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ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 4  |  Page : 809-815

Detection of the YMDD mutation responsible for lamivudine resistance in chronic hepatitis B virus-infected patients


1 Department of Medical Biochemistry, Menofia University, Menofia, Egypt
2 National Liver Institute, Faculty of Medicine, Menofia University, Menofia, Egypt

Date of Submission20-Jan-2014
Date of Acceptance17-Feb-2014
Date of Web Publication22-Jan-2015

Correspondence Address:
Nashwa M Muharram
Asem Hema Street, Shebin El Kom, Menofia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.149797

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  Abstract 

Objectives
The aim of this study was to detect the YMDD mutation responsible for lamivudine resistance in chronic hepatitis B virus (HBV)-infected patients.
Background
HBV infection is a major global public health problem. The aim of this work was to study the relation between the detection of YMDD mutation and lamivudine resistance in chronic HBV patients.
Participants and methods
This study included 50 chronic hepatitis B-infected individuals classified into two groups: group I included 25 chronic HBV patients responding to lamivudine (five female and 20 male). Group II included 25 chronic HBV patients resistant to lamivudine (one female and 24 male). All participants were selected from the National Liver Institute, Menofia University. All participants were subjected to full history taking, general examination, clinical examination, abdominal ultrasonography, laboratory investigations including serum alanine aminotransferase and aspartate transaminase, serum bilirubin, serum albumin, and detection of the YMDD mutation by PCR-RFLP.
Results
Chronic hepatitis B cannot be classified correctly with reliability on the basis of a single assessment. Serial testing for serum enzymes and HBV DNA are almost always helpful. On comparing results of the HBV DNA level and other laboratory investigations among the studied sensitive and resistant groups, it was found that the levels of HBV DNA in the pretreatment, the first, and the post-treatment PCR and the pretreatment and the post-treatment alanine aminotransferase were significantly higher in the resistant group when compared with the sensitive group (P<0.05). In the current study of 20 PCR-RFLP-tested cases, the wild type was detected in 13 cases, and the mutant type was detected in seven cases. The prevalence of mutations in the tested group was thus 35%. Mutation of codon 552 from methionine to valine (rtM204V) and from methionine to isoleucine (rtM204I) was detected in 42.9 and 28.6% of the patients, respectively, and mutation of codon 528 from leucine to methionine (rtL180M) was detected in 28.6% of the patients.
Conclusion
It could be concluded that antiviral therapy with lamivudine for chronic HBV-infected patients can be effective. However, some patients may experience resistance to lamivudine with the emergence of YMDD mutants, resulting in progressive worsening of liver disease. In this respect, detecting the YMDD mutation during therapy will help to guide antiviral treatment and to establish early stopping rules or add-on strategies to avoid antiviral resistance.

Keywords: Chronic hepatitis B infection,YMDD mutation, lamivudine resistance, PCR- RFLP


How to cite this article:
El-Sebaei HM, El-Shafie MK, Abdel Hamid AK, El-Ghobashi YA, Muharram NM, Badran HM. Detection of the YMDD mutation responsible for lamivudine resistance in chronic hepatitis B virus-infected patients. Menoufia Med J 2014;27:809-15

How to cite this URL:
El-Sebaei HM, El-Shafie MK, Abdel Hamid AK, El-Ghobashi YA, Muharram NM, Badran HM. Detection of the YMDD mutation responsible for lamivudine resistance in chronic hepatitis B virus-infected patients. Menoufia Med J [serial online] 2014 [cited 2020 Feb 17];27:809-15. Available from: http://www.mmj.eg.net/text.asp?2014/27/4/809/149797


