基因芯片检测乙醇对慢性乙型肝炎患者乙肝病毒多位点基因突变的研究

目的：研究乙醇对HBV DNA多位点基因变异的影响，为慢性乙型肝炎的临床诊断和治疗提供依据。方法:选取85例慢性乙肝患者为研究对象，分为嗜酒组和非嗜酒组，采用DNA芯片技术, 检测HBV DNA前C区G1896A及A1814C位点、BCP区 A1762T及G1764A位点、P区C528A及T552C位点的基因突变。结果:嗜酒组在BCP区A1762T及G1764A位点的突变频率明显高于非嗜酒组(均P<0.05)。在前C区G1896A及A1814C位点，P区C528A及T552C位点突变频率无明显差异（均P>0.05）。结论:乙醇导致乙肝病毒BCP区A1762T及G1764A位点发生基因突变，增强HBV基因的表达和复制，加重慢性乙型肝炎患者的病情。     

乙型肝炎病毒e系统血清转换中HBV前C、BCP基因检测及临床意义

目的：探讨乙型肝炎患者HBeAg和HBeAb双阳性状态下HBV前C区基因及BCP区基因变异情况,探讨前C区A1896及BCP区T1762与A1764变异与HBe转换的关系。方法：采用时间分辨荧光免疫分析方法定量检测乙肝“二对半”,对HBeAg／HBeAb双阳性标本采用基于膜显色的DNA芯片检测HBV nt1896、nt1762、nt1764位基因。结果：35例HBeAg、HBeAb、HBVDNA阳性的患者均存在nt1762和nt1764的突变,有14例患者出现了nt1896的突变。结论：在乙型肝炎HBe转换过程中均伴有BCP区T1762和A1764的突变,部分存在A1896位点的突变,T1762和A1764的突变早于A1896的突变,A1896的突变主要在e抗体产生过程中或产生以后。     

DNA芯片在乙型肝炎病毒基因突变分析中的应用

目的:探讨DNA芯片在乙型肝炎病毒基因突变分析中的应用及临床意义。方法:应用聚合酶链反应扩增乙型肝炎患者血清中HBVDNA,采用膜显色DNA芯片法检测HBVDNA前C区1896位,基本核心启动子区(BCP区)1762、1764位,P区528、552位突变。结果:未接受抗病毒药物拉米夫定治疗的6例HBsAg 、HBeAg 、HBeAb-的患者血清中未检测到病毒变异,13例HBsAg 、HBeAg-、HBeAb 的患者中检测到前C区1896位变异9例,BCP区1762、1764位变异11例,13例HBsAg 、HBeAg 、HBeAb 的患者中检测到前C区1896位变异4例,BCP区1762、1764位变异13例,未检测到P区基因变异;15例接受拉米夫定治疗48周后的HBVDNA阳性患者中,检测到前C区1896位变异5例,BCP区1762、1764位变异6例,P区528位变异2例,P区552位YVDD变异4例,YIDD变异7例。结论:前C区1896位、BCP区1762、1764位突变较多的出现在HBeAb 的患者中并与HBeAg的阴性表型相关,BCP区1762、1764位突变与HBeAg/HBeAb血清转换密切相关,而P区528、552位突变更多的出现在接受拉米夫定治疗后的患者中,与拉米夫定耐药相关;膜显色DNA芯片法用于乙型肝炎病毒基因突变分析,简便、快速、特异性好,适合临床推广应用。     

重型肝炎时乙型肝炎病毒基因型与基本核心启动子及前C区突变关系的研究

目的探讨重型肝炎（重肝）乙型肝炎病毒（HBV）基因型与基本核心启动子（BCP）及前C区突变的关系。方法采用聚合酶链反应（PCR）-限制性片段长度多态性分析技术（PCR-RFLP）对52例重肝和52例慢性乙肝（CHB）进行HBV基因分型。采用PCR产物直接测序技术，随机对15例B型和15例C型重肝患者的BCP区和前C区进行序列测定，分析HBV基因型与BCPT1762／A1764及前C区A1896突变的关系。结果泉州地区重肝的基因型以B型为主（48．08％），其次为C型（30．77％）和B／C混合型（17．31％），无A、E、F型存在。与CHB组比较，重肝组B型检出率明显降低，而C型和BIC混合型检出率明显升高。C型重肝患者BCPT1762／A1764双突变率显著高于B型（P〈0．05），而前C区A1896突变率在B、C型感染者中差异无统计学意义（P〉0．05）。结论C型感染易引起较重肝损伤，而B／C型混合感染可能是导致重肝发生的重要原因之一。C型重肝患者BCP T1762／A1764双突变率显著高于B型。     

