PHT HI N T BI N rtN236T V rtA181T/V KHNG ADEFOVIR DIPIVOXIL NH NG B NH NHN NHI M VIRUS HBV M N TNH B NG REALTIME PCR

H Th Thanh Th y , Nguy n Vn Hng , Nguy n B o Ton , L Huy n i Thy 2 3 Cng ty c ph n TM SX & DV Vi t , Khoa Sinh h c, i h c Khoa h c T nhin, TP. HCM, Trung tm ch n on 4 Y khoa Medic, TP. HCM, Khoa Cng ngh Sinh h c, i h c M , TP. HCM.

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M U Adefovir dipivoxil (ADV) l m t trong nm lo i thu c c FDA ch p thu n dng trong i u tr vim gan siu vi B t nm 2002. ADV, m t ng phn c a nucleotide, v i ch tc ng l enzyme reverse transcriptase c a HBV trong su t qu trnh nhn ln c a virus. V i vi c gia tng s khng lammivudine sau m t th i gian i u tr lu di, th ADV tr thnh m t l a ch n thay th . Tuy nhin, s khng ADV (ADV-R) b t u xu t hi n v gia tng theo th i gian, t 1-2% (sau 1 nm i u tr ) ln n 18% (sau 4 nm i u tr ) 1 v c th ln n 29% sau 5 nm i u tr . Nh ng t bi n c ghi nh n l nguyn nhn chnh c a 2, 3 ADV-R bao g m rtA181V/T v rtN236T , c tm th y trong vng ch c nng B v D c a gene m ha 4 cho enzyme reverse transcriptase c a virus HBV .Cc t bi n ny c th x y ra n l ho c k t h p v i nhau, v cng c th xu t hi n cng v i cc t bi n b sung nh rtI233V, rtP237H, rtN238T/D, rtV84M, rtS85A,. Nh ng t bi n b sung ny c cho l gip ch ng khng thch ng cao hn trong vi c nhn 5, 6, 7, 8 . ln C nhi u k thu t sinh h c phn t c ng d ng pht hi n cc t bi n ny. l cc k thu t gi i trnh t , k thu t PCR k t h p lai phn t , PCR k t h p vi c dng enzyme c t h n ch sau (PCR-RFLP), 8, 9, 10 hay RFMP restriction fragment mass polymorphism . Chng ti l a ch n k thu t Realtime PCR nh m pht hi n t bi n rtA181V/T v rtN236T v chnh xc, nh y cao c a k thu t. Hn n a, Realtime PCR c kh nng pht hi n c t bi n cho d n ch chi m s l ng nh trong qu n th h n h p ch a c virus d ng hoang d i v d ng t bi n. NGUYN LI U V PHNG PHP M u b nh ph m v amplicon 50 m u b nh ph m huy t thanh nhi m HBV m n tnh v ang c i u tr b ng ADV do b nh vi n i h c Y d c TP. HCM v phng xt nghi m Y khoa u L c cung c p. M i, m u d v amplicon Ba h m i, m u d ( c tm t t trong b ng 1) s d ng trong nghin c u ny c thi t k d a vo trnh t cc gene polymerase c a virus c cng b trn ngn hng gene v c t ng h p b i cng ty IDT (USA).
B ng 1. M i v m u d s d ng Tn rt181F1 rt181R1 rt181P1 rt181F2 rt181R2 rt181P2 rt236F rt236R rt236P Ch c nng M i xui M i ng c M u d M i xui M i ng c M u d M i xui M i ng c M u d Trnh t 5-CCATCATCTTGGGCTTTCGCAAG-3 5 - AACAAATGGCACTAGTAAACTGAA*- 3 5-FAM TATGGGAGTGGGCCTCAGTCCGTTTC TAMRA-3 5-CTCAGTCCGTTTCTCCTGA**-3 5-TAACTGAAAGCCAAACAGTGG-3 5-FAM AGTTTACTAGTGCCATTTGTTCAGTG TAMRA-3 5-GGGCCAAGTCTGTACAACATCT - 3 5-CCCCAWCKTTTKGTTTTRTKAGGGG***-3 5- FAM ACTCAAGATGTTGTACAGACT TGGCCCCCA TAMRA-3 Chi u di (bp) 23 24 26 19 21 26 22 25 30 103 86 Kch th c s n ph m PCR (bp) 84

* V tr b t c p nucleotide d ng t bi n ki u A/T t i codon rt181 n m trn u 3 c a m i ng c rt181R1 ** V tr b t c p nucleotide d ng t bi n ki u A/V t i codon rt181 n m trn u 3 c a m i xui rt181F2

