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Core promoter mutations 3 years after anti-hepatitis B e seroconversion in patients with chronic hepatitis B or hepatitis B and C infection and cancer remission
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2002 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 97, No. 9, 2002 ISSN 0002-9270/02/$22.00 PII S0002-9270(02)04355-1
Core Promoter Mutations 3 Years After Anti–Hepatitis B e Seroconversion in Patients With Chronic Hepatitis B or Hepatitis B and C Infection and Cancer Remission Rosa Zampino, M.D., Aldo Marrone, M.D., Peter Karayiannis, Ph.D., Grazia Cirillo, Ph.D., Emanuele Miraglia del Giudice, M.D., Giovanni Rania, M.D., Riccardo Utili, M.D., and Giuseppe Ruggiero, M.D. Chair of Internal Medicine, Department of Pediatrics, Second University of Naples, Naples, Italy; and Department of Medicine, Imperial College of Science, Technology and Medicine, St. Mary’s Hospital, London, United Kingdom
OBJECTIVES: In this study, we aimed to evaluate the persistence of hepatitis B virus (HBV) DNA and the role of HBV core promoter and precore region mutations in 28 young cancer survivor patients with HBV or HBV and hepatitis C virus (HCV) infections, and persistently normal ALT levels, after spontaneous or interferon (IFN)–induced anti– hepatitis B e (HBe) seroconversion. METHODS: Sera from 15 patients with HBV and 13 with dual HBV-HCV infection were analyzed for the presence of HBV-DNA and HCV-RNA by polymerase chain reaction 3 yr after anti-HBe seroconversion. A total of 21 patients had seroconverted spontaneously and seven did so after IFN treatment. The core promoter and the precore regions were amplified sequenced directly. RESULTS: Among patients with HBV infection, HBV-DNA was detected in five of nine (55%) with spontaneous antiHBe and in all six treated patients (p ⫽ 0.092). In the coinfected patients, four had cleared both HBV-DNA and HCV-RNA, five were HBV-DNA negative/HCV-RNA positive and four had the reverse viral pattern. Among the 15 patients with persistence of HBV-DNA, a 7– base pair nucleotide deletion in the core promoter (1757–1763) was present in seven of 10 patients with spontaneous and in one of five patients with IFN-induced seroconversion (p ⫽ 0.033). The G1896A precore stop codon mutation was never observed. HBV-DNA levels were significantly lower in patients with the core promoter deletion (p ⫽ 0.011). The 7– base pair deletion generated a truncated X protein at amino-acid position 132. CONCLUSIONS: A core promoter deletion after anti-HBe seroconversion was associated with low HBV-DNA levels, probably because of downregulation of pregenomic RNA production and truncation of the X protein. HBV-DNA persistence was a frequent event, even in the absence of
active liver disease. (Am J Gastroenterol 2002;97: 2426 –2431. © 2002 by Am. Coll. of Gastroenterology)
INTRODUCTION Hepatitis B virus (HBV) is one of the major causes of chronic liver disease worldwide (1). During chronic infection, hepatitis B e antigen (HBeAg) to anti-HBe seroconversion can lead either to clearance of serum HBV-DNA (2– 4), or to low level HBV replication, sustained by wild type or mutant viruses. Anti-HBe–positive patients may present with the G1896A substitution in the precore (PC) region abrogating synthesis and secretion of HBeAg (5–7), or with changes in the core promoter region, which can precipitate anti-HBe seroconversion (7–12), and low levels of viral replication (13), in the absence of the PC stop codon variant. The core promoter region plays an important role in HBV replication by directing the transcription of the pregenomic and pre-C RNAs. Mutations in various positions of the core promoter have been described by different authors in patients with diverse clinical presentations, such as chronic hepatitis patients (14), anti-HBe–positive patients (10, 15), asymptomatic carriers (16), patients with fulminant hepatitis (17), patients with hepatocellular carcinoma (HCC) (18), and immunosuppressed patients (19). In vitro studies have demonstrated that mutations in the core promoter and the overlapping X gene influence HBV replication, but also affect X protein function, transcription levels of the pre-C mRNA, HBeAg (19, 20), and HBcAg production (21) ␣-Interferon (IFN) treatment has been shown to induce HBeAg/anti-HBe seroconversion in 33% of cases compared with 12% of the untreated controls (22), but persistence of HBV-DNA in patients who had seroconverted has been reported in several studies (6, 23, 24). Less is known about the residual HBV activity in patients
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with HBV-HCV coinfection. This condition is common in endemic areas for both viruses and is frequent in cancer patients who have received chemotherapy and blood transfusion. Clinical and in vitro studies report a mutual biological interference between HBV and HCV (25–28). Conflicting data exist about a possible effect of HCV on HBV-DNA replication (29, 30). Accordingly, we studied HBV-DNA persistence in young patients with HBV or HBV-HCV infections who had persistently normal ALT levels after spontaneous or IFN-induced anti-HBe seroconversion. We also measured HBVDNA levels and determined the HBV basic core promoter (BCP) and PC region sequences to see whether mutations in these regions were related to HBV-DNA persistence in patients with inactive liver disease.