乙型肝炎病毒基因型C亚型和核心启动子、前C/核心区基因变异的关系

目的 研究慢性乙型肝炎病毒（HBV）感染者中HBV基因型C亚型（HBV／C）的核心启动子、前C／核心区基因变异情况，分析HBV／C亚型的病毒学特征。方法 用酶联免疫法（ELISA）筛选出79例HBV／C，再用聚合酶链反应．限制性酶长度多态性分析方法（PCR-RFLP）进行HBV／C亚型分析；同时针对HBV核心启动子、DreC／核心区基因进行半巢式PCR及PCR产物直接测序。结果 ①79例HBV／C中，33例（41．8％）为HBV／C1亚型，46例（58．2％）为HBV／C2亚型。②HBV／C1亚型仅见于来自中国南方的患者（P〈0．0001）。③A1898位点变异仅见于HBV／C1亚型（P=0．056），V1753位点变异在HBV／C1亚型中多见（P〈0．05）；HBV／C2以T1858（90％）、A1896（40％）位点变异多见（P〈0．008）。T1762／A1764位点变异在HBV／C两种亚型中均常见。④肝细胞癌（HCC）患者中，V1753和T1762／A1764变异最常见（P〈0．05）。结论 HBV／CI和HBV／C2在中国有明显的地区差异；V1753合并T1762／A1764双变异与发展为HCC相关，尤其在HBV／C1患者。     

HBV拉米夫定耐药株及BCP、Pre-C突变株芯片检测方法的应用研究

目的 构建针对HBV拉米夫定耐药株及c区启动子(basal core promoter,BCP)、前C区(Pre-C)突变株,多位点突变的基因芯片检测方法.并与DNA测序法比较,以了解该芯片的灵敏度、特异性、稳定性等性能并初步应用于临床实践.方法 该基因芯片能对HBV DNA及P区(DNA多聚酶区):180、204、207三个拉米夫定耐药突变位点;C区启动子(basal core promoter,BCP)及前C区(Pre-C):nt1896、nt1899、nt1862、nt1764、nt1762 5个突变热点,共8个HBV突变位点进行检测,并用测序法对该基因芯片进行验证.结果 ①检测HBV DNA方面,两种方法检测结果100%相符.②检测突变位点方面:总体统计32份阳性血清共有256(32×8)个突变位点.两种方法检测结果有251个突变位点完全相符;5个突变位点不完全相符,基因芯片法检测为混合型,测序法检测为野生型或突变型之一.结论 结果提示该基因芯片和DNA测序法检测结果阳性率无差异,特异性与DNA测序法相当,检测混合株有更大优势.     

我国OBI献血者中乙型肝炎病毒BCP/前C区及C区的分子特征

目的获得我国隐匿性乙型肝炎病毒感染(occult HBV infection, OBI)人群中乙型肝炎病毒(hepatitis B virus, HBV)基本核心启动子区(basic core promoter, BCP)、前C区和C区序列特征, 为进一步阐明该区域与OBI的关系奠定基础。方法收集2016—2018年全国18个省、自治区和直辖市的43家血站实验室, 经日常检测后HBV DNA阳性的血浆样本。经实验室确认后HBsAg阳性样本和OBI样本各入组不少于200例, 针对HBV BCP/前C区、C区和S区进行半巢式或巢式PCR和Sanger测序及序列分析。结果完成测序417例样本, 其中HBsAg阳性样本216例和OBI阳性201例, 均为基因型B和C。HBsAg阳性组该区域的突变频率明显高于OBI组。在B基因型中仅发现1个OBI高频突变A1896G, 在C基因型中发现T1762A/A1764G、T1803A/G、A1986G、T1866A(D22E)等OBI高频突变。在C基因型中发现的前C区ε-反式作用元件区域(1 847-1 907 nt)突变G1888T可能会改变前基因组R...     

