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)]. CONCLUSION: 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.
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)]. CONCLUSION: 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: Eric D Larson; Jose Pedrito M Magno; Matthew J Steritz; Erasmo Gonzalo D V Llanes; Jonathan Cardwell; Melquiadesa Pedro; Tori Bootpetch Roberts; Elisabet Einarsdottir; Rose Anne Q Rosanes; Christopher Greenlee; Rachel Ann P Santos; Ayesha Yousaf; Sven-Olrik Streubel; Aileen Trinidad R Santos; Amanda G Ruiz; Sheryl Mae Lagrana-Villagracia; Dylan Ray; Talitha Karisse L Yarza; Melissa A Scholes; Catherine B Anderson; Anushree Acharya; Samuel P Gubbels; Michael J Bamshad; Stephen P Cass; Nanette R Lee; Rehan S Shaikh; Deborah A Nickerson; Karen L Mohlke; Jeremy D Prager; Teresa Luisa G Cruz; Patricia J Yoon; Generoso T Abes; David A Schwartz; Abner L Chan; Todd M Wine; Eva Maria Cutiongco-de la Paz; Norman Friedman; Katerina Kechris; Juha Kere; Suzanne M Leal; Ivana V Yang; Janak A Patel; Ma Leah C Tantoco; Saima Riazuddin; Kenny H Chan; Petri S Mattila; Maria Rina T Reyes-Quintos; Zubair M Ahmed; Herman A Jenkins; Tasnee Chonmaitree; Lena Hafrén; Charlotte M Chiong; Regie Lyn P Santos-Cortez Journal: Hum Mutat Date: 2019-05-21 Impact factor: 4.878
Authors: Sandra Meršaková; Veronika Holubeková; Marián Grendár; Jozef Višňovský; Marcela Ňachajová; Michal Kalman; Erik Kúdela; Pavol Žúbor; Tibor Bielik; Zora Lasabová; Ján Danko Journal: Oncol Lett Date: 2018-09-26 Impact factor: 2.967