RESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular/virología , Antígenos del Núcleo de la Hepatitis B/genética , Hepatitis B Crónica/virología , Neoplasias Hepáticas/virología , Mutación , Adolescente , Alanina Transaminasa/sangre , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Niño , Preescolar , Codón , Análisis Mutacional de ADN , Femenino , Estudios de Seguimiento , Humanos , Modelos Logísticos , MasculinoRESUMEN
Integration of hepatitis B virus (HBV) DNA is found in most HBV-related human hepatocellular carcinomas (HCCs). In the past, construction of genomic libraries was mainly employed to study the role of viral integration. However, large amounts of tissue DNA and a laborious screening procedures were required. Inverse polymerase chain reaction (IPCR) is based on the simple procedures of digestion of DNA with restriction enzymes and circularization of cleavage products before amplification using primers synthesized in the opposite orientations to those normally employed for PCR. This technique allows the in vitro amplification of DNA flanking a region of known sequence. By employing this method, starting from nanograms of hepatoma DNA, two adjacent cellular sequences were cloned from 11 HBV integrants in three HCCs. The original configurations in the chromosomes were further confirmed. One of the flanking cellular sequences was identified as the human 28S rRNA gene, the other was not found homologous to any known human sequences. This method appears to be practical and can be improved further to clone more flanking cellular sequences, especially in early and small HCCs.
Asunto(s)
Carcinoma Hepatocelular/virología , ADN Viral/genética , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/virología , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Viral/aislamiento & purificación , Amplificación de Genes , Reordenamiento Génico , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Integración Viral/genéticaRESUMEN
The DNA of tumor tissue K1 obtained at autopsy from a case of hepatocellular carcinoma (HCC) in a 9-year-old boy contained integrated hepatitis B virus (HBV) DNA at a single site in the chromosome (case 2, Chang et al.: Hepatology 13:316-320, 1991). To characterize further the integrated viral DNA sequences, a genomic library of the K1 DNA was constructed in the lambda L47.1 vector. One phage clone, designated KTM-1, containing integrated HBV DNA and cellular flanking sequences was obtained from this library. The restriction map and DNA sequence of this clone showed that the integrated HBV DNA was partially deleted and rearranged. The most conserved viral DNA sequences were surface and X genes and arranged in the opposite orientation. The viral core gene was not present. Using chloramphenicol acetyltransferase (CAT) assay, the C-terminal truncated X open reading frame was demonstrated to retain its trans-activating ability. The result suggested that the functional integrated X gene may play a role in hepatocarcinogenesis. The study also showed that the right cellular flanking sequences were human alphoid repetitive sequences.
Asunto(s)
Carcinoma Hepatocelular/microbiología , ADN de Neoplasias/análisis , ADN Viral/análisis , Virus de la Hepatitis B/aislamiento & purificación , Neoplasias Hepáticas/microbiología , Secuencia de Bases , Carcinoma Hepatocelular/etiología , Niño , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Hepatitis B/complicaciones , Humanos , Neoplasias Hepáticas/etiología , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Ácido Nucleico , Activación Transcripcional , Integración ViralRESUMEN
A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.