HBV DNA的前C区1896 G/A变异与血清HBV DNA水平

为探讨乙型肝炎病毒（HBV）前C区1896G／A变异对血清HBV DNA水平复制及临床表现的影响，本研究报道采用酶联免疫吸附试验（EHSA）和荧光定量PCR方法（FQ-PCR）及突变特异引物PCR方法（AS-PCR）对95例诊断符合2000年修订的病毒性肝炎防治方案的HBV感染者进行了HBV血清免疫标志物和HBV DNA定量及HBV DNA1896位点G／A变异检测的结果。     

C基因型乙型肝炎病毒前C区及基本核心启动子突变与疾病进展的相关性研究

目的探讨HBV前C区（PreC）及基本核心启动子（BCP）突变与慢性乙型肝炎病毒（HBV）感染者疾病进展的关系。方法收集88例慢性HBV感染者血清标本，包括36例无症状携带者（其中24例为HBV携带者，12例为HBsAg携带者）、36例慢性乙型肝炎（慢性乙肝）和16例肝硬化患者。所有标本均经型特异性引物PCR法鉴定为HBVC基因型，并用巢氏PCR法扩增HBVPreC和BCP基因片段，用PCR产物直接测序法测序，然后用ClustalW1．8软件进行序列分析。结果在50例HBeAg阳性患者中，无症状携带者、慢性乙肝和肝硬化组的T1762／A1764双突变率和T1846突变率分别为12．5％、42．1％、100％和0％、5．3％、28．6％，差异均有统计学意义（P值分别为0．03和0．02）。在38例HBeAg阴性HBV感染者中，无症状携带者、慢性乙肝和肝硬化组的T1762／A1764双突变率分别为16．7％、58．8％和66．7％，差异无统计学意义（P=0．08）。肝硬化组的C／G1753突变率显著高于无症状携带者及慢性乙肝组（分别为55．6％、8．3％、11．8％，P=0．01），其A1896突变率也高于无症状携带者组（分别为55．6％、8．3％，P=0．01）。结论HBVT1762／A1764双突变与C基因型HBV慢性感染者的疾病进展有关。     

DNA芯片检测乙型肝炎病毒基因多态性

目的　建立DNA芯片检测乙型肝炎病毒 (hepatitisBvirus,HBV)基因多态性的研究方法并对实验条件进行优化。方法　设计多条寡核苷酸探针 ,在硅烷化芯片的特定位置上 ,用点样仪将探针固定 ,并与PCR扩增的HBV基因相应区段杂交 ,杂交结果影印至硝酸纤维素膜 ,经BCIP NBT避光显色 ,用放大镜观察杂交信号呈暗紫色圆点 ,根据特定位置上杂交信号的有无和与之相应的探针序列来判定基因突变的类型。结果　通过 1次杂交反应可检测HBV前C C区 (nt 1896 1814 )、BCP区 (nt1762 1764)和P区 (nt 52 8 552 )等多个位点的变异 ,与测序分析结果完全一致 ,具有较好的检测灵敏度和重复性。结论　DNA芯片检测HBV基因常见突变位点多态性 ,操作简便易行 ,技术要求不高 ,具有临床推广应用价值 ,而且可以方便地通过向寡核苷酸探针阵列中添加相应探针 ,扩大基因芯片的检测应用范围 ,为临床检测提供了新的方法     

Genotype and variations in core promoter and pre-core regions are related to progression of disease in HBV-infected patients from Northern Vietnam

