title: Detection of methylation of the promoter region of the MAL and CADM1 genes by pyrosequencing in cervical carcinoma.
abstract: OBJECTIVE
  Cervical cancer is the second most common cancer disease affecting the female population. A key factor in development of the disease is the human papillomavirus infection (HPV). The disease is also impacted by epigenetic changes such as DNA methylation, which causes activation or exclusion of certain genes, and simultaneously the hypermethylation of cytosines in the promoters and turn-off of previously active genes occur. In this study, we focused on the introduction of pyrosequencing for the detection of DNA methylation of the selected CADM1 and MAL genes.
  
  
  METHODS
  DNA was isolated from cytological cervical smear of patients with different types of dysplasia [L-SIL (n=14), ASC-US (n=15), H-SIL (n=1)] and four control samples from healthy women. Prepared samples were further analyzed by bisulfite conversion and subsequent pyrosequencing (Pyromark Q96 ID, Qiagen, Germany). We examined the extent of methylation of CpG islands and as control samples of this method we used a fully methylated and unmethylated DNA. Methylation level (Met level) from each sample was quantified as the mean value [sum of all methylated CpG islands in %/total number of CpG islands (MAL n=4; CADM1 n=3)].
  
  
  RESULTS
  In total, 30 clinical samples and 4 control samples from healthy women were analyzed. By means of the analysis of the CADM1promoter region, the values of the Met level were obtained [fully methylated DNA (94.83 and 88); completely unmethylated DNA (0 and 0); and control samples from healthy patients (6.825 and 0.825), L-SIL (2.107 and 2.778), ASC-US (7.313 and 3.626), H-SIL (0 and 0)]. By means of the analysis of the MAL promoter region, the values of Met level were obtained [fully methylated DNA (53.25); completely unmethylated DNA (0.875); and control samples from healthy patients (2.925), L-SIL (1.517), ASC-US (2.833), and H-SIL (4)].
  
  
  CONCLUSIONS
  We introduced a pyrosequencing method for quantification of methylation of CADM1, MAL promoter regions, and detected methylations in clinical samples and also some basal methylation in healthy women.
authors.affiliation: Department of Gynecology and Obstetrics and University Hospital, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Sandra
authors.initials: S
authors.lastname: Mersakova
authors.affiliation: Department of Gynecology and Obstetrics and University Hospital, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Jozef
authors.initials: J
authors.lastname: Visnovsky
authors.affiliation: Department of Molecular Biology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Veronika
authors.initials: V
authors.lastname: Holubekova
authors.affiliation: Department of Gynecology and Obstetrics and University Hospital, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Marcela
authors.initials: M
authors.lastname: Nachajova
authors.affiliation: Department of Gynecology and Obstetrics and University Hospital, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Erik
authors.initials: E
authors.lastname: Kudela
authors.affiliation: Department of Gynecology and Obstetrics and University Hospital, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Jan
authors.initials: J
authors.lastname: Danko
authors.affiliation: Department of Molecular Biology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia.
authors.forename: Zora
authors.initials: Z
authors.lastname: Lasabova
mesh_terms: D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins / Q000235:genetics*; D030361:Papillomavirus Infections; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology / Q000821:virology; D014626:Vaginal Smears
chemical_list: C403514:CADM1 protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; D007136:Immunoglobulins; C569026:MAL protein, human; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins