慢性乙型肝炎患者HBV C基因启动子变异的临床意义

目的:探讨慢性乙型肝炎中C基因启动子(BCP)变异的临床意义。方法:采用错配PCR与限制性长度片段多态性分析(RFLP)相结合,检测35例慢性乙型肝炎患者BCP区核苷酸(nt)1762碱基A→T和1764G→A联合突变及前C区nt1896G→A终止变异。结果:在35例慢性乙型肝炎中检出BCP区T1762A1764变异8例(23％),其中6例血清HBeAg( ),2例抗HBe( ),而7例前C区A1896变异中HBeAg( )2例,抗HBe( )5例,未见T1762A1764变异和A1896变异同时出现者。结论:提示HBV毒株BCP区T1762A1764变异可能与前C区A1896变异不同,它的出现不足以导致HBeAg(—)型的慢性肝炎。     

乙肝病毒C基因启动子变异与乙肝病情的关系

研究乙型肝炎病毒（HBV)C基因启动子（CP）变异与无症状慢性HBV携带者（AsC)肝炎发作及与慢性乙肝病情的关系。．通过PCR及其产物直接测序，检测4例AsC、27例慢性乙肝和3例慢性重型乙肝患者血清的HBV CP序列，并定量测定血清的HBV DNA。（1）CP主要变异为nt1726-1730聚集（1726A→C、1727A →T、1730C→G）和nt1762 1764双变异（1762A→T和1764G→A）。（2）CP聚集变与AsC首次肝炎 急性发作有关。11例中，8例出现CP聚集变异。且1例在AsC状态时无CP变异，肝炎发作时出现CP聚集变异。（3）CP聚集变异合并CP双突变的乙肝患者,表现为重型肝炎或迅速进展为肝硬化，HBV DNA高水平；HBeAg／抗HBe转移。CP聚集变异与AsC肝炎急性发作有关；CP聚集变异 与CP双变异同时存在，使慢性乙肝病情加重。     

慢性肝病患者乙型肝炎病毒BCP变异对HBV DNA复制水平影响的研究

目的探讨乙型肝炎病毒慢性感染中C基因启动子(BCP)变异对HBV DNA复制水平的影响及其临床意义。方法采用PCR微板核酸杂交结合ELISA检测显示技术,检测74例乙型肝炎病毒慢性感染者BCP区核苷酸(nt)1762碱基A→T和1764碱基G→A联合突变。结果在74例乙型肝炎病毒慢性感染者中检出BCP区T1762 A1764突变24例(32.4％),BCP变异阳性组的HBV DNA含量(10~(8.2992±0.8665)拷贝/ml)显著高于BCP变异阴性组的含量(10~(7.1737±1.1539)拷贝/ml ) P<0.001)。结论 BCP变异可引起HBV致病力增强,复制水平提高。     

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。     

乙型重型肝炎患者血清HBV前C/BCP变异检测及临床意义

目的探索乙型重型肝炎患者HBV前C/BCP区变异与临床病情关系。方法用荧光定量PCR法检测血清HBVDNA载量;PCR扩增前C/BCP区基因后进行序列测定。结果30例乙型重型肝炎患者中BCP变异有18例,HB-VDNA平均水平为2.71×107copies/mL,未发生BCP变异者有12例,HBVDNA平均水平为5.35×106copies/mL,两者间有显著差异。测序结果显示HBV前C区nt1896G→A终止变异和BCP变异(nt1762A→T和1764G→A)在乙型重型肝炎患者中发生率分别为43.3%(13/30)和60%(18/30),在慢性乙型肝炎患者则分别为40%(12/30)和30%(9/30),两组BCP变异(nt1762A→T和1764G→A)相比有显著差异(P=0.02)。结论BCP变异可影响病毒复制,BCP变异可能与肝病的严重程度存在着相关性。     