MATERIALS AND METHODS Study Patients We studied 28 anti-HBe–positive patients (14 men and 14 women; median age 19 yr, range 14 –31 yr) with chronic hepatitis, 15 of whom had chronic HBV infection and 13 chronic HBV-HCV coinfection. Viral infections had been acquired during infancy, when patients were treated for leukemia/lymphoma (17) or solid tumor (11). All patients were initially HBsAg/HBeAg positive and HBV-DNA positive by liquid phase hybridization. Of the 13 anti-HCV positive patients, all but one were HCV-RNA positive. During long-term follow-up, all 28 HBeAg-positive patients seroconverted to anti-HBe, 21 spontaneously and seven after IFN treatment. Median follow-up was 13 yr (range 8 –20 yr), of which 4.5 yr (range 1–16 yr) was after anti-HBe seroconversion. Sera from each patient taken 3 yr after anti-HBe seroconversion were tested for HBV-DNA and HCV-RNA by polymerase chain reaction (PCR) for evaluation of viral replication and for HBV genome analysis. All patients underwent a physical examination, and serum samples were tested for liver function and viral markers. The latter included testing for HBV, HCV, hepatitis D virus, HIV serological markers, HBV-DNA by liquid phase hybridization, and, where indicated, HCV-RNA, every 6 months or more frequently if necessary. In HCV-infected patients, HCV genotype was determined at first observation. Sera were stored at –70°C within 1 h of collection. Liver and spleen Doppler ultrasound examination and testing for serum ␣-fetoprotein were carried out every year. Histological features were scored according to the Knodell Histological Activity Index and to the Scheuer fibrosis scoring system; scores for necroinflammatory changes (grading) and for architectural alterations (staging) were considered separately (31). Eight HBeAg-positive patients (seven with HBV infection and one with HBV-HCV coinfection) were treated with ␣-2a IFN (Roferon, Roche), at a dosage of 5 MU/m2 three times weekly for 12 months. HBV response to treatment was defined on the basis of serum HBV-DNA clearance by
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liquid phase hybridization and HBeAg clearance at the end of treatment and seroconversion to anti-HBe with ALT normalization (ALT ⬍1.5 ⫻ upper normal limit) within an additional 12 months. HCV response to treatment was defined on the basis of sustained serum HCV-RNA negativity and ALT normalization at the end of therapy and after 12 months of follow-up. Anti-HBV and anti-HCV responses were evaluated separately. Serological Tests Markers for HBV, HCV, HDV, and HIV were tested in serum using commercially available immunoenzymatic assays (EIA; Abbott Laboratories, North Chicago, IL, and Ortho Diagnostic Systems, Raritan, NJ). HBV-DNA was assayed during clinical follow-up of patients by liquid phase hybridization (Abbott Laboratories). HBV-DNA quantification was carried out using the Cobas Amplicor HBV Monitor kit (Roche Diagnostics SpA, Milan, Italy). Nucleic Acid Extraction and Amplification For genome analysis, HBV-DNA was extracted from 100 l of serum mixed with 100 l of a digestion solution containing a final concentration of 25 mmol/L sodium acetate, 2.5 mmol/L of ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, 2 mg/ml of proteinase K, and 10 g/ml of yeast transfer RNA as carrier. Digestion was performed at 37°C overnight. The digests were extracted twice with phenol/chloroform, once with chloroform, precipitated with ethanol, and resuspended in 10 l of water. DNA was amplified by PCR and all samples were tested twice. Primers BP1 (5⬘-TCTGTGCCTTCTCATCTG, sense, nt 1554 –1571) and BP2 (5⬘-AATGCTCAGGAGACTCTAAG, antisense, nt 2044 –2025) were used for amplification of HBV-DNA. The reaction was performed in 100 l containing 10 mmol/L of TRIS-HCl pH 8.3, 50 mmol/L of potassium chloride, 200 mol each of dNTP, 2.5 mmol/L of magnesium chloride, and 1 U of Taq polymerase (Applied Biosystem Perkin Elmer, Rome Italy [branch of PE Europe]). Samples were subjected to 35 rounds of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The PCR products were electrophoresed in 1.5% agarose gels and visualized by UV transillumination after staining with ethidium bromide. The PCR sensitivity was ⬎100 copies/ml. HCV-RNA was detected by a qualitative test (PCR Hepatest C, ViennaLab, Vienna, Austria) and HCV genotypes were determined by reverse hybridization line probe assay (INNO LIPA, second generation; Innogenetics, Ghent, Belgium). Sequencing Analysis Amplicons were purified using Qiaquick spin columns (Qiagen, Hilden; Germany) according to the manufacturer’s instructions, and were sequenced directly using an automated capillary sequence reader (Abi Prism 310, Applied Biosystem PE Italia, branch of PE Europe). Sequence analysis was performed using the DNASIS package for Win-
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Table 1. Demographic Data on Chronically Infected Patients Who Seroconverted to Anti-HBe Spontaneously or After IFN Treatment Anti-HBe Seroconversion Spontaneous
Number of patients Mean age ⫾ SD, yr Male/Female Biopsied Liver histology Minimal changes CAH Mean follow-up
After IFN
HBV Infection
HBV-HCV Coinfection
HBV Infection
HBV-HCV Coinfection
9 17.8 ⫾ 2.5 2/7 ND
12 21.5 ⫾ 5.3 7/5 9
6 19.7 ⫾ 5.7 4/2 6
1 20* 1/0 1
4.7
5 4 7.5
2 4 3.2
1 0 5
CAH ⫽ chronic active hepatitis; ND ⫽ not done. * Single patient.
dows 95 (Microsoft, Redmond, WA) and amino acid alignments with the PROSIS one (Hitachi, Tokyo, Japan). Statistical Analysis Statistical analysis of nonparametric data was performed using Fisher’s exact test. Differences in viremia levels among the patients were compared with the Mann-Whitney U test.
RESULTS Clinical, Serological, and Viral Nucleic Acid Findings General characteristics of the study population after antiHBe seroconversion are summarized in Table 1. No significant differences in sex, mean age, and mean duration of follow-up were present among the four groups of patients. During follow-up after anti-HBe seroconversion, liver function tests were normal, with ALT levels ⬍1.5 UNL in all patients. Before anti-HBe seroconversion, 16 patients had undergone liver biopsy that showed a low degree of hepatic damage, from minimal changes to mild chronic hepatitis (Table 1). HCV genotype was determined in all HCV-RNA positive patients. The HCV genotype distribution was 1b in eight patients, 2a and 3a in one patient each, and mixed (1a ⫹ 1b) in two patients. After anti-HBe seroconversion, HBV-DNA was negative by liquid phase hybridization in both untreated and treated patients at all times. In contrast, when sera were tested for HBV-DNA by in-house PCR, we found that among patients
with spontaneous anti-HBe seroconversion, HBV-DNA persisted in five of nine patients (55%) with HBV and in four of 12 (33%) with dual HBV-HCV infection (p ⫽ 0.21; Table 2). Furthermore, during follow-up, spontaneous antiHBs seroconversion was observed in four patients with HBV-HCV infection and in one patient with HBV infection alone (p ⫽ 0.21); the latter patient was still HBV-DNA positive by PCR. In the IFN-treated group of patients who seroconverted to anti-HBe, HBV-DNA was present in all of those with HBV infection alone (six of six, 100%; see Table 2), but was absent in the patient with HBV-HCV coinfection. In the patients infected by HBV alone, the persistence of HBV-DNA after spontaneous anti-HBe seroconversion (55%) was lower than in IFN-treated patients (100%, p ⫽ 0.092). Among the 13 patients with HBV-HCV coinfection, HCV-RNA was present in five of 12 patients (42%) with spontaneous anti-HBe seroconversion and was absent in the one patient with anti-HBe seroconversion after IFN treatment. No patient showed persistence of both HBV-DNA and HCV-RNA, whereas five were HBV-DNA negative/ HCV-RNA positive and four had the reverse viral pattern. Core Promoter–Precore Sequence Analysis and HBV DNA Levels The core promoter and PC regions were analyzed in the 15 patients who were HBV DNA positive by PCR after antiHBe seroconversion, 11 with HBV infection alone (five with spontaneous anti-HBe seroconversion and six after IFN treatment), and four with HBV-HCV coinfection and spon-
Table 2. Persistence of HBV-DNA and HCV RNA 3 yr After Seroconversion to Anti-HBe Anti-HBe Seroconversion Spontaneous HBV infection HBV-DNA persistence HCV RNA persistence Anti-HBs seroconversion p ⫽ 0.092 (* vs ‡); p ⫽ 0.21 (* vs †).
5/9 (55%)* 1/9 (11%)
After IFN HBV-HCV Coinfection 4/12 (33%)† 5/12 (42%) 4/12 (33%)
HBV Infection 6/6 (100%)‡ 0/6
HBV-HCV Coinfection 0/1 0/1 0/1
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Figure 1. Nucleotide sequences of the HBV core promoter region in 15 patients who were positive for HBV-DNA by PCR, 3 yr after anti-HBe seroconversion. Top line shows wild type HBV sequence (46); –identical nucleotides/deleted nucleotides. Sequences are from patients infected with HBV, with spontaneous anti-HBe seroconversion (patients 1–5), HBV-HCV coinfected with spontaneous anti-HBe seroconversion (patients 6 –9), and HBV infected after IFN-induced anti-HBe seroconversion (patients 10 –15).
taneous anti-HBe seroconversion. The distribution of mutations in the core promoter region are summarized in Figure 1. Nucleotide deletions in the BCP affecting positions 1757– 1763 were present in four of five HBV and in three of four HBV-HCV positive patients who had spontaneously seroconverted to anti-HBe (Fig. 1). The double mutation A1762T/G1764A was observed in two patients, one with HBV infection alone and one with HBV-HCV coinfection. Among IFN-treated patients with HBV infection alone, the double A1762T/G1764A mutation and an associated mutation at position 1753 (T to C) were observed in two cases; the deletion at positions 1757–1763 was present in 1 case and wild type sequences were present in the other 3. Deletions were present more frequently in patients with spontaneous seroconversion than in patients with IFN-induced seroconversion (p ⫽ 0.033). The G1896A precore stop codon mutation was not seen in any of these patients. Median HBV DNA levels were 21.100 cp/ml (range ⬍200 to ⬎200,000). In particular, patients with the 1757– 1763 deletion or the BCP A1762T/G1764A mutations had HBV-DNA levels between ⬍200 and 37,800 cp/ml, whereas those with wild type sequences had HBV-DNA levels ⬎200,000 cp/ml (p ⫽ 0.011). The A1762T/G1764A mutations led to aminoacid substitutions at positions 130 (lysine to metheonine) and 131 (valine to isoleucine). The 7– base pair (bp) deletion generated a stop codon leading to a truncated X protein at position 132. The aminoacids 129 (leucine), 130 (lysine), and 131 (valine) were changed to proline, leucine, and tyrosine, respectively.
