乙型肝炎相关慢加急性肝衰竭患者乙型肝炎病毒前C/C区联合突变特点分析

目的分析乙型肝炎相关慢加急性肝衰竭（HBV-ACLF）患者HBV前C/C区联合突变的特征。方法入选166名慢性HBV感染者,其中ACLF 49人,慢性乙型肝炎（CHB）45人,肝硬化（LC）45人,肝细胞癌（HCC）27人,对HBV前C/C区片段进行PCR扩增,分析突变数量及联合突变状况。结果 ACLF患者多联突变比例明显高于其他组患者。4组之中,ACLF组5联突变和7联突变者比例最高,分别是20.4%（10/49）和12.2%（6/49）,6联突变者与HCC组相近分别为14.3%（7/49）和14.8%（4/27）,明显高于CHB和LC组。联合突变组合形式的分析结果提示,ACLF组患者,含nt1846和nt1913突变的联合突变组合阳性率在4组患者中最高。结论 ACLF患者HBV前C/C区多联突变比例高于其他慢性HBV感染者;以nt1846和nt1913为基础的联合突变率最高。     

HBV感染所致慢加急性肝功能衰竭患者HBV变异的纵向研究

目的明确乙型肝炎病毒（HBV）变异与慢加急性肝功能衰竭（ACLF）发病之间的关系。方法采用纵向研究的方法 ,选取6例HBV感染者为研究对象,其中ACLF4例（P1～P4）,肝硬化（LC）2例（P5、P6）。分别留取每例患者不同疾病阶段血清2份,进行HBVDNA提取、扩增、克隆和测序,测序结果采用VectorNTISuite9.0软件分析,纵向对比HBV核苷酸序列变异情况。结果 6例患者均为基因C型。ACLF患者（P1～P4）在病程中HBV的核苷酸突变位点多于发生肝硬化患者（P5、P6）。ACLF患者中,nt53（C→T或T→C）、nt1846（A→T）、nt1896（G→A）的纵向突变见于2例或2例以上患者。C1913A或T突变可见于患者P1、P2,同时患者P2可见纵向突变。结论 nt53、nt1846、nt1896和nt1913四个核苷酸的突变可能与ACLF的发病相关。     

清热利湿法对慢性乙型肝炎HBV基因突变株复制的影响

目的:研究清热利湿法对慢性乙型肝炎(CHB)患者HBV前C区基因突变株复制的影响。方法:采用基因芯片技术定量检测CHB肝胆湿热/湿热中阻证患者治疗(清热利湿法)前后的HBV前C基因变异。结果:治疗前的HBV前C区1762、1764、1896位点变异率明显高于1899位点变异率,差异有显著性意义(P<0.01);治疗后各位点变异率下降,其中治疗组1764、1896位点变异率显著下降(P<0.05)。治疗前不同位点变异株信号强度1896>1764>1762>1899,差异具有显著性意义(P<0.05);治疗后1762、1764、1896位点变异株信号强度下降,其中1896位点信号强度显著下降(P<0.05)。结论:清热利湿法在改善CHB肝胆湿热/湿热中阻证患者临床证候的同时,可在一定程度上抑制HBV前C区突变株的复制。     

慢性HBV感染者HBV前C区/基本核心启动子区基因突变与临床应用

目的:探讨2010年10月至2013年6月来我院门诊及住院慢性HBV感染者中HBV前C(Pre C)区/基本核心启动子(BCP)区基因突变与其病程进展的相关性。方法:165例慢性HBV感染者血清生化指标检测,血清HBV DNA含量和HBe Ag定性检测,HBV Pre C区/BCP区突变率比较,分析Pre C区l896、BCP区1762/1764变异在HBV携带者(As C)、慢性乙型肝炎(CHB)和慢性乙型肝炎肝硬化(CHB-LC)患者中的分布。结果:血清生化指标在慢性HBV感染者病程发展中呈现相关性。与As C组比较:CHB组、CHB-LC组患者血清ALT、AST、AFP、TBA水平升高,差异有显著性意义(p0.01),呈显著正相关,而TP、Alb、A/G水平降低,差异有显著性意义(p0.05),呈负相关。HBe Ag(+)、HBe Ag(-)的标本中,BCP的突变率高于前C区突变率,差异有非常显著性意义。BCP区1762/1764双突变的突变率高于Pre C1896突变率。As C、CHB、CHB-LC 3组患者Pre C A1896、BCP区1762/1764变异率依次升高,与As C组比较,CHB组差异有显著性意义(P0.05);CHB-LC组差异有非常显著性意义(P0.01)。结论:对165例慢性HBV感染者观察,HBe Ag(+)/HBe Ag(-)患者及其病程不同发展阶段,Pre C/BCP出现相关联的突变率。特别需要注意的是HBe Ag(-)的慢性HBV感染者BCP区的突变率高于Pre C区的突变率,需要慎防HBV复制的危害性。HBV Pre C区/BCP区突变率可以预测到慢性HBV感染者病情的发展程度,对临床此类患者的研究具有重要意义。     