  Introduction Top


Hepatitis B virus (HBV) is a blood-borne pathogen and a member of the hepadnavirus family [1]. HBV infection is a substantial public health problem, with ~400 million virus carriers worldwide [2]. Infections and cancers that are caused by HBV are important worldwide health problems with critical outcomes [3],[4]. Egypt is considered as an area of intermediate endemicity [5]. Nearly two to three million Egyptians are chronic carriers of HBV. In Egypt, HBV transmission is apparently a mixture of horizontal and perinatal transmission. However, the majority of HBV infection is acquired by the former route [6]. For persons who are chronically infected with HBV, there are two therapeutic approaches that are used to control the infection and its consequences: immunomodulatory agents and antiviral chemotherapy [7]. The nucleoside analog lamivudine (LMV) is the optimal therapeutic choice; it inhibits HBV polymerase and slows HBV replication in patients who are chronically infected with hepatitis B [8],[9]. Drug resistance remains a global public health problem [10], and resistance to LMV is emerging [7]. This phenomenon is mediated primarily by mutations in the genes of viruses that alter a drug's interaction with its corresponding target protein [10]. Typically, mutations in the YMDD motif of the polymerase gene develop after the first 6 months of treatment [11],[12]. Long-term therapy with LMV induces the emergence and the propagation of drug-resistant isolates, increasing to more than 40% after 2 years and to over 50 and 70% after 3 and 4 years, respectively [3],[4]. Several studies have reported various mutations that are induced by lamivudine therapy [12],[13]. These mutations in the YMDD motif are necessary and sufficient to confer high-level lamivudine resistance [4],[13].Through the use of sensitive, quantitative assays, the kinetics of the emergence of the YMDD-variant HBV may be evaluated more extensively and correlated with clinical aspects of the response to therapy [e.g. changes in necroinflammatory findings in liver biopsies, changes in serum alanine aminotransferase (ALT) levels, propensity for hepatitis B antigen seroconversion, etc.] [14].

Adefovir was the first acyclic nucleotide that was widely used in the treatment of lamivudine-resistant HBV infections at a dose of 10 mg/day. However, the antiviral efficacy of adefovir is rather low compared with other available antivirals and is most probably due to dose limitation, because of its nephrotoxicity. This disadvantage makes adefovir vulnerable to HBV resistance [15].

The YMDD mutation appears before increased viral load of HBV during lamivudine treatment, which makes it the most sensitive indication of the emergence of HBV resistance [3].


  Participants and methods Top


Participants

This study was carried out on 50 chronic hepatitis B-infected patients who had received lamivudine (100 mg daily) for at least 12 months: 25 chronic HBV-infected patients responding to lamivudine (five female and 20 male) and 25 chronic HBV-infected patients resistant to lamivudine (one female and 24 male). All participants were selected from the National Liver Institute, Menofia University, after taking a written medical consent. The thesis was approved by the Ethical Committee of the Faculty of Medicine, Menofia University. The chronicity was confirmed in all cases with positive results for HBsAg and anti-HBcAg-IgG and negative results for anti-HBsAg-IgM. HBV DNA levels were determined before beginning lamivudine therapy (pretreatment PCR), after 6 months (first post-treatment PCR), and after therapy (PCR 2-3 years after treatment). The participants were divided into two groups depending on sequential HBV DNA levels.

Group I (sensitive group) included 25 chronic HBV patients responding to lamivudine. It consisted of 20 male and five female individuals with a mean age of 31.6 ± 7.9 years.

Group II (resistant group) included 25 chronic HBV patients resistant to lamivudine. It consisted of 24 male and one female individuals with a mean age of 34.1 ± 8.1 years. Twenty participants of the resistant group underwent PCR-RFLP and were subdivided into the nonmutant or wild-type group (2A) and the mutant group (2B).

Group 2A (nonmutant or wild type) included 13 individuals in whom the mutation was not detected.

Group 2B (mutant) included seven individuals who developed the mutation.


  Methods Top


All participants were subjected to full history taking, general examination, clinical examination, abdominal ultrasonography, laboratory investigations including serum ALT, aspartate transaminase (AST), serum bilirubin, serum albumin determination, and detection of the YMDD mutation by PCR-RFLP.

Ten milliliters of venous blood were withdrawn from all participants and divided as follows:

(1) About 5 ml of whole blood was transferred slowly into a dry sterile centrifuge tube. The whole blood was allowed to clot at 37°C, and then centrifuged at 3000 rpm for 5 min. The serum obtained was used for ALT, AST, bilirubin, and albumin determination.

(2) The other 5 ml (plasma) was taken for the extraction of HBV DNA using the Invitek RTP DNA/RNA mini-kit. The extracted DNA was amplified by PCR primers: [RBL1: 5′-GTTCAAATGTATACCCAAAG-3′] and [FBL1: 5′-CACTGTTTGGCTTTCAGT CAT-3′] to amplify codon 552, and [RBL3: 5′-GTTCAAATGTATACCCAAAG-3′] and [FBL3: 5′-GTGGGCCTCAGTCCGTTT CTC-3′] to amplify codon 528. PCR for DNA was carried out to a total volume of 25 ml under the following conditions: 10 ml of the resuspended DNA was added to an amplification containing 2.5 ml of 5´ Taq polymerase buffer, 0.375 ml of 10 mmol/l dNTPs, 1.75 ml of 25 mmol/l MgCl 2 , 0.5 ml (2.5 U) of Taq polymerase (Promega), and 0.5 ml of each 100 pmol/l primer BL1 and BL3 (for amplification of codons 552 and 528, respectively), and 8.87 ml of H 2 O. The PCR profile was an initial 3-min denaturation at 94°C, followed by 40 cycles of amplification including denaturation for 45 s at 94°C, annealing for 55 s at 49°C, and extension for 30 s at 72°C. Strand synthesis was completed at 72°C for 6 min. As described by Allen et al. [16], RFLP analysis using restriction endonuclease digestion was carried out with 10 ml of the PCR product for 4 h after adjustment with a 10´ enzyme reaction buffer according to the manufacturer's recommendations. Reactions were carried out with 10 U of NdeI and NlaIII (Fermentas Life Sciences) at 37°C. The digested PCR products were electrophoresed on 3.0% agarose gel in 1΄ TBE buffer, and stained with 0.05% ethidium bromide.

