174 The Korean Journal of Hepatology : Vol. 5. No Key Words : Chronic hepatitis B, Lamivudine, HBV DNA polymerase, YMDD mutant :, 134,

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1 ; HBV,, 1 1 Abstract Emergence of YMDD Motif Mutant Hepatitis B Virus during Short-term Lamivudine Therapy Yong Han Paik, M.D., Kwang Hyub Han, M.D., Hyo Young Chung1 M.S. Chae Yoon Chon, M.D., Young Myoung Moon, M.D. Department of Internal Medicine, Institute of Gastroenterology, Yonsei Medical Research Center1 Yonsei University College of Medicine, Seoul, Korea Background/ Aims: T he emergence of lamivudine- resistant mutant hepatitis B virus (HBV), with aminoacid substitution in the YMDD motif of DNA polymerase, has been reported in the long- term lamivudine use group. However there is no report about the emergence of mutant viruses during the short- term lamivudine therapy. T he objective of this study was to investigate the emergence of YMDD mutant HBV during short- term lamivudine therapy. Methods: We evaluated twenty- eight chronic hepatitis B patients who were HBeAg and HBV DNA positive and treated with lamivudine 100mg p.o. daily for 12 weeks. First, we investigated the emergence of YMDD mutants by nested polymerase chain reaction (PCR) method developed by Chayama et al in 19 patients who lost HBV DNA during lamivudine therapy but showed HBV DNA re- emergence 2 weeks after the end of therapy. Second, DNA subcloning and sequencing of HBV DNA polymerase including YMDD motif was undertaken in one patient' s serial blood samples at 0, 8, 12 weeks to confirm the results of nested PCR. Res ults: YMDD motif mutation was detected in 17(90%) out of 19 patients at the end of therapy and the type of mutations were YIDD only. At the end of therapy, mutant was predominant in 5 patients, both mutant and wild type were similar in proportion in 3 patients, and wild type was predominant in 9 patients. When we carried out nested PCR serially with samples of 0, 2, 4, 8, 12, 14 weeks after initiation of therapy in 5 patients who were mutant predominant at 12 weeks, YIDD mutant began to be detected from 2 weeks in 4 patients and from 4 weeks in one patient. However, rapid turnover from mutant to wild type happened after the end of therapy, so only wild type was detected in 3 patients and wild type became predominant in 2 patients at 2 weeks after the end of therapy. All the sequencing results of serial blood samples in one patient were consistent with nested PCR data. Conclusions : The presence of YMDD motif HBV polymerase mutant may be possible before administration of lamivudine in Korean chronic hepatitis B patients. Nested PCR assay would be an useful method to detect YMDD mutant. (Korean J Hepatol 1999;5: )

2 174 The Korean Journal of Hepatology : Vol. 5. No Key Words : Chronic hepatitis B, Lamivudine, HBV DNA polymerase, YMDD mutant :, 134, (Lamivudine, (- )- - L- 2',3' - dideoxy- 3' - thiacytidine, 3T C) (cytosine nucleoside analogue) human immunodeficiency virus(hiv) B (HBV) (RNA dependent DNA polymerase) 1-4 B. 6 B HBV, HBV YMDD motif ( th) methionine isoleucine YIDD valine YVDD, YMDD.10, 11 6 HBV YMDD motif. B HBV YMDD motif Glaxo- w elcome (Zeffix ) mg 12 18, 10 38(24-60). 1 HBeAg HBV DNA anti- HCV (57%) ALT 22 (79%) HBV DNA. 4 HBV DNA 2 HBV DNA. HBV DNA HBV DNA. 12 HBV DNA , 6, 39(26-60). alanine aminotransferase (ALT ) (47-190) IU/L HBV DNA ( ) pg/ml (T able 1) , 2, 4, 8, 12, nested PCR. nested PCR , 4, 8, 12 2 nested

