Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments

doi:10.1016/j.jhep.2004.01.006

Journal of Hepatology

Volume 40, Issue 5, May 2004, Pages 837-844

https://doi.org/10.1016/j.jhep.2004.01.006 Get rights and content

Abstract

Background/Aims

Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner.

Methods

The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif.

Results

The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance.

Conclusions

The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy.

Introduction

Lamivudine [(−)-β-l-2′,3′-dideoxy thiacytidine, 3TC] has revolutionized the treatment of chronic hepatitis B and opened new options for the management of patients with decompensated cirrhosis or recurrent hepatitis B, post liver transplantation [1]. Unfortunately, long-term lamivudine therapy promotes the selection of HBV mutants with altered DNA polymerases resistant to therapy. The altered DNA polymerases frequently exhibit changes in a highly conserved tyrosine, methionine, aspartate, and aspartate (YMDD) motif, and so are named YMDD mutants. The most commonly described mutations cause the substitution of valine or isoleucine for methionine at residue 204 (revised nomenclature according to Stuyver et al.) [2], [3], [4].

Recent studies have shown that in some instances YMDD mutants may be responsible for increased liver damage in 10–25% of patients, which can be life-threatening [5], [6]. An additional concern is that individuals infected by HBV YMDD mutants pre-transplant may have an increased risk of recurrent infection post-transplant with reappearance of high level of serum HBV [7], [8]. Because the DNA polymerase mutations can be detected before viral breakthrough, advances in antiviral therapy like lamivudine have brought about an increasing need for sensitive and early detection of emerging drug-resistant mutant [9], [10], [11], [12], [13], [14].

In this study, we established a MALDI-TOF MS-based HBV genotyping assay that exploits differences in molecular weights between HBV wild type and variant oligonucleotides encoding the YMDD motifs in the viral DNA polymerase. We have compared the results of the MALDI-MS genotyping assay with DNA sequencing and RFLP assays, and measured the time course of takeover of resistant variants to determine whether they could be detected earlier with the MALDI-TOF MS-based genotyping assay than by RFLP and DNA sequencing assays.

Section snippets

Patients and sera

Sera were collected from 40 hepatitis B patients who received lamivudine (Glaxo-Wellcome, Greenford, UK) therapy for more than 3 months at the Liver Clinic of Pundang CHA General Hospital in Korea between March 1998 and November 2001 (Table 2). None of the patients was positive for either anti-hepatitis C virus antibody or anti-human immunodeficiency virus antibody. Informed consent was obtained form each patient and experimental protocol conformed to the ethical guidelines of the 1975

Genotyping strategy

The MALDI-TOF MS assay is based on mass spectrometric analysis of small DNA fragments containing sites of variation (Fig. 1). The first step requires PCR amplification using primers flanking the altered bases. The forward primer (nucleotide 712–738) was designed to introduce a FokI site (an isoschizomer of BstF5I) in the amplified product by substituting the restriction recognition sequence GGATG for T (nucleotide 730). The backward primer (nucleotides 744–771) was designed to have the second C

Discussion

HBV variants in the YMDD motif have been associated with reduced susceptibility to lamivudine [17], [18] Particularly for patients at high risk for disease progression, it is important to detect these variants early and precisely before the emergence of viral breakthrough when variant viruses represent only a minor fraction of the total viral population. Presently, the detection of HBV variants is largely performed by sequencing analysis, RFLP and hybridization-based assays [15], [19], [20].

Acknowledgements

We gratefully acknowledge the valuable assistance of Dr William Folk at the University of Missouri (Missouri, USA) for revising the manuscript.