DISCUSSION During the natural course of chronic hepatitis B or after IFN treatment, anti-HBe seroconversion is generally accompanied by a biochemical improvement and decrease in viral
replication. Several studies have shown, however, that this is not necessarily accompanied by HBV-DNA clearance (6, 23, 32). Thus, patients can harbor replicating HBV after anti-HBe seroconversion and sometimes even after antiHBs seroconversion (33, 34). Some authors have found low levels of HBV-DNA detectable in about 30% of anti-HBe– positive asymptomatic carriers (35, 36). Our findings suggest that the immunological response leading to spontaneous seroconversion may be stronger than that induced by IFN treatment, as HBV-DNA persistence was observed in 55% of patients with spontaneous anti-HBe seroconversion and in 100% of those who seroconverted after IFN treatment. The small number of patients and the short follow-up do not allow definitive conclusions to be drawn on this issue. Several authors have argued that mutual HBV-HCV interference can influence the course of liver disease and the extent of viral clearance (25–28, 37). We observed an equal distribution of patients who cleared HBV in the presence of HCV (42%) and those who cleared HCV in the presence of HBV (33%). We can confirm that viral interference can occur, but there is no evidence of one virus prevailing over the other. The patients whom we studied acquired HBV infection or HBV-HCV coinfection during an immunosuppressed state. It seems that in our patients with HBV infection alone, the behavior of HBV was not dissimilar to that observed by other investigators in immunocompentent patients (6, 38). This shows that the restoration of the immune response after immunosuppressive treatment can control the natural course of HBV infection. None of our patients presented with the PC stop codon mutation (G1896A), thus indicating that natural immunity and IFN pressure had no effect on the emergence of the “e-minus” strain in this group of patients during the follow-up period. As the e-minus mutation seems to develop in
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the late phase of the natural history of chronic hepatitis B (13), the young age and relatively short duration of the disease in our population might explain this finding. In contrast, sequence analysis of the BCP region revealed that patients with spontaneous anti-HBe seroconversion had a 7-bp deletion at positions 1757–1763, at a significantly higher rate than among IFN-treated patients. Interestingly, HBV-DNA levels were significantly lower in patients with the 7-bp deletion or the A1762T/G1764A double mutation compared to those with wild type sequences. Of note is the observation that no such variants were detected in a subset of our patients (IFN treated) who were studied during the HBeAg positive phase with high HBV-DNA serum levels (39) The 7-bp deletion in the BCP, which overlaps with the X gene open reading frame, might affect BCP function and lead to the production of a truncated X protein. We can therefore speculate that the 7-bp deletion, apart from BCP function, may also affect pre-C mRNA production and expression, and that any transactivating action the X protein may be exerted, as previously reported for other deletions in this region. Only in vitro studies would clarify this point further. These HBV mutation patterns, which are associated with low HBV-DNA levels and have been observed in patients with HBV infection alone or HBV-HCV coinfection, do not seem to be influenced by viral interference. Mutations that affect the second AT rich region of the BCP have been detected in association with low viremia levels and HBeAg negative phenotype (15). An 8-bp deletion in the BCP involving positions 1762 and 1764 has been suggested as having a negative effect on pre-C mRNA production (40). Experimental data have shown reduced pre-C mRNA synthesis and a low expression of HBeAg in vitro using constructs with this 8-bp deletion in the X gene (19). Kohno et al. (41) have demonstrated in in-vitro studies that an 8-bp deletion at positions 1768 –1775 can cause the formation of a truncated X protein with a reduced transactivating function. Horikita et al. (16) have identified an association between the 8-bp BCP deletion and low HBV DNA levels, and Preisler-Adams et al. (42) have described this association even in patients with no serological evidence of HBV infection. However, other studies have demonstrated an increase in viral replication when the double A1762T/G1764A mutation occurs (43– 45). In this respect, Baumert et al. (17) have demonstrated that substitutions at positions 1768 and 1770 identified in the BCP isolated from a HBV strain associated with fulminant hepatitis B resulted in enhanced viral replication by in vitro transfection studies. Despite HBV-DNA persistence after anti-HBe seroconversion and the appearance of mutated HBV strains, the clinical course of chronic hepatitis was mild, with normal ALT levels, in all patients during the follow-up period. It is important, however, to continue monitoring this population, to detect any possible reactivation of liver disease and to determine whether the course of chronic hepatitis evolves differently in patients with wild type or mutant viruses. The
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present study showed that deletions in HBV core promoter region led to a truncated X protein, which in turn may have been associated with persistence of HBV-DNA at low levels after anti-HBe seroconversion, and in the absence of active liver disease. Reprint requests and correspondence: Rosa Zampino, M.D., Istituto di Terapia Medica, Seconda Universita` degli Studi di Napoli, Via Cotugno, 1 (c/o Ospedale Gesu`e Maria), 80135 Napoli, Italy. Received Nov. 19, 2001; accepted Apr. 1, 2002.
