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Drug-Related Mutational Patterns in Hepatitis B Virus (HBV) Reverse Tran- scriptase Proteins From Iranian Treatment-Naïve Chronic HBV Patients Mostafa Mahabadi 1, Mehdi Norouzi 1, Seyed Moayyed Alavian 2, Katayoon Samimirad 3, Ta-lat Mokhtari Azad 1, Esmaeil Saberfar 4, Mahmood Mahmoodi 5, Fatemeh Ramezani 1, Hadi Karimzadeh 1, Reza Malekzadeh 6, Ghodrat Montazeri 6, Azim Nejatizadeh 7, Masoud Ziee 8, Farshid Abedi 9, Behrooz Ataei 13, Majid Yaran 13, Babak Sayad 11, Mohamad Hossein Somi 12, Gholamreza Sarizadeh 13, Ismaeil Sanei-Moghaddam 14, Fariborz Mansour-Ghanaei 15, Houshang Rafatpanah 16, Mohammad Amin Pourhosseingholi 17, Hossain Keyvani 18, Ebra-him Kalantari 19, Mehdi Saberfiroozi 20, Mohammad Ali Judaki 1, Shiva Ghamari 1, Maryam Daram 1, Zeinab Fazeli 1, Zahra Goodarzi 4, Abolfazl Khedive 1, Abdolvahab Moradi 21, Seyed Mohamad Jazayeri 1, *1 Hepatitis B Molecular Laboratory, Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran 11 Department of Gastroenterology, Zahedan University of Medical Sciences, Zahedan, IR Iran 12 Gastrointestinal and Liver Diseases Research Center, Guilan University of Medical Sciences, Rasht, IR Iran 13 Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, IR Iran 14 Kermanshah Liver Diseases and Hepatitis Research Center, Kermanshah, IR Iran 15 Liver and Gastrointestinal Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran 16 Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran 17 Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran 18 Department of Virology, School of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran 19 Gholhack Medical Laboratory, Tehran, IR Iran 2 Middle East Liver Diseases Center (MELD Centers), Tehran, IR Iran 20 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran 21 Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, IR Iran 3 Hepatitis C Molecular Laboratory, Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran 4 The research and Development Department of Bayerpaul Vaccine And Pharmaceutical Company, Tehran, IR Iran 5 Department of Epidemiology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran 6 Digestive Disease Research Center, Tehran University of Medical Sciences, Tehran, IR Iran Article type: Research Article; Received: 11 Jul 2012, Revised: 09 Oct 2012, Accepted: 12 Nov 2012; DOI: 10.5812/hepatmon.6712 Implication for health policy/practice/research/medical education:Hepatitis B virus DNA polymerase primary resistance mutations in treatment-naïve patients have been reported in variable fre-quencies and with unclear clinical significance in different studies Understanding Drug-Related Mutation among Treatment-Naïve Chronic HBV Patients can help to prevent viral breakthrough after treatment. Chronic HBV patients should be monitored closely prior the commencement of therapy to achieve the best regimen option.
Please cite this paper as:Mahabadi M, Norouzi M, Alavian SM, Samimirad K, Mokhtari Azad T, Saberfar E, et al. Drug-Related Mutational Patterns In Hepati-tis B Virus (HBV) Reverse Transcriptase Proteins From Iranian Treatment-Naïve Chronic HBV Patients. Hepat Mon. 2013;13(1):e6712. DOI:10.5812/hepatmon.6712 Drug-Related Mutations in HBV Reverse Transcriptase 1. Background
Nearly 400 million individuals worldwide have been infected with chronic hepatitis B virus (HBV) (1), and of these, 75% are of Asian origin. These patients are at risk of developing progressive liver diseases including fibrosis, cirrhosis, or hepatocellular carcinoma and may require liver transplantation (2). Approved HBV therapies include immune modulators interferon alpha (IFN-a), peginter- feronalpha (PegIFN-a2a), and nucleutide analogues (NAs) such as lamivudine, adefovir, entecavir, telbivudine, te- nofovir, etc. Therapy reduces HBV DNA levels as much as possible, ideally less than the lower limit of detection of molecular assays, followed by biochemical remission, a These types of AA changes have been reported to be selected during histological improvement, and prevention of complica- NA therapy and associated with replication of hepatitis b virus in vitro tions. Although the initial effect of NA in suppressing HBV replication and reducing alanine aminotransferase activ- ity is promising, the emergence of drug-resistant vari- Table 2. Mutations at AA Positions of HBV Reverse Transcriptase
ants considerably reduces the benefit of therapy (3, 4). Reported Genotypic Resistance for Drug Resistance Drug resistance has been associated with the emergence References