3 Yong Han Paik, et al. Emergence of YMDD Motif Mutant Hepatitis B Virus during Short- term Lamivudine Therapy 175 T able 1. Clinical Characteristics of 19 Patients with Chronic Hepatitis B T reated with Short- term(12 weeks) Lamivudine in Whom Nested PCR Assay Was Done Patient Age Sex ALT(IU/L) HBV DNA(pg/ ml) Nested PCR results baseline 12weeks baseline 12weeks 14weeks of YMDD motif at 12 weeks 1 35 F ND 7.6 YIDD> > YMDD 2 58 M ND 190 YIDD> > YMDD 3 42 F ND 26 YIDD> > YMDD 4 35 M ND 1300 YIDD> > YMDD 5 28 F ND 3.1 YIDD> > YMDD 6 35 M YMDD only 7 40 M ND 240 YIDD< < YMDD 8 36 M ND 180 YIDD< < YMDD 9 41 M ND 330 YIDD< < YMDD M YIDD< < YMDD F ND 27 YIDD=YMDD F ND 13 YIDD=YMDD F YIDD< < YMDD M ND 1900 YIDD< < YMDD F YMDD only M ND 1200 YIDD< < YMDD M ND 21 YIDD=YMDD M YIDD< < YMDD M YIDD< < YMDD ND: Not detectable PCR. nested PCR, 8, 12 3 PCR 5 subcloning PCR. Nested PCR HBV YMDD motif Chayama 12, HBV DNA YMDD motif HBV DNA PCR. PCR (primer) PCR YMDD (SspI for YIDD, Alw44I for YVDD ). PCR (Restriction Fragment Length Polymorphism, RFLP) HBV. 1) HBV DNA Serum 50 L buffer 350 L proteinase K(20 mg/ml) 4 L 37 incubation. DNA extraction buffer 50 mm T ris- HCl (ph 7.5), 5 mm EDT A (ph 8.0), 0.25% SDS, 150 mm NaCl. Phenol/Chloroform/Isoamyl alcohol extraction 2 chloroform extraction. 1/10 volume 3M Sodium acetate

4 volume absolute ethanol , 15,000 rpm 15 1ml 70% ethanol pellet 4, 13,000 rpm 10. Pellet 30 ul ) Nested Polymerase Chain Reaction YMDD (Ssp for YIDD, Alw 44I for YVDD ). YIDD viral DNA 10 ul (template) BF108 BR (denaturation) 35 (94 ;1, 58 ;1, 72 ;1.5 ) 72 7 (extension). PCR 1 ul primer YNSsp1, BR109 PCR. 10 ul PCR SspI 2% agarose gel. YVDD PCR BF107, T MPpu101, PCR T MApaL1, BF111 PCR YIDD. PCR Alw44I 2% agarose gel. PCR HBV DNA subcloning YMDD B. 3) Nested PCR subcloning HBV Nested PCR patient 2, 8, 12 HBV DNA PCR (automatic sequencing). PCR HBV DNA pgem- T easy vector (Promega, Madison, U.S.A.) ligation E scherichia coli XL1 blue transformation plasmid. 5 clone automatic sequencer (Amersham pharmacia Biotech, Uppsala, Sweden). 1. Nested PCR YMDD motif Nested PCR YMDD motif, 19 YMDD (89.5%) YMDD. 17 YIDD YVDD PCR band, 5, 3, 9 (T able 1). 12 5, 2, 4, 8, 12 ( ), 2 nested PCR, 5, 5 4 2, 1 4 YIDD. 2 3, 2 (T able 2).

5 4. HBV 177 T able 2. Longitudinal Data of HBV DNA and Emergence of YIDD Mutant in Patients Who Showed Predominance of YIDD Mutant at 12 Weeks after Lamivudine T herapy Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Baseline 2 weeks 4 weeks 8 weeks 12 weeks 14weeks DNA Mutant Wild Wild W> > Mi W> > Mi W< < Mi W> > Mi DNA Mutant Wild W> > Mi W> > Mi W=Mi W< < Mi Wild DNA Mutant Wild W> > Mi W> > Mi W> > Mi W< < Mi Wild DNA Mutant Wild W> > Mi W> > Mi W> > Mi W< < Mi Wild DNA Mutant Wild W> > Mi W> > Mi W=Mi W< < Mi W> > Mi DNA: HBV DNA level (pg/ml) Mutant: YIDD type mutant (Wild: w ild only, W> > Mi: mutant predominant, W=Mi: similar proportion of wild and mutant type, W< < Mi: mutant predominant) Fig ure 1. Longitudinal data of nested PCR after restriction enzyme application in the patient Nested PCR subcloning HBV Nested PCR patient 2, 8, 12 HBV DNA PCR clone 5 (automatic sequencing). nested PCR (Figure 1), 5 clone (T able 3). 8 YIDD, 5 clone 3, YIDD YIDD, 5 clone YMDD 1, YIDD 4. YMDD motif nested PCR. 3. ALT HBV DNA Mutant Patient 1 ALT 2. HBV DNA 2. YIDD 4

