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。     

HBeAg阴性慢性乙型肝炎患者出现前C和基本核启动子联合突变的意义

目的 探讨HBeAg阴性慢性乙型肝炎患者出现前C区G1896A突变和基本核启动子(BCP)区A1762T／G1764A双突变，以及两者的联合突变对患者血清病毒含量的影响。方法 收集240份HBeAg阴性、60份HBeAg阳性慢性乙型肝炎患者血清及40份阴性对照血清，采用竞争分化聚合酶链反应(CD-PCR)检测G1896A突变及A1762T/G1764A双突变，采用实时荧光定量聚合酶链反应(PCR)检测血清中病毒含量。结果 G1896A突变在HBeAg阴性和HBeAg阳性患者中的检出率分别为57．6％和6．7％；A1762T／G1764A双突变的检出率分别为37．9％和31．7％；其中两者联合突变在HBeAg阴性患者中的检出率为13．5％。在HBeAg阴性患者中，G1896A突变主要出现在血清病毒含量低的患者，而A1762T／G1764A双突变与血清病毒含量无关。联合变异株主要见于重度慢性乙型肝炎患者，与血清病毒含量无关。结论 G1896A变异株复制能力较低，而A1762T／G1764A变异株对病毒复制能力影响可能较为复杂，对HBeAg合成的影响较G1896A变异株小。联合变异株的致病力相对较强，其复制能力较单纯G1896A强，值得警惕。     

慢性乙型肝炎患者HBV前C区G1896A、核心启动子1762/1764变异与乙型肝炎自然史、血清HBsAg定量及疾病程度的相关性

目的研究HBV前C区G1896A、核心启动子1762/1764变异与HBV自然史、血清HBsAg水平间的相关性及对疾病严重程度的影响。方法分别各选取40例HBV野生株、前C区G1896A或核心启动子1762/1764变异以及两者联合变异的慢性乙型肝炎感染者为研究对象。采用PCR反向点杂交技术检测HBV前C区G1896A位点、核心启动子1762/1764变异及HBV基因分型。同时检测HBsAg定量、HBeAg、HBV DNA定量、肝脏生化指标等。结果(1)HBV前C区G1896A、核心启动子1762/1764变异患者的年龄、ALT高于野生株感染者(P0.001),Alb低于野生株感染者(P0.001)。(2)HBV自然史免疫耐受期以野生株感染为主(P0.001),免疫清除期、低(非)复制期野生株及PC G1896A或/和BCP1762/1764变异株差异无统计学意义(P0.05),再活动期以PC G1896A或/和BCP1762/1764变异株为主(P0.001)。(3)PC G1896A或/和BCP1762/1764变异株的HBsAg水平(lgIu/mL)、HBeAg阳性率较野生株组降低(P0.001),基因C型、HBV DNA水平(≥6 lg拷贝/mL)较野生株组差异无统计学意义(P0.05)。(4)HBV PC G1896A或/和BCP1762/1764变异中肝硬化患者多于野生株(P0.05)。结论慢性HBV感染者前C区G1896A、核心启动子1762/1764变异与乙型肝炎自然史、HBsAg水平、HBeAg的状态及疾病严重程度相关。     

乙型肝炎病毒X区核苷酸序列变异的检测

目的：了解乙型肝炎患者血清中乙型肝炎病毒（HBV）DNA X区核苷酸序列的变异情况。方法：采用聚合酶链反应（PCR）扩增24例乙型肝炎患者血清HBV DNA X区产物，并直接测序进行分析。结果：24例患者血清中HBV DNA X区都有程度不等的点突变（2－15），11例同时具有nt1762(A→T),nt1764(G→A）发生变异，8例患者同时在nt1636-nt1741几处位点发生变异。结论：HBV DNA X区核苷酸位于nt1762,nt1764双位变异与HBeAg阴性表型有关。     