of polymerase gene mutations that are localized within the RT region that consists of 6 functional domains (G, F, A, B, C, D and E) and 5 interdomains (F–A, A–B, B–C, C–D and D–E) (Figure 1, Tables 1 and 2). Among NAs, lamivudine and adefovir are the most commonly studied in the literature. Viral resistance emerges with both drugs, with frequen- cies as high as 30 and 80% after 4 years, respectively (5). Evidence now increasingly indicates that drug-related mutation does occur naturally and can be found in HBV N139D/K/H
carriers who have never received both therapies (6-8). The reported incidence of prevalence of YMDD mutations in treatment-naïve patients varies, ranging from 1 to 18% (9- 12) in patients with chronic hepatitis B. The prevalence of HBV in Iran ranges from1.7 to 2.5% in the general popula- tion (2, 13) and both lamivudine and adefovir have widely Figure 1. Schematic Figure Showing Polymerase Protein
L229G/V/W
A) Four main domains include the RT region, B) RT region including do- mains and inter domains, C) Mutation frequency in the F domain to the C-D inter-domain, D) Individual drug-resistance mutation to LAM (Lami- N238K/D/S/T
vudine), ADF (Adefovir), ETV (Entacavir), and LdT (Telbivudine).
Table 1. Mutations at AA Positions of HBV Reverse Transcriptase
for Drug Resistance Reported With Known Phenotypic Data a
2. Objectives
References
The aim of the present study was to explore the RT pro- tein variations between Iranian HBV chronic carriers who had not received any type of HBV therapy.
3. Materials and Methods
Three hundred and twenty-five HBS Ag-positive patients who were referred to the Iranian Hepatitis Center (Tehran IR Iran) between April 2004 and April 2010, were enrolled Drug-Related Mutations in HBV Reverse Transcriptase in a cross-sectional study. All patients were given their in- mains (A, B, C and F) divided by the number of amino acid formed consent and the study protocol was approved by residues in that particular domain. The sequences sent to the Iranian Hepatitis Network ethics committee. To cover Gen bank under accession numbers from JX565097 to all ethnic groups in the country, we studied 10 regions based on population and geographical zones. Exclusion criteria included hepatitis C virus, hepatitis D virus, and 3.4. HBV Genotyping and Phylogenetic Analysis human immunodeficiency virus infection. All patients HBV genotyping was determined using NCBI Viral Ge- were interviewed and examined by gastroenterologists notyping Tool (http://www.ncbi.nlm.nih.gov/projects/ge- to evaluate the clinical findings and the results of the notyping/formpage.cgi). Phylogenetic analysis was con- investigative workup (liver histology, ultrasonography, ducted MEGA version 4. Briefly, sequences of HBV partial and laboratory tests such as serologic, biochemical, and RT gene sequences (approximately 700-bp, 325 isolates in virological tests) to determine the clinical status of the this study) were block aligned by the CLUSRAL X program patient. All of the patients were chronic carriers of HBV. and corrected visually, the Kimuratwo-parameter algo- Next, 5 ml aliquots of whole blood samples were drawn rithm was used for genetic distance calculation. A phy- from each participant. Serum was aseptically separated logenetic tree was generated along with a different HBV in the field by centrifugation at 4,000/rpm for 5 min- genotype (A to H) sequences retrieved from the Gen Bank utes and was stored at -20°C until tested. HBV serological as reference genes by the neighbor-joining method, and markers including HBS Ag and anti-HBS were examined bootstrapped-sampling and reconstruction was carried by ELISA kits manufactured by Organon Technika, Hol- out 1000 × to confirm the reliability of the phylogenetic 3.2. DNA Extraction and Polymerase Chain Reac- 4. Results
