The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients

Cellular Immunology 276 (2012) 35–41

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The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients Sohair K. Sayed a,⇑, Mohammad A. kobeisy b a b

Department of Clinical Pathology, Assiut University School of Medicine, Egypt Department of Internal Medicine, Assiut University School of Medicine, Egypt

a r t i c l e

i n f o

Article history: Received 13 January 2012 Accepted 20 March 2012 Available online 3 April 2012 Keywords: Hepatitis B e antigen Hepatitis B virus Core promoter mutations Viral load

a b s t r a c t The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (ve) and HBV core promoter mutation. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Hepatitis B virus (HBV) infection is one of the most important infectious diseases worldwide and is a major global health problem despite the availability of effective vaccines and effective antiviral medications [1]. The natural history of chronic HBV infection can be divided into three phases: immune tolerance, immune clearance and residual or integrated phase. In addition, a fourth reactivation phase has also been proposed [2]. The HBV genome consists of four partially overlapping open reading frames: the pre-S/S gene that codes for the envelope proteins, the pre-C/C gene that codes for HBeAg and core protein, the P gene that codes for the DNA polymerase and reverse transcriptase and the X gene that codes for a protein of unclear significance. As HBV replicates asymmetrically via reverse transcription of an RNA intermediate, it is more prone to mutations than other DNA viruses [3]. The outcome of infection depends upon many factors, such as the host immune status, their age at the time of infection and the degree of viral replication that occurs. Another factor that has been postulated to affect the outcome of infection is the genetic variability of the virus, which influences expression of viral antigens [4]. Patients with CHB are typically HBeAg (+ve) with detectable HBV DNA in serum. Generally, seroconversion from HBeAg to hepatitis B e antibody (HBeAb) positive correlates with reduced HBV replication in the liver and low infectivity during the natural course ⇑ Corresponding author. Address: Department of Clinical Pathology, Faculty of Medicine, Assuit University Hospitals, Assuit, Egypt. E-mail address: [email protected] (S.K. Sayed). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.03.003

of infection [5]. In some patients, however, the immune pressure associated with seroconversion selects HBV variants that express little or no HBeAg. Although the patient may develop HBeAb, active HBV DNA replication continues with associated liver damage [6]. A previous study has also demonstrated that HBeAg may be a target antigen on HBV-infected hepatocytes. Failure to produce a target antigen may allow the infected cell to evade immune clearance [7]. Mutations in the HBV surface (S), precore (PC) and basal core promoter (BCP) genes are observed frequently in HBV infected patients, and many studies showed that these mutations are associated with the clinical outcomes of HBV disease [8,9]. The most frequent and clinically important mutations in the PC and BCP regions are G1896A and A1762T/G1764A, respectively, which were initially thought to be related to a HBeAg (ve) phenotype but recent studies showed that they may also be found in some HBeAg (+ve) patients, especially those with chronic hepatitis [4,10,11]. The predominant precore variation is a G-to-A change at A1896, which creates a premature stop codon and abolishes the synthesis of HBeAg. The most common core promoter mutations involve a two nucleotide substitution: A to T at nucleotide 1762 and G to A at nucleotide 1764 (TA) [12]. Mutations in the basal core promoter region can also reduce HBeAg production without affecting HBV replication or hepatitis B core antigen expression by selectively downregulating the transcription of the precore mRNA but without affecting the pregenomic RNA. The development of basal core promoter mutations usually occurs few years before HBeAg seroconversion. Basal core promoter mutations are serving as an alternative of the HBV to lose HBeAg and escape the host immune clearance [3].

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The aim of this study is to detect the possible association of HBV core promoter mutation (T1762/A1764), HBeAg status and viral load in CHB patients. 2. Subjects and methods This study was conducted in the Immunology unit, Department of Clinical Pathology, Assuit University Hospitals, Assuit, Egypt. It was performed on 66 adults HBV patients, 55 males and 11 females. Their age ranged from 22 to 62 years with mean value ± SE (38.5 ± 1.5) years. All patients were selected from Internal Medicine department, Assiut University Hospitals, Assuit, Egypt. The protocol of the study was approved by the ethical committee of Assiut University and informed consents were obtained from all subjects before participation. Five milliliter venous blood was collected after overnight fasting .The samples were collected in plain tube and centrifuged within 30 min at 3000 rpm for 10 min. The serum samples were collected for aspartate aminotransferase (AST), alanine aminotransferase (ALT), serological markers of HBV and HBV DNA. The remaining samples were divided into aliquots and stored at 70 °C for further analysis. 2.1. Laboratory investigations Serological markers of HBV; hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), antibodies to hepatitis B core antigen (anti-HBc IgM and IgG), HBeAg, and antibody to HBeAg (antiHBe)) and HCV-Ab were tested using commercially available micro particle enzyme immunoassay kits (Axsym, Abbott Laboratories).

