Balkan Journal of Medical Genetics

NOVEL PATTERNS OF THE EPSTEIN-BARR NUCLEAR ANTIGEN (EBNA-1) V-VAL SUBTYPE IN EBV-ASSOCIATED NASOPHARYNGEAL CARCINOMA FROM VIETNAM
Thuan LD1, Kha ND2, Minh NT3, Thuy LHA1,*
*Corresponding Author: Thuy Le Huyen Ai, Ph.D., Associate Professor, Department of Pharmaceutical and Medical Biotechnology, Faculty of Biotechnology, Room 304, 97 Vo Van Tan Street, Ward 6, District 3, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam. Tel: +84-905-784-471. E-mail: thuy.lha@ ou.edu.vn
page: 61

MATERIALS AND METHODS

Ethics Statement. The Institutional Ethics Board approval, the decision number of the permission from Ethics committee: 516/BVCR-HDDD, was obtained from the Medical Ethics Committee of the Cho Ray Hospital, Ho Chi Minh City, Vietnam. All the biopsy samples used in current study were collected from participants, who agreed and signed all the consent forms. Sample Collection and DNA Isolation. Fifty-eight NPC biopsy samples were collected from local patients at the Cho Ray Hospital, Ho Chi Minh City, Vietnam. All the sample were submitted to the histopathological diagnosis center to confirm the NPC diagnosis. Notably, all biopsies were collected from patients, who obeyed the ethical approval for study of human samples, and patients who agreed with the purpose of the study. For DNA isolation, biopsies were lysed in lysis buffer, containing 10 mM Tris-HCl pH 8, 10 mM EDTA, 150 nM NaCl, 2.0% SDS, and 0.1 mg/mL Proteinase K, incubated overnight at 56 °C. Total of genomic DNA of clinical samples was extracted using a phenol/chloroform solution, pH 8. The purification of DNA was done by a 99.0% ethanol solution. The quality and purity of DNA isolates were measured by the evaluation of A260/A280 proportion. The DNA solution was stored in Tris-EDTA 0.5M, at –20 °C for further assays. The PCR Amplification of EBNA-1 C-Terminal Domain of Clinical Samples and C-Terminal Domain Sequencing. Nested polymerase chain reaction (nested- PCR) was applied to amplify the C-terminal region of EBNA-1. The sequence and position of primers used in two stages of nested-PCR are shown in Table 1 and Figure 1, respectively. The EBNA-1-1 and EBNA-1-2 primers were used for stage 1 of nested-PCR, and EBNA-1-3 and EBNA-1-4 primers were used in stage 2 of nested-PCR. Both stages of nested-PCR were performed in a total of 15 μL, containing 100 ng genomic DNA template (stage 1) or 100 ng stage 1 PCR product (stage 2), 0.5 μM each primer, and 7.5 μL MyTaqTM Mix (Cat. No. BIO-25041; Bioline UK, London, UK). The PCR amplification was performed with an initial denaturation at 94 °C for 5 min.; followed by 45 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C (stage 1) or 65 °C (stage 2) for 30 seconds, extension at 72 °C for 30 seconds; and elongation step at 72 °C for 10 min. The nested-PCR products were visualized and analyzed by electrophoresis through stained with ethidium bromide, and 2.0% agarose gel. For EBNA-1 C-terminal sequencing, 30 μL products of the second stage of nested- PCR were sent to Nam Khoa Biotek Co. Ltd. (Ho Chi Minh City, Vietnam) for direct sequencing in both direction with primers EBNA-1-3 and EBNA-1-4. Determination and Analysis of EBNA-1 V-Val Subtypes. The results of EBNA-1 C-terminal sequencing was read by Chromas 2.6.4 (Technelysium; https:// technelysium. com.au/wp/chromas/) and checked for sequencing homology using BLAST [National Center for Biotechnology Information (NCBI); https://www.ncbi. nlm.nih.gov]. For determination of EBNA-1 C-terminal subtypes, according to the study of Bhatia et al. [14] and Gutiérrez et al. [15], amino acid at position 487 was used as the signature residue for EBNA-1 subtypes. For the current study, the valine residue at position 487, identified as V-Val subtype, was enrolled into the analysis of V-Val characteristics of Vietnamese NPC patients. All the V-Val subtype sequence data were compared with the P-Ala B95- 8 strain (Genbank accession #V10555). The alignment between data sequence and B95-8 strain sequence were analyzed using BioEdit (www.mbio.nesu.edu/BiolEdit/ bioedit.html) sequence alignment editor.

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