ORIGINAL ARTICLE
Clinical Significance of Hepatitis B Virus Precore and Core Promoter Variants in Korean Patients With Chronic Hepatitis B Sun Young Yim, MD,* Soon Ho Um, MD, PhD,* Jin Young Jung, MD,* Tae Hyung Kim, MD,* Jin Dong Kim, MD, PhD,w Bora Keum, MD, PhD,* Yeon Seok Seo, MD, PhD,* Hyung Joon Yim, MD, PhD,* Yoon Tae Jeen, MD, PhD,* Hong Sik Lee, MD, PhD,* Hoon Jai Chun, MD, PhD,* Chang Duck Kim, MD, PhD,* and Ho Sang Ryu, MD, PhD*
Background/Aim: We aimed to clarify the clinical significance of precore (preC)/core promoter (CP) variants of hepatitis B virus (HBV) in chronic hepatitis B (CHB) patients. Methods: We assessed serum HBeAg, HBV DNA levels, alanine transferase (ALT) levels, and progression of liver fibrosis in 226 Korean CHB patients, presumed to be infected with genotype C HBV, to analyze HBV variants in the preC region (G1896A) and CP regions (A1762T, G1764A). Results: CP and preC variants were more frequently found in HBeAg-negative patients than in HBeAg-positive patients (P < 0.05). HBeAg-positive patients with CP variants had higher ALT levels and more advanced fibrosis scores (all P < 0.01) than those without variants; those with preC variant had lower HBV DNA levels (P = 0.009), with no significant difference in ALT levels and fibrosis scores. However, no significant correlation was found between HBV variants and clinicopathologic findings in HBeAg-negative patients. Furthermore, multivariate analysis revealed that (1) progression of liver fibrosis (ZF2) was associated with older age in both HBeAg-positive and HBeAg-negative patients (P < 0.05) and with CP variants in the HBeAg-positive group (P = 0.007), and (2) HBV DNA levels were positively correlated with ALT levels, irrespective of HBeAg (P < 0.05), whereas they were negatively correlated with the presence of preC variant in the HBeAg-positive group (P = 0.004). Conclusions: In HBeAg-positive CHB patients infected with genotype C HBV, preC variant was associated with enhanced host immune response with lower HBV DNA levels, whereas CP variants were associated with severe liver damage and liver fibrosis progression. Key Words: chronic hepatitis B, precore variant, core promoter variants, hepatitis B virus, hepatitis B e antigen
(J Clin Gastroenterol 2015;49:61–68) Received for publication June 25, 2013; accepted November 11, 2013. From the *Department of Internal Medicine, Division of Gastroenterology and Hepatology, Korea University College of Medicine, Seoul; and wDepartment of Internal Medicine, Division of Gastroenterology and Hepatology, Cheju Halla General Hospital, Cheju, Korea. Supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea. (A102065). The authors declare that they have nothing to disclose. Reprints: Soon Ho Um, MD, PhD, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Korea (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.jcge.com. Copyright r 2014 by Lippincott Williams & Wilkins
J Clin Gastroenterol
Volume 49, Number 1, January 2015
H
epatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B (CHB) patients is associated with a loss of HBeAg in serum. The appearance of antibody against HBeAg (anti-HBe) is generally followed by disappearance of hepatitis B virus (HBV) DNA in serum and normalization of serum alanine aminotransferase (ALT) levels.1 However, some HBeAg-negative CHB patients have persistent viremia and active liver disease even after seroconversion of HBeAg.2 In 1989, it was first reported that HBeAg-negative CHB patients infected by HBV variants with mutations in the precore (preC) region failed to produce HBeAg.3 The most common preC variant was a point mutation in G1896A that converted codon 28 (TGG) to a stop codon (TAG),4 originally believed to be present only in HBeAgnegative patients with active liver disease. However, preC variant was soon determined to exist, even in asymptomatic HBV carriers during HBeAg seroconversion, replacing wild-type HBV, suggesting that wild-type HBV is easily cleared by an immune elimination mechanism, whereas variant HBV with a preC mutation manages to escape immune clearance.5 Pairing of nucleotide 1858 with nucleoside 1896 occurs in the stem-loop structure of the RNA encapsidation sequence of pregenomic RNA of HBV. The G1896A preC mutation is readily induced in HBVs with the genome arrangement of T1858, to stabilize the stem-loop structure. Thus, emergence of the