Abstract
Objectives: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. Methods: One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. Results: One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. Conclusions: HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.
Keywords: Hepatitis B virus, Lamivudine, YMDD mutant, Sequence analysis
INTRODUCTION
Hepatitis B virus (HBV) infection is a major health problem leading to around one million deaths annually worldwide (Lee, 1997). A wide range of clinical manifestations has been established for chronic hepatitis B virus infection, from asymptomatic carriers to severe chronic liver disease, including those with cirrhosis and hepatocellular carcinoma (Chen, 1993; Chu and Liaw, 1997). Lamivudine, an oral nucleoside analogue, inhibits HBV replication (Lai et al., 1997; 1998) and can markedly reduce serum HBV DNA levels and normalise alanine aminotransferase (ALT) levels associated with improvement in liver necroinflammatory activity (Lai et al., 1998), but the greatest drawback with lamivudine treatment is the emergence of drug-resistant HBV mutants, the mutation of the tyrosine-methionine-aspartate-aspartate (YMDD) motif in the C domain of the HBV DNA polymerase gene (Lai et al., 1998; Dienstag et al., 1999). The serum HBV DNA mutation of 141 patients with lamivudine resistance and 60 chronic hepatitis B patients without lamivudine treatment was analyzed by sequence detection via polymerase chain reaction (PCR) in order to investigate the relationship between HBV polymerase gene 180 and 204 sites mutation and lamivudine resistance.
MATERIALS AND METHODS
Patients
Two-hundred and one subjects were inpatients and outpatients in our hospital from June 2002 to June 2003. They were 135 men and 66 women, aged 18–53 years. Among them were 141 patients with lamivudine resistance after lamivudine treatment (100 mg/d) with mean treatment period of one year and a half; and 60 chronic hepatitis B patients without lamivudine treatment. None of the patients was ever treated with interferons and other anti-viral drugs.
Design and synthesis of primers
Upstream primer: 5′ CTCCAATCACTCACCA AC 3′; downstream primer: 5′ GGGTTTAAATGT ATACCCA 3′; sequencing primer: 5′ GTAATTCCC ATCCC 3′. All the primers above were synthesized by Sangon Company, Shanghai (China).
PCR
The amplification reaction contained 1 μl each of 25 μmol/L specific primers, 1 μl 10 mmol/L dNTP mixture (dATP, dGTP, dCTP, dTTP), 4 μl 25 mmol MgCl2, 2.5 U Taq DNA polymerase (Promaga) and 5 μl 10×PCR buffer solution. The total volume was brought to 50 μl using ddH2O. The PCR amplifications were performed in a PTC-200 peltier thermal cycler (MJ Research, USA) under the following conditions: After an initial denaturation for 5 min at 94 °C, samples were subjected to 35 cycles of amplification (94 °C 45 s, 55 °C 45 s, 72 °C 1 min), followed by a final extension of 5 min at 72 °C. The purification of PCR products was performed by QIAquick PCR purification kit according to manufacture’s instructions (QIAGEN, USA).
Sequence analysis
Sequence analysis of the PCR products was performed by DYEnamic™ ET dye terminator cycle sequencing Kit (AmershamBioscience) in a MegaBACE™ 500 according to manufacture’s instructions; sequence analysis software was used to analyze the results.
RESULTS
Types of HBV polymerase gene 180 and 204 sites mutation
There were 109 lamivudine resistance patients who had HBV YMDD mutation and 141 patients with lamivudine resistance. Among them, 45 patients (45/109; 41.28%) had rtL180M/M204V mutation that played the most important role in YMDD mutation. In addition, 28 patients (28/109; 25.70%) had rtL180M/M204I mutation and 36 patients (36/109; 33.02%) had rtM204I mutation. There were 6 patients with rtL180M mutation in the other 32 lamivudine resistance patients; 60 chronic hepatitis patients without lamivudine treatment had no mutations (Table 1).