孕妇中乙型肝炎病毒前C区和核心启动子区热点变异研究

目的研究乙型肝炎病毒（HBV）携带产妇体内HBV前C区和核心启动子（CP）区热点变异情况。方法82例无症状慢性HBV携带临产孕妇采用HBV核酸扩增荧光定量检测试剂盒抽提孕妇血清HBVDNA,血清HBsAg、HBeAg采用EIA法检测,半巢式PCR法扩增产妇HBV前C区、CP区核苷酸片段,ABI3730型DNA自动荧光测序仪对PCR产物直接测序。结果82例无症状HBV携带临产孕妇中HBsAg单阳性52例,HBsAg、HBeAg双阳性30例;单阳性孕妇中1896G→A变异的检出率为21．2％（11／52）,仅测到1例双阳性孕妇存在1896G→A变异,1899G→A变异仅发生在1例单阳性孕妇,且与1896G→A变异连锁出现。1762A→／1764G→A变异总是连锁出现,且仅发生于单阳性孕妇,单阳性孕妇中双变异的检出率为17．3％（9／52）。结论HBV前C区、CP区1896G→A及1762A→T／1764G→A热点变异在无症状HBV携带单阳性孕妇中有较高的检出率,HBV前C区和核心启动子区的热点变异可能与肝损伤的严重程度无关。     

广西慢性乙型肝炎患者HBV核心基因启动子突变的研究

目的 了解HBV核心基因启动子突变与肝损害程度或HBeAg状态的关系。方法 用套式PCR扩增59例慢性乙型肝炎患者血清HBV核心基因启动子,阳性者用直接测序法检测。结果35例HBV DNA阳性,阳性率为59.3％。无正常序列标本,最常见的突变类型是nt1762、1764发生双突(A→T、G→A),占57.1％;其次为nt1799位点突变,由C→G,占54.4%,为无义突变;nt1752位点突变,由A→G,使该密码由异亮氨酸变为缬氨酸,占37.1％;nt1753T→C,占20.0%。T_(1762)A_(1764)突变株在HBeAg阳性、阴性患者组中的分布分别为31.3％、79.0％,两者差异有显著性,x~2 8.068 8,P<0.05。结论 HBV核心基因启动子突变在广两慢性乙型肝炎患者较常见,T_(1762)A_(1764)突变株与HBeAg阴性及慢性肝炎有关。     

HBV突变与HBV相关慢加急性肝衰竭相关性研究

目的探讨HBV突变与慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)之间的相关性。方法以87例HBV相关ACLF患者为研究对象,另选取52例慢性乙型肝炎(chronic hepatitis B,CHB)患者和51例HBV肝硬化(liver cirrhosis,LC)患者为对照,应用聚合酶链反应及基因测序对患者血清标本进行HBV基本核心启动子区、前C区和C区13个位点的突变检测,分析HBV突变与ACLF发生的相关性。结果 CHB、LC、ACLF组人均核苷酸变异检出位点分别为2.31、3.55和3.86个。nt1762、nt1846、nt1899和nt1913 4个突变位点的核苷酸突变率比较,ACLF组高于CHB组;nt1764位点突变率组间比较LC组高于ACLF组,而nt1846和nt1913位点突变率比较LC组低于ACLF组;nt1753、nt1762、nt1764和nt1899 4个突变位点的核苷酸突变率比较,LC组高于CHB组。多因素分析提示A1846T和C1913(A/G)核苷酸突变是预测ACLF的有效指标。结论 CHB或LC患者HBV基因序列的A1846T和C1913(A/G)核苷酸突变可能与其发生ACLF密切相关。     

乙型肝炎病毒基因变异对慢性乙型肝炎患者病情发展及抗病毒治疗的影响

目的 探讨乙型肝炎病毒(HBV)基因变异对慢性乙型肝炎(慢乙肝)患者病情发展、严重程度以及对抗病毒治疗疗效的影响。方法 采用基因芯片技术，对选取的部分乙型肝炎患者进行HBV基因变异位点的检测。结果 α-干扰素治疗24周以上，HBeAg不发生血清转化，或出现HBeAg(-)／抗-HBe( )，但HBV—DNA定量检测仍持续阳性患者与nt1896、nt1814、nt1762、nt1764位点突变有关；拉米夫定治疗后，HBV—DNA先下降或转阴，后又再度反弹患者与aa528、aa552变异(即YMDD变异)造成拉米夫定耐药有关；拉米夫定治疗52周以上的部分患者易发生YMDD变异(26．4％)；在慢性乙肝患者中nt1896位点变异较普遍(68．5％)；慢性重症肝炎、肝硬化失代偿、原发性肝癌与nt1896、nt1762、nt1764位点突变亦有关。结论 HBV基因变异可加重患者病情，影响抗病毒治疗效果。临床上通过对乙肝患者进行HBV常见变异位点进行检测，对判断疾病预后，调整抗病毒治疗方案具有一定的参考价值。     

HBV前-C区和BCP区变异与肝细胞癌的相关性分析

目的探讨HBV前-C区G1896A和BCP区A1762T/G1764A变异与肝细胞癌(HCC)的关系及其可能的机制。方法选择于青岛市传染病医院住院的HBV DNA104拷贝/ml的慢性HBV感染者82例,其中慢性乙型肝炎(CHB)29例,乙型肝炎肝硬化(LC)27例,HBV相关肝细胞癌(HCC)26例,采用实时荧光PCR法检测其HBV前-C区1896位变异和BCP区1762/1764位变异,并采用ELISA法检测血清肿瘤坏死因子α(TNF-α)水平。结果 HCC组和LC组前-C区G1896A和BCP区A1762T/G1764A变异率、血清TNF-α水平均显著高于CHB组,但HCC组和LC组间无显著差异。前-C区G1896A和BCP区A1762T/G1764A变异者血清TNF-α水平均显著高于非变异组。结论 HBV前-C区G1896A和BCP区A1762T/G1764A变异与HCC形成有关,机制是否与HBV变异和TNF-α水平间的因果关系相关,有待进一步研究。     

Precore and core promoter mutations,hepatitis B virus DNA levels and progressive liver injury in chronic hepatitis B

BACKGROUND/AIMS: To elucidate the viral factors responsible for progressive liver injury in chronic hepatitis B. METHODS: We analyzed 179 persistently infected patients (21 asymptomatic carriers, 126 with chronic hepatitis and 32 with cirrhosis) with genotype C hepatitis B virus (HBV). HBeAg/anti-HBe, levels of HBV DNA, mutations in the basic core promoter (BCP) region at nucleotides 1762/1764 and mutation in the precore (preC) region at nucleotide 1896 were determined. Serial samples from 18 patients also were analyzed. RESULTS: HBeAg/anti-HBe and HBV DNA levels per se were not related to liver fibrosis. The frequency of BCP mutations increased with progression of liver fibrosis. Although the preC mutation was detected more often among the LC group, the role of this mutation in progression of fibrosis seems less than that of the BCP mutations. Sequential analysis showed that (1) rapidly progressing cases were positive continuously for double mutations in the BCP with a wild-type precore sequence, and (2) asymptomatic cases with anti-HBe acquired the preC mutation during their clinical course. CONCLUSIONS: Double mutations in the BCP region at nucleotide 1762/1764 are closely related to progression of chronic liver disease. Acquisition of mutation in the preC region at nucleotide 1896 may contribute to inactivation of chronic liver disease.     

The effects of hepatitis B virus core promoter mutations on hepatitis B core antigen distribution in hepatocytes as detected by laser-assisted microdissection

BACKGROUND/AIMS: The severity of liver damage in patients with chronic hepatitis B is dependent on several factors such as subcellular localization of hepatitis B core antigen (HBcAg) and mutation of hepatitis B virus (HBV) DNA. Here we studied the interrelationship between these two factors both in situ and in vitro. METHODS: Hepatocytes from liver biopsies showing expression of HBcAg only in the cytoplasm (n=6), only in the nucleus (n=4) and in both cytoplasms or nucleus of different hepatocytes (n=5) were picked up by laser-assisted microdissection and were checked for nucleotide sequences of core promoter region of HBV DNA. HepG2 and Huh7 cell lines transfected with wild and mutant HBV DNA were checked for localization of HBcAg. RESULTS: The frequencies of core promoter mutations at nucleotide (nt) 1762 and nt 1764 was significantly higher in hepatocytes with cytoplasmic expression of HBcAg compared to that of nuclear expression of HBcAg (P<0.05). Cytoplasmic expression of HBcAg was observed more frequently in HepG2 and Huh7 cells transfected with HBV mutant type (nt 1762 and 1764) than HBV wild type (P<0.05). CONCLUSIONS: Cytoplasmic localization of HBcAg was associated with HBV DNA mutations at nt 1762 and 1764.     

Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B

In chronic replicative hepatitis B the significance of mutations in the basic core promoter (BCP), core upstream regulatory sequences (CURS) and negative regulatory element (NRE) for response to interferon (IFN) is unknown. A sequence analysis of the NRE, CURS, BCP, and precore region was performed from sera of 96 patients with chronic replicative hepatitis B (64 hepatitis B e antigen [HBeAg]-positive patients and 32 HBeAg-negative patients) treated with alfa-IFN (IFN-alpha). The overall sustained response (SR) rate to IFN was 30% with no significant difference between HBeAg-positive and HBeAg-negative patients. IFN responsiveness correlated to hepatitis B virus (HBV)-DNA levels, hepatitis B surface antigen (HBsAg) levels, the number of mutations in the complete BCP, especially nucleotide (nt) region 1753 to 1766 and mutations at nt 1762 and 1764. In HBeAg-positive hepatitis, SR to IFN was associated with a high number of mutations in the BCP (P <.04) and nucleotide region 1753 to 1766 (P <.015) as well as mutations at nucleotide 1764 (P <.007). In HBeAg-negative hepatitis, SR to IFN correlated with a low number of mutations in the BCP (P <.04) and nucleotide region 1753 to 1766 (P <.02) and a wild-type sequence at nt 1764 (P <.003). Prediction of IFN response was possible on the basis of nt 1764 in 77% of HBeAg-positive patients and 78% of HBeAg-negative patients. IFN response did not correlate with the occurrence of the 1896 mutation, mutations in the CURS or NRE, disease duration, ethnic origin of the patient, alanine transaminase (ALT) levels and HBV genotype. Our data suggest that HBV genome mutations located within the BCP are determinants of a response to IFN therapy.     

Association of core promoter/precore mutations and viral load in e antigen-negative chronic hepatitis B patients

Apart from core promoter A1762T/G1764A and precore G1896A mutations, other hepatitis B virus (HBV) mutants are detected in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). The aim of this study was to determine the effects of those mutants on clinical manifestation and viral loads of genotypes B and C HBV. Seventy-nine HBeAg-negative CHB patients with hepatitis flare were enrolled in this study and their HBV precore/core region were sequenced. Serial biochemical profiles and viral loads were assessed and compared. Fifty-three patients (67%) were infected by genotype B HBV and 26 (33%) were infected by genotype C HBV. The clinical manifestation and HBV viral loads were comparable between the two groups. However, genotype B was significantly associated with precore G1896A mutation (92.5%), and more mutations within nucleotide 1809-1817 were detected in patients infected by genotype B as compared with those infected by genotype C (18.9%vs 3.8%). Most of the cases had mutations at the -2, -3 or -5 position from the precore AUG initiation codon. Triple core promoter mutations T1753C/A1762T/G1764A [corrected] appeared to be linked to genotype C rather than genotype B HBV (19.2%vs 1.9%; P = 0.013). In multivariate analysis, the presence of either triple core promoter 1753/1762/1764 mutation or nucleotide 1809-1817 mutation was the only factor associated with lower HBV viral load (<70 Meq/mL) (odds ratio = 9.01; 95% CI 1.11-71.43; P = 0.04). In conclusion, minor HBV variants with mutations in the core promoter and precore region were detectable in genotypes B and C. Such HBV variants are genotype specific and related to viraemia levels.     

Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection.

BACKGROUND/AIMS: To investigate the correlation of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations in patients with chronic hepatitis B. METHODS: HBV genotyping, nucleotide mutation, serum HBV DNA level and serological markers were analyzed in 121 patients with chronic HBV infection using INNO-LiPA HBV genotyping, polymerase chain reaction (PCR) product-based sequencing, fluorescence quantitative PCR and enzyme-linked immunosorbent assays respectively. RESULTS: Forty (33.0%), 77 (63.6%), two (1.7%) and two (1.7%) patients had genotypes B, C, B/C and D infections respectively. Significant differences were found in serum HBV DNA levels (log10 copies/ml: 6.18 vs. 5.61, P=0.042) and mutations at nucleotide (nt) 1762/1764 (71.4% vs. 42.5%, P=0.002) between genotypes C- and B-infected patients. There were significant differences in the mean age, serum biochemical parameter levels and mutation rates in BCP/PC among hepatitis e antigen (HBeAg)-positive and -negative chronic hepatitis B (CHB) and liver cirrhosis (LC) groups. CONCLUSION: Genotypes C and B are predominant in China, and the frequent nt 1762/1764 mutation, which occurs commonly in HBeAg-negative CHB, especially in genotype C patients, may be associated with the progress of chronic HBV infection.     