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Vol. 97, No. 9, 2002 ISSN 0002-9270/02/$22.00 PII S0002-9270(02)04355-1
Core Promoter Mutations 3 Years After Anti–Hepatitis B e Seroconversion in Patients With Chronic Hepatitis B or Hepatitis B and C Infection and Cancer Remission Rosa Zampino, M.D., Aldo Marrone, M.D., Peter Karayiannis, Ph.D., Grazia Cirillo, Ph.D., Emanuele Miraglia del Giudice, M.D., Giovanni Rania, M.D., Riccardo Utili, M.D., and Giuseppe Ruggiero, M.D. Chair of Internal Medicine, Department of Pediatrics, Second University of Naples, Naples, Italy; and Department of Medicine, Imperial College of Science, Technology and Medicine, St. Mary’s Hospital, London, United Kingdom
OBJECTIVES: In this study, we aimed to evaluate the persistence of hepatitis B virus (HBV) DNA and the role of HBV core promoter and precore region mutations in 28 young cancer survivor patients with HBV or HBV and hepatitis C virus (HCV) infections, and persistently normal ALT levels, after spontaneous or interferon (IFN)–induced anti– hepatitis B e (HBe) seroconversion. METHODS: Sera from 15 patients with HBV and 13 with dual HBV-HCV infection were analyzed for the presence of HBV-DNA and HCV-RNA by polymerase chain reaction 3 yr after anti-HBe seroconversion. A total of 21 patients had seroconverted spontaneously and seven did so after IFN treatment. The core promoter and the precore regions were amplified sequenced directly. RESULTS: Among patients with HBV infection, HBV-DNA was detected in five of nine (55%) with spontaneous antiHBe and in all six treated patients (p ⫽ 0.092). In the coinfected patients, four had cleared both HBV-DNA and HCV-RNA, five were HBV-DNA negative/HCV-RNA positive and four had the reverse viral pattern. Among the 15 patients with persistence of HBV-DNA, a 7– base pair nucleotide deletion in the core promoter (1757–1763) was present in seven of 10 patients with spontaneous and in one of five patients with IFN-induced seroconversion (p ⫽ 0.033). The G1896A precore stop codon mutation was never observed. HBV-DNA levels were significantly lower in patients with the core promoter deletion (p ⫽ 0.011). The 7– base pair deletion generated a truncated X protein at amino-acid position 132. CONCLUSIONS: A core promoter deletion after anti-HBe seroconversion was associated with low HBV-DNA levels, probably because of downregulation of pregenomic RNA production and truncation of the X protein. HBV-DNA persistence was a frequent event, even in the absence of
active liver disease. (Am J Gastroenterol 2002;97: 2426 –2431. © 2002 by Am. Coll. of Gastroenterology)
INTRODUCTION Hepatitis B virus (HBV) is one of the major causes of chronic liver disease worldwide (1). During chronic infection, hepatitis B e antigen (HBeAg) to anti-HBe seroconversion can lead either to clearance of serum HBV-DNA (2– 4), or to low level HBV replication, sustained by wild type or mutant viruses. Anti-HBe–positive patients may present with the G1896A substitution in the precore (PC) region abrogating synthesis and secretion of HBeAg (5–7), or with changes in the core promoter region, which can precipitate anti-HBe seroconversion (7–12), and low levels of viral replication (13), in the absence of the PC stop codon variant. The core promoter region plays an important role in HBV replication by directing the transcription of the pregenomic and pre-C RNAs. Mutations in various positions of the core promoter have been described by different authors in patients with diverse clinical presentations, such as chronic hepatitis patients (14), anti-HBe–positive patients (10, 15), asymptomatic carriers (16), patients with fulminant hepatitis (17), patients with hepatocellular carcinoma (HCC) (18), and immunosuppressed patients (19). In vitro studies have demonstrated that mutations in the core promoter and the overlapping X gene influence HBV replication, but also affect X protein function, transcription levels of the pre-C mRNA, HBeAg (19, 20), and HBcAg production (21) ␣-Interferon (IFN) treatment has been shown to induce HBeAg/anti-HBe seroconversion in 33% of cases compared with 12% of the untreated controls (22), but persistence of HBV-DNA in patients who had seroconverted has been reported in several studies (6, 23, 24). Less is known about the residual HBV activity in patients
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with HBV-HCV coinfection. This condition is common in endemic areas for both viruses and is frequent in cancer patients who have received chemotherapy and blood transfusion. Clinical and in vitro studies report a mutual biological interference between HBV and HCV (25–28). Conflicting data exist about a possible effect of HCV on HBV-DNA replication (29, 30). Accordingly, we studied HBV-DNA persistence in young patients with HBV or HBV-HCV infections who had persistently normal ALT levels after spontaneous or IFN-induced anti-HBe seroconversion. We also measured HBVDNA levels and determined the HBV basic core promoter (BCP) and PC region sequences to see whether mutations in these regions were related to HBV-DNA persistence in patients with inactive liver disease.