Table 1.
Types of HBV polymerase gene 180 and 204 sites mutation
| Group | Type | n |
| Patients with lamivudine treatment | rtL180M/M204V | 45 |
| rtL180M/M204I | 28 | |
| acrtM204I | 36 | |
| rtL180M | 6 | |
| No classification | 26 | |
| Patients without lamivudine treatment | No mutation | 60 |
Combinatorial mutation rate of M204V/I
All 45 patients with rtM204V mutation also had rtL180M mutation; the rate of this kind of combinatorial mutation was 100%. Among 64 patients with rtM204I mutation, only 28 patients also had rtL180M mutation; the occurrence rate of this kind of combinatorial mutation was 43.75%.
Combinatorial mutation rate of rtL180M
Among 79 patients with rtL180M mutation were 45 patients who had rtM204V mutation, 28 patients who had rtM204I mutation, the combinatorial mutation rate was 56.96% and 35.44%, respectively. In addition, there were only 6 patients who had rtL180M mutation without rtM204V mutation or rtM204I.
Relationship between HBV polymerase gene mutation and lamivudine treatment
Among 141 patients with lamivudine resistance, 115 patients had polymerase gene mutation, either at 204 site or at 108 site. The mutation rate was 81.56%. While in the other group, 60 chronic hepatitis patients without lamivudine treatment did not have any mutations.
DISCUSSION AND CONCLUSION
Chronic hepatitis B virus (HBV) can be treated with a nucleoside analogue, lamivudine (2′,3′-dideoxy-3′-thiacytidine). In the short term, substantial inhibition of HBV replication can be achieved. However, resistance to lamivudine emerges in approximately 14% of patients after 1 year, rising to 43% after 3 years. Resistance is associated with mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif (codons 203–206 of the reverse transcriptase (rt)), which is part of the catalytic site of the HBV polymerase (Allen et al., 1998; Chayama et al., 1998; Gutfreund et al., 2000; Kirishima et al., 2002; Kobayashi et al., 2001; Leung, 2002; Yeh et al., 2000). Three types of mutations are observed in the polymerase gene. The consensus rt domain numbering system described in Stuyver et al.(2001) has been used to describe the variants. There is the M204V associated with L180M (Group I), the M204I alone (Group II) or the M204I with L180M (Group III) (Pillay et al., 1998). The L180M and M204V mutations act synergistically to increase resistance to lamivudine (Allen et al., 1998).
One hundred and forty-one patients with lamivudine resistance and 60 chronic hepatitis B patients without lamivudine treatment were studied in this work in the Hangzhou area. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). Our results indicated 109 lamivudine resistance patients had HBV YMDD mutation. There were three main types of mutation: rtL180M/M204V, rtM204I and rtL180M/M204I. Among them, 45 patients had rtL180M/M204V mutation (41.3%), which played the most important role in YMDD mutation. In addition, 28 patients had rtL180M/M204I mutation (25.7%) and 36 patients had rtM204I mutation (33.0%).
Our results indicated that rtM204V mutation is always combined with rtL180M; the occurrence rate of this kind of combinatorial mutation was 100%; while rtM204I mutation could exist alone. These results accorded with those of other research groups’ (Pillay et al., 1998; Allen et al., 1998).
Our study revealed that there were 6 patients with rtL180M mutation among the 32 lamivudine resistance patients. This kind of mutation was novel, and requires further study to explore its clinical significance.
Though in this study, 60 chronic hepatitis patients without lamivudine treatment had no mutations, it had been reported that patients without lamivudine treatment could also have mutations; we considered that these contradictory findings had something to do with the fewer samples.
In conclusion, HBV mutations, which play an important role in lamivudine resistance, usually locate at polymerase gene 204 site; 180 site mutation was also observed in the patients. It is of importance to evaluate anti-virus therapy by surveillance of the two sites mutations.
