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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Background:  The details of liver histology of patients with precore and core promoter mutations are still not clear.

Aim:  To determine the role of precore and core promoter mutations in liver histology in Chinese patients with chronic hepatitis B.

Patients and methods:  Intrahepatic hepatitis B virus DNA (by COBAS Amplicor hepatitis B virus Monitor test) and precore and core promoter mutations (by a line probe assay) were measured in 54 chronic hepatitis B patients. Expression of hepatitis B core antigen, hepatitis B e antigen and hepatitis B surface antigen was determined by immunohistological staining. Histological activity index was scored according to Knodell's criteria.

Results:  Compared with patients without core promoter mutations, patients with core promoter mutations had more severe intrahepatic inflammation and fibrosis, and more cytoplasmic expression of hepatitis B core antigen (P = 0.028). No such differences were found in patients with and without precore mutations. Logistic regression showed that core promoter mutations were independently associated with cytoplasmic expression of hepatitis B core antigen (P = 0.026). Intrahepatic hepatitis B virus DNA levels correlated with serum hepatitis B virus DNA levels (r = 0.71, P < 0.001) and the percentage of hepatitis B core antigen-positive hepatocytes (r = 0.37, P = 0.047), but had no correlation with serum alanine aminotransferase levels nor the degree of inflammation and fibrosis.

Conclusions:  Patients with core promoter mutations had more severe inflammation and fibrosis, and more frequent cytoplasmic expression of hepatitis B core antigen. This suggested that core promoter mutations might cause more serious liver disease.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

The role of precore and core promoter mutations in the pathogenesis of chronic hepatitis B is still not clear. Hepatitis B virus (HBV) in general is not cytopathic. Liver damage is supposed to be caused by immune response against viral antigens. Studies have shown that nuclear hepatitis B core antigen (HBcAg) and cytoplasmic hepatitis B e antigen (HBeAg) are associated with high level of HBV replication.1 Cytoplasmic HBcAg, rather than cytoplasmic HBeAg, is associated with active liver disease.1, 2

The precore sequence is believed to be essential for the nuclear translocation of HBcAg.3 Precore mutations by blocking the synthesis of HBeAg may prevent nuclear translocation of HBcAg. The cytoplasmic prominent (CP) expression of HBcAg is found in a majority of patients with precore mutations in recent studies.4–6 Core promoter mutations also decrease the synthesis of HBeAg.7 Their association with cytoplasmic expression of HBcAg has not been described. Precore and core promoter mutations often co-exist in patients with chronic hepatitis B.8,9 The association of precore mutations and core promoter mutations may further complicate the intrahepatic expression of HBcAg.

This study was performed to determine the relationship of precore and core promoter mutations, intrahepatic HBV DNA levels, intrahepatic expression of HBcAg/HBeAg and liver histology.

Patients and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Patients

Fifty-four patients with chronic hepatitis B were recruited from Hepatitis Clinic, Queen Mary Hospital, The University of Hong Kong. The median age was 43.3 (range 18.3–66.6) years with male to female ratio of 43:11. Twenty-two patients were HBeAg-positive and 32 patients were anti-HBe-positive. The study has been approved by the Ethics Committee, the Institutional Review Board, The University of Hong Kong and Queen Mary Hospital, Hong Kong.

Determination of serum HBV DNA level

Serum samples were collected 1 week before or after the liver biopsy. Serum HBV DNA level was measured by COBAS Amplicor HBV Monitor test (Roche Diagnostics, Branchburg, NJ, USA) (lower limit of detection: 200 copies/mL). The serum hepatitis B surface antigen (HBsAg), HBeAg and anti-HBe were tested by micro-particle enzyme immunoassays (MEIA; Abbott Laboratories, North Chicago, IL, USA).

Determination of intrahepatic HBV DNA level

Liver biopsies were separated into two parts: one part was fixed in formalin and processed to paraffin wax for histological evaluation; another part was stored immediately in −80 °C for the determination of intrahepatic HBV DNA level and precore and core promoter mutations.

Total DNA was extracted from frozen liver biopsy using the QIAGEN QIAamp DNA extraction kit, with a final elution in 400 μL of deionized water.

