慢性乙型肝炎患者乙型肝炎病毒前C区基因突变与临床关系

为了解慢性乙型肝炎患者是否有前Ｃ区基因突变及其与临床的关系，应用快速检测乙型肝炎病毒前Ｃ区基因突变方法，对５３例患者进行了研究。结果显示：２５例ＨＢＶＤＮＡ阳性患者中１３例有突变株感染，最常见的突变形式为１８９８位或伴有１９０１位突变（１０／１３例）。     

乙型肝炎病毒前C区和/或核心启动子区突变的临床意义

目的探讨乙型肝炎病毒前C区和／或核心启动子区突变的临床意义。方法应用全自动DNA基因测序仪检测91例慢性乙型肝炎及肝硬化患者乙型肝炎病毒前C区和／或核心启动子区位点变异情况，并观察发生突变组与未发生突变组在HBVDNA载量和肝脏病理改变等方面有无差异。结果HBeAg阳性慢性乙型肝炎患者的变异率为10．7％，HBeAg阴性慢性乙型肝炎患者的变异率为65．2％（P〈0．05）；肝硬化代偿期组变异率为46．2％，失代偿期组变异率为35．7％（P〉0．05）；35例基因变异患者HBVDNA载量（取1g值）为4．98±1．38，与56例未变异患者的6．36±1．31无显著差异（P〉0．05）；基因变异的慢性乙型肝炎患者肝组织病理损害较未变异患者为重。结论乙型肝炎病毒前C区和／或核心启动子区突变多见于HBeAg阴性慢性乙型肝炎患者中，但似乎与肝硬化的发生无明显的关系。     

慢性乙型肝炎患者乙型肝炎病毒前C区基因突变与干扰素的关系

应用聚合酶链反应（ＰＣＲ）技术扩增乙型肝炎病毒（ＨＢＶ）ＤＮＡ前Ｃ和部分Ｃ区基因，快速检测ＨＢＶ前Ｃ终止密码突变方法，对慢性乙型肝炎患者干扰素治疗与ＨＢＶ前Ｃ突变关系作了初步研究。６例干扰素治疗后４例ＨＢＶＤＮＡ转阴，２例阳性者均检出突变株。１１例非干扰素治疗者中，检出突变株３例。１例干扰素治疗１个月后ＨＢＶｅ抗累（ＨＢｅＡｇ）转阴，优势病毒株已由突变株与野生株共存状态替代原有野生株，提示干扰素治疗后ＨＢｅＡｇ转阴并不意味着ＨＢＶ被清除，而有潜在ＨＢＶ前Ｃ突变的可能。     

乙型肝炎病毒前C区、C启动子区、前S2区基因突变与慢性肝病的相关性

近年研究发现，乙型肝炎病毒（HBV）前C区nt1896位G→A、C启动子区（CP）nt1762、nt1764双突变、以及前S区基因突变与慢性肝炎患者病情活动和慢性化、疾病严重程度有一定关系。为此，我们分别检测无症状HBV携带者、慢性乙型肝炎患者、肝硬化患者血清HBV DNA基因序列，对前C、CP、前S2区域的突变情况与HBV不同感染状态的关系作一探讨。     

乙型肝炎病毒相关慢性肝病前C区/BCP区基因突变的临床意义

实验采用PCR技术及芯片杂交原理，对65例乙型肝炎病毒(HBV)相关性慢性肝病患者进行前C区及BCP区A1896、A1899及nt1762／nt1764联合突变检测，探讨HBV相关性慢性肝病前C区／BCP区基因突变的临床意义。     

亚洲乙型肝炎病毒前C区突变检测的临床意义

１９８９年Ｂｒｕｎｅｔｔｏｅｔａｌ［１］和Ｃａｒｍａｎｅｔａｌ［２］先后报道抗Ｈｂｅ阳性的慢性肝炎患者中,发现了ＨＢＶ前Ｃ区突变株,即在前Ｃ区１８９６点ＴＧＧ转变为ＴＡＧ,产生终止码,阻止ＨＢｅＡｇ合成．后来在许多国家和地区都相继发现了相同的突变株．在我国也有许多关于前Ｃ区突变与慢性肝炎肝硬变有关的报道［６］．这些报道总体上说明１８９６点突变株在毒力上不但比野型株强,而且与野型株有相同的复制和感染能力．我们用双重ＰＣＲ方法对ＨＢＶ感染者血清中ＨＢＶＤＮＡ前Ｃ区１８９６点的突变率进行检测,研究其…     

