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Rate of YMDD motif mutants in lamivudine-untreated Iranian patients with chronic hepatitis B virus infection
International Journal of Infectious Diseases (2008) 12, 252—255
http://intl.elsevierhealth.com/journals/ijid
Rate of YMDD motif mutants in lamivudineuntreated Iranian patients with chronic hepatitis B virus infection Amitis Ramezani a,b, Ali Akbar Velayati c, Mohammad Reza Hasanjani Roshan d, Latif Gachkar b, Mohammad Banifazl e, Hossein Keyvani f, Arezoo Aghakhani a,* a
Clinical Research Department, Pasteur Institute of Iran, No. 69, Pasteur Ave., Tehran, Iran Infectious Diseases Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran c Masih Daneshvari Hospital, Shaheed Beheshti University of Medical Sciences, Tehran, Iran d Babol University of Medical Sciences, Babol, Iran e Iranian Society for the Support of Patients with Infectious Diseases, Tehran, Iran f Keyvan Virology Laboratory, Tehran, Iran b
Received 22 April 2007; received in revised form 31 July 2007; accepted 22 August 2007 Corresponding Editor: Jane Zuckerman, London, UK
KEYWORDS Chronic hepatitis B; Lamivudine; PCR-RFLP; YMDD motif mutants
Summary Background: Lamivudine is used for the treatment of chronic hepatitis B patients. Recent studies show that the YMDD motif mutants (resistant hepatitis B virus) occur as natural genome variability in lamivudine-untreated chronic hepatitis B patients. In this study we aimed to determine the rate of YMDD motif mutants in lamivudine-untreated chronic hepatitis B patients in Iran. Patients and methods: A total of 77 chronic hepatitis B patients who had not been treated with lamivudine were included in the study. Serum samples from patients were tested by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detection of YMDD motif mutants. All patients were also tested for liver enzymes, anti-HCV, HBeAg, and anti-HBe. Results: Of the 77 patients enrolled in the study, 73% were male and 27% were female. Mean ALTand AST levels were 124.4 73.4 and 103.1 81 IU/l, respectively. HBeAg was positive in 40% and antiHBe in 60% of the patients. Anti-HCV was negative in all of them. YMDD motif mutants were not detected in any of the patients despite the liver enzyme levels and the presence of HBeAg or anti-HBe. Conclusion: Although the natural occurrence of YMDD motif mutants in lamivudine-untreated patients with chronic hepatitis B has been reported, these mutants were not detected in Iranian lamivudine-untreated chronic hepatitis B patients. # 2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +98 21 66465147; fax: +98 21 66409467. E-mail address: [email protected] (A. Aghakhani). 1201-9712/$32.00 # 2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2007.08.003
253
YMDD mutants in untreated chronic hepatitis B patients
Introduction Hepatitis B is one of the most common infectious diseases in the world. It is estimated that more than 300 million people worldwide have chronic hepatitis B virus (HBV) infection and that 10% of these patients will die as a direct consequence of persistent viral infection.1 The global prevalence of chronic HBV infection varies widely, from high (>8%), e.g., in Africa, Asia, and the Western Pacific, to intermediate (2—7%), e.g., in Southern and Eastern Europe, to low (<2%), e.g., in Western Europe, North America, and Australia.2 Nucleoside analogue therapy allows the safe, long-term suppression of HBV, and its use marks a major milestone in the treatment of chronic hepatitis B. Lamivudine, the first of these agents approved worldwide, effectively suppresses viral replication, reduces disease activity, improves liver histology, and delays clinical progression.3,4 However, the development of lamivudine-resistant mutations occurs in 14—32% of patients after 1 year of therapy.5,6 The longer the treatment is continued, the more frequently resistance is seen (65% at 5 years).7 Lamivudine-resistant HBV is associated with mutations of the YMDD motif in the polymerase gene. The key mutations are the substitutions of methionine at the rtM204 (domain C) to either isoleucine (rtM204I, YIDD variant) or valine (rtM204V, YVDD variant).8,9 The rtM204V variant is almost always accompanied by an additional rtL180M mutation in the domain B.10—12 HBV mutants with mutations in the YMDD motif of viral polymerase have been described in patients infected with HBV who have not received lamivudine therapy.13,14 Also, cases of early emergence of mutants have been described, suggesting that mutations conferring lamivudine resistance were already present in the liver of these patients before starting therapy. This would mean that these mutant strains circulate among the population, so that a certain proportion of HBV carriers or chronic hepatitis B patients from a given geographical area might have any of them.15 The presence of a drug-resistant HBV variant before treatment could be a reason for selecting an alternative drug for therapy. Thus, routine testing for mutations involved in antiviral drug resistance might be useful for candidate patients.15 To judge whether this activity would be appropriate, however, prior knowledge of the prevalence of these variants among the HBV infected population is required. This study was performed to determine the rate of YMDD motif mutants in lamivudine-untreated chronic hepatitis B patients in Iran.
