data of the biomarker gene region of the biomarker gene region was analyzed by COBRA for 12 cases of uterine sarcoma specimens and 4 samples of sarcoma cell lines (2 samples each of human uterine sarcoma cell line SKN and human ovarian sarcoma cell line RKN). It is the result shown with the result of the hierarchical clustering in the normal myocardium sample of each 18 cases and the uterine fibroid sample which were acquired and constructed | assembled in FIG. For two new cases (specimens X and Y), CpG methylation data of the biomarker gene region was obtained by the COBRA method, and the methylation of the sarcoma, myoma, and muscle layer samples shown in FIG. This is the result of hierarchical clustering added to the data.

  Methods for collecting data for diagnosing tumors in uterine smooth muscle of the present invention (hereinafter sometimes referred to as “the collection method”) include uterine leiomyoma, uterine leiomyosarcoma, and normal uterine smooth muscle layer. Step (A) of extracting genomic DNA from the tissue; CpG sequence (site) in the biomarker gene region of the subject biomarker gene region in the genomic DNA extracted in step (A) (hereinafter also referred to as “the subject CpG site”) A step (B) of measuring the degree (level or frequency) that is methylated; a step (C) of extracting genomic DNA from a tumor tissue collected from a subject; In the step (D) of measuring the frequency with which the CpG sequence in the biomarker gene region is methylated; in the step (D), the methylation frequency measured in the step (B) Comprising the steps (A) to (E) of performing a multivariate analysis based on the determined methylation frequency and collecting data for diagnosing uterine leiomyoma and uterine leiomyosarcoma in the subject (E); There is no particular limitation as long as it is a new method. The CpG site derived from the uterine leiomyoma tissue has a different methylation frequency than the CpG site derived from the normal uterine smooth muscle layer tissue, and the CpG site derived from the uterine leiomyosarcoma tissue In order to show a different methylation frequency from this tissue-derived CpG site, multivariate analysis was performed in the above step (E), and when the present biomarker gene panel for CpG methylation was created, Whether uterine leiomyoma or uterine leiomyosarcoma can be determined. In addition, the CpG site in the present biomarker gene region in the present specification means that the gene present in the position closest to the CpG site on the genomic DNA is the gene in the present biomarker gene region. In addition, when a plurality of CpG sites are present in the vicinity of the gene and a CpG island is formed, the plurality of CpG sites may be used as a biomarker for distinguishing between uterine leiomyoma and uterine leiomyosarcoma. Although it is said that there is a correlation between the methylation frequencies of neighboring CpG sites, if the methylation frequency of a specific CpG site is increased, the CpG island formed by the CpG site It can be evaluated that the methylation frequency is also increasing.

  In the present invention, “the CpG sequence is methylated” means that the carbon at the 5th position of the cytosine base is methylated in a 2-base sequence (CG or GC sequence) in which guanine (G) appears next to cytosine (C). It means the state that was done.

  Examples of the biological species of the subject in the collection method preferably include mammals such as humans, mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats and dogs. Among them, humans and mice can be illustrated more preferably, and humans can be particularly preferably illustrated.

  The method for extracting genomic DNA in the steps (A) and (C) is not particularly limited as long as it is a method for extracting genomic DNA from a tissue. For example, MagNA PureLC DNA Isolation Kit I (Roche Diagnostics) In addition to methods using commercially available products such as Allprep DNA / RNA mini kit and QIAamp DNA FFPE Tissue kit (QIAGEN) according to the attached protocol, phenol as described in the examples below. -The usual method (refer to the protocol of Molecular Cloning 3rd edition Volume 1) using chloroform treatment and ethanol or isopropanol precipitation can be exemplified.