产前阻断对乙型肝炎病毒前C区和C启动子区的影响

目的 探讨产前阻断,即产前注射乙型肝炎免疫球蛋白(HBIG)对HBV前C区和基本C启动子(BCP)区核苷酸序列的影响.方法 无症状慢性HBV携带孕妇120例,产前阻断HBIG组67例,不产前阻断非HBIG组53例,荧光定量PCR法检测血清HBV DNA载量,ELISA检测血清HBsAg、HBeAg,套式PCR扩增HBV DNA前C区和BCP区核苷酸片段,DNA自动荧光测序仪对PCR产物直接测序.组间均数比较采用t检验,组间率比较采用卡方检验.结果 HBIG组33例孕妇收集到阻断前、后双份血清,其中23例阻断前、后HBV DNA前c区、BCP区均扩增测序成功.阻断前、后HBV前C区+BCP区、前C区、BCP区核苷酸的替代变异率分别为1.5%和1.4%、0.7%和0.6%、1.7%和1.7%(Fisher's精确检验,x2值分别为0.627、0.689、1.000,P＞0.05).阻断前、后1896G→A、1899G→A、1762A→T、1764G→A热点总变异发生率分别为27.2%和13.0%(x2=5.717,P=0.017),但各热点变异的发生率阻断前、后分别为30.4%和17.4%、17.4%和4.3%、26.1%和13.0%、34.8%和17.4%,差异无统计学意义(P＞0.05).扩增成功的53例HBIG组和47例非HBIG组孕妇临产时HBV DNA前C区+BCP区、前C区、BCP区核苷酸的替代变异率分别为0.9%和0.8%、0.3%和0.3%、1.1%和0.9%(Fisher's精确检验,x2值分别为0.434、0.839、0.340,P＞0.05);HBIG组和非HBIG组孕妇1896G→A、1899G→A、1762A→T、1764G→A热点总变异发生率及各热点变异发生率分别为5.7%和10.1%、9.4%和14.9%、0和2.1%、7.5%和10.6%、5.7%和12.8%,但差异无统计学意义(P＞0.05).结论 产前阻断可能不增加孕妇HBV DNA前C区、BCP区核苷酸变异;产前阻断可能减少孕妇体内HBV前C区、BCP区热点变异的发生.     

乙型肝炎和肝癌患者乙型肝炎病毒前C区1896位点突变的研究

为探索乙型肝炎病毒前Ｃ区突变是否与肝损程度及肝癌发生有关，对１３９例ＨＢｓＡｇ、ＨＢＶＤＮＡ和抗－ＨＢｅ阳性，ＨＢｅＡｇ阴性的慢性ＨＢＶ感染者和肝癌患者的血清标本，采用３＇碱基特异性聚合酶反应进行了第１８９６位核苷酸突变的检测分析。     

Diagnosis and management of pre-core mutant chronic hepatitis B

Chronic hepatitis due to pre-core hepatitis B virus (HBV) mutants presents as hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). HBeAg-negative CHB represents a late phase in the natural course of chronic HBV infection that develops after HBeAg loss and seroconversion to anti-HBe. It is usually associated with pre-core stop codon mutation at nucleotide 1896 (mainly selected in non-A HBV genotypes), but also with other pre-core changes or with mutations in the basic core promoter region (mainly in HBV genotype A). In chronic HBV infections, pre-core mutants can be detected both in patients with HBeAg-negative CHB and in inactive hepatitis B surface antigen (HBsAg) carriers. The diagnosis of HBeAg-negative CHB is based on HBsAg positivity, HBeAg negativity, and mainly on increased alanine aminotransferase (ALT) and serum HBV-DNA levels and exclusion of other causes of liver disease. The differential diagnosis between patients with CHB and inactive HBsAg carriers can be made only by close follow-up of aminotransferase activity and viraemia levels, although the cut-off level of serum HBV DNA has not been definitely determined. IgM anti-HBc levels have also been suggested as an index that increases the diagnostic accuracy for transient hepatitis flares, while liver biopsy confirms the diagnosis and evaluates the severity of the liver disease. Interferon-alpha (IFN-alpha) and lamivudine are the two drugs that have been tried, mainly in the management of HBeAg-negative CHB. A 12-month course of IFN-alpha achieves sustained biochemical remission in about 20% of patients, which has been associated with improvement in the long-term outcome of this subset. A 12-month course of lamivudine is rather ineffective, maintaining remission in less than 15% of patients after cessation of therapy. Long-term lamivudine is associated with progressively increasing rate of virological and subsequent biochemical breakthroughs due to YMDD mutants, with approximately 30% of patients remaining in remission in the third year of therapy. Several other antiviral agents are currently being evaluated in this setting with combined regimens being the most reasonable step for the near future.     

Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity

BACKGROUND/AIMS: We aimed to clarify the clinical relevance of hepatitis B virus pre-core mutant detection in patients with chronic hepatitis B using a newly developed assay. METHODS: Viral genotypes and pre-core mutations were studied in relation to viral persistence and liver disease severity using INNO-LiPA methodology. The study group included 151 patients with chronic hepatitis B, 85 positive for HBeAg (group I) and 66 positive for anti-HBe (group II). RESULTS: The prevalence of viral genotypes in group I was: 64% A, 1% B, 15% C, 19% D, 0% E, 0% F and in group II: 39% A, 0% B, 2% C, 56% D, 2% E, 2% F (p<0.001). The prevalence of mutations at pre-core codon 28 (M2) was lower in group I (5%) than in group II (64%) (p<0.001). The prevalence of pre-core promoter mutations was also lower in group I (21%) than in group II (61%) (p<0.001). M2 mutations were more frequently detected in genotype D than in genotype A (p<0.001), while the other mutations were not influenced by viral genotype. Serum HBV DNA levels were significantly lower in group II versus group I (p<0.001), and in patients with any of the pre-core mutations versus wild-type sequence (p<0.01). Although cirrhosis was more frequent in group II (37%) versus group I (22%) and in patients with either one of the pre-core mutation (31%) versus wild-type sequence (25%), there was no statistical difference in liver severity assessed by ALT levels and Knodell score. CONCLUSION: Pre-core mutants, whose molecular pattern is strongly dependent on viral genotypes, are associated with viral persistence in anti-HBe positive patients with ongoing chronic hepatitis B. The availability of this rapid assay should allow a precise monitoring of viral pre-core mutants during the course of chronic hepatitis B.     

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.     

Effect of multiple mutations in the core promoter and pre-core/core region of hepatitis B virus genome on the response to interferon in e antigen-positive chronic hepatitis B

BACKGROUND AND AIMS: Hepatitis B virus (HBV) genomic mutations may be one of the factors that influence the efficacy of interferon (IFN) therapy. The aim of this study was to investigate the effects of mutations in different parts of the HBV genome on IFN therapy. METHODS: We studied the baseline clinical, biochemical, serologic and virologic parameters in 17 patients with e antigen-positive chronic hepatitis B. The DNA sequence of the X gene and pre-core/core gene in serum samples of these patients was analyzed before the initiation of IFN therapy. RESULTS: All five patients with the T1762-A1764 mutation were IFN responsive, while among the 12 remaining patients, only two responded to therapy. Among five patients with both a pre-core A1896 mutation and a mutation in the epitope aa 107-118 of the core region, four were non-responders whereas the fifth responded to therapy. In three other patients with A1896 mutations, one with simultaneous mutations in five lymphocytic epitopes did not respond to therapy; the two remaining patients with concomitant mutations in one or two epitopes were responsive. Serum HBV-DNA levels were lower and titers of antibody to hepatitis B virus core antigen-immunoglobin M (anti-HBc-IgM) were higher in the responders than in the non-responders. Hepatitis B virus genotypes B and C were found to be in all these Chinese patients. CONCLUSIONS: These results suggest that HBV genomic mutations, serum viral loads and titers of anti-HBc-IgM might be predictive of the efficacy of IFN therapy. These clinical findings should be further investigated by in vivo and in vitro experiments.     

Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients

AIM: To investigate the frequency of mutations in precore(pre-C) and basic core promoter(BCP) regions of hepatitis B virus(HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCRreverse dot blot hybridization and mismatch amplification mutation assay(MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen(HBe Ag) positive patients, and 84.6% and 96.2%, respectively, in HBe Ag negative patients. The rate of pre-C mutations was significantly higher in HBe Ag negative patients than in HBe Ag positive patients(χ~2 = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients(χ~2 = 2.43, P = 0.12). The presence of mutations in the pre-C(Wilcoxon W = 1802.5, P = 0.00) and BCP regions(Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci(P 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBe Ag negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.     

Cytotoxic T lymphocytes and CD4 epitope mutations in the pre-core/core region of hepatitis B virus in chronic hepatitis B carriers in Northeast Iran

Backgrounds

Hepatitis B virus (HBV) is vulnerable to many various mutations. Those within epitopes recognized by sensitized T cells may influence the re-emergence of the virus. This study was designed to investigate the mutation in immune epitope regions of HBV pre-core/core among chronic HBV patients of Golestan province, Northeast Iran.

