1 Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden
2 Microbiology and Tumor Biology Center, Karolinska Institutet, SE-171 77 Stockholm, Sweden
3 Research Center for Gastroenterology and Liver Diseases, Shaheed Beheshti University of Medical Sciences, Taleghani Hospital, Tabnak Avenue, 19857 Tehran, Iran
Correspondence
Lars O. Magnius
Lars.Magnius{at}smi.ki.se
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences and primer sequences reported in this study are DQ102486DQ102557.
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INTRODUCTION |
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Mutations in the BCP have also been reported in patients with e-CHB or fulminant hepatitis (Okamoto et al., 1994; Kurosaki et al., 1996
; Kidd-Ljunggren et al., 1997
; Chan et al., 1999
). The most commonly detected mutations within the BCP region are an A1762T transversion and a G1764A transition. In vitro and in vivo studies have shown that these mutations decrease the transcription of pre-core mRNA and hence the secretion of HBeAg (Buckwold et al., 1996
; Günther et al., 1998
; Laras et al., 2002
).
The prevalence of pre-core and BCP mutations in HBV strains from Iranian e-CHB patients and asymptomatic carriers (ASCs) and their association with HBV DNA and aminotransferase levels are investigated in this study.
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METHODS |
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Based on clinical criteria, the patients were grouped as follows: (i) e-CHB patients with elevated aminotransferase levels above twice the normal range for more than 6 months or clinical or histological signs of chronic liver disease; and (ii) ASCs without signs of disease activity, as determined by elevated ALT or AST levels. Thirty-five patients were categorized as e-CHB, whereas 62 were classified as ASCs. All patients had given their consent for enrolling in the study.
Identification of HBV basic core promoter and pre-core mutations and HBV genotypes.
HBV DNA was extracted from 200 µl serum with a QIAamp DNA mini kit (Qiagen). Pre-core amplification was carried out with primers hepA and hep66 and was nested with primers hepA and hep68 (Arauz-Ruiz et al., 1997). For core promoter amplification, hep64 and hep70 were used in the first round of PCR and hep64 and hep54 were used in the nested PCR. Amplification of the S region was done by using primers hep75 and 73b for the first PCR and hep3/hep33 and hep4/hep34 for nesting, respectively (Norder et al., 1992
). PCR amplificates were purified with a GFX PCR DNA and gel-band purification kit (Amersham Biosciences). Purified products were used as templates in the sequencing reaction using the dideoxynucleotide chain-termination method with an ABI PRISM BigDye terminator cycle-sequencing reaction kit (version 3; Applied Biosystems) and the primers used in the PCR were used as sequencing primers. All amplification products were sequenced bi-directionally. An ABI PRISM 3100 genetic analyser (Applied Biosystems) was used for electrophoresis and data collection. Sequences obtained were edited by using the SeqMan program in the LASERGENE package (DNASTAR).
HBV DNA quantification.
HBV DNA quantification was carried out by using Affigene HBV VL kits (Sangtec Molecular Diagnostics) according to the manufacturer's instructions. In each set of PCR-ELISA, five different standards from the kit with 0, 12, 60, 600, 6x103 and 6x105 copies ml1 were run in parallel with the samples. The limit of detection was 500 copies ml1 and the quantification range was 2x1034x107 copies ml1.
Statistical analysis.
Statistical analysis was performed with 2 or Fisher's exact tests and with the independent t-test for continuous variables using the SPSS version 11.0 software package. P values (two-tailed) less than 0·05 were considered statistically significant. Logarithms of HBV DNA levels were used for the statistical comparisons.
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RESULTS |
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Pre-core mutations
The pre-core region of the infecting strain could be amplified from serum of 80 % of the patients (Table 2). A high proportion (82 %) of the patients harboured A1896 pre-core stop mutants, whereas 37 % harboured G1899A mutants with no significant differences in frequencies between e-CHB patients and ASCs. The A1899 mutation was mostly accompanied by the A1896 mutation (Fig. 1
). There was no difference in mean age between patients with A1896 mutants and those without, but the patients with A1899 mutants were older than the other patients (43 vs 35 years; P=0·014). There was a slightly, although not significantly, higher mean level of ALT in e-CHB patients with pre-core stop mutants than in patients without these mutants (190 vs 135 IU). All strains had T at nucleotide positions 1850 and 1858, which is as expected for genotype D.
