XÂY DỰNG QUY TRÌNH REAL-TIME PCR HIGH RESOLUTION MELTING XÁC ĐỊNH BIẾN THỂ DPYD*2A TRÊN GEN DYPD LIÊN QUAN CHUYỂN HOÁ THUỐC FLUOROPYRIMIDINES

Abstract

Introduction: A single nucleotide polymorphism in the DPYD gene, DPYD*2A, which leads to a deficiency of the fluoropyrimidines metabolizing enzyme dihydropyrimidine dehydrogenase (DPD), is strongly associated with fluoropyrimidine-induced severe and life-threatening toxicity. The Clinical Pharmacogenetics Implementation Consortium (CPIC) has recommended cancer patients should be identified the variant DPYD*2A before being treated with fluoropyrimidines. Despite the necessity of determining the DPYD*2A variant in individual fluoropyrimidines dose adjustment, due to its high price, most of patients cannot easily afford for genetic examinations. This disadvantageous may lead to the hesitation and procrastination in indicating molecular testing and in adjusting the fluoropyrimidines starting dose in clinical. In order to overcome the drawback, establishing a convenient, time-saving, affordable and reliable molecular procedure, real-time PCR high resolution melting (HRM), to identify the DPYD*2A variant is timely and crucial not only for medical administrations to efficiently manage the fluoropyrimidine poisonning situations but also for cancer patients to relieve economic burden of genetic examinations. Objective: Establishing a molecular procedure using real-time PCR HRM to identify the DPYD*2A variant. Method: Constructing the control recombinant plasmids to simulate the DPYD*2A variant genotypes by performing site-directed mutagenesis using overlap extension PCR and gene cloning method. Utilizing bio-informatics softwares to design a pair of primers amplifying the target DPYD gene sequence and verifying its specificity in practical laboratory condition. Evaluating the accuracy and the ability of the established real-time PCR HRM procedure in discriminating each of the DPYD*2A genotypes. Result: Successfully constructed recombinant plasmids containing G allele (wild-type) and A allele (DPYD*2A) to serve as genotype control samples. Successfully established a real-time PCR HRM procedure identifying the DPYD*2A variant with: (i) High discriminability of DPYD genotypes when using derivative melt curves, aligned melt curves and difference plot to analyze the PCR product melting temperature; (ii) High accuracy of T_m value with %CV from 0.05 to 0.06%. Conclusion: The established procedure could be applied to identify DPYD*2A variant in research. Initially applying this procedure to investigate the genetic characteristics and the frequency of DPYD*2A in the Vietnamese population.