HBV DNA was extracted from 200 µl of sera using a Qia- gen Mini Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was stored at Three hundred and twenty five HBS Ag-positive chronic -20°C. First-round amplification was conducted by RTF patients who were all were chronic carriers enrolled in CCT GCT GGT GGC TCC AGT TC as the sense primer and RTR this study, which were native residents of diverse regions CCA CAA TTC (K) TT GAC ATA CTT TCC A as the antisense of Iran, HBV DNA positive and treatment-naïve. Of total, primer. Second-round PCR was performed by RTNF GCA 46.47% (151/325) were female and 53.53% (174/325) were CAC GGA ATT CCG AGG ACT GGG GAC CCT G as the sense male with a mean age of 36 years (SD ± 20). The mean ALT primer and RTNR GAC ACC AAG CTT GGT TAG GGT TTA AAT levels were 29 IU/L (SD ± 5) and 38% (123/325) were HBeAg GTA TAC C as the antisense primer. The first-round PCR was run at 94°C for 4 minutes, followed by 35 cycles at 94°C for 30 seconds, 62°C for 35 seconds, and 72°C for 30 4.2. Genotypic and Phylogenetic Analysis seconds, followed by 72°C for 10 minutes. A similar pro- The phylogenetic tree was constructed using the align- gram was applied for the second-round PCR. Direct se- ment of HBVRT region of patients (325 isolates from this quencing of PCR products was carried out (Perkin Elmer study) along with different HBV genotype (A to H) se- ABI-3130XL DNA Sequencer, Foster city, CA, USA) using 0.5 quences retrieved from the Gen Bank as reference genes. μl of appropriate primers RTF and RTR for the first cycle, The phylogenetic tree revealed that all Iranian isolates an RTNF and RTNR for the second-round PCR products. were branched with other genotype D of HBV reference isolates with a high boot strap value, 99%, 1000 × repli- Amino acid variations within two hundred and thirty amino acid RT regions were compared with reference sequences obtained from different HBV genotype and Figure 2. Neighbor joining phylogenetic analysis based on the alignment
of the RT gene region (approximately 750-bp) of 325 HBV isolates from sequences from Iranian isolates obtained from Gen Bank Iran as well as other HBV genotypes from Gen Bank as reference. Boot- and NCBI. Comparing to the former, any amino acid strap values indicate 1000-foldreplicates. Due to clarity, the 325 isolates changes defined as “variant”. With regards to the latter from Iran and 6 reference genes of HBV genotype D was collapsed. Woolly (Iranian database sequences), amino acid differences monkey HBV was used as the out-group sequence.
defined as “mutation”. The sequences were analyzed us- ing Bio Edit Software version 7.0.5.3. Drug-related muta- 4.3. Analysis of Amino Acid Substitutions tions in hepatitis B virus reverse transcriptase sequences within RT refereed and are listed in table one and two. Compared to the reference genotypes D and accord- The amino acid mutation frequencies were obtained and ing to the above-mentioned criteria for differentiation by drug-resistance mutation found in individual RT do- between variants and mutants, all patients contained at Drug-Related Mutations in HBV Reverse Transcriptase least one amino-acid mutation in the RT region. The to- DNA polymerase mutations were obtained from direct tal number of amino-acid mutations was 365, of which sequencing techniques; therefore, the actual number of 14.5% (n = 53), 11.2% (n = 41), and 4.9% (n = 18) were found mutations might be underestimated (14). Lamivudine within the A, B and C domains, respectively. The muta- was the first effective oral HBV-replication suppressive tion frequencies in the functional domains of the partial agent to enter widespread clinical use; however, its use RTs were 0.73% (53/6825) in Domain A, 0.48% (41/8450) in had been challenged with the emergence of resistant mu- Domain B, and 0.50% (18/3575) in Domain C. The average tations, which increased from 23% to 80% following the amino-acid substitution was 0.44%, indicating the proba- first and fourth years, respectively (15, 16). The most com- bility of changes per position. In the mutational analysis monly described mutations are the substitution of valine of the RT inter domains, the substitutions and mutation or isoleucine for methionine at residue 204 (rtM204I/V). frequencies were found in A-B0.60% (141/23400), B-C0.08% These YMDD motif mutations are necessary and suffi- (3/3575), and C-D1.64% (107/6500) inter domains (Figure 1).
cient to confer a high level of lamivudine resistance. For this specific mutation, the rtL180M is the main compen- 4.4. Lamivudine-Related Resistance Mutations satory change. Other compensatory mutations include Common and frequent lamivudine-related amino acid the rtV173L and rtL80I changes (17, 18). The rtA181T change substitutions were found in this study including N53K has been reported to occur in the absence of rtM204I/V 3.68% (12/325), L80V/I 3.07% (each 5/325), L82M1.22% (4/325) and is considered a primary resistance mutation (19, 20). L91I 7.06% (23/325), T128I 1,53% (5/325), L180M3.37% (11/325), In this study we found that L180M 3.37% (11), M204I 2.76% and M204I2.76%(9/325) M204V 1.23% (4/325) see Figure 1.
