J Hepatol. 2019 Feb 19. pii: S0168-8278(19)30120-5. doi: 10.1016/j.jhep.2019.02.006. [Epub ahead of print]
Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients.
Park ES1, Lee AR2, Kim DH3, Lee JH4, Yoo JJ5, Ahn SH6, Sim H7, Park S8, Kang HS9, Won J10, Ha YN11, Shin GC12, Kwon SY13, Park YK14, Choi BS15, Lee YB16, Jeong N17, An Y18, Ju YS19, Yu SJ20, Chae HB21, Yu KS22, Kim YJ23, Yoon JH24, Zoulim F25, Kim KH26.
Author information
Abstract
BACKGROUND & AIMS:
Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance.
METHODS:
Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase (RT) was sequenced. Eleven HBV clones harboring a series of mutations in the RT gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. Relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis.
RESULTS:
Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from two patients. The mutations C, Y, and E were novel mutations associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8±0.6 µM, whereas the IC50 values for CYE and CYEI mutants were 14.1±1.8 and 58.1±0.9 µM, respectively. The IC90 value for wild-type HBV was 30±0.5 µM, whereas the IC90 values for CYE and CYEI mutants were 185±0.5 and 790±0.2 µM, respectively. All tenofovir-resistant mutants had similar susceptibility to a capsid assembly modulator, NVR 3-778 (IC50 < 0.4 µM), compared with wild-type (IC50 = 0.4 µM).
CONCLUSIONS:
Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high.
LAY SUMMARY:
Tenofovir is the most potent nucleoside analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers > 10-fold resistance to tenofovir up to 8 years. Herein, we firstly identified a quadruple mutation that confer 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir from two patients who experienced viral breakthrough during tenofovir treatment.
