Daw-Jen Tsuei

Different hepatitis B virus core gene mutations in children with chronic infection and hepatocellular carcinoma.

Background: The significance of mutations of hepatitis B virus (HBV) precore/core antigen in causing persistent infection and subsequent liver diseases is debatable. Aim: To investigate HBV core gene sequence changes in children with chronic HBV infection and their implications in hepatocellular carcinoma (HCC). Methods: Thirty one chronic HBV infected children with documented hepatitis B e antigen seroconversion selected from 415 long term carrier children and 12 HBV related HCC children were studied. Four serial serum samples before and after hepatitis B e antigen seroconversion from each of the 31 children, and one serum sample taken from the 12 HCC children were subjected to HBV core gene sequence analysis. Results: Mutations accumulated as chronic infection persisted and most frequently occurred at core gene codon 21 (29%), codon 147 (29%), codon 65 (16%), and precore stop codon 28 (74%) in the 31 chronic HBV infected children. Core gene mutation sites in HCC children were identified at core codons 74, 87, and 159. HCC children had more mutations in the core gene than those with chronic HBV infection (p=0.013). Conclusion: Accumulation of mutations of HBV core region in HCC children differ from those in chronic HBV infected children. This may be a clue to the pathogenesis of paediatric HCC.

RBMY, a male germ cell-specific RNA-binding protein, activated in human liver cancers and transforms rodent fibroblasts.

The RNA-binding motif (RRM) gene on Y chromosome (RBMY), encoding a male germ cell-specific RNA-binding protein associated with spermatogenesis, was found inserted by hepatitis B virus (HBV) DNA in one childhood hepatocellular carcinoma (HCC). This study is aimed to explore the oncogenic potential of the RBMY protein. The RBMY transcripts, expressed exclusively in the testis of normal people, were detected by reverse transcription–polymerase chain reaction in 36% of HCCs from 90 males and in 67% of hepatoblastoma from six boys. The nontumor liver counter parts, cirrhotic liver tissues from children with biliary atresia, and other types of cancers, such as bile duct, colon, stomach, lung, prostate, and kidney, were all negative for RBMY expression. One to four types of RBMY transcripts, including wild type and variants with N-terminal RRM deletion, C-terminal SRGY (serine-arginine-glycine-tyrosine) boxes deletion, or deletion of both domains, were found in the testis and liver cancer tissues. The wild-type RBMY protein was expressed in the nucleus and demonstrated its tumorigenicity by transformation of mouse fibroblast NIH3T3 cells and in vivo tumor formation. The RBMY variant protein with deletion of C-terminal exons 9–12 was trapped in the cytoplasm and showed decreased tumorigenicity. Our results suggest that RBMY is a new candidate oncogene specific for male liver cancer.

Pre-S2 deletions of hepatitis B virus and hepatocellular carcinoma in children.

The cause of early oncogenesis in hepatitis B virus (HBV)-related childhood hepatocellular carcinoma (HCC) remains unclear. This study investigated whether pre-S deletion of HBV is related to childhood HCC. By using nested polymerase chain reaction, we compared the pre-S sequence of HBV from sera of children with HCC against control children with similar chronic HBV infection. The HBV in sera of children with HCC had a significantly higher rate of pre-S deletion than that of children with chronic HBV infection (p = 0.008). All except one of the pre-S deletions from the HCC group involved the pre-S2 region, whereas no pre-S2 deletion was found in the chronic HBV group (p = 0.003). There was a trend whereby genotype-C sera had a higher rate of pre-S2 deletion than genotype-B sera (p = 0.11). A multivariate logistic regression model revealed that pre-S deletion was an independent risk factor for HCC in children (odds ratio: 36.69, p = 0.015). In conclusion, pre-S2 deletion does not need to take decades to occur; its presence in nearly half of children with HCC, in contrast to its absence in children with chronic HBV infection, suggests a link between pre-S2 deletion and HCC development in children.