Statistical analysis

Results were collected, tabulated, and statistically analyzed by an IBM personal computer and statistical package SPSS version 16. Two types of statistics were performed:

  1. Descriptive statistics: For example, percentage (%), mean, and SD.
  2. Analytical statistics: Using Student's t-test, the Mann-Whitney U-test, the c2 -test, Fisher's exact test, the Wilcoxon signed rank test, and the P value.



  Results Top


There were statistically significant differences between the studied lamivudine-sensitive and lamivudine-resistant groups regarding their ALT, whereas there was no significant difference regarding the pretreatment and the post-treatment AST, bilirubin, and albumin [Table 1].
Table 1: A statistical comparison between the studied groups regarding their laboratory investigations

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There were statistically significant differences between the studied lamivudine-sensitive and lamivudine-resistant groups regarding the pretreatment and the post-treatment PCR [Table 2].
Table 2: A statistical comparison among the pretreatment, the first, and the post-treatment PCR of the sensitive and the resistant group

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There were statistically significant differences between the studied groups regarding relapse, with the highest percent of relapse and addition of adefovir in the resistant group [Table 3].
Table 3: A statistical comparison between the studied sensitive and resistant groups regarding relapse, the addition of adefovir, and normalization

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The number and the percent of detected mutations in the studied mutant group are shown in [Table 4]. For codon 528, both homozygote and heterozygote mutations from leucine to methionine (rtL180M) were detected (14.3% for both). For codon 552, heterozygote mutations from methionine to valine (rtM204V) (28.6%) and from methionine to isoleucine (rtM204I) (28.6%) were detected [Figure 1]. A homozygote mutation from methionine to valine (rtM204V) was also detected [Table 4].
Figure 1: Detection of wild-type codons, of 120 bp for codon 552 and 192 bp for codon 528, visualized under ultraviolet light. In codon 552, the mutation (CATATG) creating the NdeI restriction site resulted in 100+20-bp, and the mutation (CATGTG) creating the NlaIII site resulted in 99+21-bp bands. In codon 528, the mutation (CATG) creating the NlaIII restriction site resulted in 136+56-bp bands. Lane 1: 100-bp ladder; lane 2: mutant codon 552 (no digestion with NdeI); lane 3: heterozygote variation of Val/Iso (partial digestion with NlaIII); lane 4: undigested amplified codon 552; lane 5: double population of codon 528 (Met/Leu) and heterozygote mutation of codon 528 (partial digestion with NlaIII); lane 6: undigested amplified codon 528.

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Table 4: Types of detected mutations in the studied mutant group

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There were statistically significant differences between the studied mutant-type and non-mutant-type subgroups regarding their post-treatment ALT, whereas there were no significant differences regarding their pretreatment and post-treatment AST, bilirubin, and albumin [Table 5].
Table 5: A statistical comparison among mutant and nonmutant subgroups regarding laboratory investigations

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There were statistically significant differences between the studied mutant subgroups regarding the pretreatment and the post-treatment PCR, and there were statistically significant differences between the studied nonmutant subgroups regarding the pretreatment and the post-treatment PCR [Table 6].
Table 6: A statistical comparison among the pretreatment, the first, and the post-treatment PCR of mutant and nonmutant subgroups

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  Discussion Top


HBV infection is a major global public health problem. Of approximately two billion people who have been infected worldwide, more than 350 million are chronic carriers of HBV [17]. Approximately 15-40% of infected patients will develop cirrhosis, liver failure, or hepatocellular carcinoma [18].

Egypt is considered as an area of intermediate endemicity [5]. Nearly two to three million Egyptians are chronic carriers of HBV. In Egypt, HBV transmission is apparently a mixture of horizontal and perinatal transmission. However, the majority of HBV infection is acquired by the former route [6].

