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Keywords:

  • core promoter mutations;
  • hepatitis B virus;
  • lamivudine resistance;
  • viral breakthrough;
  • YMDD mutation

Abstract

Background and Aim:  Virologic breakthrough (VBTH) during long-term lamivudine therapy is believed to be due to the emergence of rtYM204I/VDD mutants. We observed VBTH in 17 of 67 patients receiving long-term lamivudine therapy. The YMDD mutant at the onset of VBTH and/or subsequently was seen in eight (47%) of 17 such patients. We investigated other potential loci in the viral genome contributing to VBTH.

Methods:  Chronic hepatitis B (CHB) patients (n = 17) on long-term (≥12 months) lamivudine therapy who had at least one episode of VBTH were selected (group I). Age, sex, serology and baseline viral load matched patients on long-term lamivudine without VBTH (n = 12) served as controls (group II). Hepatitis B virus (HBV) DNA sequences were analyzed for pre-S, surface, polymerase, core promoter, precore and core regions and were compared with sequences of respective genotypes.

Results:  Group I patients with VBTH (n = 17) either had rtYM204I/VDD mutations (n = 8, group Ia YMDD) or no rtYM204I/VDD mutations (n = 9, group Ib non-YMDD). Group Ia patients had median baseline HBV DNA of 2794 pg/mL (range 3–9166 pg/mL) and a mean alanine aminotransferase (ALT) level of 86 ± 33 IU/L. In group Ib, the median baseline viral DNA was 916 pg/mL (range 8.3–5787 pg/mL) and the mean ALT was 61 ± 38 IU/L. The first VBTH in group Ia and Ib patients was noted at 21 ± 9 months and 15.2 ± 5.9 months, respectively, with a rise in HBV DNA levels from undetectable limits to 952 pg/mL (range 4.3–4875 pg/mL) and 571 pg/mL (range 1.2–1970 pg/mL), respectively. Core promoter mutations were seen in five of eight (62.5%) and in six of nine (66.6%); classic double mutations (A1762T/G1764A) of core promoter region were detected in two and three patients and novel double mutations of core promoter (G1764T/C1766G) in one patient each of group Ia and Ib patients, respectively. Compared to 11 (68%) of 17 group I patients, only three (25%) of 12 patients in group II had core promoter mutations (P < 0.05). No patient in group II had double mutations of the core promoter region. No significant difference in viral mutations was seen in any other region of the viral genome between group I and group II patients. In group I, none of the 15 patients (two died of hepatocellular carcinoma), but five (42%) group II patients achieved hepatitis B e antigen (HBeAg) seroconversion and sustained response by month 24 (P < 0.05).

Conclusion:  Both core promoter and YMDD motif mutation(s) are associated with VBTH in patients on long-term lamivudine therapy. Whether or not these promoter mutations in the absence of YMDD mutations confer drug resistance needs to be studied in an in vitro cell culture system, as they could create novel and stronger binding sites for hepatocyte nuclear factors.