Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene
The present invention relates to a kit and nucleic acid chip for diagnosing bladder cancer using a bladder cancer-specific marker gene. More particularly, the invention relates to a kit and nucleic acid chip for diagnosing bladder cancer, which can detect the promoter methylation of a bladder cancer-specific gene, the promoter or exon region of which is methylated specifically in transformed cells of bladder cancer. The use of the diagnostic kit or nucleic acid chip of the invention enables diagnosis of bladder cancer at an early stage of transformation, thus enabling early diagnosis of bladder cancer, and can diagnose bladder cancer in a more accurate and rapid manner compared to a conventional method.
1. A method for detecting CpG methylation of PENK (proenkephalin gene), the method comprising the steps of:
(a) isolating a genomic DNA from a clinical sample;
(b) treating the genomic DNA from step (a) with bisulfite; and
(c) determining hypermethylation of the CpG of the PENK gene in the bisulfite-treated genomic DNA from step (b) by using primer(s) to amplify a methylated CpG of the bisulfite-treated PENK gene, wherein the primer(s) for amplifying a methylated CpG of PENK comprises sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-185, 187-298, 300-341, 343-468, 470-579, 581-704, 706-841, 843-976, 978-1097, 1099-1210, 1212-1221.
2. The method according to claim 1 , wherein step (c) is performed by one selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing.
3. The method according to claim 1 , wherein step (c) comprises examining a CpG methylation of a promoter or exon region of PENK in the clinical sample.
4. The method according to claim 3 , wherein the promoter comprises a DNA sequence represented in SEQ ID NO: 37.
5. The method according to claim 1 , wherein the method further comprises the step of examining CpG methylation of a gene selected from the group consisting of TBX5—T-box 5; CDX2—caudal type homeobox transcription factor 2; CYP1B1—cytochrome P450, family 1, subfamily B, polypeptide 1; VSX1—visual system homeobox 1 homolog, CHX10-like (zebrafish); HOXA11—homeobox A11; T—T, brachyury homolog (mouse); PAQR9—progestin and adipoQ receptor family member IV; and LHX2—LIM Homeobox 2.
6. The method according to claim 5 , wherein the step of examining comprises examining CpG methylation of a promoter or exon region of the gene selected from the group consisting of TBX5; CDX2; CYP1B1; VSX1; HOXA11; T; PAQR9; and LHX2.
7. The method according to claim 1 , wherein the method further comprises the step of contacting at least one nucleic acid isolated from the clinical sample with an agent capable of determining a CpG methylation status of PENK gene.
8. The method according to claim 1 , wherein the primer(s) for amplifying a methylated CpG of PENK comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of PENK.
9. The method according to claim 8 , further comprising probe(s) capable of hybridizing with a methylated CpG of PENK comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of PENK.
10. The method according to claim 8 , further comprising probe(s) capable of hybridizing with a methylated CpG of PENK comprising sequence(s) selected from the group consisting of SEQ ID NOs: 45, 186, 299, 342, 469, 580, 705, 842, 977, 1098 and 1211.
11. A method for detecting CpG methylation of PENK—proenkephalin gene for bladder carcinoma or bladder cell proliferative disorder diagnosis, the method comprising the steps of:
(a) isolating a genomic DNA from a clinical sample;
(b) treating the genomic DNA from step (a) with bisulfite; and
(c) determining hypermethylation of the CpG of the PENK gene in the bisulfite-treated genomic DNA from step (b) by using primer(s) to amplify a methylated CpG of the bisulfite-treated PENK gene, wherein a bladder carcinoma or bladder cell proliferative disorder is detected in the human subject based on increased CpG methylation of the PENK gene relative to that of a control, wherein the primer(s) for amplifying a methylated CpG of PENK comprises sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-185, 187-298, 300-341, 343-468, 470-579, 581-704, 706-841, 843-976, 978-1097, 1099-1210, 1212-1221.
12. The method according to claim 11 , wherein step (c) is performed by one selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing.
13. The method according to claim 12 , wherein the clinical sample is tissue, cell, blood, urine, serum or plasma from a patient suspected of cancer or a subject to be diagnosed.
14. The method according to claim 11 , wherein step (c) comprises examining a CpG methylation of a promoter or exon region of PENK in the clinical sample.
15. The method according to claim 12 , wherein the promoter comprises a DNA sequence represented in SEQ ID NO: 37.
16. The method according to claim 11 , wherein the method further comprises the step of examining CpG methylation of a gene selected from the group consisting of TBX5—T-box 5; CDX2—caudal type homeobox transcription factor 2; CYP1B1—cytochrome P450, family 1, subfamily B, polypeptide 1; VSX1—visual system homeobox 1 homolog, CHX10-like (zebrafish); HOXA11—homeobox A11; T—T, brachyury homolog (mouse); PAQR9—progestin and adipoQ receptor family member IV; and LHX2—LIM Homeobox 2.
17. The method according to claim 16 , wherein the step of examining comprises examining CpG methylation of a promoter or exon region of the gene selected from the group consisting of TBX5; CDX2; CYP1B1; VSX1; HOXA11; T; PAQR9; and LHX2.
18. The method according to claim 11 , wherein the method further comprises the step of contacting at least one nucleic acid isolated from the clinical sample with an agent capable of determining a CpG methylation status of PENK gene.
19. The method according to claim 11 , wherein the primer(s) for amplifying a methylated CpG of PENK comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of PENK.
20. The method according to claim 19 , further comprising probe(s) capable of hybridizing with a methylated CpG of PENK comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of PENK.
21. The method according to claim 19 , further comprising probe(s) capable of hybridizing with a methylated CpG of PENK comprising sequence(s) selected from the group consisting of SEQ ID NOs: 45, 186, 299, 342, 469, 580, 705, 842, 977, 1098 and 1211.