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。     

青岛地区乙型肝炎病毒基本核心启动子区基因突变与乙型肝炎病毒所致原发性肝癌的相关性

目的通过分析山东省青岛地区HBV核心启动子基因序列的突变特征,探讨其与乙型肝炎相关原发性肝癌的相关性。方法收取慢性乙型肝炎患者和乙型肝炎相关原发性肝癌患者的血清标本各60例,然后从中提取HBV DNA,采用聚合酶链反应(PCR)扩增、纯化、克隆后测序,根据S基因区的编码序列确定患者的基因型和血清型;分别将HBV核心启动子区的各序列结果与GeneBank中的HBV标准株做对比,用DNAMAN软件对基因序列进行突变分析。采用SPSS17.0软件进行统计学分析。结果 120例标本,HBV株均为B或C基因型,以C基因型为主,其中,HBV组的C基因型所占比率为83.33%,HCC组为90.00%(χ2=0.65,P=0.42);血清型均为adw2或adrq+。HBV基因组核心启动子区常见的点突变为C1653T、T1753V、C1754T及A1762T/G1764A,发生率分别为42.73%、86.36%、71.82%、38.18%。与慢性乙型肝炎患者相比,肝癌患者中发生率比较高的突变位点为C1653T(78.85%,χ2=52.58,P<0.001)及A1762T/G1764A(73.08%,χ2=50.88,P<0.001)。结论山东青岛地区HBV基因组常见为B、C基因型,其中以C型为主;核心启动子区突变发生率高,其中,T1753V与HCC发生无关,C1653T、A1762T和G1764A位点无论单独或是联合突变均与HCC发生密切相关。     

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。     

黎族HBV感染者其病毒基因型及C基因启动子/前C区基因突变与肝硬化和肝癌的关系

HBV感染是一个严重的公共卫生问题.在中国,慢性乙型肝炎是导致肝硬化和原发性肝癌的主要危险因素之一[1].目前,根据HBV核苷酸变异＞8％,将HBV分为A～H8种基因型[2].有研究结果显示,HBV基因型、HBV基因组前C区(PreC)第1896位核苷酸G→A(G1896A)以及其启动子(BCP) 1762位核苷酸A→T和1764位核苷酸G→A的突变(BCP A1762T/G1764A)与病毒的复制、转录、抗原表达水平以及疾病的临床过程密切相关[3-4].黎族是海南岛的原住居民,目前关于海南岛黎族肝硬化及肝癌患者HBV基因型、PreC G1896A基因突变和BCP A1762T/G1764A基因突变流行病学分布的研究鲜见报道.本研究旨在了解HBV基因型、PreC G1896A基因突变和BCP A1762T/G1764A基因突变与海南岛黎族肝硬化及肝癌患者发病机制的关系.     

乙型肝炎病毒前核心区及基本启动子变异对干扰素抗病毒治疗应答的影响

乙型肝炎病毒(HBV)在慢性感染过程中具有适应性和变异性的倾向,在内源性(宿主免疫清除)和外源性(疫苗、抗病毒药物)选择压力下更易产生免疫逃逸的变异株.HBV变异常出现某些特定位点,如前核心区(前C区nt1896)及启动子(BCP,nt1762/1764)变异.在HBeAg阳性患者极大多数是野生株,而HBeAg阴性患者极大多数是变异株.我们采用微流基因芯片检测280例慢性乙型肝炎患者血清前C区nt1896及BCP变异,其中94患者予干扰素α2b抗病毒治疗,进一步探讨其对慢性乙型肝炎抗病毒治疗的影响.     

HBeAg阳性慢性乙型肝炎初治患者BCP区A1762T/G1764A变异株定量检测结果分析

目的探讨HBeAg阳性慢性乙型肝炎初治患者BCP区A1762T/G1764A变异株定量检测的临床意义。方法通过单标记探针联合选择性阻断实时荧光定量PCR法精确定量检测97例HBV DNA阳性慢性乙型肝炎初治患者BCP区A1762T/G1764A变异株,并进行统计学分析。结果 97例HBeAg阳性患者变异株的含量会随着HBV DNA水平的升高而降低(P0.01)。变异株含量同时与HBeAg水平呈显著相关,随着HBeAg水平的升高,变异株含量降低(P0.01)。结论对HBV感染者的BCP区A1762T/G1764A变异株进行定量检测,可对患者病情的可能发展进行预测,从而可以采取有效的防治措施。     

Clinical significance and evolution of core promoter and precore mutations in HBeAg-positive patients with HBV genotype B and C: a longitudinal study.