References (24)

L.J Stuyver et al.
Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region
Hepatology
(2001)
M.M Bartholomew et al.
Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation
Lancet
(1997)
R.Y Chen et al.
Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine-resistant HBV in vitro
Hepatology
(2003)
G.V Papatheodoridis et al.
Nucleoside analogues for chronic hepatitis B: antiviral efficacy and viral resistance
Am J Gastroenterol
(2002)
M Zhang et al.
Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis
Hepatology
(2002)
T Kirishima et al.
Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment
J Hepatol
(2002)
Y.H Paik et al.
Emergence of YMDD motif mutant of hepatitis B virus during short-term lamivudine therapy in South Korea
J Hepatol
(2001)
S.D Pas et al.
The dynamics of mutations in the YMDD motif of the hepatitis B virus polymerase gene during and after lamivudine treatment as determined by reverse hybridisation
J Clin Virol
(2002)
M.F Yuen et al.
Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy
Hepatology
(2001)
P Merle et al.
Therapeutic management of hepatitis B-related cirrhosis
J Viral Hepat
(2001)

M.I Allen et al.

Identification and characterization of mutations in hepatitis B virus resistant to lamivudine

Hepatology

(1998)

J.H Han et al.

YMDD motif mutants in hepatitis B virus polymerase during lamivudine therapy

Korean J Genet

(2002)

Cited by (119)