Patients and methods In this cross-sectional study, 77 chronic hepatitis B patients were tested for the presence of YMDD motif mutants. One or both of the following inclusion criteria had to be met: (1) HBsAg persistence for more than six months with liver enzymes more than 1.5 times the upper limit of normal; (2) HBsAg persistence for more than six months and liver biopsy with signs of chronic hepatitis confirmed by a pathologist according to the Knodell necroinflammatory score system.16 None of the patients had been treated with antiviral medications including lamivudine. Blood samples were collected from the patients and the serum stored at 70 8C. All samples were tested by poly-
merase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detection of YMDD motif mutants. Simultaneously, liver enzymes (ALT and AST) were also determined in all of them. Samples were also tested for hepatitis C antibody (anti-HCV), hepatitis B e antigen (HBeAg), and hepatitis B e antibody (anti-HBe) by ELISA. The commercial ELISA kits used were: anti-HCV, Bio-Rad Laboratories, Segrate, Italy; HBeAg and anti-HBe, Radim, Barcelona, Spain. All assay protocols, cut-offs, and result interpretations were carried out according to the manufacturers’ instructions.
PCR-RFLP HBV variants at M552 (nucleotides 741 or 743) were detected by PCR-RFLP. Three separate PCR assays were used to amplify three fragments around position 552 of the polymerase gene. A 274-bp fragment was amplified using the primer pair P2 (50 AAA CCT TCG GAC GGA AAC TGC-30 ) and P4 (50 -CTG GAT CCA GGG TTTAAATGTATA CCC-30 ), and the fragment was digested with FokI restriction enzyme, which cuts the sequence of the wild type YMDD and YVDD variant into 143 bp, 100 bp, and 31 bp and the sequence of the YIDD variant into 243 bp and 31 bp. The 31-bp fragment was not related to the identification of YMDD variants. A 181-bp fragment was amplified using the primer pair P5 (50 -TGG AAT TCA CCT GTA TTC CCA TCC CAT-30 ) and P6 (50 -CAG ACT TGG CCC CCA ATA CCA CAT CGT GCA-30 , which introduces an Alw441 site only into the YVDD mutant product), and the fragment was digested with Alw441 restriction enzyme, which cuts the sequence of the YVDD variant into 158 bp and 23 bp. A 138-bp fragment was amplified using the primer pair P4 (50 -CTG GAT CCA GGG TTT AAA TGT ATA CCC-30 ) and P3 (50 -TTT CCC CCA CTG TTT GGC TTT CAG TAA TAT-30 , which introduces an SspI site only into the YIDD mutant product), and the fragment was digested with SspI restriction enzyme, which cuts the sequence of the YIDD variant into 109 bp and 29 bp. Restriction enzyme SspI and Alw441 cutting sites were introduced into primers P3 and P6, respectively. For DNA extraction, 100 ml of each patient’s serum was mixed with 150 ml of TES buffer containing 10% sodium dodecyl sulfate and proteinase K, and then incubated at 60 8C for 1 hour. After digestion with proteinase K, DNA was extracted by phenol/chloroform/isoamyl alcohol. Extracted DNA was precipitated with absolute ethanol, then washed with 70% ethanol and dissolved in 30 ml of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The DNA was used for three amplification reactions of PCR. Final concentrations in the three PCR reactions were as follows: 20 mmol Tris (pH 8.3), 0.2 mmol KCl, 1.5 mmol MgCl2, 18 mmol NaCl, 0.2 mmol dNTPS, 0.5 mmol each primer, and 2.5 U Taq polymerase per 50 ml of reaction mixture. The amplification conditions in the three PCR reactions all included initial denaturation at 94 8C for 3 minutes, 35 cycles of amplification with denaturation at 94 8C for 35 seconds, primer annealing at 56 8C for 50 seconds, extension of primer at 72 8C for 50 seconds, followed by a final extension at 72 8C for 5 minutes. After amplification, aliquots of the product DNA of the three PCR reactions were digested with three enzymes, respectively, according to the manufacture’s specifications (Shanghai Biology Project Technology Company). The digestion reactions included 15 ml amplified HBV DNA, 10 U
254 enzyme, 10 restriction enzyme digestion buffer 2 ml, and 3 ml sterile ddH2O in a sterile Eppendorf tube. The tube was spun at 500 rpm for 3 seconds. The SspI and Alw441 enzyme reaction mixtures were incubated in a 37 8C water bath for 16 hours. The FokI enzyme reaction mixture was incubated in a 55 8C water bath for 3 hours .The results of PCR-RFLP were analyzed by 8.4% polypropylene acidemide gel electrophoresis.17 The detection limit of the assay was 10 000 copies per ml.
Statistical analysis Chi-square and t2 tests were used with the SPSS 11.5 program for statistical analysis. Data are presented as means stanstandard deviations or, when indicated, as absolute number and percentage. A p value of <0.05 was considered significant.
Results A total of 77 patients were enrolled in this study; 73% of them were male and 27% were female and they were aged between 11 and 50 years (mean 27.4 9.3). Chronic hepatitis B was diagnosed in patients based on HBsAg persistence for more than six months with liver enzymes more than 1.5 times the upper limit of normal and/or HBsAg persistence for more than six months with liver biopsy signs of chronic hepatitis confirmed by a pathologist. None of the patients had been treated with antiviral medications including lamivudine. In these patients ALT, AST, HBeAg, anti-HBe, and anti-HCV tests and detection of YMDD motif mutants were done simultaneously. The patients’ ALT levels were between 28 and 500 IU/l (mean 124.4 73.4) and AST levels were between 22 and 500 IU/l (mean 103.1 81). HBeAg and anti-HBe were positive in 40% and 60% of the patients, respectively. Anti-HCV was negative in all of them. In 46 patients, the diagnosis of chronic hepatitis B was based on the liver biopsy. The liver biopsy scores were between 2 and 13 (mean 5.9 2.4), grade (activity) was between 1 and 9 (mean 3.6 2.3), and stage (fibrosis) between 0 and 3 (mean 1.2 1.1) according to the Knodell necroinflammatory score system.16 PCR was ‘not determined’ in 48% (37/77) of the patients and was ‘sensitive’ in 52% (40/77) of them. No YMDD motif mutations (0) were detected in chronic hepatitis B patients by RFLP.
Discussion HBV is one of the most common infectious diseases in the world. HBV genotypes represent naturally occurring strains of HBV that have evolved over the years and reflect the geographical distribution of HBV throughout the world. Genotype D is most often found in Southern Europe, parts of Central Asia, India, Africa, and the Middle East.18 In Iran, genotype D is the only detected type found in all HBV infected patients.19 Lamivudine is an antiviral drug approved for the treatment of chronic hepatitis B.15 However, the emergence of viral mutants resistant to lamivudine is the main concern
A. Ramezani et al. with this treatment. The cause of lamivudine-resistant HBV was revealed to be the amino acid substitution from leucine to methionine at codon 180 of the B domain (rtL180M) and amino acid substitutions of the YMDD motif from methionine to valine or isoleucine at codon 204 of the C domain (rtM204V or rtM204I) of the reverse transcriptase (rt) region of the polymerase gene.20—24 Data supporting the natural occurrence of lamivudineresistant strains among the population of chronic HBV carriers have been reported recently from Japan and France.23,25 Some researchers have found the YMDD mutational strains in the serum of cases with chronic hepatitis B who did not receive lamivudine treatment.14,23,26 Yan et al. showed that 19 out of 110 chronic hepatitis B untreated cases had YMDD mutations.26 The results from a study in Spain indicate that the prevalence of these mutants is about 4% among Spanish chronic HBV carriers.15 In a study in Japan, mutations were detected in five out of 18 untreated chronic hepatitis B patients.23 In addition, Zhang et al. found that 26.2% of chronic hepatitis B cases had YMDD mutations by genetic chip determination.27 Matsuda et al. reported that a few chronic hepatitis B cases not treated with lamivudine had YMDD mutations.13 Naturally occurring YMDD motif variants were detected at a high rate in a group of lamivudineuntreated inactive HBV carriers in Turkey.28 Other studies have not reported YMDD mutations in non-lamivudine treated hepatitis B patients.22 In a study in China, the YMDD mutants were not detected in any of the five patients before lamivudine treatment.29 Kobayashi et al. found that anti-HBe was positive in all patients with YMDD mutations,23 and Ye et al. also found that anti-HBe was positive in most patients with YMDD mutations and considered that YMDD mutations might occur more easily if mutations took place in the pre-c zone.30 In a study in China, the incidence rate of YMDD mutations was 27.1% in patients with positive HBeAg and 26.7% in patients with negative HBeAg and positive anti-HBe. There was no significant difference between the two groups.31 Lee et al. detected YMDD mutants in eight of 12 HBeAg-positive patients and eight of 16 HBeAg-negative patients before lamivudine treatment.12 In our study, the YMDD motif mutants were not detected in any of the chronic hepatitis B patients before lamivudine treatment despite the liver enzyme levels and the presence of HBeAg or anti-HBe. These results do not accord with some studies20,24 but are in agreement with others.22,29 This low rate may be explained by epidemiological and geographic variations and study conditions such as the number of patients and the method used for detecting the YMDD motif mutants. YMDD mutants have significantly low replication ability. It is thought that the naturally occurring YMDD mutants make up only a very small proportion of the total HBV, and very sensitive investigative tools are required to detect them.32 RFLP often leaves out those samples with mixed virus populations and a low-component virus population, and is suited only for mono-infection.33 Further studies are needed to determine exactly the rate of YMDD motif mutants in lamivudine-untreated chronic hepatitis B patients. In conclusion, although the natural occurrence of YMDD motif mutants in lamivudine-untreated patients with chronic hepatitis B infection has been reported, these mutants were
YMDD mutants in untreated chronic hepatitis B patients not detected in Iranian lamivudine-untreated chronic hepatitis B patients. Further studies with more samples and more sensitive investigative tools should be conducted to determine this rate exactly.