BACKGROUND/AIMS: The aims of this longitudinal study were to investigate whether the clinical outcome and evolution of core promoter and precore mutations were different during hepatitis B e antigen (HBeAg) seroconversion between hepatitis B virus (HBV) genotypes B and C in HBeAg-positive patients with chronic hepatitis B. PATIENTS AND METHODS: The core promoter and precore sequences were determined from serial sera of 156 HBeAg-positive patients with chronic HBV infection. RESULTS: In HBV genotype C, the T1762/A1764 mutant was detected earlier than the A1896 mutant, and the frequency was significantly higher than in HBV genotype Ba over the entire follow-up period. In HBV genotype Ba, A1896 was found earlier than the T1762/A1764 mutant, and the frequency was significantly higher than in genotype C only before HBeAg seroconversion, and the A1896 mutant played an important role in HBeAg seroconversion in HBV genotype Ba. In addition, the T1846 variant was an independent factor associated with HBeAg seroconversion. Furthermore, HBV genotype C was associated with the development of G or C1753 and T1766/A1768 mutations, and the reactivation of hepatitis after HBeAg seroconversion. Based on Cox's regression analysis, the significant risk factors of liver cirrhosis were older age at entry [hazard ratio (HR)=1.085, 95% confidence interval (CI)=1.036-1.136, P=0.001], alanine transaminase (ALT) >80 U/l (HR=3.48, 95% CI=1.37-8.86, P=0.009), and the T1762/A1764 mutant (HR=5.54, 95% CI=2.18-14.08, P<0.001). CONCLUSIONS: Our study showed that different HBV genotypes were associated with various mutations in the core promoter and precore regions during HBeAg seroconversion. T1762/A1764 mutation could be useful in predicting clinical outcomes in HBeAg-positive patients with HBV infection.     

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。     

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.     

乙型肝炎病毒YMDD及e抗原相关多重变异及其临床意义

目的 研究拉米夫定治疗慢性乙型肝炎期间HBVYMDD基序、影响HBeAg分泌的多重变异情况与临床的关系。方法 采用基因芯片技术对拉米夫定治疗9～30个月的慢性乙型肝炎患者进行YMDD基序、G1896A、A1814C、A1762T和G1764A(BCP双突变)单碱基变异检测。结果 102例慢性乙型肝炎患者拉米夫定平均治疗18个月时,22例发生YMDD变异,其中8例发生多重变异,包括G1896A3例、A1814C2例、G1896A A1814C、BCP双突变、BCP双突变 G1896A多重变异各1例,单纯YMDD变异和前5例联合变异均为HBeAg阳性,而后3例多重变异则为HBeAg阴性,其中1例多重变异继续治疗3个月后转变为单纯YMDD野生株阳性,同时伴有HBeAg的复阳。结论 拉米夫定治疗过程中存在YMDD及HBeAg相关多重变异的优势病毒株可能是HBVDNA复阳、同时伴有HBeAg阴转的原因之一,拉米夫定治疗过程中,HBeAg阴性时应监测其可能的相关变异。     

High level of hepatitis B virus DNA after HBeAg-to-anti-HBe seroconversion is related to coexistence of mutations in its precore and basal core promoter

AIM: G1896A mutation in precore or A1762T/G1764A mutations in basal core promoter are suspected to be responsible for patients with detectable level of HBV DNA in serum after seroconversion from HBeAg to anti-HBe. However, G1896A variant has impaired, while A1762T/ G1764A variant may have intact replication ability. They themselves or their coexistence status may play different roles in such meaningless seroconversion. For these reasons, the significances of these two types of mutations were comparatively investigated in this study. METHODS: One hundred and sixty-five sera with positive anti-HBe and HBV DNA were collected from different patients. Mutations of G1896A and A1762T/G1764A among these serum samples were detected using competitively differentiated PCR. HBV DNA was demonstrated using real-time quantitative PCR. RESULTS: G1896A and/or A1762T/G1764A mutations were detected in 89.1% (147/165) out of patients with detectable HBV DNA in serum after HBeAg-to-anti-HBe seroconversion. The positive rate of G1896A variants was significantly higher than that of A1762T/G1764A mutations (77.6% vs 50.3%, X2= 26.61,P<0.01). The coexistence positive rate of these two types of mutations was 38.8% (64/165). Coexistence mutations were found in 77.1% (64/83) out of sera with A1762T/G1764A mutations, and in 50.0% (64/128) out of sera with G1896A mutation. Compared with variants with G1896A mutation only, the coexistence mutations were predominant in patients with high level of serum HBV DNA, and related to higher total bilirubin, lower serum albumin and progressive liver diseases. CONCLUSION: The coexistence of G1896A mutation and A1762T/G1764A mutations is very common, and responsible for the major cases with high level of HBV DNA in serum and progressive liver diseases after HBeAg-to-anti-HBe seroconversion. This coexistence mutation variant may have higher pathogenicity and replication ability.     