慢性HBV感染者不同病程阶段HBV前S基因缺失突变的比较分析

目的 探讨慢性HBV感染者不同病程阶段前S(pre-S)基因缺失的临床流行特点及其临床意义. 方法 采用巢式PCR方法扩增测序146例慢性乙型肝炎(chronic hepatitis B, CHB)患者(CHB组)、111例HBV相关肝硬化(liver cirrhosis, LC)患者(LC组)、146例慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)患者(ACLF组)和136例HBV相关肝细胞癌(hepatocellular carcinoma, HCC)患者(HCC组)的HBV pre-S基因(nt 2848-154).分析比较不同组pre-S基因缺失发生率、缺失热点区域和缺失片段长度. 结果 LC组和HCC组HBV pre-S基因缺失率显著高于CHB组(26.1%vs. 15.8%,P=0.040;34.6%vs. 15.8%,P<0.001);LC组pre-S1基因单独缺失率显著高于CHB组(17.1%vs. 4.8%,P=0.001);HCC组pre-S2基因单独缺失率显著高于CHB组(19.1%vs. 4.8%,P<0.001). 不同病程阶段患者发生缺失的热点区域也不相同,CHB组发生缺失的热点区域为nt 3031-3215(30.4%)和nt 24-57(30.4%);LC组为nt 2849-2866(55.2%)和nt 5-55(31.0%);ACLF组为nt 2849-2866(28.6%)和nt 1-54(25.7%);HCC组为nt 5-55(57.4%)和nt 2849-2866(12.8%).不同病程阶段患者发生pre-S基因缺失的片段长度差异无统计学意义. 结论 慢性HBV感染者中,随着疾病进展HBV pre-S基因缺失率呈上升趋势,其中pre-S1基因缺失突变在LC患者中显著增高,pre-S2基因缺失突变在HCC患者中显著增高.pre-S基因缺失可能参与驱动HBV慢性感染的疾病进展.     

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。     

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。     

慢性乙型肝炎肝肾阴虚证HBV基因突变点的分布规律

目的:研究慢性乙型肝炎肝肾阴虚证HBV基因突变点的分布规律。方法:基因芯片方法检测慢性乙型肝炎肝肾阴虚证患者HBV前C区基因变异。结果:慢性乙型肝炎肝肾阴虚证患者HBV前C区变异可见于多个位点,1896位点变异率为31.37%,1764位点变异率为25.49%,1762位点变异率为24.51%,1899位点变异率为12.75%,1862位点变异率为5.88%。1762、1764、1896和1899点的突变株信号值高于野生株信号值;1862点野生株信号值高于突变株信号值,差异有显著性意义(P<0.01)。结论:揭示慢性乙型肝炎肝肾阴虚证患者HBV基因突变点的分布规律,有利于进一步探讨中医药抑制HBV突变株复制的方法。     

海南汉族乙型肝炎病毒基因型与BCP区和前C区基因突变的关系

目的探讨海南汉族乙型肝炎病毒（HBV）基因型与前C区G1896A和BCP区A1762T／G1764A基因突变的关系。方法采用RT-PCR方法检测乙型肝炎患者的HBV基因型,PCR方法扩增包含C启动子和前C区基因核苷酸（nt1643-nt2112）,对PCR产物进行DNA测序。结果基因型C的BCP区A1762T／G1764A突变率（58．82％）显著地高于基因型B（10．53％）（P〈0．05）。基因型CG1896A突变率为29．41％,基因型BG1896A突变率为47．37％,两者比较差异无统计学意义（P〉0．05）。结论不同基因型HBV致病能力可能与病毒基因组BCP区A1762T／G1764A突变率的不同有关,而与前C区G1896A突变无关。     