Acknowledgment The authors are grateful to the Infectious Diseases Research Center, Shaheed Beheshti University of Medical Sciences for the financial support of this study. Conflict of interest: No conflict of interest to declare.
References 1. Lee WM. Hepatitis B virus infection. N Engl J Med 1997;337: 1733—45. 2. Maddrey WC. Hepatitis B–—an important public health issue. Clin Lab 2001;47:51—5. 3. Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, et al. Longterm safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology 2003;125:1714—22. 4. Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004;351:1521—31. 5. Heathcote J. Treatment of HBe antigen-positive chronic hepatitis B. Semin Liver Dis 2003;23:69—80. 6. Hadziyannis SJ, Papatheodoridis GV, Vassilopoulos D. Treatment of HBeAg-negative chronic hepatitis B. Semin Liver Dis 2003;23:81—8. 7. Wright TL. Clinical trial results and treatment resistance with lamivudine in hepatitis B. Semin Liver Dis 2004;24(Suppl 1):31—6. 8. Allen MI, Deslauriers M, Andrews CW, Tipples GA, Walters KA, Tyrrell DL, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 1998;27:1670—7. 9. Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996;24:714—7. 10. Fu L, Cheng YC. Role of additional mutations outside the YMDD motif of hepatitis B virus polymerase in L () SddC (3TC) resistance. Biochem Pharmacol 1998;55:1567—72. 11. Niesters HG, Honkoop P, Haagsma EB, de Man RA, Schalm SW, Osterhaus AD. Identification of more than one mutation in the hepatitis B virus polymerase gene arising during prolonged lamivudine treatment. J Infect Dis 1998;177:1382—5. 12. Lee CZ, Lee HS, Huang GT, Yang PM, Sheu JC. Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment. World J Gastroenterol 2006;12:5301—5. 13. Matsuda M, Suzuki F, Suzuki Y, Tsubota A, Akuta N, Hosaka T, et al. YMDD mutants in patients with chronic hepatitis B before treatment are not selected by lamivudine. J Med Virol 2004;74:361—6. 14. Shin YM, Heo J, Kim GH, Kang DH, Song GA, Cho M, et al. Natural YMDD motif mutations of HBV polymerase in the chronic hepatitis B virus infected patients. Taehan Kan Hakhoe Chi 2003;9:1—9. 15. Leon P, Pozo F, Echevarria JM. Detection of hepatitis B virus variants resistant to lamivudine and famciclovir among randomly selected chronic carriers from Spain. Enferm Infecc Microbiol Clin 2004;22:133—7. 16. Brunt EM. Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond. Hepatology 2000;31:241—6.