Consolidating the potency of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in viral diagnosis: Extrapolating its applicability for COVID diagnosis?
2022, TrAC - Trends in Analytical Chemistry
Citation Excerpt :
The MALDI-TOF MS system is a high-throughput technology with multiplex capacity. It has already been used for genotyping [5,51]. The herpes viruses infecting humans include: herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV) types A and B, cytomegalovirus (CMV), human herpesvirus 6 (HHV6) types A and B, HHV7, and HHV8 (Kaposi's sarcoma-associated herpesvirus).
MALDI-TOF-MS has essentially delivered more than expected with respect to clinical pathogens. Viruses are the most versatile entities of clinical pathogens that have challenged well-established microbiological methodologies. This review evaluates the existing scenario with respect to MALDI TOF-MS analytical technique in the successful analysis of viral pathogens. The milestones achieved with respect to detection and identification of COVID-19 has been presented. The fact that only a handful of scattered applications for COVID-19 exist has been pointed out in the review. Further, the lapses in the utilization of the available state-of-the art MALDI-TOF-MS variants/benchmark sophistications for COVID-19 analysis, are highlighted. When the world is seeking for rapid solutions for early, sensitive, rapid COVID-19 diagnosis, maybe MALDI-TOF-MS, may be the actual ‘gold standard’. Reverting to the title, this review emphasizes that there is a need for extrapolating MALDI-TOF-MS for COVID-19 analysis and this calls for urgent scientific attention.
Ternary nanocube-based “off-on” blinking-type electrochemiluminescence towards enzyme-free detection of hepatitis B virus (HBV)-related DNA
2020, Sensors and Actuators, B: Chemical
Hepatitis B virus (HBV) infection remains a global public health problem, which is directly associated with a very high risk of evolving into liver cirrhosis and hepatocellular carcinoma (HCC). So far, enzyme-linked immunosorbent assay (ELISA) is still a main detection method for HBV infection in hospital, by which it enables to identify HBV infection in vitro through detection of circulating hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). However, ELISA requires antibody-dependent and enzyme-labeling. What’s worse, ELISA is silent to the type of occult HBV infection, which does not express HBsAg. Thus, it is of significance to establish a favorable alternative to detect HBV-related specific DNA rather than proteins, as well as to avoid antibody and enzyme-labeling assay for unambiguous screening of HBV infection. Herein, we have exploited a ternary nanocube (AgAuPt NCs)-based “off-on” blinking-type electrochemiluminescence (ECL) biosensor towards detection of HBV-related DNA for the first time, integrated with Pb²⁺-requiring DNAzyme recycling amplification. By this way, the detection limit down to 65 aM is achieved. The unique “off-on” blinking-type ECL biosensor not only overcomes the drawbacks of ELISA towards detection for HBV infection but also provides a potential for clinical application.
Application of MALDI-TOF Mass Spectrometry in Clinical Virology
2018, The Use of Mass Spectrometry Technology (MALDI-TOF) in Clinical Microbiology
Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) is a diagnostic tool of microbial identification and characterization based on the detection of the mass of molecules. In the majority of clinical laboratories, this technology is currently being used mainly for bacterial diagnosis, but several approaches in the field of virology have been investigated. The introduction of this technology in clinical virology will improve the diagnosis of infections produced by viruses but also the discovery of mutations and variants of these microorganisms as well as the detection of antiviral resistance. This chapter is focused on the main current applications of MALDI-TOF MS techniques in clinical virology showing the state of the art with respect to this exciting new technology.
A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification
2017, Biosensors and Bioelectronics
We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry.
Multiplex electrochemiluminescence DNA sensor for determination of hepatitis B virus and hepatitis C virus based on multicolor quantum dots and Au nanoparticles
2016, Analytica Chimica Acta
In this work, a novel multiplex electrochemiluminescence (ECL) DNA sensor has been developed for determination of hepatitis B virus (HBV) and hepatitis C virus (HCV) based on multicolor CdTe quantum dots (CdTe QDs) and Au nanoparticles (Au NPs). The electrochemically synthesized graphene nanosheets (GNs) were selected as conducting bridge to anchor CdTe QDs₅₅₁-capture DNA_HBV and CdTe QDs₆₀₇-capture DNA_HCV on the glassy carbon electrode (GCE). Then, different concentrations of target DNA_HBV and target DNA_HCV were introduced to hybrid with complementary CdTe QDs-capture DNA. Au NPs-probe DNA_HBV and Au NPs-probe DNA_HCV were modified to the above composite film via hybrid with the unreacted complementary CdTe QDs-capture DNA. Au NPs could quench the electrochemiluminescence (ECL) intensity of CdTe QDs due to the inner filter effect. Therefore, the determination of target DNA_HBV and target DNA_HCV could be achieved by monitoring the ECL DNA sensor based on Au NPs-probe DNA/target DNA/CdTe QDs-capture DNA/GNs/GCE composite film. Under the optimum conditions, the ECL intensity of CdTe QDs₅₅₁ and CdTe QDs₆₀₇ and the concentration of target DNA_HBV and target DNA_HCV have good linear relationship in the range of 0.0005–0.5 nmol L⁻¹ and 0.001–1.0 nmol L⁻¹ respectively, and the limit of detection were 0.082 pmol L⁻¹ and 0.34 pmol L⁻¹ respectively (S/N = 3). The DNA sensor showed good sensitivity, selectivity, reproducibility and acceptable stability. The proposed DNA sensor has been employed for the determination of target DNA_HBV and target DNA_HCV in human serum samples with satisfactory results.
Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay
2014, Clinical Biochemistry
Citation Excerpt :
The RFMP assay has been confirmed as a sensitive, accurate and reliable method in many fields [16–24]. Specifically, it allows sensitive and quantitative detection of mixtures of multiple genotypes without population-based cloning and DNA sequencing [19,22,25]. In this study, we introduced an advanced RFMP strategy to detect KRAS mutations.
Mutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples.
We developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues.
Overall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~ 25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61.
The RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.

View all citing articles on Scopus

View full text

Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments

Abstract

Background/Aims

Methods

Results

Conclusions

Introduction

Section snippets

Patients and sera

Genotyping strategy

Discussion

Acknowledgements

Hepatology

Lancet

Hepatology

Am J Gastroenterol

Hepatology

J Hepatol

J Hepatol

J Clin Virol

Hepatology

Therapeutic management of hepatitis B-related cirrhosis

J Viral Hepat

Identification and characterization of mutations in hepatitis B virus resistant to lamivudine

Hepatology

YMDD motif mutants in hepatitis B virus polymerase during lamivudine therapy

Korean J Genet