(9), and M204V 1.23% (4) were more common than had been reported previously in Iran (21, 22). Our results fall 4.5. Adefovir-Related Resistance Mutation between the extremes reported by other studies around the world (7-12, 23). A Adefovir dipivoxil has been shown RT sequence changes implicated in adefovir-resistance to be effective against both wild-type and lamivudine- were detected, including S213T 5.83% (19/325), S213N 0.9% resistant HBV both in vitro and in vivo (18-20). Compared (3/325), V214E 0.61% (2/325), V214A 0.61% (2/325), Q215P 2.75% to the lamivudine, resistance to ADV usually occurs less (9/325), Q215S 7.06% (23/325), Q215H1.53% (5/325), and F221Y frequently and takes longer to emerge. Adefovir-resistant 2.76% (9/325) of isolations that previously reported adefo- HBV mutations have not been detected in patients up to vir-resistance mutation (Figure 1). 48 weeks into therapy and increase to 3%, 11%, 18%, and 29% following 2, 3, 4, and 5 years of therapy, respectively (22, 4.6. Other Drug-Related Resistance Mutation and 24). HBV genotype D is associated with an increased risk Anti-Viral Cross-Resistance Mutations of adefovir resistance (11, 25). Adefovir resistance has been Cross-resistance refers to the situation in which a de- associated with a primary mutation in the D domain at creased susceptibility to more than one antiviral drug is rtN236T and rtA181T/Vin the B domain. In addition, a conferred by the same amino acid substitution or com- number of other mutations have been detected in our bination of amino acid substitutions. Cross-resistance study and were clustered into three distinct regions of mutations to lamivudineand telbivudinewere: M204I the RT: the D and A domains (rtP237H, rtN238T/D, rtV84M, 2.76% (9/325), M204V 1.23% (4/325), M204V/I + L180M 2.45% and rtS85A) and the C-D interdomain (rtS213T/N, rtV214A, (8/325), and L80I + M204I 1.23% (4/325). Cross-resistance and rtQ215S) (26-31). The latter finding had a mutation fre- mutations to lamivudine and entecavirwere: M204I 2.76% quency of 1.64% and was higher than the other domains (9/325) and M204V/I + L180M 2.45% (8/325) (Figure 1).
studied (Figure 1). These mutations may be regarded as secondary resistance mutations, as they have been asso- 5. Discussion
ciated only with very low-level resistance in vitro (22, 26). These secondary mutations have also been detected in Hepatitis B virus DNA polymerase primary resistance the absence of rtN236T (both alone and in combination) mutations in treatment-naïve patients have been re- in patients who have either not responded to or have had ported in variable frequencies and with unclear clinical a virological breakthrough during adefovir treatment significance in different studies (9-12). The aim of this (32-34). The high number of adefovir-related mutation study was to characterize the mutational patterns of RT in this region may be indicative of an increased risk of protein in chronic HBV carriers. All 325 patients included adefovir resistance in genotype D-infected patients if in this study were treatment naïve. All had been infected they receive this drug (11, 25). Preliminary evidence indi- with genotype D. The results showed that viral muta- cates that the primary lamivudine-resistance mutation tions related to lamivudine, adefovir ,telbivudine, and rtM204V/I seems to be a prerequisite for the development entecavir resistance were discovered in patients who had of entecavir and telbivudine resistance (35). Telbivudine never been treated with these drugs, with various muta- and entecavir-related RT mutations were also found in tion frequencies ranging between 1.23% (for M204V) and this study. M204I/V and M204V/I/L180M are related to 7.06% (for L91I). However in the present study, the molec- entecavir resistance (36-39). Recently, the GLOBE-Trial ular cloning was not performed, and the results of HBV demonstrated that in vitro telbivudine resistance is con- Drug-Related Mutations in HBV Reverse Transcriptase ferred by either rtM204I or rtM204V+rtL180M and L80I/ Financial Disclosure
M204I but not by the rtM204V mutation alone, while in vivo telbivudine resistance is almost exclusively due to the presence of M204I. The reasons for the presence of Funding/Support
such mutations in treatment-naïve chronic patients are not clear. Polymerase is an essential structural protein This study was supported by Tehran University of Medi- with a high error rate resembling RNA viruses. As a quasi- cal Sciences (Grant No: 860227-5816).
species in the hepatitis B virus pool, a cocktail of variants could be found in a single patient even in each body com- References
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