Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients

Eun Sook Park, Ah Ram Lee, Doo Hyun Kim, Jeong Hoon Lee, Jeong Ju Yoo, Sung Hyun Ahn, Heewoo Sim, Soree Park, Hong Seok Kang, Juhee Won, Yea Na Ha, Gu Choul Shin, So Young Kwon, Yong Kwang Park, Byeong Sun Choi, Yun Bin Lee, Nakcheol Jeong, Yohan An, Young Seok Ju, Su Jong YuHee Bok Chae, Kyung Sang Yu, Yoon Jun Kim, Jung Hwan Yoon, Fabien Zoulim, Kyun Hwan Kim

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The 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 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC 50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC 50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC 90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC 50 <0.4 µM vs. IC 50 = 0.4 µM, respectively). Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC 50 and 26.3-fold in IC 90 . These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary: Tenofovir is the most potent nucleotide 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 identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC 50 ) and 26.3-fold (IC 90 ) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.

Original languageEnglish
Pages (from-to)1093-1102
Number of pages10
JournalJournal of Hepatology
Volume70
Issue number6
DOIs
Publication statusPublished - 2019 Jun 1

Fingerprint

Tenofovir
Chronic Hepatitis B
Hepatitis B virus
Mutation
RNA-Directed DNA Polymerase
Clone Cells
High-Throughput Nucleotide Sequencing
Capsid
Virus Diseases
Southern Blotting
Site-Directed Mutagenesis
Drug Resistance
Pharmaceutical Preparations

Keywords

  • Antivirals
  • Capsid assembly modulator
  • CYEI
  • Entecavir
  • HBV
  • Nucleotide analogue

ASJC Scopus subject areas

  • Hepatology

Cite this

Park, E. S., Lee, A. R., Kim, D. H., Lee, J. H., Yoo, J. J., Ahn, S. H., ... Kim, K. H. (2019). Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients. Journal of Hepatology, 70(6), 1093-1102. https://doi.org/10.1016/j.jhep.2019.02.006

Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients. / Park, Eun Sook; Lee, Ah Ram; Kim, Doo Hyun; Lee, Jeong Hoon; Yoo, Jeong Ju; Ahn, Sung Hyun; Sim, Heewoo; Park, Soree; Kang, Hong Seok; Won, Juhee; Ha, Yea Na; Shin, Gu Choul; Kwon, So Young; Park, Yong Kwang; Choi, Byeong Sun; Lee, Yun Bin; Jeong, Nakcheol; An, Yohan; Ju, Young Seok; Yu, Su Jong; Chae, Hee Bok; Yu, Kyung Sang; Kim, Yoon Jun; Yoon, Jung Hwan; Zoulim, Fabien; Kim, Kyun Hwan.

In: Journal of Hepatology, Vol. 70, No. 6, 01.06.2019, p. 1093-1102.