Longitudinal Study on Mutation Profiles of Core Promoter and Precore Regions of the Hepatitis B Virus Genome in Children

Precore nucleotide 1896 and core promoter mutations may account for hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection, yet the mutational profiles of the core promoter are largely unknown in children. An age-matched, case-control study enrolled 110 chronic HBV-infected children, including 55 HBeAg seroconverters and 55 nonseroconverters. Precore and core promoter genes of HBV were sequenced and the serum viral genomes were genotyped from three serial serum samples of the seroconverters and from one serum sample of the nonseroconverters. Higher frequency of A1775G and G1799C mutation rates and lower frequency of A1752G mutation rate were found in the seroconverters. Precore 1896 mutation appeared more in seroconverters than in nonseroconverters (45.5%versus 10.9%; p < 0.001). 1762 + 1764 mutation rates were not different between the seroconverters (9.1%) and the nonseroconverters (5.5%). Genotype B was the major type. Genotype C was associated with core promoter 1762 + 1764 mutations in the seroconverter group (p = 0.023). The conclusions of this study include the following:1) mutations of core promoter at nucleotide position 1752, 1775, and 1799 have significant correlations with HBeAg seroconversion; 2) core promoter 1762 + 1764 mutations play a minimal role in HBeAg seroconversion; 3) precore 1896 mutant accounted for half of childhood HBeAg seroconversion; 4) genotype C is associated with 1762 + 1764 mutations during the process of HBeAg seroconversion.

Lamivudine treatment in maternally transmitted chronic hepatitis B virus infection patients

Background : Lamivudine treatment in chronic carriers who acquired hepatitis B virus through maternal transmission were investigated.

Human Interleukin-10 Genotypes Are Associated with Different Precore/Core Gene Mutation Patterns in Children with Chronic Hepatitis B Virus Infection

OBJECTIVE To elucidate the association between human interleukin-10 (IL-10) genotypes and hepatitis B virus (HBV) precore/core gene mutation in children with chronic HBV infection. STUDY DESIGN The study group comprised of 21 children with chronic HBV infection with spontaneous hepatitis B e antigen (HBeAg) seroconversion who were followed for more than 10 years. Another nine children without HBeAg seroconversion served as the control subjects. Sera at the immune tolerance and inflammatory phase (alanine aminotransferase, >80 IU/L) were subjected to HBV precore/core sequence analysis. IL-10 -1082 polymorphism was also determined. RESULTS HBV precore/core gene mutation increased significantly more in the inflammatory phase than in the tolerance phase (G1896A, 76.2% versus 4.8%; C1913A, 33.3% versus 0%; C2189A, 28.6% versus 4.8%; G2304A, 52.4% versus 14.3%) in study group (n = 21) but not the control group (n = 9). Subjects with the G/G genotype at the IL-10-1082 polymorphism site had higher C2189A mutation rate than the A allele carriers (P = .02). C2189A mutation carriers are associated with more viral load decrement from tolerance to inflammatory phase (P = .01) and earlier spontaneous HBeAg seroconversion (P = .01). CONCLUSIONS The G/G genotype at the IL-10 -1082 polymorphism is associated with higher C2189A mutations, lower HBV viral load at immune inflammatory phase, and earlier spontaneous HBeAg seroconversion than A allele carriers.

Male germ cell-specific RNA binding protein RBMY: a new oncogene explaining male predominance in liver cancer.

Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen-peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome-specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC.

RBMY, a novel inhibitor of glycogen synthase kinase 3β, increases tumor stemness and predicts poor prognosis of hepatocellular carcinoma