72 °C for 2 min. for 35 cycles and a final elongation at 72 °C for 5 min. In the second-round PCR, the PCR mixture was as follows (1 ll of each primer eP2-1 and eP2-2 and 2 ll of amplified product in the first run and 21 ll water). The amplification profile was as follows: initial denaturation at 94 °C for 1 min., annealing at 55 °C for 1 min., and extension at 72 °C for 2 min. for 30 cycles and a final elongation at 72 °C for 5 min. A product of 307 bp was detected by electrophoresis on 2% agarose gel. Detection of core promoter mutation (T1762/A1764) of HBV was performed by incubation of the amplified product with the restriction enzyme Sau3AI (5 ll of free water, 2 ll multi-core 10 buffer, 1 ll Sau3AI enzyme and 10 ll of amplified DNA) and incubated at 37 °C for 60 min. The digested products were detected by electrophoresis on 2% agarose gel. The PCR/RFLP pattern for hepatitis B core promoter mutant (T1762/A1764) was 197 and 110 bp. In patients without hepatitis B core promoter mutantion (T1762/A1764), one band at 307 bp was detected (Fig. 1). 2.4. Statistical analysis Data were analyzed using SPSS version 17 program. Values were expressed as mean ± standard error (SE) and percentages. The levels for HBV DNA varied greatly, so it was expressed as median to normalize the data. Unpaired t-test was used to compare numerical parametric data between two groups. Chi-square test was used to determine the significance for non-parametric variable. Pearson correlation test was applied to measure correlation for numeric variables in the same group. P-values 6 0.05 were considered significant. 3. Results

2.2. Detection of HBV DNA Detection of HBV DNA was performed by real time PCR on step one instrument supplied by Applied Biosystem using ready to use PCR kit supplied by Qiagen (HBV TM PCR) kit (24)VI Lot No. 450613. The primers sequences used were: HB1 forward primer, 50 -GACCACCAAATGCCCCTAT-30 HB2 reverse primer, 50 -CCRAGAYYGAGATCTTCTGCGAC-30 The amplification was performed in a 50 ll reaction mixture containing 30 ll of mixture of HBV RG Master mix (Buffer, dNTP, Primer, Probe and enzymes) and 20 ll of DNA template to each reaction. The Real-time PCR cycling parameters consisted of denaturizing at 95 °C for 10 min followed by 45 cycles consisting of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 15 s. [13]. 2.3. Detection of core promoter mutation (T1762/A1764) of hepatitis B virus Detection of core promoter mutation (T1762/A1764) of HBV was performed by nested PCR followed by restriction fragment length polymorphism (PCR-RFLP). For nested PCR we use ReadyTo-Go PCR beads GE Health Care Kit (Lot No. 27-9557-01). The primers sequences used were: eP1-l, 50 -GCATGGAGACCACCGTGAAC-30 eP1-2, 50 -GGAAAGAAGTCAGAAGGCAA-30 eP2-1, 50 -CATAAGAGGACTCTTGGACT-30 eP2-2, 50 -GGCAAAAAAGAGAGTAACTC-30 In the first-round PCR, the PCR mixture was as follows: (1 ll of each primer eP1-l and eP1-2 and 10 ll of DNA and 13 ll water). The amplification profile was as follows: initial denaturation at 94 °C for 1 min., annealing at 55 °C for 1.5 min. and extension at

All patients were positive for HbsAg and anti-Hb core (IgG) but negative for anti- HbsAg and anti-HBc IgM. Patients’ characteristics are summarized in Table 1. Patients are classified according to the status of HBeAg and the presence or absence of core promoter mutation T1762/A1764. 3.1. Comparison between HBeAg (+ve) and HBeAg (ve) group There was no significant difference in the male/female ratio and the mean age of the two groups of patients. In HBeAg (+ve) patients ALT was significantly increased when compared to that of HBeAg (ve) patients (P = 0.007). Anti-Hbe was detected in 68.5% (37/54) of HBeAg (ve) patients. The level of HBV DNA was significantly higher in HBeAg (+ve) patients when compared to that of HBeAg (ve) patients (P = 0.023). Core promoter mutation was detected in both patients groups. The mutant detection rate was 16.7% (2/ 12) of the HBeAg (+ve) patients and 40.7% (22/54) of the HBeAg (ve) patients. The prevalence of this mutation was not significantly different between the two groups (P = 0.304) (Table 2). 3.2. Core promoter mutation, viral load, AST and ALT There was no significant difference between patients with T1762/A1764 core mutation and patients without core mutation regarding gender and age. ALT and AST were significantly higher in patients with core promoter mutation than in patients without mutation (P = 0.005 and P = 0.002, respectively). Serum level of HBV-DNA showed no significant difference between patients with core mutation and patients without core mutation. Taken together, these results suggest that T1762/A1764 core promoter mutation was associated with increased liver activity but did not affect HBV replication. Anti-HBe was detected frequently in both patients with core mutation 62.5% (15/24) and patients without core mutation 52.4% (22/42) (Table 3).

