Detecting DNA methylation of BCL2, CDKN2A and NID2 genes to predict bladder cancer in humans
The present invention provides a method of detecting DNA methylation of a plurality of genes consisting of CDKN2A, BCL2 and NID2 in a urine sample from a human. Methods and compositions are provided herein for detecting and diagnosing bladder cancer by obtaining a urine sample from a human subject suspected of bladder cancer, followed by detecting DNA methylation of CDKN2A, BCL2 AND NID2 in urine samples from the individual. The present method permits specific detection of DNA methylation of the selected gene promoters in urine as a biomarker for bladder cancer in humans.
1. A method of determining an increased risk of bladder cancer in a human, comprising the steps of:
(a) obtaining a urine sample from a human;
(b) isolating genomic DNA from said urine;
(c) exposing said isolated genomic DNA with sodium bisulfite;
(d) performing a first round PCR on said sodium bisulfite-treated genomic DNA to amplify a methylation site present on a CpG island, said CpG island is present on a plurality of genes consisting of BCL2, CDKN2A, and NID2,
said first round PCR is performed using a forward primer and a reverse primer for BCL2, CDKN2A, and NID2 respectively, said forward primers consisting of the nucleotide sequence of SEQ ID NO: 1 (for BCL2), SEQ ID NO: 5 (for CDKN2A), and SEQ ID NO: 7 (for NID2) and said reverse primers consisting of the nucleotide sequence of SEQ ID NO: 2 (for BCL2), SEQ ID NO: 6 (for CDKN2A), and SEQ ID NO: 8 (for NID2);
(e) performing a second round PCR to detect DNA methylation status on said methylation site, said second round PCR is a methylation-specific PCR; and
(f) determining an increased risk of bladder cancer in said human,
wherein the detection of DNA methylation status on said CpG island of said plurality of genes in said urine sample is indicative of an increased risk of bladder cancer in said human.
2. The method of claim 1 , after step (c), further comprising the step of: (c′) performing a real time PCR to amplify β-actin on said sodium bisulfite-treated genomic DNA, wherein a Ct value of ≦25 is indicative of a sufficient amount of sodium bisulfite treated genomic DNA in said urine.
3. The method of claim 1 , wherein said second round PCR is performed using a forward primer and a reverse primer for BCL2, CDKN2A, and NID2 respectively, said forward primers consisting of the nucleotide sequence of SEQ ID NO: 9 (for BCL2), SEQ ID NO: 15 (for CDKN2A), and SEQ ID NO: 18 (for NID2) and said reverse primers consisting of the nucleotide sequence of SEQ ID NO: 10 (for BCL2), SEQ ID NO: 16 (for CDKN2A), and SEQ ID NO: 19 (for NID2).
4. The method of claim 1 , wherein said second round PCR is performing using a probe for BCL2, CDKN2A, and NID2 respectively, said probe consisting of the nucleotide sequence of SEQ ID NO: 11 (for BCL2), SEQ ID NO: 17 (for CDKN2A) and SEQ ID NO: 20 (for NID2).
5. The method of claim 2 , wherein said first round PCR is performed using a forward primer and a reverse primer for β-actin, said forward primer consisting of the nucleotide sequence of SEQ ID NO: 3, and said reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4 (for BCL2).
6. The method of claim 2 , wherein said second round PCR is performed using a forward primer and a reverse primer for β-actin, said forward primer consisting of the nucleotide sequence of SEQ ID NO: 12, and said reverse primer having a nucleotide sequence of SEQ ID NO: 13.
7. The method of claim 2 , wherein said second round PCR is performing using a probe for β-actin, said probe consisting of the nucleotide sequence of SEQ ID NO: 14.
8. The method of claim 1 , wherein said bladder cancer is a low malignant potential bladder cancer.
9. The method of claim 8 , wherein said low malignant potential bladder cancer is transitional cell carcinoma.