In the steps (B) and (D), the method for measuring the methylation frequency of the CpG site is not particularly limited as long as it is a method capable of detecting and quantifying methylation of the CpG site. Southern hybridization using a label of a polynucleotide (probe) that can specifically hybridize to the polynucleotide sequence of the site, analysis using a methylation-sensitive restriction enzyme, bisulfite (bisulfite) A bisulfite method using treatment can be exemplified. The number of nucleotides of the polynucleotide (probe) is not particularly limited as long as it can hybridize to the present CpG site, and for example, 7 or more can be exemplified. From the viewpoint of specificity, 15 or more, more Preferably, 25 or more can be suitably exemplified. The labeling substance in the labeled product of the polynucleotide (probe) includes peroxidase (for example, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase. , Enzymes such as malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate Substance, Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Photoprotein (Blue Fluorescence Protein; BFP), yellow fluorescent protein (Yellow Fluorescence Protein; YFP), red fluorescent protein (Red Fluorescence Protein; RFP), luciferase (luciferase) fluorescent proteins, such ^{as, 3 H, 14 C, 125} I or Mention may be made of radioisotopes such as ¹³¹ I, biotin, avidin or chemiluminescent substances.

  The analysis method using the methylation-sensitive restriction enzyme utilizes a phenomenon in which DNA cannot be cleaved by cytosine in the restriction enzyme recognition sequence being methylated. The required reaction time varies depending on the type of restriction enzyme, but is about 1 to several hours. In addition, until now, the number of sequences that can be detected has been limited because of the use of restriction enzymes. Currently, however, there are more than 100 methylation-sensitive restriction enzymes available, and the recognition sequences are also diverse. It is possible to target most of the present CpG sites. After treatment with a methylation sensitive restriction enzyme, the cleavage efficiency of the present CpG site can be analyzed by methods such as Southern hybridization and PCR (polymerase chain reaction).

  The bisulfite method using the bisulfite treatment is a method using a bisulfite reaction in which only unmethylated cytosine in the present CpG site is converted into uracil by bisulfite treatment, which is currently most commonly used. It is the method that has been. Specifically, the following four methods (i) to (iv) can be exemplified.

(I) Bisulfite sequencing method By performing bisulfite treatment on the sample genomic DNA, methylated cytosine in the genomic DNA is not converted as it is, but only unmethylated cytosine is converted into uracil. Let When sequence reaction is performed on this genomic DNA, uracil is expressed as thymine. Comparing the sequence data of genomic DNA before and after bisulfite treatment, it was found that the site that was cytosine before and after bisulfite treatment was methylated cytosine. It was cytosine before bisulfite treatment and became thymine after bisulfite treatment. The site is found to be unmethylated cytosine.

(Ii) Methylation-specific PCR (MSP) method When bisulfite treatment is performed on genomic DNA, which is a sample, different base sequences are obtained between methylated CpG sites and unmethylated CpG sites. Will occur. By utilizing this fact, a PCR primer specific to each site of a different base sequence is designed in each, and a methylation state is detected by the presence or absence of a PCR product. In this MSP method, since the bisulfite-treated DNA can be used as it is for analysis, the result can be confirmed in a short time.

(Iii) COBRA (COmbined Bisulfite Restriction Analysis) method By utilizing the fact that the base sequence of a specific restriction enzyme recognition sequence changes depending on the DNA methylation state by bisulfite treatment, This is a method of measuring the methylation frequency by electrophoresis, and is also a method used in Examples described later. The methylation frequency in a specific recognition sequence can be quantitatively analyzed by the quantitative ratio of the presence or absence of cleavage of the PCR product.

(Iv) Method using HumanMethylation450BeadChip (manufactured by Illumina Co., Ltd.) This method is a genome-wide DNA methylation analysis based on the bisulfite pyrosequencing method, which is one of the bisulfite sequencing methods. Is the law. After bisulfite treatment is performed on genomic DNA extracted from the sample, the DNA is fragmented with restriction enzymes, and the methylation frequency (methylation level) at the CpG site on the DNA fragment is detected using the attached BeadChip. . In this BeadChip, a label of an oligonucleotide probe (non-methyl detection probe) that can specifically hybridize to a DNA sequence in which cytosine in each CpG site including the present CpG site is converted to uracil, Labels of oligonucleotide probes (methyl detection probes) that can specifically hybridize to unconverted DNA sequences are immobilized. By simply detecting the amount of the DNA fragment hybridized with each probe by fluorescence, the methylation frequency at the present CpG site on the genomic DNA can be measured. As a suitable index of the methylation frequency at the present CpG site, β value can be mentioned. For example, when the labeling substance is a fluorescent substance or a fluorescent protein, the β value is the fluorescence value (signal A) of the non-methyl detection probe corresponding to each CpG site obtained by measurement, and the methyl detection probe. The fluorescence value (signal B) is a value calculated by the following calculation formula.
β = (maximum value of signal B at the present CpG site) / (maximum value of signal A at the present CpG site + maximum value of signal B at the present CpG site + 100);
According to this calculation formula, the methylation frequency of each CpG site is calculated in the range of 0 (completely unmethylated) to 1 (completely methylated).

