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。     

Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe

AIM: To investigate precore/basal core promoter (PC/BCP) mutants throughout hepatitis B virus (HBV) infection and to determine their relationship to hepatitis B early antigen (HBeAg) titers.METHODS: We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012. None of the patients received antiviral therapy. HBV DNA from serum, was quantified by real-time PCR. The HBV genotype was determined by direct sequencing of the S gene. We used the Simpleprobe ultrasensitive quantitative method to detect PC/BCP mutants in each patient. We compared the strain number, percentage, and the changes in PC/BCP mutants in different phases, and analyzed the relationship between PC/BCP mutants and HBeAg by multiple linear regression and logistic regression.RESULTS: Patients with HBV infection (n = 191) were assigned to groups by phase: Immune tolerance (IT) = 55, Immune clearance (IC) = 67, Low-replicative (LR) = 49, and HBeAg-negative hepatitis (ENH) = 20. Of the patients (male, 112; female, 79) enrolled, 122 were HBeAg-positive and 69 were HBeAg-negative. The median age was 33 years (range: 18-78 years). PC and BCP mutation detection rates were 84.82% (162/191) and 96.86% (185/191), respectively. In five HBeAg-negative cases, we detected double mutation G1896A/G1899A. The logarithm value of PC mutant quantities (log₁₀ PC) significantly differed in IT, IC, and LR phases, as well as in the ENH phase (F = 49.350, P < 0.001). The logarithm value of BCP mutant quantities (log₁₀ BCP) also differed during the four phases (F = 25.530, P < 0.001). Log₁₀ PC and log₁₀ BCP values were high in the IT and IC phases, decreased in the LR phase, and increased in the ENH phase, although the absolute value at this point remained lower than that in the IT and IC phases. PC mutant quantity per total viral load (PC%) and BCP mutant quantity per total viral load (BCP%) differed between phases (F = 20.040, P < 0.001; F = 10.830, P < 0.001), with PC% and BCP% gradually increasing in successive phases. HBeAg titers negatively correlated with PC% (Spearman’s rho = -0.354, P < 0.001) and BCP% (Spearman’s rho = -0.395, P < 0.001). The negative correlation between PC% and HBeAg status was significant (B = -5.281, P = 0.001), but there was no such correlation between BCP% and HBeAg status (B = -0.523, P = 0.552).CONCLUSION: PC/BCP mutants become predominant in a dynamic and continuous process. Log₁₀ PC, log₁₀ BCP, PC% and BCP% might be combined to evaluate disease progression. PC% determines HBeAg status.     

乙型肝炎病毒基本核心启动子突变与基因型的关系

目的研究乙型肝炎病毒（HBV）基本核心启动子（BCP）突变与HBV基因型的关系。方法随机选取我院68例慢性乙型肝炎患者外周血,采用荧光定量PCR结合TaqmanMGB探针技术检测HBV基因型,并用基因扩增和DNA测序方法检测BCPT1762／A1764双突变。结果68例患者HBV分型中,B基因型20例,C基因型46例,B、C混合型1例,未分型（非B非C型）1例。66例B、C两基因型中,B基因型组T1762／A1764双突变5例,突变率25．0％（5／20）,C基因型组T1762／A1764双突变24例,突变率52．2％（24／46）,C基因型T1762／A1764双突变率明显高于B基因型（P〈0．05）。结论苏州地区慢性乙型肝炎患者基因型以C型和B型为主,C基因型比B基因型更易发生T1762／A1764双突变。     