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Fig. 1. Agarose gel electrophoresis of the PCR product after Sau3AI digestion .Lane 1 and 9: DNA molecular weight marker 100 bp. Lane 2 and 4: samples without T1762/ A1764 mutation, Lane 3, 5, 6 and 7: samples with T1762/A1764 mutation, Lane 8: negative control.

Table 1 Baseline characteristics of the 66 CHB patients included in this study. Gender Male Female Age (years) Range Mean ± SE ALT (IU/ml) Range Mean ± SE AST (IU/ml) Range Mean ± SE AST/ALT ratio Mean ± SE HBeAg (ve) Number Percentage HBeAg (+ve) Number Percentage Anti-HBe (ve) Number Percentage Anti-HBe (+ve) Number Percentage Core mutation (+ve) Number Percentage No core mutation (ve) Number Percentage HBV DNA (IU/L) Mean ± SE Range

55 (83.3%) 11 (16.7%) 22–62 38.5 ± 1.5 12–411 64.8 ± 9 11–345 52.2 ± 6.3 0.97 ± 0.04 54 81.8% 12 18.2% 27 40.9% 39 59.1% 24 36.4% 42 63.6% 42800 ± 15817 0–830000

3.3. Core promoter mutation in relation to HBeAg status, ALT, AST and HBV DNA level There was no significant difference between patients with core mutation and patients without core mutation both in HBeAg (ve) and HBeAg (+ve) patients regarding the gender and age (Table 4 and 5). In HBeAg (ve) group; AST and ALT were significantly high-

er in patients with core promoter mutation than in patients without mutation (P = 0.004 and P = 0.001, respectively), while HBV DNA showed no significant difference between the two groups of patients (P = 0.782) (Table 4). In HBeAg (+ve) group; ALT, AST and serum HBV DNA level were significantly higher in patients with core mutation than in patients without mutation (P = 0.022), (P = 0.046) and (P = 0.000), respectively (Table 5). These results revealed that core promoter mutation increase liver activity but do not affect HBV replication in the HBeAg (ve) group. 4. Discussion Chronic hepatitis B (CHB) may present either as HBeAg (+ve) or HBeAg (ve). HBeAg (+ve) CHB is due to the so-called ‘‘wild type’’ HBV. It typically represents the early phase of chronic HBV infection. HBeAg (ve) CHB is due to replication of naturally occurring HBV variants with nucleotide substitutions in the precore and/or basic core promoter regions of the genome and represents a later phase of chronic HBV infection. The prevalence of the HBeAg (ve) form of the disease has been increasing over the last decade as a result of HBV-infected population aging and represents the majority of cases in many areas [14,15]. In our study, among the CHB patients group, 12 patients (18.2%) were HBeAg (+ve) and 54 patients (81.8%) were HBeAg (ve). This finding is in agreement with those of Chan et al. and Minuk et al. who reported that the prevalence of HBeAg negativity in chronic HBV infection ranges between 70% and 100% [16,17]. Other studies reported that HBeAg negativity rates vary from 52.5% to 63.3% [18,19]. In studies of different geographical distribution that had been performed in different patient populations, considerable differences between the percentages of HBeAg (+ve) and HBeAg (ve) patients were observed. The prevalence of HBeAg (ve) was 33% in the Mediterranean area, 15% in Asia Pacific and 14% in the United States and Northern Europe [20]. Since the discovery of HBeAg by Magnius and Espmark in 1972 [21], its function has remained an enigma. There is still no solid evidence about the events that lead to seroconversion from HBeAg to anti-HBe status, but a strong contender has been the mutation in T1762 and A1764, which could prevent and reduce the production

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Table 2 Comparison between HBeAg (+ve) and HBeAg (ve) group.

a b *

Characteristics

HBeAg (+ve) (n = 12) (18.2%)

HBeAg (-ve) (n = 54)(81.8%)

P- value

Gender Male Female Age (years) Mean ± SE Range AST IU/L Mean ± SE Range ALT IU/L Mean ± SE Range AST/ALT ratio Mean ± SE HBV DNA IU/L Mean ± SE Range Median Present Absent Core mutation Positive Negative