HBV BCP区1762/1764位点变异对肝脏疾病进展及预后的影响

目的:探讨乙型肝炎病毒BCP区A1762T/G1764A变异与肝脏疾病进展的关系。方法:收集78例慢性乙型肝炎患者(CHB)和125例慢性重型乙型肝炎患者(CSHB)的血清及临床资料,并对所有入组患者进行随访,采用聚合酶链反应扩增HBV BCP区基因片段,产物纯化后直接测序,检测BCP区T1762/A1764位点变异。结果:CSHB组患者A1762T、G1764A及A1762T+G1764A的变异频率分别为64.0%(80/125)、60.0%(75/125)、60.0%(75/125),CHB患者3种变异的变异频率分别为30.8%(24/78)、28.2%(22/78)、25.6%(20/78),CSHB组3种变异的变异频率均明显高于CHB组,差异有统计学意义(P0.001)。125例CSHB患者随访48周,死亡组A1762T/G1764A位点变异频率明显高于存活组,差异有统计学意义(x~2=12.42,P0.001);A1762T/G1764A位点变异组的患者累积存活率明显低于未变异组(x~2=9.742,P0.01)。结论:HBV BCP区1762/1764位点变异可能会加重HBV感染后肝脏疾病病情,并且对慢性重型乙型肝炎的发病及预后可能起重要的作用。     

Association of Hepatitis B Virus Mutations of A1846T and C1913A/G With Acute-on-Chronic Liver Failure Development From Different Underlying Chronic Liver Diseases

Background

As most HBV-related acute-on-chronic liver failure (ACLF) have concurrent cirrhosis, it is important to clarify the association of viral factors with ACLF with or without cirrhosis.

Objectives

The aim of this study was to analyze the association of HBV genotypes and mutations with ACLF development underlying different chronic liver diseases.

Patients and Methods

Eighty-seven ACLF patients including 29 patients with chronic hepatitis (ACLF-CHB) and 58 patients with liver cirrhosis (ACLF-LC) were enrolled. Age and sex matched patients with chronic hepatitis (CHB) and liver cirrhosis (LC) were enrolled as controls. The genotypes and mutations at HBV basic core promoter (BCP), precore (PC), and partial C regions were determined by nested PCR and direct sequencing.

Results

Our results revealed significantly higher incidences (P < 0.05) of genotype B with C1913A/G or A1846T in patients with ACLF-CHB than those with CHB; genotype C with C1913A/G or A1846T in patients with ACLF-CHB and ACLF-LC than those with CHB and LC, respectively. Multivariable analysis indicated that A1846T and C1913A/G mutations were independent factors for ACLF (OR = 2.86 and 5.93, respectively), suggesting an association between the mutations and development of ACLF. In addition, there were no significant differences in mutations at T1753V, A1762T, G1764A, G1896A, and G1899A which were found between either CHB and ACLF-CHB or LC and ACLF-LC patients， suggesting no associations of these mutations with ACLF development.

Conclusions

Our findings suggest that CHB or LC patients infected with HBV A1846T and C1913A/G mutants are more susceptible to develop ACLF.     

Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B‐related acute‐on‐chronic liver failure without pre‐existing liver cirrhosis

Summary. The study was undertaken to investigate the features and clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP) and precore (PC) mutations in hepatitis B‐related acute‐on‐chronic liver failure (HB‐ACLF). Samples from 75 patients with HB‐ACLF and without pre‐existing liver cirrhosis and 328 age‐matched patients with chronic hepatitis B (CHB) were analyzed. HBV genotype and BCP/PC mutations were determined by direct sequencing. Mutations at 8 sites of the BCP/PC region were compared between the two groups of patients. A significantly higher ratio of genotype B to C was found in patients with HB‐ACLF than in patients with CHB (30.7–69.3%vs16.5–82.6%, P < 0.01). Single mutations including T1753V (C/A/G), A1762T, G1764A, G1896A and G1899A and triple mutations T1753V/A1762T/G1764A and A1762T/G1764A/C1766T (or T1768A) were more frequently detected in patients with HB‐ACLF than in patients with CHB. Correspondingly, BCP/PC wild‐type sequences were absent in patients with HB‐ACLF in contrast to 27.1% in patients with CHB. The BCP/PC mutations were found to be associated with increased HBeAg negativity, higher alanine aminotransferase level and lower viral load. Patients with HB‐ACLF infected with the PC mutant virus had a higher mortality. The findings suggest that patients with CHB infected with genotype B with BCP/PC mutations were more likely to develop HB‐ACLF than those with genotype C with wild‐type BCP/PC regions, and patients with HB‐ACLF with the PC mutation had increased risk of a fatal outcome.     