Hepatitis B virus subgenotypes and basal core promoter mutations in Indonesia

AIM： To identify the distribution of hepatitis B virus （HBV） subgenotype and basal core promoter （BCP） mutations among patients with HBV-associated liver disease in Indonesia.
METHODS： Patients with chronic hepatitis （CH, n =61）, liver cirrhosis （LC, n = 62）, and hepatocellular carcinoma （HCC, n = 48） were included in this study. HBV subgenotype was identified based on S or preS gene sequence, and mutations in the HBx gene including the overlapping BCP region were examined by direct sequencing.
RESULTS： HBV genotype B （subgenotypes B2, B3, B4, 85 and B7） the major genotype in the samples, accounted for 75.4%, 71.0% and 75.0% of CH, LC and HCC patients, respectively, while the genotype C （subgenotypes C1, C2 and C3） was detected in 24.6%, 29.0%, and 25.0% of CH, LC, and HCC patients, respectively. Subgenotypes B3 （84.9%） and C1 （82.2%） were the main subgenotype in HBV genotype B and C, respectively. Serotype adw2 （84.9%） and adrq＋ （89.4%） were the most prevalent in HBV genotype B and C, respectively. Double mutation （A1762T/G1764A） in the BCP was significantly higher in LC （59.7%） and HCC （54.2%） than in CH （19.7%）, suggesting that this mutation was associated with severity of liver disease. The T1753V was also higher in LC （46.8%）, but lower in HCC （22.9%） and CH （18.0%）, suggesting that this mutation may be an indicator of cirrhosis.
CONCLUSION： HBV genotype B/B3 and C/C1 are the major genotypes in Indonesia. Mutations in BCP, such as A1762T/G1764A and T1753V, might have an association with manifestations of liver disease.     

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。     

Detection of hepatitis B virus A1762T/G1764A mutant by amplification refractory mutation system

ObjectiveTo study the role of hepatitis B virus with A1762T/G1764A double mutation in liver cirrhosis and hepatocellular carcinoma, and create a sensitive, fast, accurate assay for detection of A1762T/G1764A double mutation.MethodsWe developed an accurate and fast real-time amplification refractory mutation system to detect A1762T/G1764A double mutation. Cloned hepatitis B virus genome was used as a control. Assay sensitivity was determined by serial dilution and mixed template experiments. Specificity was determined by cross experiments with wild and mutant hepatitis B virus. Fifty clinical samples were tested by the real-time amplification refractory mutation system and the results were compared with sequencing.ResultsThe real-time amplification refractory mutation system had a sensitivity of 100 copies of virus with these mutations, and 0.1% weak population virus with double mutation could be found in mixtures. A total of 50 randomly collected clinical samples were detected by real-time amplification refractory mutation system, and the results were consistent with those by DNA sequencing. Hepatitis B virus genotype C was more prevalent in 39 of 50 samples than genotype B (11 samples), and about 75% of genotype C carried a double mutation compared to 45% of genotype B. However, the percentage of A1762T/G1764A double mutation in hepatitis B e antigen-negative (58.3%) samples was almost the same as in hepatitis B e antigen-positive (61%) samples.ConclusionThe real-time amplification refractory mutation system is sensitive and specific for detection of hepatitis B virus double mutation.     

乙型肝炎病毒C基因启动子双突变的检测意义

目的：分析乙型肝炎病毒（HIBV）C基因启动子（BCP）双突变与乙型肝炎临床表现及HBeAg表型的关系。方法：针对BCP双突变的特点，设计一条通用捕捉探针，一条野生型和一条双突变显色探针，待测标本DNA经聚合酶链反应（PCR）扩增后分别与野生型和双突变显色探针杂交，然后用酶联免疫吸附试验（ELISA）显示杂交结果，判定BCP突变与否。结果：147例经证实为HBV DNA阳性的急慢性肝炎患者中共有51例双突变，其中42例为单纯双突变，9例为混合突变（双突变和野生型皆为阳性），117例慢性中，重型肝炎中共有36例BCP双突变，8例混合突变，78例HBeAg阳性患者中，有25例BCP双突变，其中7例为混合突变，65例HBeAg阴性患者中，有26例BCP双突变，结论：慢性肝炎BCP双突变高于高性肝炎；BCP双突变对HBeAg的表达有一定影响。PCR微板核酸杂交ELISA技术是一种简便，特异的基因突变检测的新方法。     

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.     

乙型肝炎病毒前C基因区变异与乙肝病毒复制关系的临床研究

目的探讨乙型肝炎病毒（Hepatitis B Virus,HBV）前C基因区变异与HBV-DNA载量的关系。方法通过DNA扩增、基因序列分析检测21例慢性肝炎、18例肝硬化和15例肝癌血清的HBV前C区和基本核心启动子（Basic Core Promoter,BCP）基因序列,荧光定量聚合酶链反应技术定量检测血清中的HBV-DNA。结果野生株与前C区终止变异、BCP双变异以及联合变异组HBV-DNA载量测定差异无显著性（P〉0.05）;BCP双变异HBV-DNA载量HBeAg（-）组显著高于HBeAg（＋）组（P〉0.05）。结论前C区终止变异和BCP双变异对HBV DNA复制无明显影响。HBeAg（-）的慢性肝病患者BCP变异后HBV DNA复制明显活跃。     