255 17. Yang DH, Liang WF, Xie YJ, Zhao NF, Fan J. PCR restriction fragment length polymorphism in detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine. Hepatobiliary Pancreat Dis Int 2002;1:232—7. 18. Abdo AA, Al-Jarallah BM, Sanai FM, Hersi AS, Al-Swat K, Azzam NA, et al. Hepatitis B genotypes: relation to clinical outcome in patients with chronic hepatitis B in Saudi Arabia. World J Gastroenterol 2006;12:7019—24. 19. Alavian SM, Keyvani H, Rezai M, Ashayeri N, Sadeghi HM. Preliminary report of hepatitis B virus genotype prevalence in Iran. World J Gastroenterol 2006;12:5211—3. 20. Chayama K, Suzuki Y, Kobayashi M, Tsubota A, Hashimoto M, Miyano Y, et al. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and retakeover by wild type after cessation of therapy. Hepatology 1998;27:1711—6. 21. Allen MI, Gauthier J, DesLauriers M, Bourne EJ, Carrick KM, Baldanti F, et al. Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine. J Clin Microbiol 1999;37:3338—47. 22. Kirishima T, Okanoue T, Daimon Y, Itoh Y, Nakamura H, Morita A, 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;37:259—65. 23. Kobayashi S, Ide T, Sata M. Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers. J Hepatol 2001;34:584—6. 24. Hyunjung J, Mong C, Jeong H, Hyunghoi K, Hongki J, Woowon S, et al. Oligonucleotide chip for detection of lamivudine-resistant hepatitis B virus. J Clin Microbiol 2004;42:4181—8. 25. Thibault V, Aubron-Olivier C, Agut H, Katlama C. Primary infection with a lamivudine-resistant hepatitis B virus. AIDS 2002;16:131—3. 26. Yan MH, Zhang C, Ling Q, Zhou RF. Detection of YMDD motif mutations in lamivudine-untreated patients with chronic hepatitis B. Zhonghua Gan Zang Bing Za Zhi 2003;11:430—1. 27. Zhang XH, Zhang YX, Sun LR, Wen Q, Zhou LQ, Fan GX, et al. Study of gene chips in the detection of YMDD mutations in the region of HBV polymerase. Zhonghua Yi Xue Za Zhi 2003;83:459— 62. 28. Akarsu M, Sengonul A, Tankurt E, Sayiner AA, Topalak O, Akpinar H, et al. YMDD motif variants in inactive hepatitis B carriers detected by Inno-Lipa HBV DR assay. J Gastroenterol Hepatol 2006;21:1783—8. 29. Zhang X, Liu C, Gong Q, Zhang S, Zhang D, Lu Z, et al. Evolution of wild type and mutants of the YMDD motif of hepatitis B virus polymerase during lamivudine therapy. J Gastroenterol Hepatol 2003;18:1353—7. 30. Ye XG, Wang RL, Guo HB. Detection and analysis of YMDD mutated genes in patients of chronic hepatitis B before being treated. Zhonghua Jianyan Yixue Zazhi 2002;25:248. 31. Huang ZM, Huang QW, Qin YQ, He YZ, Qin HJ, Zhou YN, et al. YMDD mutations in patients with chronic hepatitis B untreated with antiviral medicines. World J Gastroenterol 2005;11:867— 70. 32. Heo J, Cho M, Kim HH, Shin YM, Jang HJ, Park HK, et al. Detection of YMDD motif mutants by oligonucleotide chips in lamivudineuntreated patients with chronic hepatitis B virus infection. J Korean Med Sci 2004;19:541—6. 33. Ou ZY, Liu N, Chen CJ, Cheng G, He YS. Rapid and accurate genotyping of YMDD motif variants in the hepatitis B virus genome by an improved reverse dot blot method. J Clin Microbiol 2005;43:5685—9.