Vietnam is one of the countries with a high rate of hepatitis B virus (HBV) infection, but there are only a few reports about relation of HBV genotypes and mutations to clinical course in Northern Vietnam. The characteristics of HBV and its relationship to clinical outcome in patients from Northern Vietnam were analyzed. Serum samples were collected from 183 HBV-infected Vietnamese patients. They were clinically categorized into 4 groups: hepatocellular carcinoma (HCC), liver cirrhosis (LC), chronic hepatitis (CH), and asymptomatic carriers (ASC). HBV serology, alpha-fetoprotein, HBV genotypes, HBV-DNA level and mutations in the core promoter and pre-core regions of HBV-DNA were examined. The majority of sera contained HBV genotype B (67.8%) and C (27.9%). The median age was matched between genotype B and C (38.2 vs. 42.9 years). The rates of HBeAg seroconversion and G1896A for genotype B were significantly higher than those for genotype C (P<0.05). Genotype C had a higher HBV-DNA level than genotype B. C1858 was frequent, especially in genotype C (62.7%). The most prevalent genotype in ASC and CH was genotype B. The presence of the mutation A1762T/G1764A correlated with disease progression. The triple mutation T1753C/A1762T/ G1764A was quite common and was more prevalent in LC and HCC than in CH and ASC. In Northern Vietnamese, HBV genotypes B and C were prevalent. Genotype C and mutations in the core promoter region were associated with progressive, severe liver diseases.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.     

Analysis of the precore and core promoter DNA sequence in liver tissues from patients with hepatocellular carcinoma.

To investigate the role of mutant hepatitis B virus (HBV) in the development of hepatocellular carcinoma (HCC), 20 patients with HCC were studied for precore and core promoter mutations in tumorous and nontumorous tissues. The precore and core promoter region was amplified and analyzed by direct sequencing. Among the 20 tumorous and nontumorous tissues, precore mutant HBV was found in 12 (60%) and 18 (90%), respectively. Of the 12 tumorous tissues with precore mutant, nine tissues had a single mutation (1896) and one tissue had another single mutation (1899). The remaining two tissues had a double mutation (1896 and 1899). A single mutation (1896) and a single mutation (1899) were found in 11 and two of the 18 nontumorous tissues with precore mutant, respectively. Among 20 tumorous and nontumorous tissues, HBV with a C to T mutation at nucleotide (nt) 1846 was detected in six and eight, respectively, and was associated with the virus carrying a mutation (1896 or 1899) except in two tumorous tissues. Mutations at nt 1762 and 1764 in core promoter were observed in 16 (80%) tumorous tissues and 18 (90%) nontumorous tissues. Mutations in the precore and core promoter region were found frequently in nontumorous tissue and in tumorous tissue (18/20 and 12/20 in precore region, 18/20 and 16/20 in core promoter respectively). The high prevalence of precore and core promoter mutations in liver tissue from patients with HCC suggests that these mutations may contribute to the development of HCC.     

Hepatitis B e antigen‐negative mutations in the precore and core promoter regions in Korean patients

Most patients with hepatitis B e antigen (HBeAg)‐negative chronic hepatitis B have variants of the hepatitis B virus (HBV) that include mutations in the precore or core promoter regions of the HBV genome. The aim of this study was to investigate the patterns of precore and core promoter mutations and their relationship to HBeAg expression in Korean patients. Four hundred seventy‐five Korean patients with chronic HBV infection between February 1995 and December 2003 were enrolled in this study. There were 236 HBeAg‐positive and 239 HBeAg‐negative patients. Blood samples were tested for HBsAg, anti‐HBs, HBeAg, hepatitis B e antibody (anti‐HBe), liver function tests, and serum HBV DNA. Mutations in the precore and core promoter regions were determined by direct sequencing. In the core promoter region, the C1740, C1753, T1762/A1764, and T1766 mutations were associated with HBeAg escape (all; P < 0.05). In the precore region, a higher frequency of the C1802, A1828, T1846, A1850, C1858, T1862, and A1896 mutations was found in HBeAg‐negative patients (all; P < 0.05). In particular, the A1896 mutation was associated with high serum levels of ALT and HBV DNA in HBeAg‐negative patients (P = 0.014 and 0.026, respectively). Mutations around the Kozak sequence (nucleotides 1809–1812) were found in 6.7% of patients and were not associated with undetectable HBeAg (P = 0.13). In Korean patients, various mutations in the precore and core promoter regions were associated with HBeAg escape and amelioration of hepatic inflammation in HBeAg‐ negative patients. Only the A1896 mutation contributed to HBeAg‐negative chronic hepatitis B. J. Med. Virol. 81:594–601, 2009 © 2009 Wiley‐Liss, Inc.     

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 × 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 × 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.     

Prevalence of HBV core promoter/precore/core mutations in Gambian chronic carriers.

