title: Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing® assays.
abstract: DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing-based assays. Bisulfite-treated genomic DNA (bsDNA) was amplified by standard PCR and applied to novel Pyrosequencing® assays, in order to measure the methylated fraction (%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy) and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation.
authors.affiliation: Department of Laboratory Medicine, Örebro University Hospital, Örebro, Sweden.
authors.forename: Zarah M
authors.initials: ZM
authors.lastname: Löf-Öhlin
authors.forename: Sonja
authors.initials: S
authors.lastname: Levanat
authors.forename: Maja
authors.initials: M
authors.lastname: Sabol
authors.forename: Bengt
authors.initials: B
authors.lastname: Sorbe
authors.forename: Torbjörn K
authors.initials: TK
authors.lastname: Nilsson
mesh_terms: D001483:Base Sequence; D018899:CpG Islands; D019175:DNA Methylation / Q000502:physiology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005787:Gene Frequency; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016555:Matched-Pair Analysis; D008969:Molecular Sequence Data; D009375:Neoplasms, Glandular and Epithelial / Q000235:genetics* / Q000473:pathology; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011401:Promoter Regions, Genetic / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000502:physiology; D017422:Sequence Analysis, DNA / Q000379:methods*; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface