Correlation of the occurrence of YMDD mutations with HBV genotypes,HBV-DNA levels,and HBeAg status in Chinese patients with chronic hepatitis B during lamivudine treatment

Hangzhou,China

Introduction

Hepatitis B virus (HBV) infection is the main cause of hepatic cirrhosis and hepatocellular carcinoma in China.Lamivudine as a potent inhibitor of HBV reverse transcriptase activity is able to decrease serum HBV-DNA levels and improve the rate of e-seroconversion in patients with chronic hepatitis B.[1-3]Presently it has become the major therapeutic agent for the treatment of patients with chronic hepatitis B in China.Nevertheless,the occurrence of YMDD mutations may result in lamivudine resistance,causing elevated serum HBV-DNA level and ALT level.[4,5]Acute exacerbation occurs particularly in patients with hepatitis and fatal hepatic failure.[6,7]YMDD mutations or the methionine to valine or isoleucine substitution in the highly conserved tyrosine-methionine-aspartateaspartate (YMDD) motif of the HBV-DNA polymerase[8,9]are composed of the two YIDD and YVDD mutations.[10]

Viral factors,viral-host interaction,and treatment duration reportedly in fluence the occurrence of YMDD mutations.[11-13]Moreover,the incidence of YMDD mutations is higher in patients with genotype A than those with genotype D.[14]HBV genotypes are considered to play an important role in the occurrence of YMDD mutations in patients with chronic hepatitis B during lamivudine treatment.Suzuki et al[13]reported that high HBV-DNA level and HBeAg positivity are closely correlated with the occurrence of drug resistance in Japanese patients with chronic hepatitis B.However,little information is available on YMDD mutations relevant to HBV genotype,HBV-DNA level,HBeAg status,and ALT level in Chinese patients with chronic hepatitis B.

The present study aimed to explore the association of YMDD mutations with pretherapy HBV genotypes,HBV DNA levels,HBeAg status,and ALT levels in Chinese patients with chronic hepatitis B during their first year of lamivudine treatment.

Methods

Patients

A total of 319 patients with chronic hepatitis B who had received lamivudine therapy for at least 12 months between September 2008 and June 2011 were enrolled in the current study at the First Affiliated Hospital,Zhejiang University School of Medicine.The patients who had received interferon-α or other nucleotide analogs and those co-infected with hepatitis C,hepatitis D,and human immunodeficiency virus were excluded from the study.Because YMDD mutations naturally preexist in the naive patients with chronic hepatitis B,[15,16]patients whose YMDD mutations were detectable prior to lamivudine treatment were also excluded from this study.Blood samples were collected from each patient prior to lamivudine treatment to determine ALT levels,HBV-DNA levels,HBeAg and anti-HBeAg status,and YMDD mutations.Blood samples were also obtained from the patients every three months during the first year of lamivudine treatment.The patients were followed up for 12 months.

Serological tests

The blood from the patients with chronic hepatitis B was assayed for alanine transaminase and total bilirubin at our clinical laboratory using an automated analyzer.HBsAg and HBeAg as well as HBe (anti-HBe) and anti-HDV antibodies were determined using commercial radioimmunoassay kits (Abbott Laboratories,Chicago,IL).Anti-HCV antibodies were detected using enzyme immunoassay kits (Abbott Laboratories,Chicago,IL).Serum HBV-DNA levels were quantified via quantitative real-time polymerase chain reaction (PCR) with a commercial detection kit (PG Biotech,China) and the lightcycler detection system (Roche,Switzerland).The real-time PCR had a lower limit of 103copies/mL.

Genotyping of HBV

HBV genotypes were determined using an HBV genotype ELISA kit manufactured by the Institute of Immunology of Tokyo in Japan.The kit uses monoclonal antibodies against the type-specific epitopes on the product of the pre-S2 region.The assay of the HBV genotypes has been described in detail in a previous study.[17]

Detection of YMDD mutations

Serum DNA was extracted from the serum samples using a QIAamp blood kit (Qiagen,CA) according to the manufacturer's manual.The HBV-DNA fragments(589bp; nt252-840) were amplified using PCR primers.The purified PCR fragments were directly sequenced using an automated DNA sequencer.The HBV-DNA sequence was aligned with standard sequences of genotypes B and C obtained from an international DNA database using the DNAssist software (version 2.2).The amplification PCR and the sequence analysis of HBVDNA are described in detail in a previous study.[18]

Statistical analysis

Statistical analysis was performed using Student's t test or the chi-square test.Multivariate analysis was made using a stepwise logistic regression model.All data were analyzed using SPSS software (version 15.0).P values ＜0.05 were considered statistically significant.