preC variant is probably closely related to genotypes of HBV, as well as immune pressure of the host.3,6 Two point mutations in the core promoter (CP) region, A1762T and G1764A, were found in the majority of CHB patients, irrespective of their serum HBeAg status.7 HBV with these double mutations formed the major type of virus existing in HBeAg-negative HBV carriers without the preC variant, suggesting that these CP variants down regulate preC mRNA transcription and prevail over immune elimination phase through a similar mechanism in G1896A preC variant.7 The lower serum HBeAg titers in HBeAgpositive patients with A1762T/G1764A variants than in those without them support a suppressive effect of the variants on HBeAg expression.8 The biological characteristics of preC or CP variant HBV, in terms of CHB pathogenesis, remain controversial. Stimulation of HBV progeny synthesis by CP variants, as well as suppression of HBeAg expression, was proven by many in vitro studies, but not all studies confirmed this phenomenon.9–11 Some in vitro studies showed that preC protein suppresses HBV replication and that the G1896A variant stimulates progeny DNA synthesis,12 but this was not the finding in another.13 The relationship between www.jcge.com |
61
J Clin Gastroenterol
Yim et al
serum HBV titers and preC or CP variants were either dependent on HBeAg status or had no correlation at all.14–16 PreC variant HBV in HBeAg-negative CHB patients was initially considered to be related to severe liver damage, but this was contradicted by another study.17 In addition, several studies demonstrated that the presence of CP variants is associated with either elevated serum ALT levels or severe histologic damage of the liver, especially in HBeAg-positive CHB patients,18,19 unlike the results of other studies.15,20 Similarly, the correlation between hepatic fibrosis and preC/CP variants in CHB patients is controversial. Some studies showed that CP variants are related to either advanced liver fibrosis or cirrhosis, whereas others did not.16,18,21 One study suggested that the preC variant had a significant correlation with liver disease progression.15,18,21,22 Thus, many studies have reported diverse results regarding the role of preC/CP variants in relation to serum HBV DNA levels, degree of liver damage, and liver disease progression. Several factors might be responsible for these inconsistencies between studies. First, most of the studies have used a small number of patients of various ethnicities, comprising different HBV genotypes and, consequently, inconsistent clinical data. Furthermore, most studies lacked histologic assessment, disabling the evaluation of liver disease progression. In addition, accurate measurement of HBV DNA level was difficult because of low sensitivity of HBV DNA quantification methods used in earlier studies. The aim of this study was to clarify the association of preC/CP variants with serum HBeAg, serum HBV DNA levels, degree of liver damage or inflammation represented by ALT levels, and liver fibrosis progression, as well as other factors that affect these clinical parameters. Our study is different from earlier studies: firstly, this study involves a large number of patients, and these patients are of the same ethnicity, with 99% of patients with same genotype of HBV (genotype C).23 Moreover, excluding those with liver cirrhosis identified using imaging studies, patients underwent liver biopsies. Lastly, quantification of serum HBV DNA and detection of virus genotype were performed based on highly sensitive methods.
MATERIALS AND METHODS Patients A cross-sectional study was conducted. A total of 367 CHB patients without a history of alcoholic liver disease, chronic hepatitis C, or chronic liver disease of other causes, who underwent testing for HBV variants in preC/CP regions from June 2005 to March 2009 at Korea University were screened. Of the 367, 141 of the patients were excluded because of lack of biopsy (88), prior history of antiviral treatment (45), or hepatocellular carcinoma (8). Finally, 226 CHB patients who were naive for antiviral treatment at the time of diagnosis and who had detectable serum HBV DNA were analyzed. All patients were tested for preC/CP variants, HBeAg, anti-HBe, and liver function. Liver biopsies and imaging studies, such as abdominal computed tomography and ultrasonography, were performed.
