52例慢性乙型肝炎 HBV 前C区基因突变研究

本文采用多聚酶链反应－－单链构象多态分析（ＰＣＲ－ＳＳＣＰ）银染技术检测了５２例ＨＢＶＤＮＡ阳性慢性乙型肝炎口才的ＨＢＶ前Ｃ区基因突变。结果有前Ｃ区突变株感染３２例，突变株检出率为６１．５％；突变株的检出以慢性下肝炎最高，其次是慢性乙肝重度、中度口才慢性乙肝轻度患者量低；抗－ＨＢｅ阳性２１前Ｃ区突变株检出率高于ＨＢｅＡｇ阳性患者。表明ＨＢＶ前Ｃ区突变与病情轻重及ｅ系统血清标志有关。     

乙型肝炎病毒前C区基因突变的研究进展

本文综述了乙型肝炎病毒前Ｃ区基因突变的研究进展。前Ｃ区某些部位，特别是第１８９６位核到的终止突变，使ＨＢＶ失去分泌ＨＢｅＡｇ的能力，但不影响其复制与传染，前Ｃ区突变的发生除与ＨＢＶ前基因组逆转不过程中缺乏校验酶而发生碱基错配有关外，主要与宿主的免疫压力有关，前Ｃ区的突变常伴有其它基因特点是Ｃ基因的突变，共同影响突变株的生物学特性及宿主的临床转归，使某些突变株与重症肝炎的发生及干扰素的疗效关系密切，     

慢性乙型肝炎HBV前C区基因突变与中医虚实证的关系

目的:探讨慢性乙型肝炎HBV前C区基因突变与中医虚证和实证的关系。方法:采用聚合酶链反应-单链构象多态分析(PCR-SSCP)银染技术,对91例慢性乙型肝炎患者HBV前C区基因突变进行了研究。结果:53例慢性乙型肝炎实证患者,HBV DNA阳性36例,其中前C区基因突变26例,突变率为72.2％;38例虚证患者,HBV DNA阳性16例,其中前C区基因突变6例,突变率为37.5％。两组比较有显著性差异,P<0.01。实证突变组肝功能ALT、AST和SB水平显著高于虚证突变组及未突变组,P<0.01。结论:HBV前C区基因突变与中医虚证和实证有一定的关系,实证突变率高于虚证,且实证突变组的炎症活动较虚证组剧烈。     

线粒体DNA点突变与心肌病关系的研究

用聚合酶链反应（ＰＣＲ）和异源双链（ＨＥＴ）凝胶电泳检测了１５例原发性扩张型心肌病（ＤＣＭ）、１３例急性心肌炎患儿及１个肥厚型心肌病（ＨＣＭ）家系中１７例成员 的外周血线粒体ＤＮＡ（ｍｔＤＮＡ）点突变。结果显示，ＤＣＭ患儿中，６例在ｍｔＤＮＡ保守区３１０８￣３７１７位存在点突变，其中１例家族性ＤＣＭ患儿及其母亲均检出点突变，提示ｍｔＤＮＡ点突变在ＤＣＭ发病中起一定作用；１３例急性心肌炎患儿中有１例     

肥厚性心肌病患者心脏β－肌凝蛋白重链基因点突变的研究

应用聚合酶链式反应（ＰＣＲ）与ＤＮＡ单链构象多态（ＳＳＣＰ）技术，分析３２例肥厚性心肌病患者心脏β－肌凝蛋白重链（β－ＭＨＣ）基因第１３、１６、２０和２３外显子区域，发现１例患者第１３外显子ＰＣＲ产物的单链电泳带与正常对照有明显的差异；对其核苷酸顺序分析表明，该患者β－ＭＨＣ基因第３８３密码子位置发生Ｇ→Ｔ颠换，导致赖氨酸（Ｌｙｓ）变成天冬酰胺（Ａｓｎ）。由于此突变发生在β－ＭＨＣ基因种系进化中高度保守的区域，故提示其突变是引起患者发生心肌肥厚的主要病因。家系调查显示，该患者父母、姐姐、妹妹及女儿心脏超声及ＰＣＲ－ＳＳＣＰ检查均未见异常。在β－ＭＨＣ基因第３８３密码子区域合成一对正常及发生Ｇ→Ｔ突变的等位基因特异性寡核苷酸探针，分别与患者、其家系成员及对照个体相应区域的ＰＣＲ产物进行杂交，结果显示仅患者的ＰＣＲ产物能与突变探针杂交，说明此患者的点突变为发生在胚细胞水平的新生突变，患者的肥厚性心肌病为散发性。这是在中国人群中发现心脏β－ＭＨＣ基因突变的首例报道，亦是一种新的β－ＭＨＣ基因突变类型     