6 178 The Korean Journal of Hepatology : Vol. 5. No T able 3. Comparison between Nested PCR Results and Automatic Sequencing Result of HBV DNA Including YMDD Motif Sampling Nested PCR Subcloning and automatic sequencing time result of HBV DNA(5 clones for each sample) Baseline YMDD wild only clone A T TAT /ATG/ GAT /GAT A- 3 YMDD clone A T TAT /ATG/ GAT /GAT A- 3 YMDD clone A T TAT /ATG/ GAT /GAT A- 3 YMDD clone A T TAT /ATG/ GAT /GAT A- 3 YMDD clone A T TAT /ATG/ GAT /GAT A- 3 YMDD 8 weeks Wild type = Mutant clone B C TAT/ ATT /GAT/ AAT G- 3 YIDD clone B T TAT /ATT/ GAT /AAT A- 3 YIDD clone B T TAT /ATG/ GAT /AAT A- 3 YMDD clone B T TAT /ATG/ GAT /AAT A- 3 YMDD clone B T TAT /ATG/ GAT /AAT A- 3 YMDD 12 weeks Wild type << Mutant clone C T TAT/ ATT /GAT/ GAT G- 3 YIDD clone C T T AT/ATT /GAT/GAT G- 3 YIDD clone C T T AT/ATT /GAT/GAT G- 3 YIDD clone C T T AT/ATT /GAT/GAT G- 3 YIDD clone C T T AT/ATG/GAT/GAT G- 3 YMDD (Figure 3). Fig ure 2. Longitudinal data of serum ALT and HBV DNA and nested PCR results after restriction enzyme application in patient YIDD. 2 (Figure 2). Patient 2,3,4,5 ALT HBV DNA 2. Mutant 2 12 YIDD B 7-10% B.13 B,.. B % HBeAg,14, 15 B 25-30% B.19, 20 B

7 Yong Han Paik, et al. Emergence of YMDD Motif Mutant Hepatitis B Virus during Short- term Lamivudine Therapy 179 Fig ure 3. Longitudinal data of serum ALT and HBV DNA and nested PCR results after restriction enzyme application in patient 2,3,4,5. DNA.1, 21, 22 Lai mg 143 B 16% HBeAg 98% HBV DNA, 72% ALT HBV, B 100 mg % HBV DNA 87.8% ALT. HBV DNA ALT B YMDD motif( th) YIDD YVDD Ling T ipple.5, 6 HBV hepadnavirus HIV (viral RNAdependent DNA polymerase) YMDD (tyrosine, methionine, aspartate, aspartate) motif nucleotide.25 HBV RNA- dependent DNA polymerase YMDD motif 552 methionine isoleucine YIDD valine YVDD HIV in vitro.10, 11, 26 6 B 14-29% YIDD YVDD.8, 9, 12, 27 6 HBV YMDD motif. YMDD PCR DNA

8 ,. Nested PCR Chayama HBV DNA PCR YMDD primer PCR (Restriction Fragment Length Polymorphism, RFLP) HBV 1:100 1:1, nested PCR, (89.5%) 12 YIDD , , ,,, 2., 19 nested PCR.. HBV YMDD motif., HIV 1 28 HBV. Chayama YIDD HIV HIV Guanine Adenine YIDD YVDD 28, 29 HBV YIDD,. Chayama nested PCR PCR DNA (direct DNA sequencing) YMDD motif.12 3 HBV 5 colony DNA nested PCR nested PCR. nested PCR YMDD motif. HBV DNA, ALT YIDD YIDD 2 3, 2.

9 4. HBV 181,.11, 30 HBV DNA HBV DNA ( pg/ml). ALT 4 12 ALT. HBV DNA ALT HBV.... : Nucleoside analogue B B. 6 1 YMDD motif(a.a ) 550 methionine isoleucine (YIDD mutant) valine (YVDD mutant). HBV YMDD motif. Chayama nested PCR B HBV YMDD motif. : Glaxo- welcome (Zeffix) B mg nested PCR HBV. PCR primer (Ssp for YIDD mutant, ApaL for YVDD mutant) (restriction fragment length polymorphism, RFLP). Nested PCR PCR subcloning. : 1) 12 nested PCR 89.5% 17 (YIDD mutant). 2) 1) 5 2 nested PCR 2 4, 4 1, ) 1 HBV nested PCR. 4) ALT ALT. : HBV