Liver Biopsy Liver biopsies were performed on 186 patients, and stages of fibrosis were scored from F0 to F4, according to Batts and Ludwig’s scoring system.24 Forty patients with
62 | www.jcge.com
Volume 49, Number 1, January 2015
liver cirrhosis, identified by performing abdominal computed tomography or ultrasonography, were graded F4.
Laboratory Tests Complete blood count, serum ALT levels, bilirubin levels, albumin levels, and prothrombin time were measured by standard methods. HBsAg, HBeAg, and anti-HBe were examined by an Abbott assay (Abbott Laboratories, North Chicago, IL). Quantification of serum HBV DNA levels was performed by the Cobas Amplicor Monitor Test (Roche Diagnostics, Rotkreuz, Swiss) and Hybrid Capture II HBV DNA Test (Chemiluscent Molecular Hybridization Assay; Digene Corporation, MD) from 2005 to June 2006, with HBV DNA levels ranging from 2.0 102 to 2.0 105 copies/mL and 1.4 105 to 1.7 109 copies/mL, respectively. We began using TaqMan real-time polymerase chain reaction quantification (Roche) in July 2007, and this improved our detection range to 1.0 102 to 8.5 108 copies/mL. Nucleotide sequence positions of the HBV genome were numbered according to Ono et al.25 CP and preC variants (nt 1762, nt 1764, and nt 1896) were detected using restriction fragment mass polymorphism method based on matrix-assisted laser desorption ionization timeof-flight mass spectrometry26 (Supplementary Fig. 1, Supplemental Digital Content 1, http://links.lww.com/JCG/ A121).
Statistical Analysis Data were analyzed using the SPSS version 20.0 software (SPSS Inc., Chicago, IL). P-values of
Clinical Significance of Hepatitis B Virus Precore and Core Promoter Variants in Korean Patients With Chronic Hepatitis B Sun Young Yim, MD,* Soon Ho Um, MD, PhD,* Jin Young Jung, MD,* Tae Hyung Kim, MD,* Jin Dong Kim, MD, PhD,w Bora Keum, MD, PhD,* Yeon Seok Seo, MD, PhD,* Hyung Joon Yim, MD, PhD,* Yoon Tae Jeen, MD, PhD,* Hong Sik Lee, MD, PhD,* Hoon Jai Chun, MD, PhD,* Chang Duck Kim, MD, PhD,* and Ho Sang Ryu, MD, PhD*
Background/Aim: We aimed to clarify the clinical significance of precore (preC)/core promoter (CP) variants of hepatitis B virus (HBV) in chronic hepatitis B (CHB) patients. Methods: We assessed serum HBeAg, HBV DNA levels, alanine transferase (ALT) levels, and progression of liver fibrosis in 226 Korean CHB patients, presumed to be infected with genotype C HBV, to analyze HBV variants in the preC region (G1896A) and CP regions (A1762T, G1764A). Results: CP and preC variants were more frequently found in HBeAg-negative patients than in HBeAg-positive patients (P < 0.05). HBeAg-positive patients with CP variants had higher ALT levels and more advanced fibrosis scores (all P < 0.01) than those without variants; those with preC variant had lower HBV DNA levels (P = 0.009), with no significant difference in ALT levels and fibrosis scores. However, no significant correlation was found between HBV variants and clinicopathologic findings in HBeAg-negative patients. Furthermore, multivariate analysis revealed that (1) progression of liver fibrosis (ZF2) was associated with older age in both HBeAg-positive and HBeAg-negative patients (P < 0.05) and with CP variants in the HBeAg-positive group (P = 0.007), and (2) HBV DNA levels were positively correlated with ALT levels, irrespective of HBeAg (P < 0.05), whereas they were negatively correlated with the presence of preC variant in the HBeAg-positive group (P = 0.004). Conclusions: In HBeAg-positive CHB patients infected with genotype C HBV, preC variant was associated with enhanced host immune response with lower HBV DNA levels, whereas CP variants were associated with severe liver damage and liver fibrosis progression. Key Words: chronic hepatitis B, precore variant, core promoter variants, hepatitis B virus, hepatitis B e antigen