乙型肝炎病毒基因组变异对干扰素治疗的影响

目的 研究乙型肝炎病毒基因组Ｘ区和前Ｃ／Ｃ区多部位变异对干扰素（ＩＦＮ）治疗的影响。方法 利用套式聚合酶链反应产物直接测序技术，测定１７例慢性乙型肝炎患者ＩＦＮ治疗前病毒核苷酸序列。结果 ５例Ｔ＾１７６２－Ａ＾１７６４变异株感染者均对ＩＦＮ治疗有应答，而在１２例无Ｔ＾１７６２－Ａ＾１７６４突变患者中仅３例有应答。在８例Ａ＾１８９６变异株感染者中，５例同时伴Ｃ区Ｂ细胞表位ａａ１０７￣１１８突变，其中     

乙肝病毒基因前C区突变株感染的检测及临床意义

应用突变特异性ＰＣＲ（ｍｕｔａｔｉｏｎｓｐｅｃｉｆｉｃＰＣＲ简称ｍｓＰＣＲ）技术对１１７例１４２份临床各型乙肝病毒（ＨＢＶ）感染者的血清标本进行了ＨＢＶ基因前ｃ区１８９６位已知Ｇ→Ａ点突变株的检测，发现２４例３０份标本有突变株感染，检出率为２０．５１％。其中重肝组３９．１３％（９／２３），慢活肝组１９．６４％（１１／５６），慢迁肝组１３．３３％（２／１５），无症状携带者组８．６９％（２／２３）。分析结果发现ＨＢＶ基因前Ｃ区突变株感染与重肝的发生、发展有一定的关系，对干扰素的疗效似有影响，与年龄，性别差异关系不大。     

HBV前C区突变株对干扰素治疗的应答反应

目的评价干扰素对ＨＢＶ前Ｃ区突变株的应答反应。方法对１０４例慢性乙型肝炎患者，应用克隆测序和快速检测ＨＢＶ前Ｃ区终止密码突变的方法，共检出２１例突变株，其中１６例曾应用干扰素治疗；野生株２１／２５例曾应用干扰素治疗。结果１６例ＨＢＶ前Ｃ区突变株对干扰素治疗有应答者６例；野生株２１例，对干扰素有应答者９例，二组相比差异无显著性。结论ＨＢＶ前Ｃ区突变株及野生株对干扰素均有部分应答，应答率分别为３７．５％和４２．９％，二组相比差异无显著性     

乙型肝炎病毒前C区突变株感染的临床和病理

探讨乙型肝炎病毒（ＨＢＶ）前Ｃ区突变株（１８９６位点Ｇ→Ａ点突变）慢性感染患者的临床特点、病理特征及与干扰素疗效的关系。方法采用突变特异性ＰＣＲ检测１０８份慢性ＨＢＶ感染患者血清和／或肝组织中ＨＢＶ前Ｃ区突变株，按Ｋｎｏｄｅｌｌ方法评价肝组织病理损伤。结累ＨＢＶ前Ｃ区突变株在不同ｅ系统均存在，但ＨＢｅＡｂ（＋）组中单纯突变株感染或突变株感染占优势的混台感染（１４．２９％，４６．２３％）显著地高于ＨＢｅＡｇ（＋）组（０％，６．４５％）；突变株感染与肝脏疾病严重程度相关，慢性肝炎重度组检出率（１３．６４％，４０．９１％）极显著地高于慢性无症状携带者（０％，１０．２６％）。５例前Ｃ区突变株感染者应用干扰素治疗可产生应答和无应答２种结果。临床症状较轻的ＨＢＶ前Ｃ区突变株感染者的肝脏病理损伤程度无明显加重。结论前Ｃ区突变株感染普遍存在，但更常见于ＨＢｅＡｂ阳性者。【关键词】##4乙型肝炎病毒;;突变;;病理学;;治疗     

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。     

A novel primer-extension assay for the detection of a G to A mutation in the distal precore region of hepatitis B virus DNA

The roles of genetic heterogeneity of the hepatitis B virus (HBV) precore gene in the pathogenesis of HBV infection are unclear. Various methods have been used to detect nucleotide (nt) 1896 precore mutants. We established a new primer-extension assay to facilitate the detection of these mutants. This assay is based upon the fact that there is no adenine in the distal precore region of wild-type HBV. Polymerase chain reaction (PCR)-amplified template DNA was denatured and annealed to the [γ-³²P]-labelled primer. During primer extension in the presence of DNA polymerase and dCTP, dGTP, dTTP and ddATP, the reaction terminates if there is a nucleotide A. When mixtures of different ratios of wild-type and nt 1896 precore mutants were analysed in the primer-extension assay, correlation between the percentage known amounts and the percentage measured amounts of nt 1896 precore mutants was excellent (r²=0.9669). When the primer-extension assay and direct sequencing were compared in hepatitis B e antigen (HBeAg)-positive and -negative chronic active hepatitis B patients, the primer-extension assay detected a greater number of nt 1896 precore mutants than direct sequencing and thus most HBV infections were found to be mixed infections. In conclusion, the primer-extension assay is a reliable and sensitive method for the detection of nt 1896 precore mutants.     

Mutations of Basal Core Promoter and Precore Regions in Hepatitis B Virus Genotypes B and C

Background:

Mutations in basal core promoter (BCP) and precore regions of hepatitis B virus (HBV) are associated with course and treatment outcomes of chronic HBV infection. While BCP and precore mutation analysis have been carried out in adult patients between different genotypes, this analysis has rarely been performed for chronically infected children.

Objectives:

The aim of this study was to assess the mutation profiles of BCP and precore regions in different HBV genotypes in chronically infected children.

Patients and Methods:

A cohort of 245 children and 92 adults with chronic HBV infection was included in this study. BCP and precore regions were analyzed by PCR amplification and sequenced.

Results:

Ten nucleotide positions, including nt1679, nt1721, nt1753, nt1757, nt1758, nt1762, nt1764, nt1775, nt1856 and nt1858 in BCP/precore regions of HBV genome, showed obviously higher frequencies of mutation in genotype C subjects than in genotype B subjects among children, while there were only three positions, including nt1679, nt1758 and nt1775 showing higher mutation frequencies in genotype C subjects than in genotype B subjects in adults. Several combined mutations were obviously highly distributed in children with chronic HBV genotype C infection, such as G1721A/A1775G/T1858C triple mutation; a novel combined mutation type, exclusively detected in children with chronic HBV genotype C infection. In addition, G1721A/A1775G/T1858C combined mutation was associated with higher viral load and lower age distribution.

Conclusions:

The mutation ratio difference between genotypes B and C in children was higher than that of adults and several combined mutations were exclusively detected in children with chronic HBV genotype C infection associated with higher viral load.     

乙型肝炎病毒基因前C区变异的动态观察

以错配聚合酶链反应产物作限制片段长度多态性（ＲＦＬＰ）分析，检测５４例经肝活检确诊的慢性乙型肝炎血清前Ｃ区变异株，总检出率为６６．７％；在慢性活动性肝炎中的检出率（８０．０％），明显高于慢性迁延性肝炎（４６．７％）；在乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）阳性和抗－ＨＢｅ（＋）中分别为４１．２％和６３．３％；在抗－ＨＢｅ（＋）且丙氨酸转氦酶正常血清中检出率８２．４％。前Ｃ区变异株可在病程中出现亦可消失。结果提示，前Ｃ区变异株与原型株的混合感染广泛存在于慢性乙型肝炎病毒感染者，其发生可能与机体免疫压力有关。     

Precore and X region mutants in hepatitis B virus infections among renal dialysis patients

SUMMARY. Hepatitis B virus (HBV) variants containing mutations within the X and the precore regions of the viral genome were demonstrated by polymerase chain reaction (PCR) amplification and DNA sequencing in renal dialysis patients with different serological patterns of HBV infection. Among carriers, X region deletion mutants predominated in patients who lost hepatitis B e antigen (HBeAg), or developed anti-HBe, but not in persistently HBeAg-positive patients. The precore region remained wild type in all carriers whether or not they seroconverted from HBeAg to anti-HBe. The frequency of precore and X region mutants was greatest among non-carrier patients with viral antibodies as the only indication of infection and among patients with non-A, non-B hepatitis (NANBH), suggesting an inverse relationship between the presence of wild type HBV markers and the presence of HBV mutants. Furthermore, the detection of one but not the other mutation in many serum samples suggests that these mutations are independently selected for during infection. Finally, the absence of HBV DNA in 21 'uninfected' dialysis patients with normal transaminases and no viral serology, suggests that replication of these mutants is associated with hepatitis. These results have important implications for HBV screening and treatment, as well as for the pathogenesis of chronic infection.     

Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissues

The development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) frequently follows persistent HBV infection and may arise in individuals who are hepatitis B e antigen (HBeAg) negative, indicating the possible presence of precore/core mutants. It is unclear whether precore/core mutants are associated with tumour development or are selected for after chromosomal integration of the wild-type viral DNA. We studied the status and sequence variation of the precore/core region of HBV in 56 patients with HBV-associated HCC and in various corresponding non-tumour tissues by Southern blot analysis, polymerase chain reaction and direct sequencing. Southern blot showed that integrated HBV DNA existed in 43 of 56 HCC tissues. Sequence analysis revealed mutations in 65% of the HCC (26/40) and 45% (14/31) of the corresponding non-tumour tissues. The mutation at nucleotide (nt) 1896, known to prevent HBeAg synthesis, was detected in 40% (16/40) of the tumours and in 35.4% (11/31) of the non-tumour tissues. Other mutations were found at nt 1899 (eight of 40 in HCC; three of 31 in non-tumour tissues), nt 1898 (seven of 40 in HCC; two of 31 in non-tumour tissues), nt 1912 (seven of 40 in HCC; none of 31 in non-tumour tissues) and nt 1886 (three of 40 in HCC; none of 31 in non-tumour tissues). To determine whether this finding merely reflected the prevalence of such mutants in this geographical region, HBV DNA from the sera of patients (also in this region) with acute and chronic hepatitis were sequenced. The nt 1896 mutant was found in 5.6% (one of 18) of patients with acute hepatitis B and in 22.8% (nine of 35) of patients with chronic hepatitis B. However, the nt 1898 mutation was not found in any of these sera. The precore/core mutant was observed with increasing frequency from acute hepatitis to chronic hepatitis, non-tumour and HCC, and this difference in frequency was significant between HCC and acute hepatitis B groups (P < 0.01), suggesting that the precore/core mutant or hepatocytes harbouring this mutant may be under immune selection and that such mutations may facilitate integration and subsequent tumour development.     

Clinical relevance of mutations in the precore genome of the hepatitis B virus.

A stop codon in the precore genome of the hepatitis B virus (HBV) in anti-HBe positive HBV carriers may be associated with a more progressive form of HBV infection. Earlier studies, however, were mainly performed in patients from the Mediterranean area who had severe infection. The aim of this study was to evaluate the prevalence of precore mutants in an unselected population living in northern Europe. Twenty of 42 of these patients are infected predominantly with a virus strain, which has the typical stop codon in the precore genome, characterised by a mutation at base 83. In six patients there was an additional G to A mutation at base 86 of the precore genome. Statistical analysis showed no difference between the patients with or without a stop codon in the precore genome. When patients with a double mutation at base 83 and 86 of the precore genome were compared with the other anti-HBe positive HBV carriers, however, the corresponding clinical data were worse. Therefore we suggest, that it is not the stop codon in the precore gene itself, but the occurrence of a double mutation at bases 83 and 86, which is associated with a more severe course of disease in anti-HBe positive HBV carriers.     

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HBV感染前C区变异与病毒复制水平关系

探讨HBV前C基因变异与病毒复制水平的关系在慢性HBV感染者中的意义。应用错配聚合酶链反应（PCR）－限制性片段长度多态性（RFLP）分析与荧光定量聚合酶链反应检测HBVDNA相结合,对30例HBsAg（＋）、抗－HBe( )及抗－HBc( )慢性HBV感染者,其中无症状携带者（AsC)9例、慢性乙型肝炎（CHB）12例及慢性重症肝炎（CHF）9例进行前C区基因变异与HBVDNA水平关系进行分析。AsC组3例（33．33％）,CHB组9例（75％）及CHF组8例（88．89％）有A83(nt1896)变异。荧光定量PCR结果表明HBVDNA含量在CHF组中最高。HBV前C变异与HBV不同感染状态中都可见,其病毒复制水平与肝病活动相关。     

Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood

AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood. METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected. RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively. CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.