Methods for determining methylation of the TWIST1 gene for bladder cancer detection
View Patent ↗Disclosed are methods of detecting a predisposition to, or the incidence of, bladder cancer in a sample comprising detecting an epigenetic change, such as methylation, in at least one gene selected from TWIST1, NID2, RUNX3, BMP7, CCNA1, PDLIM4, TNFRSF25, APC, RASSF1A, LOXL1, TUBB4, NTRK2, ARFGAP3, OSMR and TJP2, or a panel thereof. Also disclosed are kits for performing the disclosed methods, such as kits for detecting a predisposition to, or the incidence of, bladder cancer in a sample comprising at least one primer pair for determining the methylation status of each of NID2, TWIST1 and RUNX3. The kits may contain means for processing a urine sample.
1. A method of detecting incidence or recurrence of bladder cancer or a predisposition to the incidence or recurrence of bladder cancer in a sample comprising bladder cells, the method comprising:
(a) reacting the sample with a reagent which selectively modifies unmethylated cytosine residues in DNA contained in the sample to produce detectable modified residues but which does not modify methylated cytosine residues;
(b) determining methylation status of the promoter region of the TWIST1 gene in the sample comprising the primer binding sites for SEQ ID NO:22 and SEQ ID NO:23 and the probe binding site for SEQ ID NO:24, wherein determining methylation status comprises hybridizing to the promoter region of the TWIST1 gene comprising the primer binding sites for SEQ ID NO:22 and SEQ ID NO:23 and the probe binding site for SEQ ID NO:24 a primer and/or probe that hybridizes to the promoter region of the TWIST1 gene comprising the primer binding sites for SEQ ID NO:22 and SEQ ID NO:23 and the probe binding site for SEQ ID NO:24; and
(c) detecting hypermethylation of the promoter region of the TWIST1 gene comprising the primer binding sites for SEQ ID NO:22 and SEQ ID NO:23 and the probe binding site for SEQ ID NO:24 to indicate incidence or recurrence of bladder cancer or a predisposition to the incidence or recurrence of bladder cancer.
2. The method of claim 1 further comprising determining the methylation status of the NID2 gene.
3. The method of claim 2 further comprising determining the methylation status of the RUNX3 gene.
4. The method of claim 1 wherein the sample comprises a bladder tissue sample or a urine sample.
5. The method of claim 4 wherein a stabilising buffer is added to the urine sample once collected, thus permitting the sample to be stored for a period of up to 72 hours at room temperature without the need for centrifugation of the sample.
6. The method of claim 1 further comprising performing urinary cytology or mutation analysis.
7. The method of claim 6 wherein the mutation analysis is FGFR3 gene mutation analysis.