Male predominance of hepatocellular carcinoma (HCC) occurs particularly among young children aged 6‐9 years, indicative of a possible role of the Y chromosome–encoded oncogene in addition to an androgenic effect. The discovery of oncogenic activation of RBMY (RNA‐binding motif on Y chromosome), which is absent in normal hepatocytes but present in male HCC tissues, sheds light on this issue. Herein, we report on a critical hepatocarcinogenic role of RBMY and its ontogenic origin. During liver development, the Ser/Thr phosphorylated RBMY is expressed in the cytoplasm of human and rodent fetal livers. It is then silenced in mature hepatocytes and restricted to scarce expression in the bile ductular cells. Upon hepatocarcinogenesis, a noteworthy increase of cytoplasmic and nuclear RBMY is observed in HCC tissues; however, only the former is expressed dominantly in hepatic cancer stem cells and correlates significantly to a poor prognosis and decreased survival rate in HCC patients. Cytoplasmic expression of RBMY, which is mediated by binding to nuclear exporter chromosome region maintenance 1 and further enriched upon Wnt‐3a stimulation, confers upon tumor cells the traits of cancer stem cell by augmenting self‐renewal, chemoresistance, cell‐cycle progression, proliferation, and xenograft tumor growth. This is achieved mechanistically through increasing Ser9 phosphorylation‐inactivation of glycogen synthase kinase 3β by RBMY, thereby impeding the glycogen synthase kinase 3β–dependent degradation of β‐catenin and eventually inducing the nuclear entry of β‐catenin for the transcription of downstream oncogenes. Conclusion: RBMY is a novel oncofetal protein that plays a key role in attenuating glycogen synthase kinase 3β activity, leading to aberrant activation of Wnt/β‐catenin signaling, which facilitates malignant hepatic stemness; because of its absence from normal human tissues except the testis, RBMY represents a feasible therapeutic target for the selective eradication of HCC cells in male patients. (Hepatology 2015;62:1480–1496)

Differential integration rates of hepatitis B virus DNA in the liver of children with chronic hepatitis B virus infection and hepatocellular carcinoma

Background and Aim:  Integration of hepatitis B virus‐DNA (HBV‐DNA) into the host genome, a phenomenon found frequently in hepatocellular carcinomas (HCC) and causally linked to oncogenesis, has not been well characterized in children. The aim of the present study was to determine the prevalence of HBV integration more accurately and to decide whether the integration rate varies at different stages of chronic HBV infection in children.

Mutations of T-Cell Epitopes in the Hepatitis B Virus Surface Gene in Children With Chronic Infection and Hepatocellular Carcinoma

BACKGROUND AND OBJECTIVES:Hepatitis B virus (HBV) infection induces an interaction of host immune responses against virus antigen-presenting hepatocytes. The emergence of mutants is a strategy through which the virus can escape host attacks and produce chronic infection. In this study, we aimed to investigate mutations of the human leukocyte antigen-A2-restricted T-cell epitope (TCE) in chronic HBV-infected children.METHODS:In total, 441 chronic HBV-infected children were longitudinally followed-up every 6 months. They were divided into hepatitis B e antigen (HBeAg) (−) (N = 60) and HBeAg (+) (N = 381) groups according to this seromarker at their enrollment. The HBeAg (+) group was further divided into HBeAg (+/−) (N = 229) and HBeAg (+/+) (N = 152) groups, depending on the occurrence of spontaneous HBeAg seroconversion. Twenty-five children with HBV-related hepatocellular carcinoma (HCC) were also recruited. TCE mutations were examined using the latest serum samples, and serum samples at enrollment were used if TCE mutations were present in the latest serum samples in the seroconverters. HBV genotypes and liver enzymes were also analyzed.RESULTS:The HBeAg (+/+) group had a lower TCE mutation rate (7.9%) than that of the HBeAg (+/−) (29.2%), HBeAg (−) (26.7%), and HCC (28.0%) groups. In the HBeAg (+/−) group, TCE mutations were present before HBeAg seroconversion in 11.9% of the subjects. Those with TCE mutations showed HBeAg seroconversion at an older age (16.1 ± 5.3 yr vs 12.7 ± 5.7 yr, P < 0.001) and with higher peak alanine aminotransferase (ALT) levels (median 175 U/L vs 119 U/L, P = 0.03) than those without TCE mutations.CONCLUSIONS:TCE mutations tended to be positively associated with HBeAg seroconversion and higher ALT levels in chronic HBV-infected children.