Results

Of the 319 patients with chronic hepatitis B,122 were seropositive for HBeAg and 197 were seropositive for anti-HBeAg.In the 319 patients,173 were male,with a median age of 43 years (range 23-67),and 146 were female,with a median age of 45 years (range 27-65).YMDD mutations were not detected in these patients at baseline.

HBV genotypes and YMDD mutations

Among the 319 patients,137 (42.95%) were infected with genotype B and 182 (57.05%) with genotype C.No significant differences in mean age,male-to-female ratio,ALT levels,HBeAg positivity,HBV-DNA levels were observed between the patients with genotypes B and C before lamivudine treatment (Table 1).YMDD mutations occurred in 94 patients (29.47%) after one year of lamivudine treatment.Fifty patients with HBVgenotype B and 44 patients with genotype C showed YMDD mutations.Significant differences were observed in the rate of YMDD mutations between patients with genotypes B and C (36.50% vs 24.18%,respectively,P＜0.05).In 50 patients with HBV genotype B,there were 21 with YIDD mutations and 29 patients with YVDD mutations.In 44 patients with genotype C,there were 19 patients with YIDD mutations and 25 patients with YVDD mutations.The rate of YIDD mutations was not significantly different between patients with genotype B and C (42.00% vs 43.18%,P＞0.05),and the rate of YVDD mutations was also not significantly different between patients with genotype B and C(58.00% vs 56.82%,P＞0.05).Furthermore,logistic regression analysis showed that HBV genotypes at baseline were highly associated with the occurrence of YMDD mutations (HBV genotype:OR=2.159,95% CI 1.291-3.609,P=0.003; Table 2).

Table 1.Characteristics of patients with chronic HBV genotypes B and C before lamivudine treatment

Table 2.Logistic regression analysis on the occurrence of YMDD mutations and the clinical characteristics including pretreatment HBV genotype,HBV-DNA levels,HBeAg status,and ALT levels during lamivudine treatment

HBV-DNA levels and YMDD mutations

Significant differences in HBV-DNA levels were observed between the patients with or without YMDD mutations prior to lamivudine treatment (6.71±0.98 vs 6.44±0.77 Lg copies/mL,respectively,P＜0.05).Logistic regression analysis also showed that the pretreatment HBV-DNA level was closely related to the occurrence of YMDD mutations (HBV-DNA:OR=1.653,95% CI 1.231-2.218,=0.001; Table 2).

Table 3.Baseline characteristics of chronic hepatitis B patients with and without YMDD mutations after one year of lamivudine treatment

HBeAg-positive and YMDD mutations

HBeAg status of the patients revealed that 45 YMDD mutations occurred in 122 HBeAg-positive patients and 49 YMDD mutations in 197 HBeAg-negative patients after lamivudine treatment for a year.The HBeAg-positive patients had a significantly higher rate of YMDD mutations than the HBeAg-negative patients (47.87% vs 34.22%,respectively,P＜0.05,Table 3).Logistic regression analysis also showed that the HBeAg status at baseline was highly associated with the occurrence of YMDD mutations (HBeAg:OR=2.021,95% CI 1.201-3.399,P=0.008; Table 2).

ALT levels and YMDD mutations

No significant differences in the ALT levels were observed between patients with or without YMDD mutations prior to lamivudine treatment (108.09±6.51 vs 110.14±14.9 U/L,P＞0.05,Table 3).Logistic regression analysis also showed that the pretreatment ALT levels were not related to the occurrence of YMDD mutations(ALT:OR=0.981,95% CI 0.949-1.013,P=0.238; Table 2).