Research output: Contribution to journalArticle

Park, ES, Lee, AR, Kim, DH, Lee, JH, Yoo, JJ, Ahn, SH, Sim, H, Park, S, Kang, HS, Won, J, Ha, YN, Shin, GC, Kwon, SY, Park, YK, Choi, BS, Lee, YB, Jeong, N, An, Y, Ju, YS, Yu, SJ, Chae, HB, Yu, KS, Kim, YJ, Yoon, JH, Zoulim, F & Kim, KH 2019, 'Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients', Journal of Hepatology, vol. 70, no. 6, pp. 1093-1102. https://doi.org/10.1016/j.jhep.2019.02.006
Park, Eun Sook ; Lee, Ah Ram ; Kim, Doo Hyun ; Lee, Jeong Hoon ; Yoo, Jeong Ju ; Ahn, Sung Hyun ; Sim, Heewoo ; Park, Soree ; Kang, Hong Seok ; Won, Juhee ; Ha, Yea Na ; Shin, Gu Choul ; Kwon, So Young ; Park, Yong Kwang ; Choi, Byeong Sun ; Lee, Yun Bin ; Jeong, Nakcheol ; An, Yohan ; Ju, Young Seok ; Yu, Su Jong ; Chae, Hee Bok ; Yu, Kyung Sang ; Kim, Yoon Jun ; Yoon, Jung Hwan ; Zoulim, Fabien ; Kim, Kyun Hwan. / Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients. In: Journal of Hepatology. 2019 ; Vol. 70, No. 6. pp. 1093-1102.
@article{2b06bb82090a4e1aaad5d4c044a95cbf,
title = "Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients",
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 was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The 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 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC 50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC 50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC 90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC 50 <0.4 µM vs. IC 50 = 0.4 µM, respectively). Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC 50 and 26.3-fold in IC 90 . These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary: Tenofovir is the most potent nucleotide 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 identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC 50 ) and 26.3-fold (IC 90 ) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.",
keywords = "Antivirals, Capsid assembly modulator, CYEI, Entecavir, HBV, Nucleotide analogue",
author = "Park, {Eun Sook} and Lee, {Ah Ram} and Kim, {Doo Hyun} and Lee, {Jeong Hoon} and Yoo, {Jeong Ju} and Ahn, {Sung Hyun} and Heewoo Sim and Soree Park and Kang, {Hong Seok} and Juhee Won and Ha, {Yea Na} and Shin, {Gu Choul} and Kwon, {So Young} and Park, {Yong Kwang} and Choi, {Byeong Sun} and Lee, {Yun Bin} and Nakcheol Jeong and Yohan An and Ju, {Young Seok} and Yu, {Su Jong} and Chae, {Hee Bok} and Yu, {Kyung Sang} and Kim, {Yoon Jun} and Yoon, {Jung Hwan} and Fabien Zoulim and Kim, {Kyun Hwan}",
year = "2019",
month = "6",
day = "1",
doi = "10.1016/j.jhep.2019.02.006",
language = "English",
volume = "70",
pages = "1093--1102",
journal = "Journal of Hepatology",
issn = "0168-8278",
publisher = "Elsevier",
number = "6",

}

TY - JOUR

T1 - Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients

AU - Park, Eun Sook

AU - Lee, Ah Ram

AU - Kim, Doo Hyun

AU - Lee, Jeong Hoon

AU - Yoo, Jeong Ju

AU - Ahn, Sung Hyun

AU - Sim, Heewoo

AU - Park, Soree

AU - Kang, Hong Seok

AU - Won, Juhee

AU - Ha, Yea Na

AU - Shin, Gu Choul

AU - Kwon, So Young

AU - Park, Yong Kwang

AU - Choi, Byeong Sun

AU - Lee, Yun Bin

AU - Jeong, Nakcheol

AU - An, Yohan

AU - Ju, Young Seok

AU - Yu, Su Jong

AU - Chae, Hee Bok

AU - Yu, Kyung Sang

AU - Kim, Yoon Jun

AU - Yoon, Jung Hwan

AU - Zoulim, Fabien

AU - Kim, Kyun Hwan

PY - 2019/6/1

Y1 - 2019/6/1

N2 - 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 was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The 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 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC 50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC 50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC 90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC 50 <0.4 µM vs. IC 50 = 0.4 µM, respectively). Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC 50 and 26.3-fold in IC 90 . These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary: Tenofovir is the most potent nucleotide 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 identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC 50 ) and 26.3-fold (IC 90 ) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.

AB - 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 was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The 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 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC 50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC 50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC 90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC 50 <0.4 µM vs. IC 50 = 0.4 µM, respectively). Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC 50 and 26.3-fold in IC 90 . These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary: Tenofovir is the most potent nucleotide 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 identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC 50 ) and 26.3-fold (IC 90 ) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.

KW - Antivirals

KW - Capsid assembly modulator

KW - CYEI

KW - Entecavir

KW - HBV

KW - Nucleotide analogue

UR - http://www.scopus.com/inward/record.url?scp=85063609690&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85063609690&partnerID=8YFLogxK

U2 - 10.1016/j.jhep.2019.02.006

DO - 10.1016/j.jhep.2019.02.006

M3 - Article

C2 - 30794889

AN - SCOPUS:85063609690

VL - 70

SP - 1093

EP - 1102

JO - Journal of Hepatology

JF - Journal of Hepatology

SN - 0168-8278

IS - 6

ER -