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Core promoter mutations
The core promoter regions of the infecting strains could be sequenced from serum for 74 % of the patients (Table 2). The sequences of the region bound by nt 1742 and 1791 for these strains are shown in Fig. 1
. A high proportion (81 %) of the patients had strains with at least one nucleotide substitution in the BCP region. The most common mutation was C1766G (38 % patients), followed by G1764A (32 %), G1764T (31 %), A1762T (29 %) and T1753C (20 %), with no significant difference in frequencies between e-CHB patients and ASCs; a G1757A substitution was found in 71 % of the strains and was considered to be a genotype D wild-type substitution.
Nineteen patients (26 %) had strains with T1762A1764 mutations in the core promoter regions. The T1762A1764 double mutation was found more frequently in conjunction with G1757 than with A1757 (Table 4; P<0·001, Fisher's exact test). The 12 patients with this double mutation plus G1757 had the highest virus loads and also significantly higher virus loads than six of seven patients with this double mutation plus A1757 [105·2±1·8 vs 103·2±0·8 copies ml1; P=0·004 (Table 3
)]. One patient in the latter group had deletions in the internal part of the core region and had a high virus titre (107·6 copies ml1) and was not included in the comparison. Patients with G1757 were older than patients with A1757 (50 vs 29 years; P=0·002). A mutation to C1753 was significantly more often present in strains with T1762A1764 than in strains lacking this double mutation, irrespective of A/G1757 (P<0·001). Nine patients (three e-CHB and six ASCs) harbouring strains with the mutational pattern C1753T1762A1764 accompanied by G1757 had higher HBV DNA levels (105·2 vs 103·3 copies ml1) and were older (52 vs 28 years) than the three patients, all ASCs, with the pattern C1753T1762A1764 accompanied by A1757. This difference in age was significant (P=0·003).
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Two e-CHB patients had the triple mutational pattern T1762T1766A1768 (Fig. 1). One of them had a high virus load, 105·7 copies ml1. None of the ASCs had this mutational pattern.
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DISCUSSION |
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Despite a high prevalence of hepatitis B in the Middle East, few studies have been conducted on the relative role of pre-core and core promoter mutants in e-CHB patients in this region. In a recent study of 23 HBV strains all belonging to genotype D from Iranian anti-HBe-positive patients, 56 % were reported to have A1896 pre-core mutations (Amini-Bavil-Olyaee et al., 2005).
In our study, the overall prevalence of pre-core mutants was 87 %, including the two other mutants in the pre-core region, one of which abolished the pre-core start codon. A similar prevalence of pre-core mutants has been reported in Turkey (Bozdayi et al., 1999). The e-CHB patients with A1896 mutants did not have higher levels of HBV DNA or aminotransferases compared with the e-CHB patients who lacked these mutants, which is in agreement with other studies (Okamoto et al., 1990
; Takeda et al., 1990
; Gandhe et al., 2003
). There are, however, studies showing that e-CHB with pre-core mutants frequently take a more aggressive course, with the development of cirrhosis and/or hepatocellular carcinoma (HCC) (Bonino et al., 1986
; Brunetto et al., 1991
; Naoumov et al., 1992
).
The A1899 mutant, detected in 37 % of patients, has been associated with increased severity of liver disease (Carman et al., 1989; Tillmann et al., 1995
; Chan et al., 2000
). In our study, patients with A1899 mutants were older than the other patients, suggesting that these mutants evolve in patients with a longer history of infection and who therefore will have more advanced liver disease more frequently. In a study from Spain, all e-CHB patients with T1762 and A1899 mutations associated with A1764 and A1896 mutations had liver cirrhosis and it was concluded that the increased risk of severe liver disease was due to enhanced HBV replication (Jardi et al., 2004
). In our study, both T1762 and A1899 mutants were found in older patients, which may explain why they have been found more frequently in cirrhotic patients in the Spanish study.