HBsAg携带者与HBeAg阴性慢性乙型肝炎患者病毒学特征的比较

目的了解非活动性HBsAg携带者（Inactive HBsAg carrier,HBsAg-IaC）和HBeAg阴性慢性乙型肝炎患者（e^--CHB）病毒学特点的异同。方法对连续收集的HBsAg-IaC（n=187）和e^--CHB（n=99）采用直接序列测定法检测G1896和核心基因启动子突变;结合聚合酶链反应-限制性片段长度多态性方法判断HBV基因型。结果113例HBsAg-IaC者血清HBVDNA阳性,其中103份完成前C区测序。HBsAg-IaC群体中HBV基因B型比例高于e^--CHB患者（84/113对46/99,P〈0.001）;HBsAg-IaC的G1896A突变比例高于e^--CHB（69/103对39/99,P〈0.001）,但A1762/G1764突变株比例低于后者（38/103对65/99,P〈0.001）。男性（OR=7.681,95%CI=2.693～20.992,P〈0.001）、年龄超过40岁（OR=24.421,95%CI=5.187～114.969,P〈0.001）、基因C型感染（OR=2.695,95%CI=1.240～5.859,P=0.012）以及A1762/G1764突变（OR=2.116,95%CI=1.012～4.425,P=0.046）是与e^--CHB相关的危险因素。结论HBsAg-IaC在病毒学特征上与e^--CHB明显不同;HBV基因C型感染、发生A1762/G1764突变、年龄大的男性HBsAg-IaC群体可能需要更多关注。     

Role of hepatitis B virus base core and precore/core promoter mutations on hepatocellular carcinoma in untreated older genotype C Chinese patients

The aim of the study was to investigate the prevalence of mutations of basal core promoter (BCP) and precore (PreC) region of hepatitis B virus (HBV) and their association with hepatocellular carcinoma. A total of 341 untreated older HBV patients were divided into three groups: chronic hepatitis B (CHB, 185), cirrhotic hepatocellular carcinoma (LC-HCC, 113) and non-cirrhotic hepatocellular carcinoma (non-LC-HCC, 43). HBV BCP and PreC mutations and genotypes were determined by direct sequencing. Using univariate analysis, age (≥ 45 years), single mutations including A1896 and A1899 and multiple mutations T1762/A1764 + A1896, T1762/A1764 + A1899 and T1762/A1764 + A1896 + A1899 were more frequently detected in LC-HCC and non-LC-HCC patients than in CHB patients. BCP T1762/A1764 mutations were highly detected in LC-HCC patients than in CHB patients. Multivariate logistic regression analysis (adjusted for age and gender) revealed that among HBeAg-positive patients, BCP T1762/A1764 mutations (OR, 5.975; P = 0.05), PreC A1899 mutation (OR, 4.180; P = 0.013) and multiple mutations T1762/A1764 + A1899 (OR, 6.408; P = 0.006) were independently associated with the development of LC-HCC; PreC A1899 mutation (OR, 7.347; P = 0.034) was also independently associated with the development of non-LC-HCC. On the other hand, among HBeAg-negative patients, PreC A1896 mutation (OR, 5.176; P = 0.002) and multiple mutations T1762/A1764 + A1896 (OR, 4.149; P = 0.007) were independently associated with the development of non-LC-HCC. These results indicated that older age (≥ 45 years) was associated with LC-HCC and non-LC-HCC development. BCP T1762/A1764 mutations and PreC A1899 mutation were associated with the LC-HCC development in HBeAg-positive patients. PreC A1896 mutation was associated with the non-LC-HCC development in HBeAg-negative patients.     