http://intl.elsevierhealth.com/journals/ijid
Rate of YMDD motif mutants in lamivudineuntreated Iranian patients with chronic hepatitis B virus infection Amitis Ramezani a,b, Ali Akbar Velayati c, Mohammad Reza Hasanjani Roshan d, Latif Gachkar b, Mohammad Banifazl e, Hossein Keyvani f, Arezoo Aghakhani a,* a
Clinical Research Department, Pasteur Institute of Iran, No. 69, Pasteur Ave., Tehran, Iran Infectious Diseases Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran c Masih Daneshvari Hospital, Shaheed Beheshti University of Medical Sciences, Tehran, Iran d Babol University of Medical Sciences, Babol, Iran e Iranian Society for the Support of Patients with Infectious Diseases, Tehran, Iran f Keyvan Virology Laboratory, Tehran, Iran b
Received 22 April 2007; received in revised form 31 July 2007; accepted 22 August 2007 Corresponding Editor: Jane Zuckerman, London, UK
KEYWORDS Chronic hepatitis B; Lamivudine; PCR-RFLP; YMDD motif mutants
Summary Background: Lamivudine is used for the treatment of chronic hepatitis B patients. Recent studies show that the YMDD motif mutants (resistant hepatitis B virus) occur as natural genome variability in lamivudine-untreated chronic hepatitis B patients. In this study we aimed to determine the rate of YMDD motif mutants in lamivudine-untreated chronic hepatitis B patients in Iran. Patients and methods: A total of 77 chronic hepatitis B patients who had not been treated with lamivudine were included in the study. Serum samples from patients were tested by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detection of YMDD motif mutants. All patients were also tested for liver enzymes, anti-HCV, HBeAg, and anti-HBe. Results: Of the 77 patients enrolled in the study, 73% were male and 27% were female. Mean ALTand AST levels were 124.4 73.4 and 103.1 81 IU/l, respectively. HBeAg was positive in 40% and antiHBe in 60% of the patients. Anti-HCV was negative in all of them. YMDD motif mutants were not detected in any of the patients despite the liver enzyme levels and the presence of HBeAg or anti-HBe. Conclusion: Although the natural occurrence of YMDD motif mutants in lamivudine-untreated patients with chronic hepatitis B has been reported, these mutants were not detected in Iranian lamivudine-untreated chronic hepatitis B patients. # 2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +98 21 66465147; fax: +98 21 66409467. E-mail address: [email protected] (A. Aghakhani). 1201-9712/$32.00 # 2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2007.08.003
253
YMDD mutants in untreated chronic hepatitis B patients
Introduction Hepatitis B is one of the most common infectious diseases in the world. It is estimated that more than 300 million people worldwide have chronic hepatitis B virus (HBV) infection and that 10% of these patients will die as a direct consequence of persistent viral infection.1 The global prevalence of chronic HBV infection varies widely, from high (>8%), e.g., in Africa, Asia, and the Western Pacific, to intermediate (2—7%), e.g., in Southern and Eastern Europe, to low (<2%), e.g., in Western Europe, North America, and Australia.2 Nucleoside analogue therapy allows the safe, long-term suppression of HBV, and its use marks a major milestone in the treatment of chronic hepatitis B. Lamivudine, the first of these agents approved worldwide, effectively suppresses viral replication, reduces disease activity, improves liver histology, and delays clinical progression.3,4 However, the development of lamivudine-resistant mutations occurs in 14—32% of patients after 1 year of therapy.5,6 The longer the treatment is continued, the more frequently resistance is seen (65% at 5 years).7 Lamivudine-resistant HBV is associated with mutations of the YMDD motif in the polymerase gene. The key mutations are the substitutions of methionine at the rtM204 (domain C) to either isoleucine (rtM204I, YIDD variant) or valine (rtM204V, YVDD variant).8,9 The rtM204V variant is almost always accompanied by an additional rtL180M mutation in the domain B.10—12 HBV mutants with mutations in the YMDD motif of viral polymerase have been described in patients infected with HBV who have not received lamivudine therapy.13,14 Also, cases of early emergence of mutants have been described, suggesting that mutations conferring lamivudine resistance were already present in the liver of these patients before starting therapy. This would mean that these mutant strains circulate among the population, so that a certain proportion of HBV carriers or chronic hepatitis B patients from a given geographical area might have any of them.15 The presence of a drug-resistant HBV variant before treatment could be a reason for selecting an alternative drug for therapy. Thus, routine testing for mutations involved in antiviral drug resistance might be useful for candidate patients.15 To judge whether this activity would be appropriate, however, prior knowledge of the prevalence of these variants among the HBV infected population is required. This study was performed to determine the rate of YMDD motif mutants in lamivudine-untreated chronic hepatitis B patients in Iran.
Patients and methods In this cross-sectional study, 77 chronic hepatitis B patients were tested for the presence of YMDD motif mutants. One or both of the following inclusion criteria had to be met: (1) HBsAg persistence for more than six months with liver enzymes more than 1.5 times the upper limit of normal; (2) HBsAg persistence for more than six months and liver biopsy with signs of chronic hepatitis confirmed by a pathologist according to the Knodell necroinflammatory score system.16 None of the patients had been treated with antiviral medications including lamivudine. Blood samples were collected from the patients and the serum stored at 70 8C. All samples were tested by poly-
merase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detection of YMDD motif mutants. Simultaneously, liver enzymes (ALT and AST) were also determined in all of them. Samples were also tested for hepatitis C antibody (anti-HCV), hepatitis B e antigen (HBeAg), and hepatitis B e antibody (anti-HBe) by ELISA. The commercial ELISA kits used were: anti-HCV, Bio-Rad Laboratories, Segrate, Italy; HBeAg and anti-HBe, Radim, Barcelona, Spain. All assay protocols, cut-offs, and result interpretations were carried out according to the manufacturers’ instructions.