The amount of total intrahepatic HBV DNA in the extract was directly measured by the COBAS Amplicor HBV Monitor test. Instead of using 100 μL of serum for the test, 100 μL of diluted DNA extract was applied directly to the sample lyses buffer. The rest of the procedures were performed according to the instructions of the manufacturer. The final HBV DNA quantity was expressed in copies of HBV DNA/mL of sample extract.

Beta-actin gene was used as an internal control for the normalization of the amount of human genomic (hg) DNA in the extract. A TaqMan polymerase chain reaction (PCR) Reagent Kit (Applied Biosystems, Foster City, CA, USA) was used to measure the amount of hgDNA in the samples. Real-time PCR amplification and detection were performed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The amount of intrahepatic HBV DNA measured by COBAS Amplicor Monitor test was normalized by the amount (in ng) of hgDNA in the biopsy samples. The average copy number of HBV DNA per hepatocyte was calculated when the average hgDNA per hepatocyte was assumed to be 6.667 pg.

Determination of precore and core promoter mutations in the liver tissue

Total DNA was extracted from liver biopsy as mentioned above. Precore and core promoter mutations were determined using the line probe assay (INNO-LiPA HBV Precore; Innogenetics NV, Ghent, Belgium). The LiPA assay was performed as described previously.10 The lower detection limit of the LiPA assay is about 500 copies/mL of mutants in a mixed viral population. The LiPA assay can detect mutants when they are presenting in >5% among a mixed viral population.

Histological evaluation and immunostaining of HBcAg and HBeAg

The liver tissues available for histological evaluation were at least 1 cm in length and with six and more portal tracts.

Immunostaining for HBcAg, HBeAg and HBsAg were performed on 5-μm sections from the formalin-fixed, paraffin-embedded liver tissue. After blocking endogenous peroxidase in 3% H2O2 solution and non-specific protein binding sites with associated normal second-antibody animal serum, sections were incubated with first antibodies at 4 °C overnight. After washing with Tris Buffer Saline, the sections were incubated with biotin-labelled second antibody at 37 °C for 30 min. Then the antibody binding was detected using standard streptavidin ABC method (Strept ABComplex/HRP, K0377; Dako, Glostrup, Denmark) with 3,3-diaminobenzidine tetrahydrochloride (DAB) as chromogen. The first antibodies used were rabbit anti-HBc polyclonal antibody (B0586, 1:200; Dako), monoclonal anti-HBe antibody E6 (gift of Dr B. Ferns and Prof. R. Tedder) 11 and goat anti-HBs monoclonal antibody (3E7, M3506, 1:50; Dako). The second antibodies were biotin-swine anti-rabbit antibody (X0453, 1:50; Dako) or biotin-goat anti-mouse antibodies (E0433, 1:200; Dako).

The expression pattern of HBcAg was classified into CP, nuclear and cytoplasmic (NC), and nuclear prominent (NP). The cases showing cytoplasmic expression in more than 90% of HBcAg-positive hepatocytes were taken as CP. The cases with nuclear expression in more than 90% of HBcAg-positive hepatocytes were taken as NP. The cases with both cytoplasmic and nuclear expression of HBcAg, but both <90% of HBcAg-positive cells, were taken as NC. The above scoring system was also adapted to HBeAg-positive hepatocytes.

Histological activity index (HAI) was scored according to the criteria of Knodell et al.12 HAI for inflammation (HAI-i) was the sum of the score for portal, periportal and lobular inflammation. Liver tissue with HAI-i higher than 3 was regarded as case with active liver disease.

Statistical analysis

The Statistical Program for Social Sciences (SPSS 11.0 for windows; SPSS Inc, Chicago, IL, USA) was used for all statistical analyses. The data were analysed with Chi squared test, Fisher's exact test and Mann–Whitney test. Logistic regression was performed to determine the association of precore mutations, core promoter mutations and the frequency of cytoplasmic expression of HBcAg, and the association of the amount of HBcAg-positive hepatocytes, the expression pattern of HBcAg and the frequency of active liver disease. Results were considered statistically significant if P-values were <0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Demographic and serologic data

The demographic, serologic data and HBV DNA level were listed in Table 1. HBeAg-positive patients were younger than anti-HBe-positive patients (P = 0.005). HBeAg-positive patients and anti-HBe-positive patients had comparable male to female ratio (P = 0.1), prevalence of precore (P = 0.40) and core promoter (P =0.77) mutations.

