COMPOSITION USING CPG METHYLATION CHANGES IN SPECIFIC GENES TO DIAGNOSE BLADDER CANCER, AND USE THEREOF

Abstract

The present invention relates to a composition, a kit, a nucleic acid, and a method capable of diagnosing bladder cancer by detecting the methylation level of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3, and BOLL. According to the present invention, since the hypermethylation of a CpG site of a gene is specifically exhibited in bladder cancer, a composition, a kit, a chip, or a method according to the present invention can be used to accurately and rapidly diagnose bladder cancer and to be used for early diagnosis and monitoring recurrence.

Claims

1. A composition for diagnosing bladder cancer comprising an agent for measuring the methylation level of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL.

2. The composition of claim 1, wherein the CpG site is located between +/−3000 bases (3 kb) from a transcription start site of the gene.

3. The composition of claim 1, wherein the agent for measuring the methylation level of the CpG site of the gene is selected from the group consisting of: a compound of modifying an unmethylated cytosine or methylated cytosine base; a primer specific to a methylated sequence of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL from the sample; and a primer specific to the unmethylated sequence.

4. The composition of claim 3, wherein the compound of modifying the unmethylated cytosine base is bisulfite or a salt thereof and the compound of modifying the methylated cytosine base is a Tet protein.

5. A kit for diagnosing bladder cancer comprising a primer pair for amplifying fragments including a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL.

6. A nucleic acid chip for diagnosing bladder cancer in which a probe capable of hybridizing with fragments including a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL is immobilized.

7. A method for providing information for diagnosing bladder cancer comprising: measuring the methylation level of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL from a sample of a patient suspected of having bladder cancer; and comparing the measured methylation level with the methylation level of a CpG site of the same gene in a normal control sample.

8. The method of claim 7, wherein the method for measuring the methylation level is selected from the group consisting of a bisulfite-free detection method, methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing.

9. The method of claim 7, wherein the sample is selected from the group consisting of tissues, cells, blood, plasma, serum, feces, and urine.

10. A method for measuring the methylation level of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL in a biological sample isolated from a subject, in order to provide information required for diagnosing the recurrence or progression of bladder cancer.

11. The method of claim 10, wherein the subject is a patient diagnosed with bladder cancer or a patient who has been treated for bladder cancer.

12. The method of claim 10, wherein the biological sample is urine.

13. Use of an agent for measuring the methylation level of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL for preparing an agent for diagnosing bladder cancer.

14. A method for diagnosing bladder cancer comprising the steps of: a) obtaining a sample from a subject; b) measuring the methylation level of a CpG site of at least one gene selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL from the sample; and c) comparing the measured methylation level with the methylation level of a CpG site of the same gene in a normal control sample.

Description

DESCRIPTION OF DRAWINGS

[0093] FIG. 1 is a result of confirming a difference in methylation between a tumor tissue (tumor) group and a non-tumor tissue (normal) group of a total of six genes selected according to the present invention.

[0094] FIG. 2 is a result confirming methylation information of a total of six genes selected according to the present invention in a tumor tissue (tumor) cell line group.

[0095] FIG. 3 is a result of confirming methylation information of a total of six genes selected according to the present invention in peripheral blood mononuclear cells.

[0096] FIG. 4 is a result of confirming methylation information of a total of six genes selected according to the present invention for each type of peripheral blood mononuclear cells.

[0097] FIG. 5 is a result of confirming the diagnosis accuracy of bladder cancer of a total of 6 genes selected according to the present invention.

MODES FOR THE INVENTION

[0098] Hereinafter, preferred Examples will be proposed in order to help in understanding of the present invention. However, the following Examples are just provided to more easily understand the present invention and the contents of the present invention are not limited by Examples.

Example 1: Selection of bladder cancer-specific methylated gene

[0099] To select a methylated gene specifically found in bladder cancer, a comparative study on large-scale methylation of cancer and normal tissues obtained from cancer surgery in bladder cancer patients was performed using large-scale methylation microarray chip data. The tumor tissue used in this study means cancer tissue of bladder cancer, and the non-tumor tissue means tissues including normal tissue other than the cancer tissue. The number of bladder cancer tissues used in the analysis was 412 and the number of non-tumor tissues was 21.

[0100] In order to select bladder cancer-specific methylated genes, DNA was extracted from each tissue, and the methylation level of a gene region was confirmed using an Infinium Human Methylation 450 Beadchip microarray.

[0101] DNA extracted from each tissue was converted through bisulfite treatment, and as a result, a cytosine base was modified depending on whether a DNA site was methylated. A probe used in the corresponding microarray experiment was specifically designed for methylation and unmethylation in order to confirm whether a cytosine base in a methylated region of a gene was modified.