PCR-RFLP HBV variants at M552 (nucleotides 741 or 743) were detected by PCR-RFLP. Three separate PCR assays were used to amplify three fragments around position 552 of the polymerase gene. A 274-bp fragment was amplified using the primer pair P2 (50 AAA CCT TCG GAC GGA AAC TGC-30 ) and P4 (50 -CTG GAT CCA GGG TTTAAATGTATA CCC-30 ), and the fragment was digested with FokI restriction enzyme, which cuts the sequence of the wild type YMDD and YVDD variant into 143 bp, 100 bp, and 31 bp and the sequence of the YIDD variant into 243 bp and 31 bp. The 31-bp fragment was not related to the identification of YMDD variants. A 181-bp fragment was amplified using the primer pair P5 (50 -TGG AAT TCA CCT GTA TTC CCA TCC CAT-30 ) and P6 (50 -CAG ACT TGG CCC CCA ATA CCA CAT CGT GCA-30 , which introduces an Alw441 site only into the YVDD mutant product), and the fragment was digested with Alw441 restriction enzyme, which cuts the sequence of the YVDD variant into 158 bp and 23 bp. A 138-bp fragment was amplified using the primer pair P4 (50 -CTG GAT CCA GGG TTT AAA TGT ATA CCC-30 ) and P3 (50 -TTT CCC CCA CTG TTT GGC TTT CAG TAA TAT-30 , which introduces an SspI site only into the YIDD mutant product), and the fragment was digested with SspI restriction enzyme, which cuts the sequence of the YIDD variant into 109 bp and 29 bp. Restriction enzyme SspI and Alw441 cutting sites were introduced into primers P3 and P6, respectively. For DNA extraction, 100 ml of each patient’s serum was mixed with 150 ml of TES buffer containing 10% sodium dodecyl sulfate and proteinase K, and then incubated at 60 8C for 1 hour. After digestion with proteinase K, DNA was extracted by phenol/chloroform/isoamyl alcohol. Extracted DNA was precipitated with absolute ethanol, then washed with 70% ethanol and dissolved in 30 ml of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The DNA was used for three amplification reactions of PCR. Final concentrations in the three PCR reactions were as follows: 20 mmol Tris (pH 8.3), 0.2 mmol KCl, 1.5 mmol MgCl2, 18 mmol NaCl, 0.2 mmol dNTPS, 0.5 mmol each primer, and 2.5 U Taq polymerase per 50 ml of reaction mixture. The amplification conditions in the three PCR reactions all included initial denaturation at 94 8C for 3 minutes, 35 cycles of amplification with denaturation at 94 8C for 35 seconds, primer annealing at 56 8C for 50 seconds, extension of primer at 72 8C for 50 seconds, followed by a final extension at 72 8C for 5 minutes. After amplification, aliquots of the product DNA of the three PCR reactions were digested with three enzymes, respectively, according to the manufacture’s specifications (Shanghai Biology Project Technology Company). The digestion reactions included 15 ml amplified HBV DNA, 10 U
254 enzyme, 10 restriction enzyme digestion buffer 2 ml, and 3 ml sterile ddH2O in a sterile Eppendorf tube. The tube was spun at 500 rpm for 3 seconds. The SspI and Alw441 enzyme reaction mixtures were incubated in a 37 8C water bath for 16 hours. The FokI enzyme reaction mixture was incubated in a 55 8C water bath for 3 hours .The results of PCR-RFLP were analyzed by 8.4% polypropylene acidemide gel electrophoresis.17 The detection limit of the assay was 10 000 copies per ml.
Statistical analysis Chi-square and t2 tests were used with the SPSS 11.5 program for statistical analysis. Data are presented as means stanstandard deviations or, when indicated, as absolute number and percentage. A p value of <0.05 was considered significant.
Results A total of 77 patients were enrolled in this study; 73% of them were male and 27% were female and they were aged between 11 and 50 years (mean 27.4 9.3). Chronic hepatitis B was diagnosed in patients based on HBsAg persistence for more than six months with liver enzymes more than 1.5 times the upper limit of normal and/or HBsAg persistence for more than six months with liver biopsy signs of chronic hepatitis confirmed by a pathologist. None of the patients had been treated with antiviral medications including lamivudine. In these patients ALT, AST, HBeAg, anti-HBe, and anti-HCV tests and detection of YMDD motif mutants were done simultaneously. The patients’ ALT levels were between 28 and 500 IU/l (mean 124.4 73.4) and AST levels were between 22 and 500 IU/l (mean 103.1 81). HBeAg and anti-HBe were positive in 40% and 60% of the patients, respectively. Anti-HCV was negative in all of them. In 46 patients, the diagnosis of chronic hepatitis B was based on the liver biopsy. The liver biopsy scores were between 2 and 13 (mean 5.9 2.4), grade (activity) was between 1 and 9 (mean 3.6 2.3), and stage (fibrosis) between 0 and 3 (mean 1.2 1.1) according to the Knodell necroinflammatory score system.16 PCR was ‘not determined’ in 48% (37/77) of the patients and was ‘sensitive’ in 52% (40/77) of them. No YMDD motif mutations (0) were detected in chronic hepatitis B patients by RFLP.
