宿主免疫基因多态性与乙肝病毒优势突变株选择的关联研究

Chronic hepatitis B virus(HBV) infection is a major cause of hepatocellular carcinoma(HCC) but its mechanisms of oncogenesis remain obscure.It has been documented that HBV core promoter double mutations (T1762A1764) is associated with the development of HCC.Viral resistance in antiviral therapy and failure of vaccination may also occurred by mutations that arise in the viral genome. HBV gene mutations have been attributed to the lack of a proof-reading activity in the viral polymerase, and antiviral therapy and vaccine immunisation may increase the mutation rate. However, the role of host factors in the prevalence of the mutations is not understood. ..Taking the unique advantage of our Long An cohort, which was established in 2004 in the Zhuang ethnic minority area, Long An county, Guangxi with funds from the Wellcome Trust, we will select study subjects from the cohort to establish a 1:1 case-control study. Cases will be those with HBV core promoter double mutations (T1762A1764) and controls will be those with the wild type sequence. Human DNA will be genotyped using the ImmunoChip and the data will be analysed using the program PLINK to search for single-nucleotide polymorphisms (SNPs) associated with the selection of the HBV double mutations. Meanwhile, we will carry out a replication study, in which cases and controls will be selected from the majority Han population. ..The results of the proposed study will not only reveal the relationship between host factors and viral mutations and selection of the mutants but also help to understand further the mechanisms of HBV oncogenesis. The results may also provide a clue to study the relationship between host factors and viral resistance in antiviral therapy and so guide new strategies to search for the cause of drug resistance.

慢性乙肝病毒（HBV）感染是原发性肝癌的主要病因，但其致癌机理仍未明了。HBV核心基因启动子双突变(T1762A1764)与肝癌发生有关，其它位点突变可导致耐药和免疫失败。人们一直认为HBV基因突变归因于病毒聚合酶缺乏纠错能力，抗病毒治疗和疫苗免疫提高突变率，却忽视了宿主因素的作用。.本研究将利用2004年英国Wellcome 基金会在壮族地区隆安县资助建立的研究队列的独有优势，按匹配病例对照研究原理，选队列中HBV双突变株(T1762A1764)、野毒株感染者为病例和对照，用ImmunoChip做人类基因分型，用plink分析资料，探索与双突变株选择有关的人类基因位点。另选汉族HBV感染者做重复试验，验证初筛结果。.本研究不仅能阐明宿主因素与病毒基因突变、优势突变株选择的关系,而且有助于进一步认识HBV致癌机理，有助于启发研究人类基因与抗病毒治疗耐药的关系，为耐药原因研究提供新思路。

慢性乙肝病毒（HBV）感染是原发性肝癌的主要病因，但其致癌机理仍未明了。HBV 核心基因启动子双突变(T1762A1764)与肝癌发生有关，其它位点突变可导致耐药和免疫失败。人们一直认为HBV 基因突变归因于病毒聚合酶缺乏纠错能力，抗病毒治疗和疫苗免疫提高突变率，却忽视了宿主因素的作用。. 为查明人类基因位点突变与HBV 核心基因启动子双突变(T1762A1764)的发生是否有关，本研究按匹配病例对照研究原理，从隆安研究队列选择病例和对照，用Human Core基因芯片对人类基因进行初筛，用plink对初筛结果进行统计分析，用Sequenom技术对初筛阳性位点进行验证，用X2检验对验证结果进行统计分析。用qRT-PCR技术对经初筛及验证均有关联的阳性基因位点对应的基因表达量进行测定。. 初筛结果发现2个基因区域有差异趋势，集中在第5、10号染色体上，验证46个基因位点，发现其中3个（rs7717457，rs670011，rs2071611）突变与HBV 核心基因启动子双突变(T1762A1764)的发生有关，选择差异最大的位点rs7717457进行分析，发现该位点突变影响了其调控的基因C7的表达（t=2.045，P=0.048）。这3个基因位点突变率有随年龄增长而增加趋势，纯合突变与年龄关系更明显，但与HBV血清学标记物如HBeAg、anti-HBe和anti-HBc是否阳性、AFP（甲胎蛋白）是否阳性和ALT（转氨酶）是否正常无相关关系。本研究还发现基因位点rs198828、rs198843、rs5024431、rs1165159、rs3094228、rs7738548、rs9348385、rs2523822突变与HBV慢性感染有关，它们集中在第6号染色体HLA区域。. 这些研究结果说明HBV核心基因启动子双突变(T1762A1764)不仅与病毒本身有关，而且与宿主本身也有关，由于HBV核心基因启动子双突变与肝癌有关，这一发现不仅有助于阐明宿主因素与病毒基因突变关系,而且有助于进一步认识HBV 致癌机理。由于人类基因参与病毒基因突变，我们的发现还有助于启发研究人类基因与抗病毒治疗耐药的关系，为耐药原因研究提供新思路。本项目已产生3篇学术论文，其中SCI论文2篇，培养硕士研究生2名。本研究成果处在国际领先地位。