  The method for performing multivariate analysis based on the DNA methylation frequency in the step (E) is not particularly limited as long as it is a technique that can statistically analyze the similarity of the methylation state of the extracted genomic DNA of each tissue. However, hierarchical clustering, self-organizing mapping, principal component analysis, and the like can be suitably exemplified, and it is preferable to use hierarchical clustering that is generally widely recognized.

In the steps (B) and (D), the region for measuring the frequency of methylation in the present CpG site is not particularly limited, but preferably,
Nucleotide sequence 144-145 of the nucleotide sequence shown in SEQ ID NO: 1,
Nucleotide sequence from positions 146 to 147 of the nucleotide sequence shown in SEQ ID NO: 2,
Nucleotide sequence 181 to 182 of the nucleotide sequence shown in SEQ ID NO: 3,
Nucleotide sequences 67-68 of the nucleotide sequence shown in SEQ ID NO: 4,
Nucleotide sequences 171 to 172 of the nucleotide sequence shown in SEQ ID NO: 5,
Nucleotide sequences 377-378 of the nucleotide sequence shown in SEQ ID NO: 6,
Nucleotide sequence from positions 159 to 160 of the nucleotide sequence shown in SEQ ID NO: 7,
Nucleotide sequence 65-66 of the nucleotide sequence shown in SEQ ID NO: 8,
Nucleotide sequence 259-260 of the nucleotide sequence shown in SEQ ID NO: 9,
Nucleotide sequences 102 to 103 of the nucleotide sequence shown in SEQ ID NO: 10,
It is preferable to measure the methylation frequency.

  The above step (E) is “a step of producing a biomarker gene panel by performing multivariate analysis on the methylation frequency at the present CpG site measured in step (B) based on the DNA methylation frequency”. Here, the sample tissue used in the step (A) and the sample tissue used in the step (C) are preferably the same type of tissue. Moreover, it is preferable to use the specimen tissue of the same biological species as the subject (or human if the subject is human).

  As described above, based on the methylation frequency at the CpG site in the genomic DNA extracted from the specimen tissue, it is determined whether the specimen tissue corresponds to uterine leiomyoma, uterine leiomyosarcoma, or uterine smooth muscle layer. be able to. More specifically, the methylation frequency data in the subject tissue measured in the step (D) is subjected to multivariate analysis in the step (E) together with the methylation frequency data measured in the reference step (B). Thus, it can be distinguished whether the subject is a uterine leiomyoma, uterine leiomyosarcoma, or uterine leiomyosarcoma layer.

  As a kit for diagnosing a tumor in uterine smooth muscle of the present invention (hereinafter sometimes referred to as “the present diagnostic kit”), a primer or a probe for measuring the methylation frequency of a CpG sequence in the present biomarker gene region, Or a kit for diagnosing a tumor in uterine smooth muscle provided with such a label, wherein the diagnostic kit is a use invention relating to a kit for diagnosing a tumor in uterine smooth muscle In addition to the components generally used in this type of diagnostic kit, for example, carriers, pH buffers, stabilizers, etc., this kit is accompanied by instruction manuals, instructions for diagnosing tumors in uterine smooth muscle, etc. Documents are usually included.