Discussion

The current study was designed to explore the association of the occurrence of YMDD mutations with pretreatment HBV genotype,HBV-DNA level,HBeAg status,and ALT level in Chinese patients with chronic hepatitis B receiving lamivudine treatment.The study showed that HBV genotype,HBV-DNA level,and HBeAg status at baseline were independent factors related to the occurrence of YMDD mutations in patients with chronic hepatitis B receiving lamivudine treatment.Sex and age of the patients were not independent factors in this study (as shown in Table 3),as reported previously.[11]But sex,body mass index and HBV-DNA levels were reported to be associated with occurrence of YMDD mutations.[19]

Lamivudine resistance is associated with mutations in the highly conserved YMDD motif of reverse transcriptase,specifically YMDD mutations including YIDD and YVDD mutations.YMDD mutations reportedly increase from 14% in 1 year to 38%,49%after 2 and 3 years of treatment,respectively.[2,4]In the current study,the rate of YMDD mutations was 29.48%after one year of lamivudine treatment,which is higher than that in a previous study.[2]However,it is lower than that in another study,which had YMDD mutations in 57.4% of Japanese patients with chronic hepatitis B after one year of lamivudine treatment.[20]

Eight HBV genotypes have been classified to date.[21-23]Genotypes A and D are the major genotypes in European countries,whereas genotypes B and C are dominantly prevalent in Asian countries,especially in China.[21,24]The correlation of HBV genotypes with the occurrence of YMDD mutations is different in different studies.A study[14]showed that HBV genotype A was also related to a high incidence of YMDD mutations after 12 months of lamivudine treatment.Other studies[12,25]found that HBV genotype did not play an important role in the occurrence of YMDD mutations.However,these studies suggested that large-scale prospective investigation is necessary to explore the role of genotypes in YMDD mutations after lamivudine treatment.In the current study,the rate of YMDD mutations was significantly higher in the patients with HBV genotype B than in those with HBV genotype C.This finding indicates that HBV genotype B patients had a higher incidence of YMDD mutations than genotype C patients.

Moreover,logistic regression analysis in the current study proved that pretreatment HBV genotype is an independent factor for YMDD mutations,suggesting that HBV genotype is a determinant for YMDD mutations.These findings are in consistent with those of previous studies showing that YMDD mutations are more likely related to certain HBV genotypes.[14,26]In addition,HBV genotypes are important for the development of YMDD mutations,and patients receiving lamivudine treatment should be monitored for HBV genotypes.

The current study also aimed to determine the association of pretreatment HBV-DNA levels and HBeAg status at baseline with the occurrence of YMDD mutations.Logistic regression analysis demonstrated that high pretreatment HBV-DNA levels are independent factors for the occurrence of YMDD mutations.These findings are in accordance with the results of other studies.[11,13,19,27]And HBeAg status at baseline is recognized as an independent factor for YMDD mutations.Thus HBeAg status at baseline is one of the predictors.[13]But the results are not consistent with those of another study suggesting that the rate of YMDD mutations in HBeAg positive patients was similar to that in HBeAg negative patients.[28]The present study suggest that pretreatment HBV-DNA levels and HBeAg status should be considered for patients with chronic HBV after lamivudine treatment.

High ALT levels are thought to be related to a more rapid selection of YMDD mutations.[5,28-30]The current study revealed no significant differences in ALT levels in patients with or without YMDD mutations prior to lamivudine treatment.Logistic regression analysis in our study also showed that pretreatment ALT levels are not predictors of YMDD mutations.Studies[14,31]including the current one showed that high ALT levels are not related to the rapid selection of YMDD mutations.Thus,there are different findings concerning the correlation of ALT levels with YMDD mutations.However,ALT levels are very important parameters for host factors,and they should be monitored carefully during lamivudine treatment.

In conclusion,HBV genotype,HBV-DNA levels and HBeAg status at baseline are important factors for the development of YMDD mutations.Hence,patients with chronic hepatitis B should be monitored for these factors prior to lamivudine treatment.

Contributors:ZB proposed the study.WF and ZB performed research and wrote the first draft.WMJ,ZGXL and ZSM collected and analyzed the data.All authors contributed to the design and interpretation of the study and to further drafts.ZB is the guarantor.

Funding:None.

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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