(J Clin Gastroenterol 2015;49:61–68) Received for publication June 25, 2013; accepted November 11, 2013. From the *Department of Internal Medicine, Division of Gastroenterology and Hepatology, Korea University College of Medicine, Seoul; and wDepartment of Internal Medicine, Division of Gastroenterology and Hepatology, Cheju Halla General Hospital, Cheju, Korea. Supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea. (A102065). The authors declare that they have nothing to disclose. Reprints: Soon Ho Um, MD, PhD, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Korea (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.jcge.com. Copyright r 2014 by Lippincott Williams & Wilkins
J Clin Gastroenterol
Volume 49, Number 1, January 2015
H
epatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B (CHB) patients is associated with a loss of HBeAg in serum. The appearance of antibody against HBeAg (anti-HBe) is generally followed by disappearance of hepatitis B virus (HBV) DNA in serum and normalization of serum alanine aminotransferase (ALT) levels.1 However, some HBeAg-negative CHB patients have persistent viremia and active liver disease even after seroconversion of HBeAg.2 In 1989, it was first reported that HBeAg-negative CHB patients infected by HBV variants with mutations in the precore (preC) region failed to produce HBeAg.3 The most common preC variant was a point mutation in G1896A that converted codon 28 (TGG) to a stop codon (TAG),4 originally believed to be present only in HBeAgnegative patients with active liver disease. However, preC variant was soon determined to exist, even in asymptomatic HBV carriers during HBeAg seroconversion, replacing wild-type HBV, suggesting that wild-type HBV is easily cleared by an immune elimination mechanism, whereas variant HBV with a preC mutation manages to escape immune clearance.5 Pairing of nucleotide 1858 with nucleoside 1896 occurs in the stem-loop structure of the RNA encapsidation sequence of pregenomic RNA of HBV. The G1896A preC mutation is readily induced in HBVs with the genome arrangement of T1858, to stabilize the stem-loop structure. Thus, emergence of the preC variant is probably closely related to genotypes of HBV, as well as immune pressure of the host.3,6 Two point mutations in the core promoter (CP) region, A1762T and G1764A, were found in the majority of CHB patients, irrespective of their serum HBeAg status.7 HBV with these double mutations formed the major type of virus existing in HBeAg-negative HBV carriers without the preC variant, suggesting that these CP variants down regulate preC mRNA transcription and prevail over immune elimination phase through a similar mechanism in G1896A preC variant.7 The lower serum HBeAg titers in HBeAgpositive patients with A1762T/G1764A variants than in those without them support a suppressive effect of the variants on HBeAg expression.8 The biological characteristics of preC or CP variant HBV, in terms of CHB pathogenesis, remain controversial. Stimulation of HBV progeny synthesis by CP variants, as well as suppression of HBeAg expression, was proven by many in vitro studies, but not all studies confirmed this phenomenon.9–11 Some in vitro studies showed that preC protein suppresses HBV replication and that the G1896A variant stimulates progeny DNA synthesis,12 but this was not the finding in another.13 The relationship between www.jcge.com |
61
J Clin Gastroenterol
Yim et al
serum HBV titers and preC or CP variants were either dependent on HBeAg status or had no correlation at all.14–16 PreC variant HBV in HBeAg-negative CHB patients was initially considered to be related to severe liver damage, but this was contradicted by another study.17 In addition, several studies demonstrated that the presence of CP variants is associated with either elevated serum ALT levels or severe histologic damage of the liver, especially in HBeAg-positive CHB patients,18,19 unlike the results of other studies.15,20 Similarly, the correlation between hepatic fibrosis and preC/CP variants in CHB patients is controversial. Some studies showed that CP variants are related to either advanced liver fibrosis or