BACKGROUND/AIMS: Considering the incidence of prevailing hepatitis B virus (HBV) genotypes in neighboring nations, the predominance of genotype C in Korea is exceptional and needs to be confirmed by nationwide investigation. METHODS: A total of 510 HBsAg (+) or HBeAg (+) serum samples was collected from subjects in several cities and harbors throughout the Korean peninsula for genotype (A-G)-specific multiplex PCR analysis. Another 40 serum samples from chronic HBV carriers from Iksan city were selected for sequencing of the entire HBV genome. Phylogenetic analysis was performed with 22 whole genomic sequences of Korean HBV strains enrolled in GenBank. RESULTS: An amplicon was found in 377 specimens and genotype C occupied 98.1% (370 cases); none of the other genotypes were found. A mixed pattern of genotypes B and C was seen in seven specimens (1.9%), of which five were tested using PCR targeting the X fragment; no genotype B bands were found. With the exception of 1 case, which was subgenotype A2, whole sequences of Korean HBV strains (n=62) belonged to subgenotype C2. CONCLUSIONS: The prevailing HBV genotype in Korea is C2; the other genotypes occur only rarely. Future studies should include confirmation of the detection of genotypes other than C.
Genotype ; Hepatitis B Surface Antigens/blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/classification/*genetics ; Humans ; Korea/epidemiology ; Phylogeny ; Protein Precursors/analysis/genetics ; Sequence Analysis, DNA ; Viral Envelope Proteins/analysis/genetics

OBJECTIVETo study the association between hepatitis B virus (HBV) mutants and the pathogenesis of severe hepatitis B by full-length HBV genome.

METHODSSerum samples from 10 severe hepatitis B patients were collected in our hospital. Serum HBV DNAs were extracted using DNA mini Kit, and amplified by LA Taq DNA polymerase to yield full-length HBV DNA. PCR products were isolated and cloned into vector pUCm-T, then transfected into DH-5 alpha cells. Positive clones were selected and checked by digestion, and full-length HBV DNAs were sequenced.

RESULTS4 cases were cloned into vector pUCm-T successfully and completed the full-length sequencing. Among them, 3 cases had a G to A mutation at nucleotide 1896 in pre-C region and 1 had a double mutation of T1762-A1764 in the core promoter region. Some amino acid changes occurred within the known CTL, B or T cell epitopes of the PrS2 and C regions.

CONCLUSIONSThis method could serve to study the relationship between HBV genome and the pathogenesis of severe hepatitis B.

Adult ; B-Lymphocytes ; metabolism ; Cloning, Molecular ; Epitopes ; genetics ; Genome, Viral ; genetics ; Hepatitis B ; etiology ; virology ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Protein Precursors ; genetics ; Sequence Analysis ; T-Lymphocytes, Cytotoxic ; metabolism

Biomarkers ; blood ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; diagnosis ; genetics ; Early Diagnosis ; Humans ; Insulin-Like Growth Factor II ; analysis ; Liver Neoplasms ; blood ; diagnosis ; genetics ; Protein Precursors ; blood ; Prothrombin ; RNA, Messenger ; analysis ; alpha-Fetoproteins ; metabolism

OBJECTIVEThis study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection.

METHODSSera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive.

RESULTSDNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.

CONCLUSIONSOccult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.

Base Sequence ; Blood Donors ; DNA, Viral ; analysis ; Genotype ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Antibodies ; Hepatitis B Core Antigens ; isolation & purification ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; Protein Precursors ; genetics

OBJECTIVETo investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R), and their role in the development of rat testis.

METHODSOligonucleotide primers were designed from the sequences of rat hypothalamus prepro TRH (ppTRH) and pituitary TRH-R cDNA for reverse transcription polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed, and expression was found in E. coli DH5-alpha. ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats at different developmental stages by real time quantitative RT-PCR.

RESULTSThe quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). ppTRH and TRH mRNA signals were detected on day 15 and increased progressively on days 20, 35, 60 and 90.

CONCLUSIONOur results suggest that rat testis could specifically express TRH and TRH-R, and the transcriptions of ppTRH and TRH-R genes in the rat testis were development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on the physiological significance of TRH and TRH-R expression in rat testis.