乙型肝炎病毒家族聚集性感染者的基因型和基因变异特征分析

目的:研究HBV家族聚集性感染者的基因型和基因变异特征．方法:选择HBV聚集性感染家族成员内部基因型完全相同的共27个家族84人,其中HBV 携带者(ASC)29例;慢性活动性肝炎(CAH)23 例;肝硬化(LC)21例;肝细胞癌(HCC)11例;用基因芯片法检验前C1896和BCP1762,1764双突变;用测序法检验HBV基因型,并设相应基因型的HBV感染对照组．结果:B型HBV家族聚集感染者ASC率显著大于C型家族(X2=5．0734,P=0．0243,P<0．05); 其BCP1762．1764双突变的发生率也显著低于 C基因型(X2=22．4000,P=0．0000,P<0．001)．两种基因型的家族聚集感染者组与相应的对照组相比在疾病临床类型方面均无显著差异．结论:检验HBV家族聚集感染者的基因型和／或BCP区1762、1764基因的双突变可以协助疾病的预测分析．     

慢性乙型肝炎病毒感染者病毒前C区和基本核心启动子区变异检测及意义

目的 探讨乙型肝炎病毒（HBV）前C区和基本核心启动子（BCP）区变异与基因型及疾病进展间的关系。方法 收集HBV携带者（ASC）、慢性乙型肝炎（CHB）、肝炎肝硬化（LC）、肝细胞肝癌（HCC）患者血清148份，用半巢式聚合酶链反应扩增HBV前C／C基因部分片段，产物纯化后直接测序，检测前C区A1896及BCP区T1762／A1764变异。用S基因聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP）方法确定HBV基因型。结果 有128份血清能够成功分型和测序，其中B基因型60份，C基因型68份。在B基因型感染者中前C区A1896变异检出率（48．33％）明显高于C基因型感染者（29．41％，X^2=4．83，P〈0．05）；而BCP区T1762／A1764变异检出率却明显低于C基因型感染者，差异亦有统计学意义（30．00％：73．54%，X^2=24．25。P〈0．05）。前C区A1896变异在CHB、LC、HCC中的阳性检出率分别为46．88％（15/32）、39．39％（13／33）、51．52％（17／33）。与ASC的13．33％（4／30）相比，P分别〈0．05，差异有统计学意义。BCP区T1762／A1764变异检出率在HCC、LC组分别为87．88％（29／33）和72．73％（24／33）．明显高于CHB组的37．50％（12／32）及ASC组10.00％（3／30）（P〈0．05）。结论 前C区A1896变异常见于B基因型感染者，而BCP区T1762/A1764变异C基因型感染者多见。除ASC外．前C区A1896变异与疾病进展关系不大．而BCP区T1762/A1764变异与乙型肝炎进展及顶后相关。     

An outbreak of fulminant hepatitis B in immunocompromised hemodialysis patients

Background We aimed to clarify the pathogenesis of an outbreak of fulminant hepatitis B in hemodialysis (HD) patients whose compromised cell-mediated immunity in turn contributed to chronic hepatitis B virus (HBV) carriage.Methods Five consecutive adult HD patients with acute hepatitis B were evaluated. Viral genotype, mutations, and HBV-DNA levels were studied in relation to viral clearance, liver disease severity, and liver histology by immunostaining.Results All five patients had hepatitis B surface antigen (HBsAg) genotype C, a G-to-A stop codon mutation at nucleotide (nt) 1896 in the precore region, an A-to-T mutation at nt 1762 and an G-to-A mutation at nt 1764 in the basal core promoter. The possible index patient, who suffered from liver cirrhosis, had HBsAg genotype C, anti-hepatitis B envelope (HBe), and these mutations. The level of HBV-DNA declined by about 10 percent per week and no difference in viral kinetics between the patients who died and the survivor was found, irrespective of therapies. The amount of liver cell apoptosis, as assessed by single-stranded DNA, was scarce. The risk of fulminant hepatic failure did not correlate with the preexistent liver histopathological changes. Acute HBV superinfection was associated with hepatitis C virus (HCV) elimination and increased mortality.Conclusions This outbreak of fulminant hepatitis B suggests that HD patients can foster highly virulent HBV strains (possibly owing to their compromised immune responses), which may place others at risk of severe, life-threatening acute liver damage and at increased risk of mortality if chronic carriers of HCV should be infected.