Typically, mutations in the YMDD motif of the polymerase gene develop after the first 6 months of treatment [11],[12].

On comparing results of the HBV DNA level and other laboratory investigations among the studied sensitive and resistant groups, it was found that the levels of HBV DNA in the pretreatment, the first, and the post-treatment PCR and the pretreatment and the post-treatment ALT were significantly higher in the resistant group when compared with the sensitive group, whereas there was no significant statistical difference between them regarding the pretreatment and the post-treatment AST, bilirubin, and albumin.

These results are in agreement with Carman et al. [19]. This could be explained by the fact that after infection, the immune system attempts to clear the HBV by destroying infected hepatocytes. This leads to increasing circulatory blood levels of ALT [20],[21].

Puchhammer-Stockl et al. [21] found that HBV DNA levels were lower among responders than among nonresponders.

On comparing results of HBV DNA of both the sensitive and the resistant groups [Table 2], it was found that the levels of HBV DNA in the pretreatment PCR were significantly higher when compared with the first and the post-treatment PCR, and on comparing results of patients included in the wild-type and the mutant subgroups [Table 6], it was found that the levels of HBV DNA in the pretreatment PCR were significantly lower when compared with the first and the post-treatment PCR.

These results are in agreement with Magnus et al. [22] who studied HBV DNA levels before, during, and after lamivudine therapy using COBAS AMPLICOR and real-time PCR. Initially, there was a reduction of viremia in patients responding to treatment that was documented similarly by both assays. After 21 months of lamivudine therapy, the HBV DNA level had increased to 300 000 copies/ml, and it then increased gradually further to three million copies per ml. During this period, the aminotransferase levels remained elevated and liver function did not improve, confirming the emergence of resistance.

As per this result, Papatheodoridis et al. [23] explained that HBV resistance has to be expected when an increase in HBV DNA of greater than 1 log 10 during antiviral treatment is observed.

In the current study, breakthrough occurred in 25 patients included in the resistant group: 20 of them underwent PCR-RFLP to detect the presence of the YMDD mutation. The breakthrough rate was 11 (44%), 10 (40%), two (8%), and two (8%) patients in the first, the second, the third, and the fourth years, respectively.

These results are in accordance with Naglaa et al. [24] who reported that 40% of their patients suffered from relapse after initial response: 16 during the second year of therapy, three during the third year, and one patient during the fourth year of therapy.

In the current study of the 20 tested cases, the wild type was detected in 13 cases, and the mutant type was detected in seven cases. The prevalence of mutations in the tested group was thus 35%.

These results are in agreement with Masumeh et al. [25] who studied lamivudine resistance mutations (YMDD) among chronic hepatitis B patients and stated that the usual HBV mutation, which plays an important role in lamivudine resistance, is rtM204V/I; mutations at rt180 were also observed in these patients. Natural YMDD mutations occur at a rate of 10%.

Si-Yue et al. [26] found that the pattern of rtM204I alone was observed predominantly, followed by rtM204V+rtL180M (36.26 and 33.91%), respectively.

In the current study, HBV DNA levels in patients with the wild-type virus were significantly lower than in the mutants.

These results are in agreement with Liu et al. [27] and Locarnini [28] who found that the HBV DNA level increases with continuous therapy, especially after the first and the second years since the emergence of the primary mutation, and they explained this result by a phenotypic analysis: the rtM204V (codon 552) and the rtL180M (codon 528) mutations induce a 1000-fold decrease in the susceptibility to lamivudine in comparison with the wild-type (wt) HBV [Figure 2].
Figure 2: Lane 2: wild-codon 552 (no digestion with NdeI; lane 3: homozygote mutation of isoleucine of codon 552 (no digestion with NlaIII); lane 4: undigested amplified codon 552; lane 5: homozygote mutation of codon 528 (digestion with NlaIII); lane 6: undigested amplified codon 528.

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  Conclusion Top


It could be concluded that antiviral therapy with lamivudine for chronic HBV-infected patients can be effective. However, some patients may experience resistance to lamivudine with the emergence of YMDD mutants resulting in progressive worsening of the liver disease. In this respect, detecting the YMDD mutation early during therapy will help to guide the antiviral treatment and to establish early stopping rules or add-on strategies to avoid antiviral resistance.

Recommendations

Further larger scale studies are needed to evaluate lamivudine used in Egyptian patients and its association with the response rate and the incidence of YMDD mutation.

Studies are also recommended to examine the possible associations of YMDD mutation among clinical, serologic, and genetic patterns of HBV infections in Egypt.

Sequencing is recommended to detect further mutations of HBV during lamivudine and adefovir therapy.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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