10 (83.3%) 2 (16.7%)

45 (83.3%) 9 (16.7%)

0.644a

39.1 ± 3.2 23-55

38.4 ± 1.7 22-62

0.837b

78.1 ± 0.7 41- 167

46.5 ± 7.1 11 - 345

0.083b

115.4 ± 22.9 41- 343

53.6 ± 9.1 12- 411

0.007*

0.7 ± 0.1

1.0 ± 0.4

0.011*

136000 ± 73505 105-650000 850 12 (100%) 0

22100 ± 16562 0-830000 0.00 15 (27.8%) 39 (72.2%)

0.023*

2 (16.7%) 10 (83.3%)

22 (40.7%) 32 (59.3%)

0.304a

Chi-square test. Independent samples t-test. Statistical significant difference (P < 0.05).

Table 3 Relation between core mutation, HBV DNA, ALT and AST.

a b *

Characteristics

Core mutation (n = 24) (36.5%)

No core mutation (n = 42)(63.6%)

p-value

Gender Male Female Age (years) Mean ± SE Range AST IU/L Mean ± SE Range ALT IU/L Mean ± SE Range AST/ALT ratio Mean ± SE HBV DNA IU/L Mean ± SE Range Median Anti-Hbe Positive Negative

21 (87.5%) 3 (12.5%)

34(81.0%) 8 (19.0 %)

0.374a

37.9 ± 2.5 22- 62

38.8 ± 1.8 22- 60

0.778b

75.0 ± 13.9 14 – 345

39.2 ± 5.0 11-178

0.005*

100.1 ± 19.8 19 – 411

44.6 ± 6.9 12-230

0.002*

0.9 ± 0.3

1.0 ± 0.1

0.063b

69300 ± 39294 0-650000 145

27700 ± 20834 0-830000 0.0

0.308b

15 (62.5%) 9 (37.5%)

24 (57.1%) 18 (42.9%)

0.296a

Chi-square test. Independent samples t-test. Statistical significant difference (P < 0.05).

of HBeAg [22–24]. Alfaresi et al. found that the majority of the HBV-positive patients had probably been infected for a long time and had therefore likely developed mutations in the core region. Therefore, some patients were probably HBeAg (ve) but antiHBe (+ve) [4]. In this study, among the HBeAg (ve) patients, 72.2% (39/54) were HBeAb (+ve). This findings is in agreement with those of Funk et al. who found that in HBeAg (ve) patients, 79.6% were HBeAb(+ve), which indicates either seroconversion and suppression of HBV DNA or emergence of the precore mutant virus infection [20]. In our study, ALT and HBV-DNA levels were higher in HBeAg (+ve) patients than in HBeAg (ve) patients. These findings are supporting those of Alfaresi et al. who mentioned that the most important predictor of an elevated ALT level and a high HBV DNA level was HBeAg status, as HBeAg (+ve) patients were more

likely to have higher ALT levels and HBV-DNA levels than HBeAg (ve) patients [4]. Cacoub et al., Pungpapong et al. and Elgouhari et al. also found that there was an elevated ALT level in HBeAg (+ve) patients, they attributed this elevation to immune clearance phase in which an immune mediated process aims at clearing the viral infection, but it also leads to concomitant hepatocellular injury [25–27] . The frequency of core promoter mutation T1762/A1764 among the CHB patients in the present study was 36.5% (24/66). This finding is in agreement with those of Chen et al., who reported that the frequency of mutation at core promoter nucleotides T1762/A1764 was 38.5% [24]. Also, Alfaresi et al. found that the frequency of this mutation ranged from 23.2% to 37.5% [4]. Core promoter mutation may augment the host immune response to HBV-infected

39

S.K. Sayed, M.A. kobeisy / Cellular Immunology 276 (2012) 35–41 Table 4 Core mutation in relation to HBV DNA, ALT, AST in HBeAg (ve).

a b *

Characteristics

Core mutation (n=22) (40.7%)

No core mutation (n=32)(59.3%)

p-value

Gender Male Female Age (years) Mean±SE Range AST IU/L Mean±SE Range ALT IU/L Mean±SE Range AST/ALT Mean ± SE HBV DNA IU/L Mean ± SE Range Median Anti-Hbe Positive (n = 26) Negative (n = 9)

19 (86.4 %) 3 (13.6 %)

26 (81.2 %) 6 (18.8%)

0.457a

38.4 ± 2.7 22 -62

38.3 ± 2.2 22- 60

0.997b

70.8 ± 14.5 14- 345

29.8 ± 5.0 11-178

0.004*

87.6 ± 18.2 19- 411

30.1 ± 6.6 12- 230

0.001*

0.9 ± 0.1

1.1 ± 0.1

0.031*

16500 ± 15882 0- 350000 62.5

25900 ± 25937 0-830000 0.0

0.782b

15 (68.2%) 7 (31.8%)

22 (68.8%) 10 (31.2%)

0.447a

Chi-square test. Independent samples t-test. Statistical significant difference (P < 0.05).