Methods

In 120 chronic HBV carriers, HBV DNA was extracted from blood plasma samples and PCR was done using specific primers. Direct sequencing and alignment of the pre-core/core region were applied using reference sequence from Gene Bank database (Accession Number AB033559).

Results

The study showed 27 inferred amino acid substitutions, 9 of which (33.3%) were in CD4 and 2 (7.4%) in cytotoxic T lymphocytes’ (CTL) epitopes and 16 other mutations (59.2%) were observed in other regions.

Conclusions

CTL escape mutations were not commonly observed in pre-core/core sequences of chronic HBV carriers in the locale of study. It can be concluded that most of the inferred amino acid substitutions occur in different immune epitopes other than CTL and CD4.

     

Core promoter/pre-core mutations are associated with lamivudine-induced HBeAg loss in chronic hepatitis B with genotype C

BACKGROUND/AIMS: To clarify the factors associated with the efficacy of lamivudine. METHODS: Variables including basic core promoter (BCP) and pre-core (PreC) mutations were evaluated in 60 chronic hepatitis B e antigen (HBeAg)-positive patients with genotype C. Thirty patients were treated with lamivudine and the remaining 30 patients were age- and sex-matched controls. RESULTS: Severe fibrosis was significantly more frequent in patients with the BCP-mutant/PreC-wild (MW) and BCP-mutant/PreC-mutant (MM) patterns compared to BCP-wild/PreC-wild (WW) pattern (P=0.02). The cumulative rates of HBeAg loss at 6, 12 and 18 months were significantly higher in the lamivudine group (14.2, 36.3, and 60.9%) compared with the control group (17.6, 17.6, and 24.5%, P=0.03), and was especially pronounced in patients with the MW pattern (P=0.04). The rate of lamivudine-related HBeAg loss was significantly lower in patients with the WW pattern (P=0.03). Factors correlating with HBeAg loss were histological fibrosis and activity, hepatitis B virus-DNA levels, BCP/PreC mutation and lamivudine therapy. Multivariate analysis revealed BCP/PreC mutations and fibrosis were independent factors for HBeAg loss. CONCLUSIONS: With specific reference to the genotype C, we found earlier HBeAg loss was expected in patients carrying MM and MW patterns, while the efficacy of lamivudine was limited in patients with the WW pattern.     

Late onset hepatic failure due to hepatitis B virus with mutations in the pre-core region

A 60-year-old man complained of severe general fatigue on October 11, 1992. Pertinent laboratory findings were: aspantate aminotransferase (AST) 1920 IU, alanine aminotransferase (ALT) 2050 IU, and total bilirubin (T.Bil) 124 micromol/l (normal range, 0–17 micromol/l). Virological assay revealed that hepatitis B surface antigen (HBsAg), anti-hepatitis B e (HBe), anti-HBc, and immunoglobulin M (IgM) anti-HBc were positive, and anti-HBs, HBeAg, and anti-delta antibody were negative. A diagnosis of acute hepatitis due to hepatitis B virus was made. Despite a decrease in transaminase, jaundice worsened and prothrombin time was prolonged. On the 60th day of hospitalization, massive ascites developed, but the patient's consciousness was not impaired. Although albumin and diuretics were given, the ascites further increased. Paracentesis of 2000ml of ascitic fluid was performed twice a week. On the 120th day of hospitalization, the patient passed black stools and he exhibited renal failure 3 weeks later. Although severe jaundice persisted, he was still alert. On the 150th day of hospitalization, massive gastrointestinal bleeding occurred, due to hemorrhagic gastritis. Despite receiving intensive care, the patient died. Determination of the HBV DNA sequence revealed two point mutations in the pre-core region; these have not been reported elswwhere.     

乙型肝炎病毒C基因启动子和前C基因变异与HBeAg含量和肝炎病情的临床研究

研究乙型肝炎病毒(HBV)C基因启动子(CP)和前C基因变异对HBeAg表达和病情的影响。通过DNA扩增、基因序列分析检测48例慢性乙肝和12例慢性重型乙肝患者血清的HBV CP和前C基因序列,及通过微粒子发光法定量检测血清中HBeAg的含量。(1)前C终止变异(nt1896G→A)在重型乙型肝炎病例中的发生率显著升高(66.7％);CP双变异(nt1762A→T和1764G→A)则在慢性乙型肝炎中度和重度的病例中的发生率显著升高(分别为52.6％和54.5％)。(2)双变异组和终止变异组的HBeAg含量均显著下降,P<0.01。但终止变异组HBeAg含量的下降较双变异组更为明显,P<0.05,且eAb阳性率也显著升高,P相似文献