Hepatitis B virus subgenotype C2- and B2-associated mutation patterns may be responsible for liver cirrhosis and hepatocellular carcinoma, respectively

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.

Precore and Core Promoter Mutations of the Hepatitis B Virus Gene in Chronic Genotype C -Infected Children

The precore (G1896A) and core promoter (A1762T, G1764A) mutations of the hepatitis B virus gene are known to be associated with changes in immunologic phase or the progression to complicated liver disease in adults. We analyzed these mutations in chronically HBV-infected children. Serum was collected from 37 children with chronic HBV infection from March 2005 to September 2008. HBV DNA extraction and nested PCR were followed by sequencing of the PCR products. The children were 6.7 +/- 4.6 yr old. All of 37 children had HBV genotype C. Of the cohort, 31 (83.8%) were HBeAg-positive and 6 (16.2%) were HBeAg-negative; the former group comprised 18 (48.6%) who were in the immune-tolerance phase (ITP) and 13 (35.2%) in the immune-clearance phase (ICP). Most of the patients had HBV DNA levels of > 1.0 x 10(8) copies/mL. In the ITP group, only 1 (5.5%) had core promoter mutations, and none had the precore mutation. In the ICP group, only 2 (15.4%) had core promoter mutations; the remaining 6 patients had HBV DNA levels of < 2.0 x 10(3) copies/mL and no core promoter/precore mutations. The very low incidence of the precore/core promoter gene mutation, in children, suggests that these mutations may be the result of life-long chronic HBV infection.

Prevalência de genótipos e de mutantes pré-core A-1896 do vírus da hepatite B e suas implicações na hepatite crônica, em uma população da Amazônia oriental / Prevalence of hepatitis B virus genotypes and the occurrence of precore mutation A-1896 and to correlate them with the clinical presentation of chronic hepatitis, in a population group of the eastern Amazon region

A infecção pelo virus da hepatite B apresenta amplo espectro de manifestações clínicas. Objetivando conhecer os genótipos do HBV mais prevalentes e determinar a ocorrência da mutação pré-core A-1896, em uma população da Amazônia oriental, correlacionando com o diagnóstico clínico, foram selecionados 51 pacientes portadores crônicos de HBsAg e HBV-DNA positivos e divididos em três grupos: grupo A (n=14, pacientes assintomáticos); grupo B (n=20, sintomáticos HBeAg positivos) e grupo C (n=17, sintomáticos HBeAg negativos), sendo usado o sequenciador automático ABI modelo 377 para identificação de genótipos e mutantes pré-core. Os resultados evidenciaram o genótipo A como o mais prevalente, 81,8%, 89,5% e 93,7%, nos grupos A, B e C, respectivamente. A mutação pré-core A-1896 foi encontrada em 11,5% (3/26), sendo todos assintomáticos. Concluiu-se que na população estudada o genótipo A foi o mais prevalente e houve baixa ocorrência do mutante pré-core A-1896, ambos não se constituindo fatores agravantes da doença hepática.

Hepatitis B virus (HBV) infection presents itself with a variety of clinical manifestations. The present work aims to describe the prevalence of HBV genotypes and the occurrence of precore mutation A-1896 in a population group of the Eastern Amazon region of Brazil and to correlate them with the clinical presentation of chronic HBV infection. 51 HBsAg carriers (HBV-DNA positive) were selected and divided into three groups: A (14 asymptomatic subjects), B (20 HBeAg positive symptomatic patients) and C (17 HBeAg negative symptomatic patients). Using an automa ed DNA sequencer ABI model 377 by sequencing for determined of genotypes and precore mutation. The results showed that the genotype A was the most commonly found (81,1%, 89,5% and 93,7% in groups A, B and C, respectively) and precore mutation A-1896 was described in 11,5% (3/26) of group A subjects. Genotype A of HBV was the most prevalent (89,1%) and low occurrence of precore mutation A-1896, both not associate with the worst outcome of the chronic infection of HBV.

The frequency of pre-core gene mutations in chronic hepatitis B infection: a study of Malaysian subjects.

A retrospective study was carried out to determine the frequency of the pre-core stop codon mutant virus in a group of chronic hepatitis B carriers: 81 cases were considered [33 hepatits B e antigen (HBe) positive and 48 HBe negative]. All of the HBe positive cases had detectable viral DNA by hybridization analysis; in the case of the HBe negative cases, one third had detectable viral DNA by hybridization analysis and two thirds had HBV DNA detectable by polymerase chain reaction (PCR) amplification. Pre-core stop codon mutant detection was carried out on all specimens using allele-specific oligonucleotide hybridization following PCR amplification of the target sequence. The pre-core mutant was detected in 13/33 (39.4%) of HBe positive cases and in 32/48 (66.7%) of HBe negative cases. Sequence analysis was carried out on 8 of the 16 HBe negative specimens that did not carry the pre-core mutant virus to determine the molecular basis for the HBe minus phenotype in these cases: the 1762/1764 TA paired mutation in the second AT rich region of the core promoter was detected in five cases; a start codon mutation was detected in one case. The predominant mutation resulting in the HBe minus phenotype in our isolates was the 1896A pre-core ("pre-core stop codon") mutation; other mutations responsible for the phenotype included the core promoter paired mutation and pre-core start codon mutation. In view of the high frequency of the pre-core mutant virus, sequence analysis was performed to determine the virus genotype on the basis of the nucleotide sequence of codon 15. The sequences of 21 wild type virus (14 HBe positive and 7 HBe negative cases) were examined: 15 were found to be codon 15 CCT variants (71.4%); the frequency in the HBe positive group was 12/14 (85.7%), while that in the HBe negative group was 3/7 (42.9%). The high frequency of the codon 15 CCT variant in association with the frequent occurrence of the pre-core mutant in our isolates concurs with the results of other studies.

Core promoter and precore mutants of hepatitis B virus: prevalence and clinical relevance in chronic hepatitis patients.

The present study was conducted to determine prevalence and exact type, as well as nucleotide position of the precore/core mutations of hepatitis B virus found in Thai patients diagnosed with chronic hepatitis and/or cirrhosis in relation to the clinical parameters established with the respective patients. To that end, 24 HBeAg-positive and 56 HBeAg-negative individuals were selected at random from a cohort of altogether 256 chronic liver disease patients. DNA was extracted from their blood sera, amplified by polymerase chain reaction using semi-nested primers and subjected to direct sequencing. Clinically, the HBeAg-positive chronic hepatitis patients displayed significantly higher transaminase levels than those negative for HBeAg. Our results showed 2 of the 7 (28.6%) PCR-positive HBeAg-positive sera displaying double mutations in the core promoter region at position 1762/64. The nucleotide sequences obtained from the 24 PCR-positive HBeAg-negative sera revealed 18 (75%) mutations in the core promoter region (1762/64), and/or 7 (29.2%) mutations at position 1753, and/or 6 (25%) mutations of the start codon (1814), and/or 8 of (33.3%) nucleotide 1896 turning codon 28 into a stop codon and one sample (4.2%) displaying a deletion between nucleotides 1758-1772. It is suggested that the mutations observed have an impact on the DNA secondary structure in such a way that successful transcription of the HBeAg gene is rendered impossible. To what extent this mutation influences the severity of chronic liver disease remains to be elucidated.