Pyrosequencing-based Quantitative Evaluation of p16 Methylation in Diffuse Large B-cell Lymphoma

Norafiza Zainuddin, Lailatul Jalilah Mohd. Ridah, Aziah Ismail, Norlelawati A. Talib, Naznin Muhammad, Faezahtul Arbaeyah Hussain

Abstract


Introduction: Methylation of promoter region of p16 leading to gene silencing has been implicated in a wide range of malignancies including lymphomas. In diffuse large B cell lymphoma (DLBCL) particularly, a varying percentage of epigenetic inactivation of p16 promoter region was observed ranging from 16 - 54%. However, quantitative analysis of p16 promoter methylation in DLBCL has not been extensively studied in Malaysia. Objective: This study aims to quantitatively analyse p16 methylation in DLBCL samples pyrosequencing technique. Methods: Genomic DNA was extracted from 16 formalin-fixed paraffin-embedded lymphoma tissue blocks from patients diagnosed with DLBCL. Primers were designed to amplify bisulfite-treated DNA targeting p16 promoter region. Methylation status of 7 CpG sites was determined by pyrosequencing. Results: All the 16 samples studied showed promoter methylation of p16. The range of mean methylation percentage was 18 - 81%. Conclusion: The present study has successfully identified methylation of p16 in all 7 CpG sites though there is limitation in the sample size. Our experience shows that pyrosequencing is relatively inexpensive, is able to analyse up to 96 samples in a single assay that is reproducible, with simple data extraction from displayed pyrogram, and more importantly it helps overcome the complexities related DNA methylation research.

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