*** V tr b t c p nucleotide d ng t bi n t i codon rt236 n m trn m i ng c rt236R.

Tch chi t DNA DNA t m u huy t thanh b nh nhn c ly trch b ng phng php s d ng phenol/chloroform, ph ng theo phng php c a Chomczynski & Sacchi (1987): 200 l huy t thanh c thm vo 900 l Trizol, pH8. DNA sau c t a b ng Isopropanol v i ch t tr t a GlycoBlue. DNA c gi trong dung d ch TE 1X o (Tris-EDTA) v b o qu n -20 C cho n khi s d ng. Realtime PCR 0 Ph n ng c th c hi n trn my Mxpro-Mx3005P (Stratagene) v i chng trnh nhi t bao g m 95 C- 5 0 0 (1 chu k) v 40 chu k l p l i v i 95 C- 15, 60 C- 1. Th tch h n h p ph n ng l 50 l bao g m: 1 dung d ch m PCR, 100 nmol/L m i v m u d, 400 mol/L m i lo i dATP, dGTP, dCTP v dTTP, 1.5 units hot-start Taq DNA polymerase, 3 mmol/L MgCl2. M i t bi n c th c hi n trong m i ph n ng ring bi t. Gi i trnh t S n ph m PCR ch a cc v tr t bi n rtA181V/T v rtN236T c xc nh b ng phng php realtime PCR sau g i n cng ty Macrogen, Hn qu c tinh s ch v gi i trnh t . K T QU & TH O LU N c hi u c a m i v m u d Trong th c nghi m ki m tra c hi u c a cc h m i v m u d, cc amplicon mang cc t bi n c thi t k d a vo trnh t cc gene polymerase c a virus c cng b trn ngn hng gene v c t ng h p b i cng ty IDT (USA) c s d ng.

rt N236T Mut rt A181V Mut rt A181T Mut rt N236T WT rt A181V WT rt A181T WT

Hnh 1. K t qu kh o st kh nng khu ch i c a m i v m u d Bi u cho th y m u HBV cc d ng t bi n (k hi u Mut) c tn hi u hunh quang v t trn tn hi u n n, cc m u HBV d ng hoang d i (k hi u WT) c tn hi u hunh quang th p hn tn hi u n n. Nh v y h primer-probe-amplicon h at ng hi u qu v c hi u cho cc t bi n rtA181V/T v rtN236T. nh y c a ph n ng Bi u cho th y c ba d ng th bi u th cho cc d ng t bi n rt181T, rt182V v rt235T u cho tn hi u 7 hunh quang v t trn tn hi u nn t m u c n ng amplicon 10 copy/ml v i chu k ng ng (Ct) gi m 2 d n n m u c n ng 10 copy/ml. M u c n ng 10 copy/ml th c tn hi u hunh quang th p hn tn hi u n n.
107coppies/ml 106coppies/ml 105coppies/ml 104coppies/ml 103coppies/ml 102coppies/ml 101coppies/ml

GHI CH: : rt N236T Mut : rt A181V Mut : rt A181T Mut

Hnh 2. K t qu kh o st nh y c a ph n ng

K t qu Realtime PCR trn b nh ph m

Hnh 3. K t qu Realtime PCR trn m u b nh ph m B ng 2. K t qu Realtime PCR trn 50 m u b nh ph m t bi n rtN236T STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x x x x x x x x x x x x x x x x x x x x x x x x c khng x x x x x x x x x x x x x x x x x x c t bi n rtA181T khng x x x x x c t bi n rtA181V khng x x x x x x x x x x x x x x x x x x x K t Lu n khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny tm th y t bi n t i v tr rtA181T v rtN236T khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny tm th y t bi n t i v tr rtA181T tm th y t bi n t i v tr rtA181V khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 x x

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x x x x

x x x x x x x x x x x x x x x x x x x x x x x x x

khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny tm th y t bi n t i v tr rtN236T khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny tm th y t bi n t i v tr rtA181V khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny khng tm th y t bi n t i 2 v tr ny tm th y t bi n t i v tr rtA181T khng tm th y t bi n t i 2 v tr ny tm th y t bi n t i v tr rtN236T khng tm th y t bi n t i 2 v tr ny

x x x x x x x x x x x x x x x x x x x x x x x

x x x x x x x x x x x x x x x x x x x x

x x

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Quy trnh c th nghi m trn 50 m u b nh ph m nhi m HBV m n tnh. K t qu cho th y c 3/50 m u xu t hi n t bi n rt236T; 3/50 m u xu t hi n t bi n rt181T v 2/50 m u xu t hi n t bi n rt181V. Trong , c 1 tr ng h p (m u s 6, b ng 2) xu t hi n ng th i hai t bi n rt181T v rt236T. Cc gi tr Ct c a cc m u xu t hi n t bi n l n l t l 31,8; 29,2 v 30,1( i v i cc t bi n rt236T, tng ng l n l t cc m u s 6, 26 v 49); 28,9; 21,9 v 19,2( i v i cc t bi n rt181T, tng ng l n l t cc m u s 6, 19 v 47) v 16,2; 27,4 ( i v i cc t bi n rt181V, tng ng l n l t cc m u s 20, 37 ). Cc k t qu Realtime PCR ny c ki m ch ng l i b ng gi i trnh t s n ph m PCR v k t qu c a c hai phng php cho th y hon ton trng kh p trong pht hi n cc t bi n. Tm l i, phng php Realtime PCR m chng ti v a xy d ng hon ton ph h p trong pht hi n t bi n khng thu c ADV HBV. TI LI U THAM KH O
(1) Locarnini, S. 2005. Molecular virology and the development of resistant mutants: implications for therapy. Semin. Liver Dis. 25(Suppl. 1):919.