  The primer in the present diagnostic kit may be any primer that can measure the methylation frequency of the present CpG site and can be amplified by PCR. For example, a primer sequence complementary to the nucleotide sequence upstream of the present CpG site. A primer set consisting of a forward primer that anneals and a reverse primer that anneals to the nucleotide sequence downstream of the CpG site, or a complementary sequence of the nucleotide sequence upstream of the CpG site, and the 3 ′ end with cytosine of the CpG site A primer set of a forward primer that anneals with a reverse primer that anneals to a nucleotide sequence downstream of the CpG site, a forward primer that anneals to a complementary sequence of a nucleotide sequence upstream of the CpG site, and the CpG site A primer set with a reverse primer that anneals at the 3 ′ end with cytosine, or a uracil site that anneals to the complementary sequence of the nucleotide sequence upstream of the CpG site and the cytosine of the CpG site is converted to uracil. A primer set of a forward primer that anneals at the 3 ′ end and a reverse primer that anneals to the nucleotide sequence downstream of the CpG site, a forward primer that anneals to a complementary sequence of the nucleotide sequence upstream of the CpG site, and the CpG site A primer set with a reverse primer that anneals at the 3 'end at the uracil site when the cytosine is converted to uracil. Further, the length of the primer sequence, the site to be annealed, etc. can be appropriately selected in consideration of the DNA amplification efficiency and specificity. For example, 10 to 30 bases can be selected as the length of the primer sequence. The 5 'terminal side of the nucleotide sequence of the present biomarker gene registered in the NCBI database is upstream, and the 3' terminal side is downstream.

  Examples of the probe in the present diagnostic kit include a probe that hybridizes to a nucleotide sequence including the present CpG site, and a probe that hybridizes to the nucleotide sequence obtained by converting cytosine of the present CpG site to uracil. The length, the site to hybridize, and the like can be appropriately selected in consideration of the efficiency and specificity of hybridization.

As described above, the labeling substance in the labeling product of the diagnostic kit includes peroxidase (for example, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenation. Enzymes, malic acid dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, acetylcholinesterase and other enzymes, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride, tetramethylrhodamine isothiocyanate, etc. Fluorescent substance, Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Fluorescence Protein Park protein (Blue Fluorescence Protein; BFP), yellow fluorescent protein (Yellow Fluorescence Protein; YFP), red fluorescent protein (Red Fluorescence Protein; RFP), luciferase (luciferase) fluorescent proteins, such ^{as, 3 H, 14 C, 125} I or Mention may be made of radioisotopes such as ¹³¹ I, biotin, avidin or chemiluminescent substances.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Sample tissue collection and DNA extraction]
Tissue samples were obtained from patients who underwent total hysterectomy due to medical indications, with the approval of the Ethics Review Committee on Genetic Analysis Research at Yamaguchi University School of Medicine and Hospitals. The specimens used were 18 specimens of normal muscle layer, 18 specimens of uterine fibroids, and 12 specimens of uterine sarcoma. In addition, two human leiomyosarcoma-derived cell lines (human uterine sarcoma cell line SKN and human ovarian sarcoma cell line RKN) (purchased from JCRB Cell Bank, Incorporated Administrative Agency) were used.
Genomic DNA was extracted from the tissue specimen. Ten to 30 mg of tissue pieces were frozen in liquid nitrogen and then pulverized into powder. DNA extraction was performed by a general method. The powdered tissue piece was treated with Proteinase K (QIAGEN), extracted with phenol / chloroform, and purified by ethanol precipitation.

[Selection of candidate genes for myoma-specific biomarkers]
To date, the applicant has extracted 120 genes with specific methylation mutations based on DNA methylome data of 3 samples each of uterine fibroids and normal muscle layers (PLoS One, 8 (6): e66632, 2013). The DNA methylation pattern is specific for each tissue / cell type. Since both uterine fibroids and uterine sarcomas are derived from uterine smooth muscle but are essentially different tumors, the applicant has identified a group of genes with uterine fibroid-specific methylation mutations as uterine fibroids and uterine sarcomas. We thought that there was a high possibility that the biomarker could be clearly distinguished. Therefore, of the 120 genes, 33 genes that were successfully constructed as primers for DNA methylation analysis were selected as candidates for uterine fibroid-specific methylation mutant genes. 33 genes are shown in Table 1.

