A NEW HRM QPCR ASSAY FOR EVALUATION OF DNA METHYLATION RATES IN THE P15 GENE AMONG ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS

Kewan Kamal Ahmad^{1, *}, Zrar Saleem Kareem², Ari Qadir Nabi³

¹Department of Biology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region – F.R. Iraq - 2Department of Biology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq - ²Department of Biology, Faculty of Science and Health, Koya University Koya KOY45, Kurdistan Region - F.R. Iraq - ³Department of Biology, College of Science, Salahaddin University-Erbil, Kurdistan Region, Iraq

Introduction: Acute Lymphoblastic Leukaemia (ALL) is the most common childhood cancer. The prevalence of this cancer in the Kurdistan Region has increased noticeably, yet no research has been done recently in the region to investigate the related epigenetic modifications to the genes. This study aims to evaluate methylation status of the promoter region in the tumor suppressor gene of Cyclin-Dependent Kinase Inhibitor 2B (CDKN2B) known as P15 in ALL patients in the Iraqi Kurdistan. 

Material and methods: Nonaberrant DNA methylation plays a significant role in human homeostasis and is essential for molecular studies. To understand the methylation ratio, a real-time PCR-based high-resolution melting (HRM) assay was developed to amplify both methylated and unmethylated nucleotide sequences in the promoter region of the P15 gene. Methylated and un-methylated cytokines in the p15 promoter region were bisulfite-modified for all samples and then analyzed by qPCR and HRM. 

Results: The assay outcomes indicated that despite genetic abnormalities in ALL patients, the P15 gene was hypermethylated in the CpG island of the promoter region in ALL patients compared to healthy controls. 

Conclusion: Therefore, developing an HRM-qPCR assay and sequencing the promoter region of CDKN2B could better describe its expression.