Table 5 Core mutation related to HBV DNA, ALT, AST in HBeAg (+ve).

a b *

Characteristics

Core mutation(n = 2)(16.7%)

No core mutation(n = 10)(83.3%)

p-value

Gender Male Female Age (years) Range Mean ± SE AST IU/L Mean ± SE Range ALT IU/L Mean ± SE Range ALT/AST Mean ± SE HBV DNA IU/L Mean ± SE Range Median Anti-Hbe Positive Negative

2(100 %) 0(0%)

8(80.0%) 2(20.0%)

0.682a

23 -45 33.5 ± 10.5

29- 55 40.3 ± 3.4

0.453b

121.5 ± 45.5 76-167

69.4 ± 8.5 34-114

0.066b

237.5 ± 105.5 132-343

91.0 ± 11.3 41-135

0.009*

0.53 ± 0.1

0.8 ± 0.1

0.015*

650000 ± 0.0 650000 325000

33500 ± 29758 105-300000 850

0.000*

0 (0.0%) 2 (100.0%)

2 (20.0%) 8 (80.0%)

0.536a

Chi-square test. Independent samples t-test. Statistical significant difference (P < 0.05).

hepatocytes by diminishing circulating HBeAg and increasing hepatocyte apoptosis and regeneration, thus leading to liver injury [28–29]. In our study, ALT and AST levels showed significant increase in patient with core promoter mutation (T1762/A1764) when compared to patients without core promoter mutation. These findings are supporting a study which demonstrated that the core promoter mutation T1762/A1764 located at the HBV X gene diminishes HBeAg production and is associated with more active liver disease [30]. Similar results were observed by Tangkijvanich et al. who found an association between this mutation and the appearance of higher ALT levels. They also reported that these mutant strains showed more liver inflammation and fibrosis, as measured by histology activity index (HAI) scores [31]. Infection with HBeAg (ve) HBV strain usually does not occur de novo,but rather emerging during immune clearance of a wild type strain, when increased immune pressure on the wild type

strain leads to selection of HBeAg (ve) mutant [32]. In the present study, the frequency of HBV core promoter mutation (T1762/ A1764) in patients with HBeAg (ve) patients was 40.7% (22/54) and in HBeAg (+ve) was 16.7% (2/12). These findings are in agreement with Tangkijvanich et al. who reported that the core promoter mutations were found more frequently in HBeAg (ve) than in HBeAg (+ve) CHB [31]. Another studies were conducted by Parekh et al. and Mahtab et al. reported that core promoter region (nucleotides 1742 to 1849) is located upstream of the precore region (nucleotides 1814 to 1901). It has an important role in HBV replication as well as in HBeAg production. Mutations in these regions downregulate precore mRNA transcription and HBeAg synthesis [33,34]. Contrary to our findings, Chen et al. suggested that the T1762 and A1764 mutation appears to be insufficient to lead to the HBeAg negative phenotype [24].