cirrhosis, whereas others did not.16,18,21 One study suggested that the preC variant had a significant correlation with liver disease progression.15,18,21,22 Thus, many studies have reported diverse results regarding the role of preC/CP variants in relation to serum HBV DNA levels, degree of liver damage, and liver disease progression. Several factors might be responsible for these inconsistencies between studies. First, most of the studies have used a small number of patients of various ethnicities, comprising different HBV genotypes and, consequently, inconsistent clinical data. Furthermore, most studies lacked histologic assessment, disabling the evaluation of liver disease progression. In addition, accurate measurement of HBV DNA level was difficult because of low sensitivity of HBV DNA quantification methods used in earlier studies. The aim of this study was to clarify the association of preC/CP variants with serum HBeAg, serum HBV DNA levels, degree of liver damage or inflammation represented by ALT levels, and liver fibrosis progression, as well as other factors that affect these clinical parameters. Our study is different from earlier studies: firstly, this study involves a large number of patients, and these patients are of the same ethnicity, with 99% of patients with same genotype of HBV (genotype C).23 Moreover, excluding those with liver cirrhosis identified using imaging studies, patients underwent liver biopsies. Lastly, quantification of serum HBV DNA and detection of virus genotype were performed based on highly sensitive methods.
MATERIALS AND METHODS Patients A cross-sectional study was conducted. A total of 367 CHB patients without a history of alcoholic liver disease, chronic hepatitis C, or chronic liver disease of other causes, who underwent testing for HBV variants in preC/CP regions from June 2005 to March 2009 at Korea University were screened. Of the 367, 141 of the patients were excluded because of lack of biopsy (88), prior history of antiviral treatment (45), or hepatocellular carcinoma (8). Finally, 226 CHB patients who were naive for antiviral treatment at the time of diagnosis and who had detectable serum HBV DNA were analyzed. All patients were tested for preC/CP variants, HBeAg, anti-HBe, and liver function. Liver biopsies and imaging studies, such as abdominal computed tomography and ultrasonography, were performed.
Liver Biopsy Liver biopsies were performed on 186 patients, and stages of fibrosis were scored from F0 to F4, according to Batts and Ludwig’s scoring system.24 Forty patients with
62 | www.jcge.com
Volume 49, Number 1, January 2015
liver cirrhosis, identified by performing abdominal computed tomography or ultrasonography, were graded F4.
Laboratory Tests Complete blood count, serum ALT levels, bilirubin levels, albumin levels, and prothrombin time were measured by standard methods. HBsAg, HBeAg, and anti-HBe were examined by an Abbott assay (Abbott Laboratories, North Chicago, IL). Quantification of serum HBV DNA levels was performed by the Cobas Amplicor Monitor Test (Roche Diagnostics, Rotkreuz, Swiss) and Hybrid Capture II HBV DNA Test (Chemiluscent Molecular Hybridization Assay; Digene Corporation, MD) from 2005 to June 2006, with HBV DNA levels ranging from 2.0 102 to 2.0 105 copies/mL and 1.4 105 to 1.7 109 copies/mL, respectively. We began using TaqMan real-time polymerase chain reaction quantification (Roche) in July 2007, and this improved our detection range to 1.0 102 to 8.5 108 copies/mL. Nucleotide sequence positions of the HBV genome were numbered according to Ono et al.25 CP and preC variants (nt 1762, nt 1764, and nt 1896) were detected using restriction fragment mass polymorphism method based on matrix-assisted laser desorption ionization timeof-flight mass spectrometry26 (Supplementary Fig. 1, Supplemental Digital Content 1, http://links.lww.com/JCG/ A121).
Statistical Analysis Data were analyzed using the SPSS version 20.0 software (SPSS Inc., Chicago, IL). P-values of