[Measurement of DNA methylation frequency]
The COBRA method was used to measure the DNA methylation frequency. When bisulfite-PCR is performed after bisulfite treatment of genomic DNA, a reaction occurs in which unmethylated cytosine is converted to thymine, while methylated cytosine remains cytosine and not converted. By utilizing the fact that the base sequence of a specific restriction enzyme recognition sequence changes depending on the methylation state by this reaction, the methylation frequency is analyzed by electrophoresis of the PCR product treated with the enzyme.
Bisulfite treatment was performed using EpiTect ^(R) Bisulfite kit (manufactured by Qiagen) based on the instructions attached to the kit. Bisulfite reaction was performed on 1 μg of genomic DNA under the conditions of 95 ° C. for 5 minutes, 65 ° C. for 85 minutes, 95 ° C. for 5 minutes, and 65 ° C. for 175 minutes. Bisulfite-PCR uses 1/100 amount of bisulfite DNA as a template and Biotaq HS DNA polymerase (manufactured by Bioline) using 95 ° C for 10 minutes [95 ° C for 0.5 minutes, 60 ° C for 0.5 minutes. , 72 ° C., 0.5 minute] × 35 cycles, 72 ° C. for 7 minutes. The restriction enzymes used were TaqI that recognizes the sequence TCGA and HpyCH4IV that recognizes the sequence ACGT. Half of the PCR product was used for restriction enzyme treatment, and TaqI reacted at 37 ° C and HpyCH4IV at 65 ° C for 2 hours or longer. PCR products treated with restriction enzymes were separated by agarose gel electrophoresis, and the methylation rate was calculated.

  First, based on the methylation analysis of one gene by the COBRA method for the genes listed in Table 1, it was examined whether normal muscle layer and uterine fibroids could be distinguished. It became clear that it was difficult to distinguish clearly. FIG. 1 shows the results of analyzing the methylation frequency of the ALX1 gene using 18 normal muscle layer specimens and uterine fibroid specimens as an example.

[Selection of myoma-specific biomarkers]
Based on Example 1, in constructing a myoma-specific biomarker gene panel, the number of genes that can minimize the labor of methylation analysis while maintaining discrimination accuracy was examined.
For the 33 genes selected as candidates for uterine fibroid-specific methylation mutant genes, the methylation frequency of one gene was analyzed by the COBRA method using three cases of uterine fibroid specimens. As a result, 17 genes having hypermethylation mutations or hypomethylation mutations in all myoma specimens were identified as myoma-specific biomarker genes. Furthermore, the number of uterine fibroid specimens was increased to 11 cases, and the same analysis was performed. Ten genes (ALX1, CBLN1, CORIN, FOXP1) in which 70% or more of the myoma specimens had hypermethylation mutations or hypomethylation mutations commonly occurred. , GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4).
Table 2 shows the PCR primers used for bisulfite-PCR for the identified myoma-specific biomarker gene. The primer sequences shown in Table 2 are designed so that a sequence in which unmethylated cytosine is converted to uracil (thymine) by bisulfite treatment can be amplified. Moreover, the nucleotide sequence of the genomic region amplified by bisulfite-PCR of the present biomarker gene is shown in SEQ ID NOs: 1 to 10 (SEQ ID NO: 1 ALX1, SEQ ID NO: 2: CBLN1, SEQ ID NO: 3: CORIN, sequence) No. 4: FOXP1, SEQ ID NO: 5: GATA2, SEQ ID NO: 6: IGLON5, SEQ ID NO: 7: NPTX2, SEQ ID NO: 8: NTRK2, SEQ ID NO: 9: PRL, SEQ ID NO: 10: STEAP4). The CpG sites analyzed for methylation frequency by the COBRA method are as follows.
144-145th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 146-147th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 2 181-182nd nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 3 SEQ ID NO: Nucleotide sequence 67-68 of the nucleotide sequence shown in 4 nucleotide sequence 171-172 of the nucleotide sequence shown in SEQ ID NO 5 nucleotide sequence 377-378 of the nucleotide sequence shown in SEQ ID NO 6 Nucleotide sequence 159-160 of the nucleotide sequence shown nucleotide sequence 65-66 of the nucleotide sequence shown in SEQ ID NO: 8 nucleotide sequence 259-260 of the nucleotide sequence shown in SEQ ID NO: 9 102-103 nt sequence of the nucleotide sequence shown in ID NO: 10