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The core promoter mutation may enhance replication capacity and reduce virion secretion leading to increase viral load in the liver, thus triggering liver damage either directly or indirectly through the immune response [35]. Also, core promoter mutation appears to enhance the efficacy of viral replication either by modulating the relative levels of precore and core RNAs or by creating a transcription factor binding site for hepatocyte nuclear factor 1 [36]. However, reports of the association of core promoter mutation with virus replication have reached conflicting conclusions: increased virus replication [37], no effect on virus replication [12,38] or reduced virus replication [39]. In our study, the core promoter mutation was not significantly linked to HBV DNA levels, as there was no significant difference in HBV- DNA level between patients with core promoter mutation and patients without mutation. The replicative phase of CHB infection is characterized by the presence of HBeAg and HBV DNA. The non replicative phase is characterized by the absence of HBeAg and HBV DNA. Several HBeAg (ve) patients have persistent high HBV replication associated with liver inflammation and ongoing fibrosis. This replicative stage of chronic HBV infection is the time of maximal infectivity and liver injury [40–43]. In this study, 27.8% of HBeAg (ve) patients had detectable HBV DNA level. This suggests that a significant number of patients with HBeAg (ve) CHB continue to replicate the virus. When our subset analysis focused on such individuals, a relatively large proportion was found to have core promoter mutation associated with HBeAg negativity, high ALT level and low viral load. This finding is in accordance with previous studies [44–46]. It might be speculated that the core promoter mutant viruses with enhanced viral replication and reduced HBeAg expression were more likely to activate immune responses, leading to ALT elevation and viral load decline in patients with CHB. Contrary to our finding, other studies found that core promoter mutation was associated with high viral load in HBeAg (ve) patients [7,37,47] . Although the core promoter mutation does not prevent the synthesis of HBeAg, they suppress its levels, so that samples with both HBeAg (+ve) and core promoter mutation are uncommon [48]. In our study, among the HBeAg (+ve) patients, core promoter mutation was observed only in two patients. Although the number of samples is limited, it is clear that the core promoter mutation in HBeAg (+ve) patients was associated with high viral replication levels and evidence of liver disease. These findings are in keeping with the results of other investigators who showed that delayed HBeAg seroconversion may prolong the inflammation process and subsequently result in more severe liver damage [17]. Comparable results were revealed by Chen et al. who showed a relationship between core mutation and the clinical manifestations of HBV infection in HBeAg (+ve) patients [9]. In contrast to our study, other studies found that in HBeAg (+ve) patients, T1762/A1764 mutation was associated with low viral load [7,45,46]. In conclusion: The frequency of HBeAg (ve) patients is higher than HBeAg (+ve) patients in chronic hepatitis B patients in Upper Egypt. The T1762/A1764 mutation is frequent in both the HBeAg (ve) and HBeAg (+ve) patients and is associated with a lower viral load in HBeAg (ve) patients than in HBeAg (+ve) patients. The T1762/A1764 mutation appears to be responsible for low HBV DNA levels in HBeAg (ve) patients and the high viral replication levels in HBeAg (+ve) patients. References [1] J.H. Kao, D.S. Chen, Global control of hepatitis B virus infection, Lancet Infect Dis. 2 (2002) 395–403. [2] C.M. Chu, S.J. Hung, J. Lin, D.I. Tai, Y.F. Liaw, Natural history of hepatitis B e antigen to antibody seroconversion in patients with normal serum aminotransferase levels, Am. J. Med. 116 (2004) 829–834.