抗-HBe阳性乙型肝炎病毒C基因启动子和前C基因变异

研究抗 -HBe阳性乙型肝炎病毒C基因启动子 (CP)和前C基因变异。用基因序列分析检测基因变异。(1)抗 -HBe阳性患者的前C终止变异 (nt1896G→A)的发生率显著高于抗 -HBe阴性组 (P <0 0 0 1) ;而CP双变异(nt176 2A→T和 176 4G→A)则在两组中无明显差异。 (2 )同抗 -HBe阳性慢性乙肝组比较 ,抗 -HBe阳性重型乙肝患者的前C终止变异和CP双变异发生率无明显差异 ,而CPnt175 2A→G变异发生率则显著增高 (P <0 0 5 )。前C终止变异与抗 -HBe阳性乙型肝炎密切相关 ;而CPnt175 2变异则可能与抗 -HBe阳性重型乙型肝炎发病有关     

乙型肝炎病毒基本核心启动子及前C区突变对疾病进展的影响

目的　研究乙型肝炎病毒 (HBV)基本核心启动子 (BCP)T176 2 /A176 4双突变及前C区A1896突变与肝脏损伤程度的相关性。方法　 2 0 0 0～ 2 0 0 2年哈尔滨医科大学附属第二医院及广东省廉江市医院随机选取 113例慢性HBV感染者 ,采用INNO -LiPA法测定HBVBCPT176 2 /A176 4双突变及前C区A1896突变 ,同时测定HBVS基因序列明确基因型。结果　CHB组、LC组、HCC组BCPT176 2 /A176 4双突变率明显高于AsC组 (分别为 2 4 . 1%比 2. 8% ,χ2 =5 .93,P <0 . 0 5 ;71. 4 %比 2 .8% ,χ2 =2 3 .83,P <0 .0 1和 5 5 . 6 %比 2 .8% ,χ2 =13. 0 9,P <0 . 0 1) ,LC组BCPT176 2 /A176 4双突变率显著高于CHB组 (71 .4 %比 2 4 . 1% ,χ2 =9 .12 ,P <0 .0 1) ;单一C基因型感染者CHB、LC和HCC组BCPT176 2 /A176 4双突变率明显高于AsC组 (分别为 33 .3%比 5 . 3% ,χ2 =3 .89,P <0 .0 5 ;6 9 .2 %比5 . 3% ,P <0 . 0 1和 5 0 .0 %比 5. 3% ,P <0 . 0 5 )。各组前C区A1896突变率均较低 ,在CHB组和LC组无一例发生 ;HCC组前C区A1896突变率与AsC组比较差异无显著性 (11 .1%比 8. 3% ,χ2 =0 . 0 0 ,P >0 . 0 5 )。结论　在慢性HBV感染者中 ,BCPT176 2 /A176 4双突变与慢性肝病进展有关。     

Mutations of polymerase,precore and core promoter gene in hepatitis B virus during 5-year lamivudine therapy

BACKGROUND/AIMS: The effects of long-term lamivudine therapy on changes in polymerase and precore/core promoter mutations are unknown. The aim of this study was to determine the changes in these regions in patients with chronic hepatitis B virus (HBV) infection treated with lamivudine for 5 years. METHODS: Serum samples obtained from 16 patients at the beginning of and during therapy were polymerase chain reaction-amplified, and nucleotide sequences of HBV analyzed. RESULTS: By the end of 5-year therapy, mutations in YMDD motif emerged in ten patients. Mutations in L526M, M550V and M550I in polymerase were found in seven, six and six patients, respectively. The L526M mutant was found at the time or after detection of M550V/I mutant in six of seven patients. At baseline, precore and core promoter mutations were found in eight and 12 of 16 patients, respectively. Mutants of precore and core promoter were replaced by wild-type virus in each of three patients infected with mutants at 1 year. However, at 5 years of treatment, precore and core promoter mutations reappeared in some patients. CONCLUSIONS: Our data showed that lamivudine initially selected from precore/core promoter mutants to wild-type virus, but precore mutation reappeared during prolonged therapy.     