MATERIALS AND METHODS Study Patients We studied 28 anti-HBe–positive patients (14 men and 14 women; median age 19 yr, range 14 –31 yr) with chronic hepatitis, 15 of whom had chronic HBV infection and 13 chronic HBV-HCV coinfection. Viral infections had been acquired during infancy, when patients were treated for leukemia/lymphoma (17) or solid tumor (11). All patients were initially HBsAg/HBeAg positive and HBV-DNA positive by liquid phase hybridization. Of the 13 anti-HCV positive patients, all but one were HCV-RNA positive. During long-term follow-up, all 28 HBeAg-positive patients seroconverted to anti-HBe, 21 spontaneously and seven after IFN treatment. Median follow-up was 13 yr (range 8 –20 yr), of which 4.5 yr (range 1–16 yr) was after anti-HBe seroconversion. Sera from each patient taken 3 yr after anti-HBe seroconversion were tested for HBV-DNA and HCV-RNA by polymerase chain reaction (PCR) for evaluation of viral replication and for HBV genome analysis. All patients underwent a physical examination, and serum samples were tested for liver function and viral markers. The latter included testing for HBV, HCV, hepatitis D virus, HIV serological markers, HBV-DNA by liquid phase hybridization, and, where indicated, HCV-RNA, every 6 months or more frequently if necessary. In HCV-infected patients, HCV genotype was determined at first observation. Sera were stored at –70°C within 1 h of collection. Liver and spleen Doppler ultrasound examination and testing for serum ␣-fetoprotein were carried out every year. Histological features were scored according to the Knodell Histological Activity Index and to the Scheuer fibrosis scoring system; scores for necroinflammatory changes (grading) and for architectural alterations (staging) were considered separately (31). Eight HBeAg-positive patients (seven with HBV infection and one with HBV-HCV coinfection) were treated with ␣-2a IFN (Roferon, Roche), at a dosage of 5 MU/m2 three times weekly for 12 months. HBV response to treatment was defined on the basis of serum HBV-DNA clearance by
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liquid phase hybridization and HBeAg clearance at the end of treatment and seroconversion to anti-HBe with ALT normalization (ALT ⬍1.5 ⫻ upper normal limit) within an additional 12 months. HCV response to treatment was defined on the basis of sustained serum HCV-RNA negativity and ALT normalization at the end of therapy and after 12 months of follow-up. Anti-HBV and anti-HCV responses were evaluated separately. Serological Tests Markers for HBV, HCV, HDV, and HIV were tested in serum using commercially available immunoenzymatic assays (EIA; Abbott Laboratories, North Chicago, IL, and Ortho Diagnostic Systems, Raritan, NJ). HBV-DNA was assayed during clinical follow-up of patients by liquid phase hybridization (Abbott Laboratories). HBV-DNA quantification was carried out using the Cobas Amplicor HBV Monitor kit (Roche Diagnostics SpA, Milan, Italy). Nucleic Acid Extraction and Amplification For genome analysis, HBV-DNA was extracted from 100 l of serum mixed with 100 l of a digestion solution containing a final concentration of 25 mmol/L sodium acetate, 2.5 mmol/L of ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, 2 mg/ml of proteinase K, and 10 g/ml of yeast transfer RNA as carrier. Digestion was performed at 37°C overnight. The digests were extracted twice with phenol/chloroform, once with chloroform, precipitated with ethanol, and resuspended in 10 l of water. DNA was amplified by PCR and all samples were tested twice. Primers BP1 (5⬘-TCTGTGCCTTCTCATCTG, sense, nt 1554 –1571) and BP2 (5⬘-AATGCTCAGGAGACTCTAAG, antisense, nt 2044 –2025) were used for amplification of HBV-DNA. The reaction was performed in 100 l containing 10 mmol/L of TRIS-HCl pH 8.3, 50 mmol/L of potassium chloride, 200 mol each of dNTP, 2.5 mmol/L of magnesium chloride, and 1 U of Taq polymerase (Applied Biosystem Perkin Elmer, Rome Italy [branch of PE Europe]). Samples were subjected to 35 rounds of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The PCR products were electrophoresed in 1.5% agarose gels and visualized by UV transillumination after staining with ethidium bromide. The PCR sensitivity was ⬎100 copies/ml. HCV-RNA was detected by a qualitative test (PCR Hepatest C, ViennaLab, Vienna, Austria) and HCV genotypes were determined by reverse hybridization line probe assay (INNO LIPA, second generation; Innogenetics, Ghent, Belgium). Sequencing Analysis Amplicons were purified using Qiaquick spin columns (Qiagen, Hilden; Germany) according to the manufacturer’s instructions, and were sequenced directly using an automated capillary sequence reader (Abi Prism 310, Applied Biosystem PE Italia, branch of PE Europe). Sequence analysis was performed using the DNASIS package for Win-
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Table 1. Demographic Data on Chronically Infected Patients Who Seroconverted to Anti-HBe Spontaneously or After IFN Treatment Anti-HBe Seroconversion Spontaneous
Number of patients Mean age ⫾ SD, yr Male/Female Biopsied Liver histology Minimal changes CAH Mean follow-up
After IFN
HBV Infection
HBV-HCV Coinfection
HBV Infection
HBV-HCV Coinfection
9 17.8 ⫾ 2.5 2/7 ND
12 21.5 ⫾ 5.3 7/5 9
6 19.7 ⫾ 5.7 4/2 6
1 20* 1/0 1
4.7
5 4 7.5
2 4 3.2
1 0 5
CAH ⫽ chronic active hepatitis; ND ⫽ not done. * Single patient.
dows 95 (Microsoft, Redmond, WA) and amino acid alignments with the PROSIS one (Hitachi, Tokyo, Japan). Statistical Analysis Statistical analysis of nonparametric data was performed using Fisher’s exact test. Differences in viremia levels among the patients were compared with the Mann-Whitney U test.