Hepatitis B virus genotype B and mutations in basal core promoter and pre-core/core genes associated with acute-on-chronic liver failure: a multicenter cross-sectional study in China

Background and aim

In China, acute-on-chronic liver failure (ACLF) is mostly caused by hepatitis B virus (HBV). However, the mechanism remains unclear. This study aims to investigate the association between both HBV genotype and mutations in basal core promoter (BCP) and pre-core/core (pre-C/C) regions with the development of HB-ACLF.

Methods

A multicenter cross-sectional study was performed in China. Serum samples from 522 patients were analyzed, including 231 patients with mild-chronic hepatitis B (CHB-M), 84 with severe-chronic hepatitis B (CHB-S) and 207 with HB-ACLF. HBV genotype and related mutations in the BCP and pre-C/C regions were determined by direct sequencing.

Results

A significantly higher ratio of HBV genotype B to C was detected in HB-ACLF patients than in CHB-M or CHB-S patients. The A1762T/G1764A, A1846T and G1896A mutations were significantly more common in HB-ACLF patients infected with either genotype B or C as compared with CHB-M, whereas the C1913A/G and A2159G mutations were more associated with HB-ACLF in genotype C patients. Comparing with CHB-S, the A1762T/G1764A mutation in genotype B and the A2159G mutation in genotype C were significantly more common in HB-ACLF patients. A multivariate analysis showed that factors such as HBV genotype B, age ≥40 years and A1762T/G1764A, A1846T and G1896A mutations were independently associated with the development of HB-ACLF.

Conclusion

Chronic HBV infection with genotype B, A1762T/G1764A, A1846T and G1896A mutations has a higher possibility to develop HB-ACLF. These virological factors could serve as possible molecular markers for prediction of the clinical outcomes of chronic HBV infection.     

慢性重型乙型肝炎患者HBVDNA前C／BCP区突变基因分析

目的分析慢性重型乙型肝炎（慢重乙肝）患者HBVDNA前C区和基本核心启动子（前C／BCP）区突变特点与意义。方法收集87例慢重乙肝和196例慢性乙型肝炎（慢乙肝）患者血清，提取HBVDNA，用巢式PCR扩增HBVDNA前C／BCP区基因，PCR产物进行DNA测序，用NBI软件比对结果，重点分析G1896、G1862、G1899、A1762、G1764、T17536个位点突变。结果慢重乙肝组和慢乙肝组6个位点突变全阴率分别为3．4％和28．1％（P〈0．01）；慢重乙肝组在其中5个位点上的突变检出率显著高于慢乙肝组。此外，慢重乙肝组和慢乙肝组≥三联突变检出率分别为56．3％和35．2％（P〈0．01），≥四联突变检出率分别为25.3％和8．7％（P〈0．01），插入／缺失突变检出率分别为10．3％和1．0％（P〈0．01）。结论HBVDNA前c／BcP区基因突变发生频率的增加与慢乙肝发生重症化相关，结合临床资料分析突变的意义将有助于认识慢乙肝重症化的发生机制。     

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.     

Combinations of eight key mutations in the X/preC region and genomic activity of hepatitis B virus are associated with hepatocellular carcinoma

Accumulation of eight key mutations located in the X/preC regions of the hepatitis B virus (HBV) genome (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) is a risk marker for the development of hepatocellular carcinoma (HCC). In this study, we analysed the 8 key mutations in 442 serum samples collected from 310 non‐HCC and 132 HCC patients to identify the combinations linked to HCC. After the patients were stratified according to the age groups and mutation combinations, clinical parameters were compared between the HCC and the non‐HCC groups. Analyses were focused on patient ≥40 years of age infected by HBV genotype C with A1762T and G1764A mutations in the basal core promoter region (BCP double mutation). In patients with ≥6 mutations, the combination of [G1613A + C1653T + A1846T + G1896A] mutations was closely linked to HCC, whereas no specific single or double mutation combination was associated with HCC. In patients with ≤5 mutations, HBeAg and HBV DNA serum titres were lower in the HCC group than those in the non‐HCC group. Unlike the number of mutations, no specific combination correlated with advanced clinical stage in HCC. Of the BCP double mutation–based HBV mutant types, combinations of ≥6 mutations that include G1613A + C1653T + A1846T + G1896A, and combinations of ≤5 mutations with reduced HBeAg production, may be more specific indicators of HCC risk than only the number of mutations or any specific combination(s).