[3] H.L-Y. Chan, Significance of hepatitis B virus genotypes and mutations in the development of hepatocellular carcinoma in Asia, J. Gastroenterol. Hepatol. 26 (2011) 8–12. [4] M. Alfaresi, A. Elkoush, H. Alshehhi, A. Alzaabi, A. Islam, Hepatitis B virus genotypes and precore and core mutants in UAE patients, J. Virol. 7 (2010) 160–167. [5] H.J. Yim, A.S. Lok, Natural history of chronic hepatitis B virus infection: what we knew in 1981 and what we know in, Hepatology 43 (2006) (2005) S173– S181. [6] S.J. Hadziyannis, Hepatitis B e antigen negative chronic hepatitis B. From clinical recognition to pathogenesis and treatment, Viral. Hepat. Rev. 1 (1995) 7–36. [7] A. Utama, M.-D. Siburian, S. Purwantomo, M.D.B. Intan, et al., Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg (+) and HBeAg () patients, World J. Gastroenterol. 17 (2011) 708–716. [8] K. Kidd-Ljunggren, E. Myhre, J. Blackberg, Clinical and serological variation between patients infected with different Hepatitis B virus genotypes, J. Clin. Microbiol. 42 (2004) 5837–5841. [9] C.H. Chen, C.M. Lee, C.H. Hung, T.H. Hu, J.H. Wang, J.C. Wang, S.N. Lu, C.S. Changchien, Clinical significance and evolution of core promoter and precore mutations in HBeAg-positive patients with HBV genotype B and C: a longitudinal study, Liver Int. 27 (2007) 806–815. [10] F. Tacke, M.P. Manns, C. Trautwein, Influence of mutations in the hepatitis B virus genome on virus replication and drug resistance–implications for novel antiviral strategies, Curr. Med. Chem. 11 (2004) 2667–2677. [11] G. Bahramali, M. Sadeghizadeh, S. Amini-Bavil-Olyaee, S. Alavian, A. BehzadBehbahani, A. Adeli, M.R. Aghasadeghi, S. Amini, F. Mahboudi, Clinical, virologic and phylogenetic features of hepatitis B infection in Iranian patients, Gastroenterology 14 (2008) 5448–5453. [12] B. Yoo, J. Park, H.J. Kim, D.H. Lee, Y.J. Chan, S.M. Park, Precore and core promotor mutations of hepatitis B virus and hepatitis b e antigen- negative chronic hepatitis B in Korea, J. Hepatol. 38 (2003) 98–103. [13] G.S. Chopra, P.K. Gupta, A.C. Anand, P.P. Varma, V. Nair, R. Ramji, Real time-PCR HBV-DNA analysis: significance and first experience in armed forces, Medical Journal Armed Forces India 61 (2005) 234–237. [14] J.P. Zarski, P. Marcellin, V. Leroy, C. Trepo, D. Samuel, N. Ganne- Carrie, et al., Characteristics of patients with chronic hepatitis B in France. predominant frequency of HBe antigen negative cases, J. Hepatol. 45 (2006) 355–360. [15] M. Rizzetto, A. Ciancio, Chronic HBV-related liver disease, Mol. Aspects Med. 29 (2008) 72–84. [16] H.L. Chan, N.W. Leung, M. Hussain, M.L. Wong, A.S. Lok, Hepatitis B e antigennegative chronic hepatitis B in Hong Kong, Hepatology 31 (2000) 763–768. [17] G.Y. Minuk, P.S. Orr, R. Brown, S. Macdonald, R.K. Chaudhary, P. Temple, Precore mutant infections in the Canadian inuit, J. Hepatol. 33 (2000) 781–784. [18] H. Tsugeno, G. Yamada, M. Kinoshita, H. Shimomura, Y. Iwasaki, T Tsuji, Quantitative analysis of wild-type and precore mutant hepatitis B virus in carriers, Hepatol. Res. 23 (2002) 48–54. [19] C.K. Lim, J.T. Tan, J.B. Khoo, A. Ravichandran, H.M. Low, Y.C. Chan, S.H. Ton, Correlations of HBV genotypes, mutations affecting HBeAg expression and HBeAg/anti-HBe status in HBV carriers, Int. J. Med. Sci. 3 (2006) 14–20. [20] M.L. Funk, D.M. Rosenberg, A.S. Lok, World-wide epidemiology of HBeAgnegative chronic hepatitis B and associated precore and core promoter variants, J. Viral. Hepat. 9 (2002) 52–61. [21] L.O. Magnius, J.A. Espmark, New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants, J. Immunol. 109 (1972) 1017– 1021. [22] H. Okamoto, F. Tsuda, Y. Akahane, Y. Sugai, M. Yoshiba, K. Moriyama, T. Tanaka, Y. Miyakawa, M. Mayumi, Hepatitis B virus with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e antigen, J. Virol. 68 (1994) 8102–8110. [23] M. Kurosaki, N. Enomoto, Y. Asahina, I. Sakuma, T. Ikeda, S. Tozuka, N. Izumi, F. Marumo, C. Sato, Mutations in the core promoter region of hepatitis B virus in patients with chronic hepatitis B, J. Med. Virol. 49 (1996) 115–123. [24] C.H. Chen, C.M. Lee, S.N. Lu, C.S. Changchun, H.L. Eng, C.M. Huang, J.H. Wang, C.H. Hung, T.H. Hu, Clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations affecting HBV e antigen expression in Taiwan, J. Clin. Microbiol. 43 (2005) 6000–6006. [25] P. Cacoub, D. Saadoun, M. Bourlière, et al., Hepatitis B virus genotypes and extrahepatic manifestations, J. Hepatol. 43 (2005) 764–770. [26] S. Pungpapong, W.R. Kim, J.J. Poterucha, Natural history of hepatitis B virus infection: an update for clinicians, Mayo Clin. Proc. 82 (2007) 967–975. [27] H.M. Elgouhari, T.I. Tamimi, W.D. Carey, Hepatitis B virus infection: understanding its epidemiology, course, and diagnosis, Clevel. Clin. J. Med. 75 (2008) 881–889. [28] M.J. Tong, L.M. Blatt, J.H. Kao, J.T. Cheng, W.G. Corey, Basal core promoter T1762/A1764 and precore A1896 gene mutations in hepatitis B surface antigen-positive hepatocellular carcinoma: a comparison with chronic carriers, Liver Int. 27 (2007) 1356–1363. [29] J.M. Yuan, A. Ambinder, Y. Fan, Y.T. Gao, M.C. Yu, J.D. Groopman, Prospective evaluation of hepatitis B 1762(T)/1764(A) mutations on hepatocellular carcinoma development in Shanghai, China, Cancer Epidemiol. Biomarkers Prev. 18 (2009) 590–594. [30] C. Hannoun, P. Horal, M. Lindh, Long-term mutation rates in the hepatitis B virus genome, J. Gen. Virol. 81 (2000) 75–83. [31] P. Tangkijvanich, A. Theamboonlers, P. Jantaradsamee, P. Hirsch, V. Mahachai, P. Suwangool, Y. Poovorawan, Core promoter and precore mutants of hepatitis