(2) Angus, P., R. Vaughan, S. Xiong, H. Yang, W. E. Delaney, C. S. Gibbs, C. L. Brosgart, D. Colledge, R. Edwards, A. Ayres, A. Bartholomeusz, and S. Locarnini. 2003. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 125:292297. (3) Hadziyannis, S. J., N. C. Tassopoulos, J. Heathcote, T.-T. Chang, G. Kitis, M. Rizzetto, P. Marcellin, S. G. Lim, Z. Goodman, J. Ma, S. Arterburn, S. Xiong, G. Currie, and C. L. Brosgart. 2005. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic Hepatitis B. N. Engl. J. Med. 352: 26732681. (4) Bartholomeusz, A., B. G. Tehan, and D. K. Chalmers. 2004. Comparisons of the HBV and HIV polymerase, and antiviral resistance mutations. Antivir. Ther. 9:149160. (5) Marc, G., T. Jake Liang. Drug targets and molecular mechanism of drug resistance in chronic hepatitis B. Gastroenterology. 2007; 132: 1574-1585 (6) Marcello, P; Asselah, T. Resistance to adefovir: A new challenge in the treatment of chronic hepatitis B. Journal of Hepatology. 2005; 43: 920-923 (7) Locarnini, S. Primary resistance, multidrug resistance, and cross-resistance pathways in HBV as a consequence of treatment failure. Hepatology International. 2008; 2: 147-151 (8) Shaw, T; Bartholomeusz, A; Locarnini, S. Review : HBV drug resistance : Mechanism, detection and interpretation. Journal of Hepatology. 2006; 44: 593-606 (9) Osiowy, C; Villeneuve, J. P; Heathcote, E. J; Iles, E; Borlang, J. Detection fo rtN236T and rtA181V/T mutations associated with resistance to adefovir dipivoxil in samples from patients with chronic hepatitis B virus infection by INNOLiPA HBV DR line probe assay (version 2). Journal of Clinical Microbiology. 2006; 44: 1994-1997 (10) Sablon, E; Shapiro, F. Advantages in molecular diagnosis of HBV infection and drug resistance. International Journal of Medical Sciences. 2005; 2(1): 8-16

Tc gi ch u trch nhi m chnh: L Huy n i Thy: Khoa Cng ngh Sinh h c, i h c M , TP. HCM, email: thuy.lha@ou.edu.vn, i n tho i: 08-39300086, mobile: 0905784471
SUMMARY

DETECTION OF rtN236T AND rtA181V/T MUTATIONS ASSOCIATED WITH RESISTANCE TO ADEFOVIR DIPIVOXIL IN SAMPLES FROM PATIENTS WITH CHRONIC HEPATITIS B VIRUS INFECTION BY REALTIME-PCR
Ho Thi Thanh Thuy , Nguyen Van Hung , Nguyen Bao Toan , Le Huyen Ai Thuy 1 2 3 Viet A Trading Production & Service Corp, Faculty of Biology, Natural Sciences University of HoChiMinh city, 4 Medic medical center of HoChiMinh city, Faculty of Biotechnology, Open University of HoChiMinh city.
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Background: The nucleotide analog adefovir dipivoxil (ADV) is an effective antiviral treatment for chronic hepatitis B virus (HBV) infection, with resistance to ADV estimated to occur less frequently than resistance to lammivudine treatment. The detection of ADV resistance mutations is necessary during therapy to monitor and anticipate possible treatment failure. Objectives: To develop realtime-PCRs in order to detection of rtN236T and rtA181V/T mutations associated with resistance to ADV. Study design: We have established the realtime-PCR methods using universal amplicons and Taqman probes to detect rtA181V/T and/or rtN236T mutants by individual reactions. The realtime-PCR protocols were compared with sequencing and were applied on a further 50 clinical samples. Results: The performance of these techniques were assessed by analyzing serial dilutions of amplicons of 2 known concentrations and the lower limit of detection were found to be 10 DNA copies/ml. Evaluation of these techniques to detect ADV resistance mutations was performed by analyzing 50 patients treated with ADV. 3/50 patients (for rt236T mutant), 3/50 patients (for rt181T mutant), and 2/50 patients (for rt181V mutant) were found. This result was confirmed by DNA sequencing. These real-time PCR protocols are rapid and accurate methods for quantification of rt181 and rt236 mutants of HBV virus in adefovir-treated patients.