Discussion HBV is one of the most common infectious diseases in the world. HBV genotypes represent naturally occurring strains of HBV that have evolved over the years and reflect the geographical distribution of HBV throughout the world. Genotype D is most often found in Southern Europe, parts of Central Asia, India, Africa, and the Middle East.18 In Iran, genotype D is the only detected type found in all HBV infected patients.19 Lamivudine is an antiviral drug approved for the treatment of chronic hepatitis B.15 However, the emergence of viral mutants resistant to lamivudine is the main concern
A. Ramezani et al. with this treatment. The cause of lamivudine-resistant HBV was revealed to be the amino acid substitution from leucine to methionine at codon 180 of the B domain (rtL180M) and amino acid substitutions of the YMDD motif from methionine to valine or isoleucine at codon 204 of the C domain (rtM204V or rtM204I) of the reverse transcriptase (rt) region of the polymerase gene.20—24 Data supporting the natural occurrence of lamivudineresistant strains among the population of chronic HBV carriers have been reported recently from Japan and France.23,25 Some researchers have found the YMDD mutational strains in the serum of cases with chronic hepatitis B who did not receive lamivudine treatment.14,23,26 Yan et al. showed that 19 out of 110 chronic hepatitis B untreated cases had YMDD mutations.26 The results from a study in Spain indicate that the prevalence of these mutants is about 4% among Spanish chronic HBV carriers.15 In a study in Japan, mutations were detected in five out of 18 untreated chronic hepatitis B patients.23 In addition, Zhang et al. found that 26.2% of chronic hepatitis B cases had YMDD mutations by genetic chip determination.27 Matsuda et al. reported that a few chronic hepatitis B cases not treated with lamivudine had YMDD mutations.13 Naturally occurring YMDD motif variants were detected at a high rate in a group of lamivudineuntreated inactive HBV carriers in Turkey.28 Other studies have not reported YMDD mutations in non-lamivudine treated hepatitis B patients.22 In a study in China, the YMDD mutants were not detected in any of the five patients before lamivudine treatment.29 Kobayashi et al. found that anti-HBe was positive in all patients with YMDD mutations,23 and Ye et al. also found that anti-HBe was positive in most patients with YMDD mutations and considered that YMDD mutations might occur more easily if mutations took place in the pre-c zone.30 In a study in China, the incidence rate of YMDD mutations was 27.1% in patients with positive HBeAg and 26.7% in patients with negative HBeAg and positive anti-HBe. There was no significant difference between the two groups.31 Lee et al. detected YMDD mutants in eight of 12 HBeAg-positive patients and eight of 16 HBeAg-negative patients before lamivudine treatment.12 In our study, the YMDD motif mutants were not detected in any of the chronic hepatitis B patients before lamivudine treatment despite the liver enzyme levels and the presence of HBeAg or anti-HBe. These results do not accord with some studies20,24 but are in agreement with others.22,29 This low rate may be explained by epidemiological and geographic variations and study conditions such as the number of patients and the method used for detecting the YMDD motif mutants. YMDD mutants have significantly low replication ability. It is thought that the naturally occurring YMDD mutants make up only a very small proportion of the total HBV, and very sensitive investigative tools are required to detect them.32 RFLP often leaves out those samples with mixed virus populations and a low-component virus population, and is suited only for mono-infection.33 Further studies are needed to determine exactly the rate of YMDD motif mutants in lamivudine-untreated chronic hepatitis B patients. In conclusion, although the natural occurrence of YMDD motif mutants in lamivudine-untreated patients with chronic hepatitis B infection has been reported, these mutants were
YMDD mutants in untreated chronic hepatitis B patients not detected in Iranian lamivudine-untreated chronic hepatitis B patients. Further studies with more samples and more sensitive investigative tools should be conducted to determine this rate exactly.
Acknowledgment The authors are grateful to the Infectious Diseases Research Center, Shaheed Beheshti University of Medical Sciences for the financial support of this study. Conflict of interest: No conflict of interest to declare.
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