References
- 1.Allen MI, Deslauriers M, Andrews CW, Tipples GA, Walters KA, Tyrell DL, Brown N, Condreay LD. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Hepatology. 1998;27(6):1670–1677. doi: 10.1002/hep.510270628. [DOI] [PubMed] [Google Scholar]
- 2.Chayama K, Suzuki Y, Kobayashi M, Tsubota A, Hashimoto M, Miyano Y, Koike H. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild-type after cessation of therapy. Hepatology. 1998;27(6):1711–1716. doi: 10.1002/hep.510270634. [DOI] [PubMed] [Google Scholar]
- 3.Chen DS. From hepatitis to hepatoma: Lessons from type B viral hepatitis. Science. 1993;262(5132):369–370. doi: 10.1126/science.8211155. [DOI] [PubMed] [Google Scholar]
- 4.Chu CM, Liaw YF. Natural history of chronic hepatitis B virus infection: An immunopathological study. J Gastroenterol Hepatol. 1997;12(9-10):S218–S222. doi: 10.1111/j.1440-1746.1997.tb00503.x. [DOI] [PubMed] [Google Scholar]
- 5.Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann HW, Goodman Z. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med. 1999;341(17):1256–1263. doi: 10.1056/NEJM199910213411702. [DOI] [PubMed] [Google Scholar]
- 6.Gutfreund KS, Williams M, George R, Bain VG, Ma MM, Yoshida EM. Genotypic succession of mutations of the hepatitis B virus polymerase associated with lamivudine resistance. J Hepatol. 2000;33(3):469–475. doi: 10.1016/s0168-8278(00)80284-6. [DOI] [PubMed] [Google Scholar]
- 7.Kirishima T, Okanoue T, Daimon Y, Itoh Y, Nakamura H, Morita A. Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment. J Hepatol. 2002;37(2):259–265. doi: 10.1016/s0168-8278(02)00145-9. [DOI] [PubMed] [Google Scholar]
- 8.Kobayashi S, Ide T, Sata M. Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers. J Hepatol. 2001;34(4):584–586. doi: 10.1016/s0168-8278(00)00023-4. [DOI] [PubMed] [Google Scholar]
- 9.Lai CL, Ching CK, Tung AKM, Li E, Young J, Hill A. Lamivudine is effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface antigen carriers: A placebo-controlled trial. Hepatology. 1997;25(1):241–244. doi: 10.1002/hep.510250144. [DOI] [PubMed] [Google Scholar]
- 10.Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI. A one-year trial of lamivudine for chronic hepatitis B. N Engl J Med. 1998;339(2):61–68. doi: 10.1056/NEJM199807093390201. [DOI] [PubMed] [Google Scholar]
- 11.Lee WM. Hepatitis B virus infection. N Engl J Med. 1997;337(24):1733–1745. doi: 10.1056/NEJM199712113372406. [DOI] [PubMed] [Google Scholar]
- 12.Leung N. Treatment of chronic hepatitis B: Case selection and duration of therapy. J Gastroenterol Hepatol. 2002;17(4):409–414. doi: 10.1046/j.1440-1746.2002.02767.x. [DOI] [PubMed] [Google Scholar]
- 13.Pillay D, Bartholomeusz A, Cane PA, Mutimer D, Schinazi RF, Locarnini SA. Mutations in the hepatitis B virus DNA polymerase associated with antiviral resistance. Int Antiviral News. 1998;6:167–169. [Google Scholar]
- 14.Stuyver LJ, Locarnini SA, Lok A, Richmann DD, Carman WF, Dienstag JL. Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region. Hepatology. 2001;33(3):751–757. doi: 10.1053/jhep.2001.22166. [DOI] [PubMed] [Google Scholar]
- 15.Yeh CT, Chien RN, Chu CM, Liaw YF. Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy. Hepatology. 2000;31(6):1318–1326. doi: 10.1053/jhep.2000.7296. [DOI] [PubMed] [Google Scholar]