Table 1.  Demographic, serological data and HBV DNA levels of patients
 HBeAg-positiveAnti-HBe-positiveP-value
  1. HBV, hepatitis B virus; HBeAg, hepatitis B e antigen.

Age [median (range) years]38.0 (18.5–53.6)44.8 (24.9–66.6)0.005
Male to female ratio15:728:40.1
Prevalence of precore mutations7/22 (31.8%)15/32 (46.9%)0.40
Prevalence of core promoter mutations15/22 (68.2%)23/32 (71.9%)0.77
Serum HBV DNA levels [Median (range) copies/mL]4.1 × 108 (1.86 × 106–1.1 × 1013)1.0 × 107(<2.0 × 102–1.4 × 109)<0.001
Intrahepatic HBV DNA level [median (range) copies of HBV DNA per hepatocyte)45.8 (1–370)4.0 (0–49)<0.001

No significant differences were found in serum HBV DNA levels between patients with and without precore or core promoter mutations in both HBeAg-positive and anti-HBe-positive patients (all P > 0.05).

Histological evaluation

Patients were stratified according to serum HBeAg status. Intrahepatic inflammation was evaluated in portal, periportal and lobular area. It was found that HBeAg-positive patients with core promoter mutations had more severe portal and periportal inflammation but similar lobular inflammation compared with HBeAg-positive patients without core promoter mutations [median portal inflammation (range): 3(1–4) vs. 1(1–3), P = 0.021; median periportal inflammation (range): 3(1–4) vs. 1(0–3), P = 0.009; median lobular inflammation (range): 1(1–3) vs. 1(0–3), P = 0.162, respectively]. There was no significant difference in the score of fibrosis between HBeAg-positive patients with and without core promoter mutations [median HAI-f (range): 1(0–4) vs. 1(0–3) respectively, P = 0.16].

In anti-HBe-positive patients, patients with core promoter mutations had more severe periportal inflammation compared with those without core promoter mutations [median periportal inflammation (range): 3(0–4) vs. 1(0–3), P = 0.043]. There were no difference in the other inflammation scores, total inflammation score and the score of fibrosis.

No significant differences were found in the scores of inflammation and fibrosis between patients with and without precore mutations in either HBeAg-positive or anti-HBe-positive patients.

Intrahepatic HBV DNA levels

Intrahepatic HBV DNA levels were determined in 40 patients. HBeAg-positive patients had significantly more copies of HBV DNA per hepatocyte compared with anti-HBe-positive patients (Table 1, P < 0.001). The copies of HBV DNA per hepatocyte correlated significantly with serum HBV DNA level (r = 0.71, P < 0.001), but it had no significant correlation with serum alanine aminotransferase level (P = 0.31), the score of inflammation (r = 0.34) and the score of fibrosis (r = 0.88) in the liver tissue.

There was no significant difference in the copies of HBV DNA per hepatocyte between patients with and without precore or core promoter mutations in both HBeAg-positive and anti-HBe-positive patients (all P > 0.05).

Two of six anti-HBe-positive patients with HBV DNA levels lower than 105 copies/mL had mildly active liver disease (HAI-i of 3 out of 18). These two patients had 0.002 and 1 copy of HBV DNA per hepatocyte respectively.

Intrahepatic expression of HBcAg and HBeAg

Liver tissues from 34 patients were available for the immunostaining of HBcAg, HBeAg and HBsAg. The demographics, serum HBeAg status, serum and intrahepatic HBV DNA levels, core promoter and precore mutations, histology and immunostaining patterns of these 34 patients are listed in Table 2. The different immunostaining patterns from six different patients are illustrated in Figure 1.

Table 2.  Demographics, HBV DNA levels, precore/core promoter mutations, intrahepatic expression of HBcAg and HBeAg, and histology of 34 patients
 Core promoterPrecore
Wild typeMutantWild typeMutant
  1. HBV, hepatitis B virus; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; CP, cytoplasmic prominent; NC, nuclear and cytoplasmic; NP, nuclear prominent; Neg, negative; Pos, positive.