Eighty-one per cent of our patients harboured strains with at least one substitution in the region bound by nt 1742 and 1791, which is important in the control of pre-C RNA transcription (Yuh et al., 1992). Clinical studies have correlated core promoter mutants with virus load, liver disease and response to antiviral therapy (Chan et al., 1999
; Baptista et al., 1999
; Erhardt et al., 2000
). Most of these have focused on T1762A1764 mutants and have shown an association between these mutants and the severity of liver disease (Sato et al., 1995
; Baumert et al., 1996
; Scaglioni et al., 1997
; Kao et al., 2003
). In our study, the T1762A1764 double mutant was more frequent in older patients, which is in accordance with a recent study from Albania suggesting that longer duration of infection could be important in the emergence of these mutants (Kondili et al., 2005
). e-CHB patients harbouring strains with T1762A1764 mutations associated with G1757 were found to have higher HBV DNA levels than patients with T1762A1764 mutations associated with A1757. A mutation to C1753 was present significantly more often in strains with T1762A1764 than in strains lacking this double mutation. The triple C1753T1762A1764 mutant has previously been isolated from HBeAg-positive patients and yielded highly replicating clones, but showed only a minor reduction in HBeAg expression, which is similar to isolates with only the T1762A1764 double mutation (Parekh et al., 2003
). When patients harbouring strains with C1753T1762A1764 associated with G1757, which is less prevalent in genotype D, were compared with patients harbouring strains with C1753T1762A1764 associated with A1757, the latter had lower virus load, indicating that the T1762A1764 mutation was less efficient when associated with A1757 and in conjunction with C1753.
The T1764G1766 mutant has previously been found in a child with HCC and was shown to give a 2·8-fold increase in core promoter activity. This mutant was suggested to form a putative new binding site for the transcription factor hepatocyte nuclear factor 3 (HNF3) (Gerner et al., 1999). Although this previously seemed to be a rare mutation, it was found in almost one-third of our patients. These patients had higher virus load and aminotransferase levels than patients lacking core promoter mutations, although these differences were not significant.
Two e-CHB patients were found to harbour a unique triple mutant, T1762T1766A1768, in one case associated with a high virus load. The T1766A1768 double mutant has previously been reported in an outbreak of fulminant hepatitis and was shown to display a more than 10-fold enhancement in promoter activity, whereas the T1762A1764 mutant showed just about threefold enhancement compared with wild-type clones (Baumert et al., 1996; Gerner et al., 1999
).
The T1762A1764 mutant changes two codons in the X protein with the substitutions Met130Ile131, which would decrease the transactivating activity of the X gene product on both pre-core and core transcription (Li et al., 1999). It has been suggested that the T1762A1764 mutant will restore core transcription, but not pre-core transcription, by creating an HNF1 site (Li et al., 1999
). The T1764G1766 mutant will create a substitution to Leu131 in the X gene product, which might also suppress both pre-core and core transcription. The latter could then be restored by the T1764G1766 mutation by creating an HNF3 site (Gerner et al., 1999
). The combined putative mutational patterns T1762A1764G1766 or T1762T1764G1766 would not generate compensatory HNF1- or HNF3-binding sites and would therefore not be selected for, which explains their absence in this study.
Most interestingly, only patients harbouring strains with A1757 had T1764G1766 mutations. As the vast majority of HBV strains belonging to genotypes A to C have G1757 and it is only in genotypes D and E that an A is more common than G at this position (Table 5), the T1764G1766 mutant is likely to occur mainly in genotype D and possibly genotype E. As G1757 forms the 5' end of the HNF1-binding site, T1762A1764 mutations are more efficient when there is a G1757 instead of an A1757. However, when there is an A1757, the emergence of the T1764G1766 mutation will create an HNF3 site and will be more efficient than the T1762A1764 mutation in conjunction with A1757 in compensatory upregulation of HBcAg production. The most likely reason for the complete absence of the T1764G1766 mutation in association with G1757 is the high efficiency of the T1762A1764 mutation in this molecular context. This view is supported by HBV DNA levels being highest for T1762A1764 associated with G1757, intermediate for T1764G1766 and lowest for T1762A1764 associated with an A1757.
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ACKNOWLEDGEMENTS |
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Received 10 March 2005;
accepted 23 May 2005.
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