5 3 1999 ; 173 183 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;5:173 183)
174 The Korean Journal of Hepatology : Vol. 5. No. 3. 1999 Key Words : Chronic hepatitis B, Lamivudine, HBV DNA polymerase, YMDD mutant :, 134, 1999. (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 ( 549-552th).5-9 550 methionine isoleucine YIDD valine YVDD, YMDD.10, 11 6 HBV YMDD motif. B HBV YMDD motif. 1. 1998 1 1999 2 28 Glaxo- w elcome (Zeffix ) 3 100 mg 12 18, 10 38(24-60). 1 HBeAg HBV DNA anti- HCV. 12 16 (57%) ALT 22 (79%) HBV DNA. 4 HBV DNA 2 HBV DNA. HBV DNA 22 13 2 HBV DNA. 12 HBV DNA 6 2 13 19. 13, 6, 39(26-60). alanine aminotransferase (ALT ) 123.8 44.9(47-190) IU/L HBV DNA 3305.3 5325.4(24-17000) pg/ml (T able 1). 2. 19, 2, 4, 8, 12, 2. 19 12 nested PCR. nested PCR 12 5 2, 4, 8, 12 2 nested
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 168 36 130 ND 7.6 YIDD> > YMDD 2 58 M 184 32 400 ND 190 YIDD> > YMDD 3 42 F 47 26 24 ND 26 YIDD> > YMDD 4 35 M 122 50 82 ND 1300 YIDD> > YMDD 5 28 F 64 13 81 ND 3.1 YIDD> > YMDD 6 35 M 144 81 540 21 3500 YMDD only 7 40 M 166 49 2200 ND 240 YIDD< < YMDD 8 36 M 122 92 370 ND 180 YIDD< < YMDD 9 41 M 58 47 90 ND 330 YIDD< < YMDD 10 31 M 132 49 2900 4.6 8600 YIDD< < YMDD 11 35 F 180 30 83 ND 27 YIDD=YMDD 12 27 F 89 33 4000 ND 13 YIDD=YMDD 13 39 F 148 54 2000 2.9 1200 YIDD< < YMDD 14 28 M 88 79 1600 ND 1900 YIDD< < YMDD 15 60 F 106 90 17000 32 17000 YMDD only 16 56 M 135 131 17000 ND 1200 YIDD< < YMDD 17 42 M 190 23 1900 ND 21 YIDD=YMDD 18 51 M 67 41 2800 3.9 470 YIDD< < YMDD 19 36 M 142 110 9600 7.8 7900 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
176 5 3 1999 2-3 volume absolute ethanol - 70 5. 4, 15,000 rpm 15 1ml 70% ethanol pellet 4, 13,000 rpm 10. Pellet 30 ul - 20. 2) Nested Polymerase Chain Reaction YMDD (Ssp for YIDD, Alw 44I for YVDD ). YIDD viral DNA 10 ul (template) BF108 BR112 94 5 (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 12 19 17 (89.5%) YMDD. 17 YIDD YVDD. 12 17 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).
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 130 5.8 - - - 7.6 Mutant Wild Wild W> > Mi W> > Mi W< < Mi W> > Mi DNA 400 - - - - 190 Mutant Wild W> > Mi W> > Mi W=Mi W< < Mi Wild DNA 24 - - - - 26 Mutant Wild W> > Mi W> > Mi W> > Mi W< < Mi Wild DNA 82 - - - - 1300 Mutant Wild W> > Mi W> > Mi W> > Mi W< < Mi Wild DNA 81 - - - - 3.1 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 2. 2. 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 2. 12 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
178 The Korean Journal of Hepatology : Vol. 5. No. 3. 1999 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- 1 5 - - - - - - T - - - - TAT /ATG/ GAT /GAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD clone A- 2 5 - - - - - - T - - - - TAT /ATG/ GAT /GAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD clone A- 3 5 - - - - - - T - - - - TAT /ATG/ GAT /GAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD clone A- 4 5 - - - - - - T - - - - TAT /ATG/ GAT /GAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD clone A- 5 5 - - - - - - T - - - - TAT /ATG/ GAT /GAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD 8 weeks Wild type = Mutant clone B- 1 5 - - - - - - C- - - - TAT/ ATT /GAT/ AAT- - - - - - - - - - - - - - - - - - - - - - - G- 3 YIDD clone B- 2 5 - - - - - - T - - - - TAT /ATT/ GAT /AAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YIDD clone B- 3 5 - - - - - - T - - - - TAT /ATG/ GAT /AAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD clone B- 4 5 - - - - - - T - - - - TAT /ATG/ GAT /AAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD clone B- 5 5 - - - - - - T - - - - TAT /ATG/ GAT /AAT- - - - - - - - - - - - - - - - - - - - - - - A- 3 YMDD 12 weeks Wild type << Mutant clone C- 1 5 - - - - - - T- - - - TAT/ ATT /GAT/ GAT - - - - - - - - - - - - - - - - - - - - - - - G- 3 YIDD clone C- 2 5 - - - - - - T- - - - T AT/ATT /GAT/GAT - - - - - - - - - - - - - - - - - - - - - - - G- 3 YIDD clone C- 3 5 - - - - - - T- - - - T AT/ATT /GAT/GAT - - - - - - - - - - - - - - - - - - - - - - - G- 3 YIDD clone C- 4 5 - - - - - - T- - - - T AT/ATT /GAT/GAT - - - - - - - - - - - - - - - - - - - - - - - G- 3 YIDD clone C- 5 5 - - - - - - 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 1. 12 YIDD. 2 (Figure 2). Patient 2,3,4,5 ALT HBV DNA 2. Mutant 2 12 YIDD. 2 2 5 B 7-10% B.13 B,.. B. 30-40% HBeAg,14, 15 B 25-30%.16-18 B.19, 20 B
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 23 1 100mg 143 B 16% HBeAg 98% HBV DNA, 72% ALT HBV, 24 45 B 100 mg 1 79.5% HBV DNA 87.8% ALT. HBV DNA ALT B YMDD motif( 551-554th) 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
180 5 3 1999,. Nested PCR Chayama HBV DNA PCR YMDD primer PCR (Restriction Fragment Length Polymorphism, RFLP) HBV 1:100 1:1,000.12 nested PCR, 19 17 (89.5%) 12 YIDD. 17 12 5 2, 4 2 1 4. 5-9, 27 6. 2,,, 2., 19 nested PCR.. HBV YMDD motif., HIV 1 28 HBV. Chayama 12 5 8. 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 2 4 12 YIDD 2 3, 2.
4. HBV 181,.11, 30 HBV DNA 2 5 4 HBV DNA 1 4 2 5 (3.1-1300 pg/ml). ALT 4 12 ALT. HBV DNA ALT HBV.... : Nucleoside analogue B B. 6 1 YMDD motif(a.a 549-552) 550 methionine isoleucine (YIDD mutant) valine (YVDD mutant). HBV YMDD motif. Chayama nested PCR B HBV YMDD motif. : Glaxo- welcome (Zeffix) B 19. 100mg 12 2. 4 12. 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, 2 5. 3) 1 HBV nested PCR. 4) ALT ALT. : HBV
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