  2. 1 Serum HBV DNA level expressed as log 10 of the measured value. ‘0’ represents serum HBV DNA level lower than 200 copies/mL.

  3. 2 Intrahepatic HBV DNA level expressed as copy of HBV DNA per hepatocyte.

Sex (male/female)7/220/517/410/3
Age [median (range)]34.2 (18–50)44.4 (27–67)42.0 (10–50)45.9 (28–67)
Serum HBeAg (Pos/Neg)5/410/1511/104/9
Score of inflammation
 Periportal (0/1/2/3/4)1/6/0/2/00/7/0/9/91/7/0/9/40/6/0/2/5
 Lobular (0/1/2/3/4)2/6/0/1/01/21/0/3/02/16/0/3/01/11/0/1/0
 Portal (0/1/2/3/4)0/7/0/2/00/6/0/17/20/7/0/14/00/6/0/5/2
Score of fibrosis (0/1/2/3/4)5/2/0/2/05/7/0/11/25/7/0/8/15/2/0/5/1
Serum HBV DNA1 [median (range)]7.82 (6.73–9.56)7.25 (0–9.37)7.25 (5.99–9.56)7.54 (0–8.76)
Intrahepatic HBV DNA2 [median (range)]10 (0–370)19 (0–151)25 (0–370)10 (0–151)
Intrahepatic HBcAg
 Percentage1 (0–55)7 (0–90)7 (0–90)5 (0–55)
 Expression pattern (CP/NC/NP/Neg)2/0/5/29/6/4/68/4/4/53/2/5/3
Intrahepatic HBeAg (Pos/Neg)4/59/168/135/8
image

Figure 1. Expression of hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in the liver tissue. (a) Cytoplasmic prominent expression of HBcAg (patient with core promoter mutation and precore wild type); (b) Nuclear and cytoplasmic expression of HBcAg (patient with core promoter mutation and precore wild type); (c) Nuclear prominent expression of HBcAg (patient with core promoter wild type and precore mutation); (d) Cytoplasmic expression of HBeAg (patient with core promoter wild type and precore wild type); (e) Nuclear expression of HBeAg (patient with core promoter mutation and precore wild type); (f) Expression of HBsAg (patient with core promoter wild type and precore mutation).

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Twenty-six patients (76.5%) were positive for HBcAg in the liver tissue. The percentage of HBcAg-positive hepatocytes correlated moderately with intrahepatic HBV DNA level (r = 0.37, P = 0.047) and serum HBV DNA level (r = 0.37, P = 0.03). But there were no significant differences in the intrahepatic and serum HBV DNA levels between patients with CP, NC and NP expression of HBcAg (median intrahepatic HBV DNA level: 47.55, 22.23 and 31.91 copies/cell in patients with CP, NC and NP expression of HBcAg respectively, P = 0.42; median serum HBV DNA levels: 2.0 × 107, 8.4 × 107 and 3.1 × 109 copies/mL in patients with CP, NC and NP expression of HBcAg respectively, P = 0.70). No significant difference was found in the percentage of HBcAg-positive hepatocytes between patients with and without precore or core promoter mutations (P > 0.05).

The percentage of HBcAg-positive hepatocytes was not significantly correlated with the score of inflammation and fibrosis (P = 0.13 and 0.11). Patients with CP, NC and NP expression of HBcAg had comparable score of inflammation and fibrosis (P = 0.11 and 0.40). Logistic regression analysis after adjustment for the percentage of HBcAg-positive hepatocytes and serum HBeAg status showed that cytoplasmic expression of HBcAg was significantly associated with active liver disease (HAI-i > 3) [Odds Ratio = 9.0, 95% confidence interval (CI) = 1.11–71.04, P = 0.037].

As shown in Table 2, cytoplasmic HBcAg expression was more frequently found in patients with core promoter mutations compared with patients without core promoter mutations [78.9% (15/19) vs. 28.6% (2/7), P = 0.028]. Patients with and without precore mutations had comparable frequency of cytoplasmic HBcAg expression in liver tissue. Multiple logistic regressions were performed to determine the association of precore mutations, core promoter mutations and the cytoplasmic expression of HBcAg. Core promoter mutations were found to be independently associated with cytoplasmic expression of HBcAg (odds ratio = 9.4, 95% CI = 1.3–67.6, P = 0.026) after adjusting for precore mutations and serum HBeAg status.