[Construction of DNA methylation mutant uterine fibroid-specific biomarker gene panel]
Clustering was performed using Pearson correlation as a measure to measure the similarity of methylation status of each case, and using average linkage as a clustering algorithm. In the actual hierarchical clustering, open resource software MultiExperiment Viewer (MeV) was used to perform case-direction clustering.
FIG. 2 shows the results of hierarchical clustering in the normal muscle layer specimen and the uterine fibroid specimen in each of 18 cases. Based on the selected 10 genes, 18 cases of normal muscle layers and uterine fibroids were distinguished into completely different clusters by clustering. On the other hand, when the myoma specimen is cut at about half the height (dotted line) of the dendrogram, it is classified into three subgroups (L1, L2, and L3) with slightly different methylation patterns. Was also shown.

[Separation of uterine sarcoma specimens by clustering]
12 cases of uterine sarcoma specimens and 4 samples of sarcoma cell lines (2 samples each of human uterine sarcoma cell line SKN and human ovarian sarcoma cell line RKN), normal muscle layer of 18 cases each constructed in Example 2 FIG. 3 shows the results shown together with the results of the hierarchical clustering in the specimen and the uterine fibroid specimen.
Eight cases of sarcoma specimens and 4 sarcoma cell lines were separated into clusters different from the myoma / muscle layer. On the other hand, the sarcoma specimens of 4 cases were separated into the myoma subgroup L2. Among specimens classified as clusters that do not include myoma, those not included in the muscle layer cluster can be judged as sarcomas. Among the samples classified into clusters containing myomas, those clustered into subgroups L1 and L3 can be judged as myomas, and those clustered into subgroup L2 have a 40% probability of being a meat species It can be judged.

[Diagnosis of uterine sarcoma by hierarchical clustering]
It can be diagnosed whether the case is a sarcoma by investigating to which cluster the added new case data is classified into sarcoma, myoma, and muscle layer specimen. For two new cases (specimens X and Y), the methylation data of the 10 gene regions described above were obtained by the COBRA method, and the methylation data of the sarcoma, myoma and muscle layer samples shown in Example 3 were used. In addition to the above, hierarchical clustering was performed. Samples X and Y are diagnosed as uterine fibroids and uterine sarcomas, respectively, by pathological diagnosis. The result of hierarchical clustering is shown in FIG.
Samples X and Y were classified into hysteromyoma and uterine sarcoma, respectively, by hierarchical clustering. From this, it was shown that this myoma specific biomarker gene panel is applicable to the diagnosis of uterine sarcoma.

  According to the present invention, it is possible to accurately diagnose whether a tumor in uterine smooth muscle is a benign tumor (uterine leiomyoma) or a malignant tumor (uterine leiomyosarcoma). More accurate classification and effective treatment become possible, and the effects such as improvement of QOL (Quality of Life), reduction of medical expenses, and elimination of the declining birthrate due to uterus preservation that can be expected are expected.

Claims (4)