RESULTS Clinical, Serological, and Viral Nucleic Acid Findings General characteristics of the study population after antiHBe seroconversion are summarized in Table 1. No significant differences in sex, mean age, and mean duration of follow-up were present among the four groups of patients. During follow-up after anti-HBe seroconversion, liver function tests were normal, with ALT levels ⬍1.5 UNL in all patients. Before anti-HBe seroconversion, 16 patients had undergone liver biopsy that showed a low degree of hepatic damage, from minimal changes to mild chronic hepatitis (Table 1). HCV genotype was determined in all HCV-RNA positive patients. The HCV genotype distribution was 1b in eight patients, 2a and 3a in one patient each, and mixed (1a ⫹ 1b) in two patients. After anti-HBe seroconversion, HBV-DNA was negative by liquid phase hybridization in both untreated and treated patients at all times. In contrast, when sera were tested for HBV-DNA by in-house PCR, we found that among patients
with spontaneous anti-HBe seroconversion, HBV-DNA persisted in five of nine patients (55%) with HBV and in four of 12 (33%) with dual HBV-HCV infection (p ⫽ 0.21; Table 2). Furthermore, during follow-up, spontaneous antiHBs seroconversion was observed in four patients with HBV-HCV infection and in one patient with HBV infection alone (p ⫽ 0.21); the latter patient was still HBV-DNA positive by PCR. In the IFN-treated group of patients who seroconverted to anti-HBe, HBV-DNA was present in all of those with HBV infection alone (six of six, 100%; see Table 2), but was absent in the patient with HBV-HCV coinfection. In the patients infected by HBV alone, the persistence of HBV-DNA after spontaneous anti-HBe seroconversion (55%) was lower than in IFN-treated patients (100%, p ⫽ 0.092). Among the 13 patients with HBV-HCV coinfection, HCV-RNA was present in five of 12 patients (42%) with spontaneous anti-HBe seroconversion and was absent in the one patient with anti-HBe seroconversion after IFN treatment. No patient showed persistence of both HBV-DNA and HCV-RNA, whereas five were HBV-DNA negative/ HCV-RNA positive and four had the reverse viral pattern. Core Promoter–Precore Sequence Analysis and HBV DNA Levels The core promoter and PC regions were analyzed in the 15 patients who were HBV DNA positive by PCR after antiHBe seroconversion, 11 with HBV infection alone (five with spontaneous anti-HBe seroconversion and six after IFN treatment), and four with HBV-HCV coinfection and spon-
Table 2. Persistence of HBV-DNA and HCV RNA 3 yr After Seroconversion to Anti-HBe Anti-HBe Seroconversion Spontaneous HBV infection HBV-DNA persistence HCV RNA persistence Anti-HBs seroconversion p ⫽ 0.092 (* vs ‡); p ⫽ 0.21 (* vs †).
5/9 (55%)* 1/9 (11%)
After IFN HBV-HCV Coinfection 4/12 (33%)† 5/12 (42%) 4/12 (33%)
HBV Infection 6/6 (100%)‡ 0/6
HBV-HCV Coinfection 0/1 0/1 0/1
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Figure 1. Nucleotide sequences of the HBV core promoter region in 15 patients who were positive for HBV-DNA by PCR, 3 yr after anti-HBe seroconversion. Top line shows wild type HBV sequence (46); –identical nucleotides/deleted nucleotides. Sequences are from patients infected with HBV, with spontaneous anti-HBe seroconversion (patients 1–5), HBV-HCV coinfected with spontaneous anti-HBe seroconversion (patients 6 –9), and HBV infected after IFN-induced anti-HBe seroconversion (patients 10 –15).
taneous anti-HBe seroconversion. The distribution of mutations in the core promoter region are summarized in Figure 1. Nucleotide deletions in the BCP affecting positions 1757– 1763 were present in four of five HBV and in three of four HBV-HCV positive patients who had spontaneously seroconverted to anti-HBe (Fig. 1). The double mutation A1762T/G1764A was observed in two patients, one with HBV infection alone and one with HBV-HCV coinfection. Among IFN-treated patients with HBV infection alone, the double A1762T/G1764A mutation and an associated mutation at position 1753 (T to C) were observed in two cases; the deletion at positions 1757–1763 was present in 1 case and wild type sequences were present in the other 3. Deletions were present more frequently in patients with spontaneous seroconversion than in patients with IFN-induced seroconversion (p ⫽ 0.033). The G1896A precore stop codon mutation was not seen in any of these patients. Median HBV DNA levels were 21.100 cp/ml (range ⬍200 to ⬎200,000). In particular, patients with the 1757– 1763 deletion or the BCP A1762T/G1764A mutations had HBV-DNA levels between ⬍200 and 37,800 cp/ml, whereas those with wild type sequences had HBV-DNA levels ⬎200,000 cp/ml (p ⫽ 0.011). The A1762T/G1764A mutations led to aminoacid substitutions at positions 130 (lysine to metheonine) and 131 (valine to isoleucine). The 7– base pair (bp) deletion generated a stop codon leading to a truncated X protein at position 132. The aminoacids 129 (leucine), 130 (lysine), and 131 (valine) were changed to proline, leucine, and tyrosine, respectively.