S.K. Sayed, M.A. kobeisy / Cellular Immunology 276 (2012) 35–41

[32] [33]

[34]

[35] [36] [37]

[38]

[39]

[40]

B virus: prevalence and clinical relevance in chronic hepatitis patients, South east Asian J. Trop. Med. Public Health 31 (2000) 627–635. S.J. Hadziyannis, D. Vassilopoulos, Immunopathogenesis of hepatitis B e antigen negative chronic hepatitis B infection, Antiviral Res. 8 (2001) 91–98. S. Parekh, F. Zoulim, S.H. Ahn, A. Tsai, J. Li, S. Kawai, N. Khan, C. Trépo, J. Wands, S. Tong, Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants, J. Virol. 77 (2003) 6601–6612. M.A. Mahtab, S. Rahman, M. Khan, A.A. Mamun, M. Kamal, Pre-core/core promoter mutant hepatitis B virus produces more severe histologic liver disease than wild type hepatitis B virus, Hung. Med. J. 1 (2007) 41–46. S. Tong, K.H. Kim, C. Chante, J. Wands, J. Li, Hepatitis B virus e antigen variants, Int. J. Med. Sci. 2 (2005) 2–7. Y.H. Shi, C.H. Shi, Molecular characteristics and stages of chronic hepatitis B virus infection, World J. Gastroenterol. 15 (2009) 3099–3105. Z.L. Fang, C.A. Sabin, B.Q. Dong, S.C. Wei, Q.Y. Chen, K.X. Fang, J.Y. Yang, X.Y. Wang, T.J. Harrison, The association of HBV core promoter double mutations (A1762T and G1764A) with viral load differs between HBeAg positive and antiHBe positive individuals: a longitudinal analysis, J. Hepatol. 50 (2009) 273– 280. E. Orito, M. Mizokami, H. Sakugawa, K. Michitaka, K. Ishikawa, T. Ichida, T. Okanoue, H. Yotsuyanagi, S. Iino, A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group., Hepatology 33 (2001) 218–223. W.N. Chen, C.J. Oon, Mutations and deletions in core promoter and precore stop codon in relation to viral replication and liver damage in Singaporean hepatitis B virus carriers, Eur. J. Clin. Invest. 30 (2000) 787–792. C.Q. Pan, J.X. Zhang, Natural history and clinical consequences of hepatitis B virus infection, Int. J. Med. Sci. 2 (2005) 36–40.

41

[41] M.J. Tong, L.M. Blatt, J.H. Kao, J.T. Cheng, W.G. Corey, Precore/basal core promoter mutants and hepatitis B viral DNA levels as predictors for liver deaths and hepatocellular carcinoma, World J. Gastroenterol. 12 (2006) 6620– 6626. [42] S. Jammeh, F. Tavner, R. Watson, H.C. Thomas, P. Karayiannis, Effect of basal core promoter and pre-core mutations on hepatitis B virus replication, J. Gen. Virol. 89 (2008) 901–909. [43] E. Sablon, F. Shapiro, Advances in molecular diagnosis of HBV infection and drug resistance, Int. J. Med. Sci. 2 (2005) 8–16. [44] X. Ren, Z. Xu, Y. Liu, X. Li, S. Bai, N. Ding, Y. Zhong, L. Wang, P. Mao, F. Zoulim, D. Xu, Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute on- chronic liver failure without preexisting liver cirrhosis, J. Viral. Hepat. 17 (2010) 887–895. [45] C.J. Chu, M. Hussain, A.S. Lok, Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection, Hepatology 36 (2002) 1408– 1415. [46] H.J. Yuan, M.F. Yuen, D. Ka-Ho Wong, S.M. Sum, E. Sablon, I. Oi-Lin Ng, C.L. Lai, Impact of precore and core promoter mutations on hepatic histology in patients with chronic hepatitis B, Aliment. Pharmacol. Ther. 22 (2005) 301– 307. [47] X.M. Peng, G.M. Huang, J.G. Li, Y.S. Huang, Y.Y. Mei, Z.L. Gao, High level of hepatitis B virus DNA after HBeAg-to-anti- HBe seroconversion is related to coexistence of mutations in its precore and basal core promoter, World J. Gastroenterol. 11 (2005) 3131–3134. [48] A. Pang, M.F. Yuen, H.J. Yuan, C.L. Lai, Y.L. Kwong, Real-time quantification of hepatitis B virus core-promoter and pre-core mutants during hepatitis E antigen seroconversion, J. Hepatol. 40 (2004) 1008–1017.