[0102] The microarray experiment measured the methylation level of the gene through about 450,000 (450 k) probes representing the methylated region of each gene, and a result of each probe derived from the experiment was presented as a beta value. The beta value had a value of 0 to 1, and it was determined that the closer to 1, the higher the methylation level of the corresponding gene region.

[0103] In order to identify differentially methylated regions (DMRs) between the tumor group and the non-tumor group, by using an empirical Bayes t-test, a Linear Models for Microarray Data (Limma) method, a gene region having a statistically significant methylation difference between the groups was identified.

[0104] The Limma method has been known to be least affected by an outlier among several methylation statistical analysis methods to identify the difference between the groups. Therefore, the method was a suitable method for finding cancer-specific markers because the method was less affected by abnormal measurement values of some samples. In the present experiment, it was determined that there was a significant difference in methylation between the two groups as an adjusted p-value derived through the Limma method was smaller.

[0105] In particular, in order to search for tumor-specific methylated regions, among gene regions with a significant difference in beta values between tumor and non-tumor groups, gene regions with higher methylation in tumor tissue than in non-tumor tissue were selected as cancer-specific biomarker candidates.

[0106] As a result, as a result of limma analysis in a dataset, compared with the non-tumor group, when comparing the tumor groups, gene regions having a low p value within the 10,000-th among 450,000 probes, and a large difference in beta value of 0.3 or more between the groups were selected as tumor-specific hypermethylated regions. As a result, among about 450,000 gene regions, 352 gene regions exhibiting tumor-specific hypermethylation in the dataset were selected as biomarker candidates.

[0107] Among these gene regions, when the corresponding region was not a pseudogene, existed in a CpG island site, was located in a gene region selected between +/−3000 bases (3 kb) from a transcription start site (TSS) of the gene, and existed in an autosome, the corresponding region was selected as a bladder cancer-specific hypermethylated gene.

[0108] As a result, as shown in Table 2 below, a total of 6 genes were selected (see FIG. 1).

TABLE-US-00002 TABLE 2 Location CpG Symbol Name (Chromosome) Island IFFO1 intermediate filament family 12 Island orphan 1 (IFFO; HOM-TES- 103) MARCH11 membrane associated ring-CH- 5 Island type finger 11 (RNF226; MARCHF11; MARCH-XI) BARHL2 BarH like homeobox 2 1 Island NR2E1 nuclear receptor subfamily 2 6 Island group E member 1 KCNA3 potassium voltage-gated 1 Island channel subfamily A member 3 (MK3; HGK5; HLK3; PCN3; HPCN3; KV1.3; HUKIII) BOLL boule homolog, RNA binding 2 Island protein (BOULE)

Example 2: Confirmation of Bladder Cancer Specificity of Bladder Cancer Diagnostic Gene in Cell Line

[0109] In order to confirm whether the high methylation of the six selected genes was caused by bladder cancer cells, methylated patterns in 20 cancer cell lines derived from bladder cancer were largely analyzed using a public database. For the corresponding data, the Infinium Human Methylation 450 Beadchip microarray experiment was performed on DNA extracted from each cell line according to a manufacturer's standardized methylation analysis test procedure.

[0110] In the result values of the performed experiment, the methylation levels of genes were measured through about 450,000 probes as in Example 1, and the methylation value of each probe was presented as a beta value. The beta value had a value of 0 to 1, and it was determined that the closer to 1, the higher the methylation level of the corresponding gene region.

[0111] The 20 bladder cancer cell lines were as follows: 5637 (comic ID: 687452), 639-V (comic ID: 906798), 647-V (comic ID: 906797), BFTC-905 (comic ID: 910926), CAL-29 (comic ID: 1290730), DSH1 (comic ID: 753552), HT-1197 (comic ID: 907065), HT-1376 (comic ID: 907066), J82 (comic ID: 753566), KU-19-19 (comic ID: 907312), LB831-BLC (comic ID: 753584), RT-112 (comic ID: 909704), RT4 (comic ID: 687455), SCABER (comic ID: 1299051), SW1710 (comic ID: 909749), SW780 (comic ID: 687457), T-24 (comic ID: 724812), TCCSUP (comic ID: 687459), UM-UC-3 (comic ID: 724838), VM-CUB-1 (comic ID: 909780)

[0112] As a result of confirming the methylation level in the bladder cancer cell line of the six selected genes, a median methylation value of each gene was 0.93 or higher, which showed a high level of methylation value as in the high methylation level of the selected gene in the tumor tissue (see FIG. 2). Therefore, it was confirmed that the selected gene had specific methylation in bladder cancer.