DISCUSSION During the natural course of chronic hepatitis B or after IFN treatment, anti-HBe seroconversion is generally accompanied by a biochemical improvement and decrease in viral
replication. Several studies have shown, however, that this is not necessarily accompanied by HBV-DNA clearance (6, 23, 32). Thus, patients can harbor replicating HBV after anti-HBe seroconversion and sometimes even after antiHBs seroconversion (33, 34). Some authors have found low levels of HBV-DNA detectable in about 30% of anti-HBe– positive asymptomatic carriers (35, 36). Our findings suggest that the immunological response leading to spontaneous seroconversion may be stronger than that induced by IFN treatment, as HBV-DNA persistence was observed in 55% of patients with spontaneous anti-HBe seroconversion and in 100% of those who seroconverted after IFN treatment. The small number of patients and the short follow-up do not allow definitive conclusions to be drawn on this issue. Several authors have argued that mutual HBV-HCV interference can influence the course of liver disease and the extent of viral clearance (25–28, 37). We observed an equal distribution of patients who cleared HBV in the presence of HCV (42%) and those who cleared HCV in the presence of HBV (33%). We can confirm that viral interference can occur, but there is no evidence of one virus prevailing over the other. The patients whom we studied acquired HBV infection or HBV-HCV coinfection during an immunosuppressed state. It seems that in our patients with HBV infection alone, the behavior of HBV was not dissimilar to that observed by other investigators in immunocompentent patients (6, 38). This shows that the restoration of the immune response after immunosuppressive treatment can control the natural course of HBV infection. None of our patients presented with the PC stop codon mutation (G1896A), thus indicating that natural immunity and IFN pressure had no effect on the emergence of the “e-minus” strain in this group of patients during the follow-up period. As the e-minus mutation seems to develop in
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the late phase of the natural history of chronic hepatitis B (13), the young age and relatively short duration of the disease in our population might explain this finding. In contrast, sequence analysis of the BCP region revealed that patients with spontaneous anti-HBe seroconversion had a 7-bp deletion at positions 1757–1763, at a significantly higher rate than among IFN-treated patients. Interestingly, HBV-DNA levels were significantly lower in patients with the 7-bp deletion or the A1762T/G1764A double mutation compared to those with wild type sequences. Of note is the observation that no such variants were detected in a subset of our patients (IFN treated) who were studied during the HBeAg positive phase with high HBV-DNA serum levels (39) The 7-bp deletion in the BCP, which overlaps with the X gene open reading frame, might affect BCP function and lead to the production of a truncated X protein. We can therefore speculate that the 7-bp deletion, apart from BCP function, may also affect pre-C mRNA production and expression, and that any transactivating action the X protein may be exerted, as previously reported for other deletions in this region. Only in vitro studies would clarify this point further. These HBV mutation patterns, which are associated with low HBV-DNA levels and have been observed in patients with HBV infection alone or HBV-HCV coinfection, do not seem to be influenced by viral interference. Mutations that affect the second AT rich region of the BCP have been detected in association with low viremia levels and HBeAg negative phenotype (15). An 8-bp deletion in the BCP involving positions 1762 and 1764 has been suggested as having a negative effect on pre-C mRNA production (40). Experimental data have shown reduced pre-C mRNA synthesis and a low expression of HBeAg in vitro using constructs with this 8-bp deletion in the X gene (19). Kohno et al. (41) have demonstrated in in-vitro studies that an 8-bp deletion at positions 1768 –1775 can cause the formation of a truncated X protein with a reduced transactivating function. Horikita et al. (16) have identified an association between the 8-bp BCP deletion and low HBV DNA levels, and Preisler-Adams et al. (42) have described this association even in patients with no serological evidence of HBV infection. However, other studies have demonstrated an increase in viral replication when the double A1762T/G1764A mutation occurs (43– 45). In this respect, Baumert et al. (17) have demonstrated that substitutions at positions 1768 and 1770 identified in the BCP isolated from a HBV strain associated with fulminant hepatitis B resulted in enhanced viral replication by in vitro transfection studies. Despite HBV-DNA persistence after anti-HBe seroconversion and the appearance of mutated HBV strains, the clinical course of chronic hepatitis was mild, with normal ALT levels, in all patients during the follow-up period. It is important, however, to continue monitoring this population, to detect any possible reactivation of liver disease and to determine whether the course of chronic hepatitis evolves differently in patients with wild type or mutant viruses. The
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present study showed that deletions in HBV core promoter region led to a truncated X protein, which in turn may have been associated with persistence of HBV-DNA at low levels after anti-HBe seroconversion, and in the absence of active liver disease. Reprint requests and correspondence: Rosa Zampino, M.D., Istituto di Terapia Medica, Seconda Universita` degli Studi di Napoli, Via Cotugno, 1 (c/o Ospedale Gesu`e Maria), 80135 Napoli, Italy. Received Nov. 19, 2001; accepted Apr. 1, 2002.
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