A method for collecting data for diagnosing a tumor in uterine smooth muscle, comprising the following steps (A) to (E):
(A) extracting genomic DNA from the collected uterine leiomyoma, uterine leiomyosarcoma and normal uterine leiomyosarcoma tissue;
(B) Regions encoding ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 genes in the genomic DNA of each tissue extracted in step (A), or transcriptional control of the respective genes Measuring the methylation frequency of the CpG sequence in the region;
(C) a step of extracting genomic DNA from tumor tissue in uterine smooth muscle collected from a subject;
(D) A region encoding the ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 genes in the genomic DNA extracted in the step (C) or the transcriptional control region of each gene is encoded. Measuring the methylation frequency of the CpG sequence in the region to be treated;
(E) In order to collect data for diagnosing uterine leiomyoma and uterine leiomyosarcoma in the subject, the methylation frequency data of the CpG sequence measured in step (D) is used as a reference in step (B). Performing hierarchical clustering with the measured methylation frequency data of the CpG sequence ; In steps (B) and (D),
The CpG sequence in the region encoding the ALX1 gene or the transcriptional regulatory region of the gene is the nucleotide sequence 144th to 145th of the nucleotide sequence shown in SEQ ID NO: 1,
The CpG sequence in the region encoding the CBLN1 gene or the transcription control region of the gene is the nucleotide sequence of positions 146 to 147 of the nucleotide sequence shown in SEQ ID NO: 2,
The CpG sequence in the region encoding the CORIN gene or the transcriptional regulatory region of the gene is the nucleotide sequence 181 to 182 of the nucleotide sequence shown in SEQ ID NO: 3,
The CpG sequence in the region encoding the FOXP1 gene or the transcription control region of the gene is the 67th to 68th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 4, and the region encoding the GATA2 gene or the transcription control region of the gene The CpG sequence in 171 to 172 of the nucleotide sequence shown in SEQ ID NO: 5,
The CpG sequence in the region encoding the IGLON5 gene or the transcription control region of the gene is the nucleotide sequence of positions 377 to 378 of the nucleotide sequence shown in SEQ ID NO: 6,
The CpG sequence in the region encoding the NPTX2 gene or the transcriptional regulatory region of the gene is the nucleotide sequence at positions 159 to 160 of the nucleotide sequence shown in SEQ ID NO: 7,
The region encoding the NTRK2 gene or the CpG sequence in the transcription control region of the gene is the 65-66th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 8, and the region encoding the PRL gene or the transcription control region of the gene The CpG sequence is the nucleotide sequence 259-260 of the nucleotide sequence shown in SEQ ID NO: 9, and the CpG sequence in the region encoding the STEAP4 gene or the transcription control region of the gene is the nucleotide sequence shown in SEQ ID NO: 10 claim 1 Symbol mounting method characterized in that it is a 102-103 nt sequences.   A primer or probe for measuring the methylation frequency of the CpG sequence in the region encoding the ALX1, CBLN1, CORIN, FOXP1, GATA2, IGLON5, NPTX2, NTRK2, PRL, and STEAP4 genes, or in the transcription control region of each of the genes, or A kit for diagnosing a tumor in uterine smooth muscle, comprising the marker. The CpG sequence in the region encoding the ALX1 gene or the transcriptional regulatory region of the gene is the nucleotide sequence 144th to 145th of the nucleotide sequence shown in SEQ ID NO: 1,
The CpG sequence in the region encoding the CBLN1 gene or the transcription control region of the gene is the nucleotide sequence of positions 146 to 147 of the nucleotide sequence shown in SEQ ID NO: 2,
The CpG sequence in the region encoding the CORIN gene or the transcriptional regulatory region of the gene is the nucleotide sequence 181 to 182 of the nucleotide sequence shown in SEQ ID NO: 3,
The CpG sequence in the region encoding the FOXP1 gene or the transcription control region of the gene is the 67th to 68th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 4, and the region encoding the GATA2 gene or the transcription control region of the gene The CpG sequence in 171 to 172 of the nucleotide sequence shown in SEQ ID NO: 5,
The CpG sequence in the region encoding the IGLON5 gene or the transcription control region of the gene is the nucleotide sequence of positions 377 to 378 of the nucleotide sequence shown in SEQ ID NO: 6,
The CpG sequence in the region encoding the NPTX2 gene or the transcriptional regulatory region of the gene is the nucleotide sequence at positions 159 to 160 of the nucleotide sequence shown in SEQ ID NO: 7,
The region encoding the NTRK2 gene or the CpG sequence in the transcription control region of the gene is the 65-66th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 8, and the region encoding the PRL gene or the transcription control region of the gene The CpG sequence is the nucleotide sequence 259-260 of the nucleotide sequence shown in SEQ ID NO: 9, and the CpG sequence in the region encoding the STEAP4 gene or the transcription control region of the gene is the nucleotide sequence shown in SEQ ID NO: 10 The kit according to claim 3 , wherein the nucleotide sequence is from 102 to 103.