XÂY DỰNG QUY TRÌNH GIẢI TRÌNH TỰ SANGER CHẨN ĐOÁN CÁC BIẾN THỂ DI TRUYỀN TRÊN GEN DPYD LIÊN QUAN ĐẾN TÍCH LŨY ĐỘC TÍNH THUỐC FLUOROPYRIMIDINES

Abstract

Introduction: Several genetic variants in the DPYD gene have been shown to reduce or completely eliminate the activity of dihydropyrimidine dehydrogenase (DPD), markedly increasing the risk of severe, and sometimes fatal, toxicity in cancer patients treated with fluoropyrimidines. These variants are distributed across multiple regions of the gene, requiring numerous individual PCR reactions prior to Sanger sequencing. This study proposes a Multiplex PCR combined with Sanger sequencing approach to simultaneously detect key DPYD variants. This strategy simplifies the workflow, shortens turnaround time, reduces overall cost, and provides timely clinical information to ensure patient safety. Objective: To establish a Sanger sequencing protocol for the detection of DPYD gene variants associated with fluoropyrimidine-induced toxicity. Materials and Methods: (i) Experimental optimization of four pairs of specific primers and development of a Multiplex PCR reaction amplifying four DPYD regions harboring clinically relevant variants: rs3918290 (DPYD 2A), rs55886062 (DPYD 13), rs67376798 (c.2846A>T), and rs56038477 (c.1236G>A); (ii) optimization of PCR product quantity for high-quality Sanger sequencing; (iii) application of the complete protocol to five genomic DNA samples from healthy volunteers for validation. Results: Four specific primer pairs were successfully designed to amplify the target DPYD regions containing the variants of interest. The optimized 25 µL Multiplex PCR reaction consisted of: Q5® High-Fidelity Master Mix (1×), 50 ng genomic DNA template, and primer concentrations as follows: HaploB3, 0.125 µM; DPYD2A, 0.25 µM; DPYD13, 0.5 µM; D949V, 0.125 µM; with nuclease-free water added to final volume. The thermal cycling conditions were: initial denaturation at 98°C for 30 s; 35 cycles of 98°C for 10 s, 58°C for 15 s, and 72°C for 30s; followed by a final extension at 72°C for 60s. A minimum of 20 ng Multiplex PCR product was sufficient to achieve high-quality Sanger sequencing results. All five volunteer samples produced sequencing chromatograms meeting manufacturer quality standards, with variant positions achieving QV20+ scores, enabling unambiguous genotype determination. Conclusion: A Sanger sequencing workflow was successfully established and optimized for the detection of four clinically significant DPYD variants. This method offers a practical and efficient approach for preemptive pharmacogenetic testing in fluoropyrimidine therapy.