Example 3: Confirmation of Low Methylation Level of Bladder Cancer Diagnostic Gene in Peripheral Blood Mononuclear Cells

[0113] Peripheral blood mononuclear cells (PBMC) were peripheral blood cell with a spherical nucleus. These peripheral blood mononuclear cells include immune-related cells such as T cells, B cells, macrophages, dendritic cells, natural killer cells (NK cells, natural killer cells), and the like.

[0114] In order to confirm the methylation levels of the selected six genes, a step of obtaining cancer samples was required. These samples refer to a wide range of body fluids, including all cancer-related biological fluids obtained from individuals, bodily fluids, cell lines, tissue cultures, etc., depending on a type of analysis to be performed. In the process of obtaining the samples, contamination of peripheral blood mononuclear cells was a cause for inhibiting measurement of methylation occurring most frequently, and affected the gene methylation levels in cancer samples by methylation of genes to be measured in peripheral blood mononuclear cells.

[0115] In order to determine whether the selected six genes were affected by the contamination of the peripheral blood mononuclear cells, methylated patterns of the peripheral blood mononuclear cells were analyzed from a total of 110 subjects. For the data, DNA extracted from the peripheral blood mononuclear cells was subjected to an Infinium Human Methylation 450 Beadchip microarray experiment according to a standardized manufacturer's methylation analysis test process as in Examples 1 and 2. In the result values of the performed experiment, the methylation levels of genes were measured through about 450,000 probes, and the methylation value of each probe was presented as a beta value. The beta value had a value of 0 to 1, and it was determined that the closer to 1, the higher the methylation level of the corresponding gene region.

[0116] As a result of confirming the methylation levels in the peripheral blood mononuclear cells of the six selected genes, a median methylation value of each gene was less than 0.10, which showed a low level of methylation value as in the high methylation level of the selected gene in the tumor tissue (see FIG. 3). Therefore, it was confirmed that the selected gene had specific methylation in bladder cancer.

[0117] In addition, methylation analysis was performed by cell group constituting the peripheral blood mononuclear cells to analyze whether a specific cell group affected the measurement of the methylation level in the sample. In a total of six normal blood samples, the cell groups were divided into whole blood, peripheral blood mononuclear cells (PBMC), and granulocytes, CD4+ T cells, CD8+ T cells, CD56+NK cells, CD19+B cells, CD14+ cells, monocytes, neutrophils, and eosinophils constituting the PBMCs to analyze the DNA methylation levels. The methylation level was measured through the Infinium Human Methylation 450 Beadchip microarray experiment as in Examples 1 and 2.

[0118] As a result of confirming the levels for each sub-cell group of peripheral blood mononuclear cells of the six selected genes, a median methylation value of each gene was at most less than 0.16, and the selected genes also showed low methylation level values even in each of the peripheral blood mononuclear cells (see FIG. 4).

[0119] These results mean that since the selected genes exhibited low methylation levels in the peripheral blood mononuclear cells, the effect from the contamination of the peripheral blood mononuclear cells on the measurement of methylation of the corresponding gene from the sample is extremely limited.

Example 4: Evaluation of Diagnostic Performance of Bladder Cancer Diagnostic Genes

[0120] In order to confirm the usefulness of the selected genes as a diagnostic marker in bladder cancer, the accuracy of diagnosis of bladder cancer according to the methylation level was evaluated.

[0121] In order to evaluate the accuracy of diagnosis, sensitivity and specificity were used. A receiver operating characteristic (ROC) curve that presented changes in sensitivity and specificity according to a cut-off value may be shown through the calculation of the sensitivity and specificity values for feasible cut-off values of consecutive diagnostic test measurement values. The accuracy of diagnosis may be measured by an area under the ROC curve (AUC). The AUC value has a value between 0.5 and 1, and it is evaluated that the higher the value, the higher the diagnostic accuracy. If the AUC value is 1, it is meant that the diagnostic result is a perfectly accurate test, but if the AUC value is 0.5, it is determined that the diagnostic result is the same as the random result.

[0122] As a result of analyzing the cancer classification accuracy according to the methylation level between the non-tumor tissue and the tumor tissue using the selected genes by using a collected methylation dataset, as illustrated in FIG. 7, it was confirmed that all the selected genes had area under curve (AUC) values of 0.906 or higher to have high diagnostic accuracy, so that the selected genes were useful for diagnosing bladder cancer (see FIG. 5).

INDUSTRIAL APPLICABILITY

[0123] As described above, since hypermethylation of the CpG site of at least one selected from the group consisting of IFFO1, MARCH11, BARHL2, NR2E1, KCNA3 and BOLL is specifically exhibited in bladder cancer, it is possible to accurately and quickly diagnose bladder cancer, and also to monitor for early diagnosis and recurrence after treatment using a composition, a kit, a chip or a method according to the present invention.