Methylation status of p16INK4a and p14ARF gene in saliva of smokeless tobacco users: A pilot descriptive study

Abstract

ObjectivesThe tumor suppressor genes p16INK4a and p14ARF are frequently silenced through hypermethylation in oral potentially malignant disorders (OPMDs) and oral cancers. This study aimed to evaluate the methylation status of p16INK4a and p14ARF in saliva of smokeless tobacco users as an early diagnostic marker of dysplasia. MethodsUnstimulated whole saliva with exfoliated buccal cells were collected from 30 non-tobacco users and 30 smokeless tobacco users without any clinically evident tobacco associated oral lesions from South Indian population. DNA extraction, bisulfite modification and methylation specific polymerase chain reaction were done to identify methylation with respect to p16INK4a and p14ARF. The amplified sequences were visualized in 1% agarose gel electrophoresis. ResultsOf the 60 samples collected, 26 samples were rejected owing to the poor quality and insufficient quantity of DNA. A total of 34 DNA samples were assessed for methylation status of three sets of primers for p16INK4a and one set of primer for p14ARF. Methylation was not detected in saliva from non-tobacco users. In case of smokeless tobacco users, partial methylation was detected in 33.3 % samples corresponding to set II primer of p16INK4a. ConclusionDetection of methylation in p16INK4a in oral mucosa of smokeless tobacco users without any associated oral lesions, indicates its role in early dysplastic changes.