Age Factors ; Animals ; Male ; Protein Precursors ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Thyrotropin-Releasing Hormone ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Testis ; growth & development ; metabolism ; Thyrotropin-Releasing Hormone ; genetics

OBJECTIVETo evaluate the effects of the Syk mRNA expression in human breast cancer on tumor growth and metastasis, and to study the correlation of expression of the Syk gene with ER, PR, p53 and HER2/neu.

METHODSSpecimens from 40 breast cancer patients (tumor tissues, adjacent normal tissues), 15 fibroadenoma were detected for their expression of the Syk gene and level of Syk mRNA by semi-RT-PCR technique. Meanwhile, ER, PR, p53, HER2/neu were detected in 40 tumor tissues from breast cancer with immunohistochemical staining.

RESULTSAll normal breast tissues were detected the expression of the Syk gene. Unlike normal breast tissue, 31 out of 40 breast cancer tissue did not show any detectable Syk mRNA expression, there were significant difference in two groups (chi(2) = 47.4, P < 0.05). The level of Syk mRNA in the primary breast cancer tissues were significantly lower than that in the adjacent non-cancerous breast tissues (t = 3.41, P < 0.05). Furthermore, only one breast cancer tissue in 18 patients with lymph node metastasis had the Syk mRNA expression, the rate and level of Syk mRNA expression in the patients with lymph node metastasis were lower than those without lymph node metastasis (chi(2) = 3.77, P < 0.05, t = 2.74, P < 0.05). Syk expression was correlated to p53 expression.

CONCLUSIONThe expression of the Syk gene may play an important role in suppressing growth and metastasis of breast cancer.

Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Enzyme Precursors ; genetics ; Estrogens ; analysis ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Intracellular Signaling Peptides and Proteins ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; genetics ; RNA, Messenger ; genetics ; metabolism ; Receptor, ErbB-2 ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Syk Kinase ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo identify the HBV genotype-specific tag sequence.

METHODSThe large S region sequences from 930 HBV genomes were aligned to identify the genotype-specific tag sequences. PCR was used to check the genotyping effect of these tags.

RESULTSTwo tag sequences, sequence between 149-169 and sequence between 461-483, were identified in the large S region. Using primers specific to these tag sequences, the genotype of HBV can be specifically identified.

CONCLUSIONThese tag sequences can be used for HBV genotyping.

Base Sequence ; DNA Primers ; Gene Library ; Genes, Viral ; Genotype ; Hepatitis B virus ; classification ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Protein Precursors ; genetics ; Sequence Analysis, DNA

Disappearance of hepatitis B surface antigens (HBsAg) in chronic hepatitis B usually indicates clearance of hepatitis B virus (HBV) infection. However, false HBsAg negativity with mutations in pre-S2 and 'a' determinant has been reported. It is also known that YMDD mutations decrease the production of HBV and escape detection of serum HBsAg. Here, we report overlapping gene mutations in a patient with HBsAg loss during the lamivudine therapy. After 36 months of lamivudine therapy in a 44-yrold Korean chronic hepatitis B patient, serum HBsAg turned negative while HBV DNA remained positive by a DNA probe method. Nucleotide sequence of serum HBV DNA was compared with the HBV genotype C subtype adr registered in NCBI AF 286594. Deletion of nucleotides 23 to 55 (amino acids 12 to 22) was identified in the pre-S2 region. Sequencing of the 'a' determinant revealed amino acid substitutions as I126S, T131N, M133T, and S136Y. Methionine of rtM204 in the P gene was substituted for isoleucine indicating YIDD mutation (rtM204I). We identified a HBV mutant composed of pre-S2 deletions and 'a' determinant substitutions with YMDD mutation. Our result suggests that false HBsAg negativity can be induced by combination of overlapping gene mutations during the lamivudine therapy.
Adult ; Amino Acid Sequence ; Anti-HIV Agents/therapeutic use ; Base Sequence ; Comparative Study ; DNA Mutational Analysis ; DNA, Viral/blood/chemistry/genetics ; DNA-Directed DNA Polymerase/genetics ; Gene Deletion ; Genes, Overlapping/*genetics ; Hepatitis B Surface Antigens/blood/*genetics ; Hepatitis B virus/*genetics ; Hepatitis B, Chronic/blood/*drug therapy/virology ; Humans ; Lamivudine/*therapeutic use ; Male ; Molecular Sequence Data ; *Mutation ; Protein Precursors/genetics ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid ; Viral Proteins/genetics