10 182 The Korean Journal of Hepatology : Vol. 5. No HBV. 1. Doong SL, T sai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of hepatitis B virus in vitro by 2',3' - dideoxy- 3' - thiacytidine and related analogues. Proc Natl Acad Sci USA 1991;19: Chang CN, Doong SL, Zhou JH, Cheng YC. Biochemical pharmacology of (+)- and (- )- 2',3' - dideoxy- 3' thiacytidine as a anti- hepatitis B virus agents. J Biol Chem 1992;31: Mansour T S, Jin H, Wang W, et al. Anti- human immunodeficiency virus and anti- hepatitis B virus activities and toxicities of the enantiomers of 2' - deoxy- oxa- 4' - thiocytidine and their 5- fluoro analogues in vitro. J Med Chem 1995;1: Xie H, Voronkov M, Liotta DC, et al. Phosphatidyl- 2',3' - dideox y- 3' thiacytidine: synthesis and activity in hepatitis B and HIV- 1 infected cells. Antiviral Res 1995;28: Ling R, Mutimer D, Ahmed M, et al. Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recepients with lamivudine. Hepatology 1996;3: T ipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, T yrrell DL. Mutation in HBV RNA- dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996;3: Bartholomew MM, Jansen RW, Jeffers LJ, et al. Hepatitis- B- virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation. Lancet 1997; 9044: Honkoop P, Niesters HG, de Man RA, Osterhaus AD, Schalm SW. Lamivudine resistance in immunocompetent chronic hepatitis B. J Hepatol 1997;26: Lai Cl, Liaw YF, Leung NWY, et al. Genotypic resistance to lamivudine in a prospective, placebo- controlled multicenter study in Asia of lamivudine therapy for chronic hepatitis B infection: incidence, kinetics of emergence, and correlation with disease parameters. Hepatology 1997;26: 259A. 10. Ladner SK, Miller T J, King RW. T he M539V polymerase variant of human hepatitis B virus demonstrates resistance to 2' - deoxy- 3' - thiacytidine and a reduced ability to synthesize viral DNA. Antimicrob Agents Chemother 1998:42(8): Allen MI, Deslauriers M, Andrews W, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Hepatology 1998;27: Chayama K, Suzuki Y, Kobayashi M, et al. Emergence and takeover of YMDD motif mutant hepatitis B virus during long- term lamivudine therapy and retakeover by wild type after cessation of therapy. Hepatology 1998;27: ,,. B. 1992;14(1): Hoofnagel JH, Peters M, Mullen KD, et al. Randomized controlled trial of recombinant human alpha- interferon in patients with chronic hepatitis B. Gasroenterology 1988;95: Wong DK, Cheung AM, O' Rourke K, Naylor CD, Detsky AS, Heathcote J. Effects of alpha- interferon treatment in patients w ith hepatitis B e antigen positive chronic hepatitis B: A meta- analysis. Ann Intern Med

11 Yong Han Paik, et al. Emergence of YMDD Motif Mutant Hepatitis B Virus during Short- term Lamivudine Therapy ;119: ,,. B interferon alpha. 1989;21: ,,. B. 1990; 22:310-3l6. 18.,,. B. 1991;41: Zoulim F, T repo C. Nucleoside analogs in the treatment of chronic viral hepatitis. Efficiency and complications. J Hepatol 1994; 21: Schalm SW, de Man RA, Heijtink RA, Niesters HGM. New nucleoside analogues for chronic hepatitis B. J Hepatol 1995; 22(suppl 1): T yrrell DLJ, Mitchell MC, de Man RA, et al. Phase II trial of lamivudine for chronic hepatitis B [Abstract]. Hepatology 1993;18: 112A. 22. Dienstag JL, Perillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin M. A preliminary trial of lamivudine for chronic viral hepatitis 1995;333: B infection. N Engl J Med 23. Lai CL, Chein RN, Leung WY, et al. A one year trial of lamivudine for chronic hepatitisb. N Engl J Med 1998;339(2): ,,. B lamivudine. 1999;5(1):S Poch O, Sauvaget I, Delarue m, T ordo N. Identification of four conserved motifs among the RNA- dependent polymerase dependent polymerase encoding elements. EMBO J 1989;8: T isdale M, Kemp SD, Parry NR, Larder BS. Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3' - thiacytidine inhibitors due to mutation in the YMDD region of reverse transcriptase. Proc Natl Acad Sci U S A 1993;90: T assopoulos NC, Volpes R, Pastore G, et al. Efficacy of lamivudine in patients with hepatitis B e antigen- negative/hepatitis B virus DNA- positive (precore mutant) chronic hepatitis B. Hepatology 1999;29: Schuurman R, Nijhuis M, Leeuw en RV, et al. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug- resistant virus populations in persons treated with lamivudine(3t C). JID 1995;171: Ji J, Loeb LA. Fidelity of HIV- 1 reverse transcriptase copying a hypervariable region of the HIV- 1 env gene. Virology 1994;199: Melegari M, Scaglioni PP, Wands JR. Hepatitis B virus mutants associated with 3T C and famciclovir administration are replication defective. Hepatology 1998;27:

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歯5-강성호.PDF 7 200 ; 5-33 B Lamivudine B YDD motif, WHO Abstract Lamivudine-Resistance in Patients with Chronic Hepatitis B and/or Cirrhosis and Detection of utations in YDD otif of Hepatitis B Virus Genome Sung Ho