慢加急性乙型肝炎肝衰竭患者乙型肝炎病毒前C/CP区变异研究进展*

慢加急性肝衰竭（ACLF）是我国肝衰竭的主要类型,其病情发展迅速、预后不良、病死率较高。在我国,乙型肝炎病毒（HBV）感染是导致ACLF的主要病因,其中HBV前C（PC）/核心启动子（CP）区基因变异与ACLF的发生密切相关。本文重点介绍HBV PC/CP区基本结构和功能、PC/CP区变异后生物学特性的改变、ACLF的免疫病理损伤机制、PC/CP区变异与ACLF疾病进展的临床意义等方面的研究进展。     

The STAT4 and not the IFNL3 variant is associated with hepatitis B virus clearance in a population from the Khyber Pakhtunkhwa region of Pakistan

Background and study aimsHost genetic modifiers of the risk and persistence of hepatitis B virus (HBV) infection in the Pakistani population have not been clearly elucidated. Recently, two genome-wide association studies described that STAT4 and IFNL3 variants are associated with different aspects of the course of HBV infection. However, the roles of these variants in the persistence of HBV infection have not been investigated in the HBV-infected population of Pakistan. Therefore, we examined the roles of the STAT4 and IFNL3 variants in a chronic HBV-infected population from the Khyber Pakhtunkhwa (KPK) region of Pakistan.Patients and methodsSTAT4 rs7574865 and IFNL3 rs12979860 genotyping were performed in 297 subjects (240 infected with HBV and 57 controls). Statistical analyses were performed using the chi-squared test, Student’s t-test, Hardy–Weinberg equilibrium tests and logistic regression models.ResultsAmong the 297 subjects, compared with the IFNL3 rs12979860 genotype [odds ratio (OR) = 0.7, 95% confidence interval (CI) = 0.39–1.29, p = 0.2), the STAT4 rs7574865 genotype was independently associated with the risk of developing chronic HBV infection [OR = 1.9, 95% CI = 1.09–3.50, p = 0.02].ConclusionThe STAT4 rs7574865 and not the IFNL3 rs12979860 variant is associated with persistence of HBV infection in a Pakistani population from the KPK region.     

焦磷酸测序技术检测HBV基本核心启动子变异的研究

目的建立HBV基本核心启动子（BCP）区变异的快速检测方法。方法采用焦磷酸测序（pyrose-quencing）技术,设计一对引物,其中一条经生物素标记,PCR扩增产物含HBVBCPA1762T／G1764A双突变的区域,借用生物素包被葡聚珠纯化单链PCR产物,设计一条测序引物,运用焦磷酸测序技术对A1762T／G1764A两个变异点进行变异分析。通过对已知变异类型的HBV分离株进行测定分析,评价所建立方法的检测敏感性和特异性。结果PCR扩增后,可在2h内得到A1762T／G1764A两个变异点的突变情况。所建立方法的检测灵敏度达到HBV．DNA血清中的含量为10^3copies／ml;在所分析的HBVBCP区均可检测出变异株、非变异株及混合株,且所检测的结果与直接测序方法结果符合率为95％。结论Pyrosequencing技术用于检测病毒基因突变有高通量、自动化程度高和结果准确等特点,适用于对HBVBCP区突变进行快速高通量检测。     

乙型肝炎病毒HBeAg上调S100钙结合蛋白A11基因启动子表达活性的研究

目的 探讨乙型肝炎病毒e抗原(HBeAg)对S100钙结合蛋白A11(calgizzarin S100A11)启动子转录的激活作用。方法 以我室构建的HBeAg反式调节基因的cDNA文库抑制性消减杂交(SSH)筛选结果为基础，利用生物信息学技术确定S100A11的启动子区域(S100A11-P)，聚合酶链反应(PCR)扩增S100A11-P，克隆至真核报告载体PCAT3中，构建pCAT3-S100-p报告载体，以该质粒转染肝癌细胞系HepG2细胞系，用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性，并与pcDNA3．1(-)-HBeAg共转染HepG2细胞系，用ELISA法检测CAT的表达活性。结果pCAT3-S100-P在HepG2细胞中能够指导CAT的表达；共转染实验中pCAT3-S100-P-pcDNA3.1(-)-HBeAg组CAT的表达活性是pCAT3-S100-P组的6．1倍。结论 我室克隆的S100A11启动子有顺式激活下游基因的活性，HBeAg具有对S100A11的反式激活作用。本实验进一步验证了我室利用SSH技术研究HBeAg反式激活作用的结果。     

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。