Thirteen patients (38.2%) showed detectable expression of HBeAg in liver tissue: 11 of 15 HBeAg-positive patients (73.3%) and two of 19 anti-HBe-positive patients (10.5%). Both anti-HBe-positive patients with HBeAg expression had NC HBcAg expression in the liver and HAI score for inflammation of 8. They had serum HBV DNA levels of 5.06 × 107 and 1.17 × 108 copies/mL respectively. The LiPA assay showed that both patients had core promoter mutants and precore mutants without detectable core promoter or precore wild type virus in the liver tissues.

Thirty-three patients (97.1%) showed HBsAg expression in liver tissue (Figure 1). The expression of HBsAg did not show any significant difference in HBeAg-positive and anti-HBe-positive patients, in patients with and without precore or core promoter mutations.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

In accordance with previous studies,13,14 this study demonstrated that patients with core promoter mutations had more severe inflammation and fibrosis compared with patients without core promoter mutations.

Patients with and without core promoter mutations had comparable serum HBV DNA levels, intrahepatic HBV DNA levels and amount of HBcAg-positive hepatocytes. An interesting finding was that presence of core promoter mutations was independently associated with cytoplasmic expression of HBcAg. It has been reported that core promoter mutations could increase the production of core protein (15-fold).15 Furthermore cytoplasmic expression of HBcAg is supposed to be the target of HBV-specific cytotoxic T lymphocyte. Subcellular localization of HBcAg is also supposed to be cell cycle-regulated.16, 17 Although the number of patients in this study is relatively small, it is possible that core promoter mutations induce more severe liver damage through modulating the subcellular localization of HBcAg. In vitro study should be performed to confirm the effect of core promoter mutations on the subcellular distribution of HBcAg.

There are conflicting reports about the association of precore mutations and severity of liver disease.18 This study showed that patients with and without precore mutations had comparable degree of inflammation and fibrosis, comparable serum and intrahepatic HBV DNA levels. Contrary to previous studies,4 presence of precore mutations also had no significant association with cytoplasmic expression of HBcAg in this cohort of patients. One possible reason is that the effect of the simultaneously existed core promoter mutations has not been studied in previous studies. Another possible explanation is the method used in this study. The LiPA assay is sensitive enough to detect 5–10% of precore mutations in the mixed virus population. The small population of precore mutations might be not sufficient to significantly affect the synthesis of precore protein and therefore subcellular distribution of HBcAg. Further quantitative analysis of the relationship of proportion of precore and core promoter mutants among the mixed virus population with expression pattern of HBcAg and liver damage is indicated.

Another interesting finding was that two anti-HBe patients had detectable HBeAg expression in the liver tissue. The LiPA assay showed that both patients had precore mutations alone and core promoter mutations alone in the liver tissue. As the presence of HBeAg expression in the liver tissue indicates precore wild type virus,19 our finding confirmed a previous study conducted by Schneider et al.20 that a very low level (0.1%) of precore wild type virus could sustain persistent secretion of HBeAg. Immunostaining of HBeAg may be a useful tool for the determination of existence of precore wild type virus.19 Both anti-HBe patients also had high HBV DNA level and cytoplasmic expression of HBcAg and HAI-i of 8. This is compatible with the observation that the expression of HBeAg in anti-HBe-positive patients is associated with more severe liver disease.21, 22

In conclusion, core promoter mutations are associated with cytoplasmic expression and more severe liver disease. Patients with and without precore mutations had comparable severity of liver damage.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References
  • 1
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    Villari D, Pollicino T, Spinella S, et al. Hepatitis B E antigen detection in formalin-fixed liver biopsy specimens. A tool to investigate wild-type and E-minus variant HBV infection. Am J Clin Pathol 1995; 103: 13640.
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    Lindh M, Horal P, Dhillon AP, Furuta Y, Norkrans G. Hepatitis B virus carriers without precore mutations in hepatitis B e antigen-negative stage show more severe liver damage. Hepatology 1996; 24: 494501.
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