One hundred forty-two precore/core sequences were obtained from Gambian chronic hepatitis B virus (HBV) carriers and the predominant variants defined. The two point mutations, from A to T and G to A at nt positions 1762 and 1764 in the basic core promoter region, were found in only 7/99 (7%) of the samples where this region was sequenced. These mutations were found in both HBeAg-positive and -negative patients. The precore stop-codon mutation at nt position 1896 was found in 14/51 (27%) of HBeAg-negative samples, which is a lower prevalence rate in comparison with other parts of the world with high carrier rates. In HBeAg-positive patients the core amino acid sequences were conserved, but after seroconversion to anti-HBe significantly more changes were apparent. Several of the amino acid substitutions found have been described previously been in wild-type viruses of other genotypes.     

Clinical and molecular characterization of chronic hepatitis B in Albania: a country that is still highly endemic for HBV infection

Albania is a Mediterranean country, still with a high endemicity level of hepatitis B virus (HBV) infection. The chronic hepatitis B profile was characterized in this geographical area and used as a model to investigate the impact of endemicity level on the prevalence of the two major forms of chronic hepatitis B (HBeAg-positive and HBeAg-negative chronic hepatitis B). A cross-sectional study was conducted among 62 chronic hepatitis B patients consecutively admitted to the most important tertiary health care center for the diagnosis and treatment of liver disease in Albania. HBV-DNA was measured with an in-house PCR with a sensitivity of 10(4) copies/ml which uses primers encompassing the pre-core/core region. PCR products were subjected to sequencing and oligohybridization assay. Of the 62 patients, 75.8% had HBeAg-negative chronic hepatitis B. Genotype D was found in all 39 patients with detectable HBV viremia, for whom the heterogeneity of the region modulating HBeAg expression was assessed. Basic core promoter (BCP) mutations (1762/1764) were observed more often in anti-HBe-positive and older patients. In more than 90% of the HBeAg-negative chronic hepatitis B patients with detectable viremia, HBV that carries the G to A pre-core mutation at nucleotide 1896 was found. Patients with HBeAg-positive chronic hepatitis B were younger than HBeAg-negative chronic hepatitis B patients, and for symptomatic and asymptomatic liver-disease patients, the age of peak prevalence was at least 10 years lower for HBeAg-positive chronic hepatitis B patients. In conclusion, the virological and clinical pattern of chronic hepatitis B in Albania is similar to that observed in other Mediterranean countries; it seems to be independent of the HBV endemicity level.     

Variations in the core promoter/pre-core region in HBV genotype C in Japanese and Northern Vietnamese patients

Hepatitis B virus (HBV) subgenotypes Cs (C1) and Ce (C2) are common in East Asia. To investigate the genomic difference of HBV genotype C between two separated regions, 50 subgenotype Cs-infected Vietnamese and 70 subgenotype Ce-infected Japanese patients were enrolled for analysis. The patients were categorized to either a hepatocellular carcinoma group (HCC) or a non-HCC group including liver cirrhosis, chronic hepatitis, and asymptomatic carriers. HBV serology, HBV-DNA level, and variations in core promoter/pre-core region were examined. Phylogenetic analysis based on the full genome sequences and nucleotide sequences partly in the S gene and in the P gene revealed that all Japanese strains (70/70) were subgenotype Ce, and nearly all of the Vietnamese strains (50/51) were subgenotype Cs, excluding one subgenotype C5. C1858 and G1775 were common in the Vietnamese (64% and 40%) but not in the Japanese (0%). The prevalence of C/A1753 in Vietnamese was higher than that in the Japanese (32% vs. 17.1%), however the frequency of A1896 in the Japanese was significantly higher (32.9% vs. 12%, P < 0.05). Most of the Vietnamese patients with HCC had a high level of HBV-DNA, the Japanese HCC had a relatively low level. In the Vietnamese, C/A1753 and C1858 were associated closely with T1762A1764, higher HBV-DNA levels and higher HCC incidence. The multivariate analysis revealed that male, T1653 and C/A1753 were independent risk factors for HCC. The subgenotypes and unique mutations of HBV genotype C in the Vietnamese and Japanese differed, and C/A1753 and C1858 variants might play a role in the pathogenesis of liver disease in Vietnamese patients.