Comparison of sequence changes of precore and core promoter regions in HBeAg-positive chronic hepatitis B patients with and without HBeAg clearance in lamivudine therapy

BACKGROUND/AIMS: The aim of this study was to compare the serial sequence changes of precore and core promoter regions in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients with and without HBeAg clearance in lamivudine treatment. METHODS: Precore and core promoter genes of the hepatitis B virus (HBV) were sequenced from five serial serum samples of 74 HBeAg-positive CHB patients received lamivudine for 9-12 months (34 complete responders and 40 non-responders). RESULTS: Before lamivudine therapy, stepwise logistic regression analysis disclosed that ALT level > or =300 U/L, A1896 mutant, and log HBV DNA levels were the major determinants for complete response. In addition, Cox regression showed that age < 35 years and G1752 mutant were independent factors for sustained response. Compared with complete responders, a higher frequency of mutation in nucleotides 1773, 1802, 1803, 1845, 1850, and 1858 was found in the non-responders during therapy. Lamivudine therapy resulted in a further increase in T1762/1764 mutants and a further decrease in A1896 mutant during treatment and after HBeAg clearance in complete responders. CONCLUSIONS: T1762/A1764 mutation (not A1896) played an important role in lamivudine-induced HBeAg clearance. Moreover, T1773, C1802, G1803, T1846, A1850, and C1858 mutations might have significant correlation with HBeAg nonseroconversion.     

Mutations of pre-core and basal core promoter before and after hepatitis B e antigen seroconversion

AIM: To investigate the role of pre-core and basal core promoter(BCP) mutations before and after hepatitis Be antigen(HBe Ag) seroconversion.METHODS: The proportion of pre-core(G1896A) and basal core promoter(A1762T and G1764A) mutant viruses and serum levels of hepatitis B virus(HBV) DNA, hepatitis B surface antigen(HBs Ag), and HB core-related antigen were analyzed in chronic hepatitis B patients before and after HBe Ag seroconversion(n = 25), in those who were persistently HBe Ag positive(n = 18), and in those who were persistently anti-HBe positive(n = 43). All patients were infected with HBV genotype C and were followed for a median of 9 years.RESULTS: Although the pre-core mutant became predominant(24% to 65%, P = 0.022) in the HBe Ag seroconversion group during follow-up, the proportion of the basal core promoter mutation did not change. Median HBV viral markers were significantly higher in patients without the mutations in an HBe Ag positive status(HBV DNA: P = 0.003; HBs Ag: P < 0.001; HB core-related antigen: P = 0.001). In contrast, HBV DNA(P = 0.012) and HBs Ag(P = 0.041) levels were significantly higher in patients with the pre-core mutation in an anti-HBe positive status.CONCLUSION: There is an opposite association of the pre-core mutation with viral load before and after HBe Ag seroconversion in patients with HBV infection.     

A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group

Clinical and molecular virological differences were evaluated in 50 Japanese patients chronically infected with HBV of genotype B and C who were matched for age and sex as well as the severity of liver disease in a case-control study. Hepatitis B e antigen (HBeAg) was significantly less frequent (16% vs. 42%, P <.01), whereas antibody to HBeAg (anti-HBe) was significantly more common (84% vs. 56%, P <. 01) in genotype B than C patients. The predominance of mutants with G-to-A mutation at nucleotide (nt) 1896 in the precore region (A1896) over the wild-type was comparable between genotype B and C patients (60% and 62%, respectively), and it correlated with anti-HBe. The double mutation in the basic core promoter (A-to-T at nt 1762 and G-to-A at nt 1764), however, was significantly more frequent in genotype C than B patients (58% vs. 16%, P <.01), and it did not correlate with anti-HBe or HBeAg. By the multiple logistic regression analysis, the double mutation in the basic core promoter (T1762/A1764) was significantly associated with genotype C [odds ratio (OR), 9.3; 95% confidence interval (CI), 3.4-25.1]], age > or = 35 years (OR, 5.5; CI, 1.5-20.5), and more advanced liver disease (OR, 4.1; CI, 1.6-10.2), but it was not associated with sex, HBeAg, HBV DNA, or the precore mutation (A1896). These results suggest a role of the double mutation in the basic core promoter in association with genotype C and a longer duration of infection in the aggravation of chronic hepatitis B.