超深焦磷酸测序分析HBV准种异质性及其与拉米夫定抗病毒治疗应答的关系

发布时间：2018-03-01 19:28

  本文关键词： 肝炎病毒 乙型 准种 超深焦磷酸测序 克隆测序 遗传异质性　出处：《上海交通大学》2014年博士论文　论文类型：学位论文

【摘要】：背景与目的：本课题组之前采用克隆测序（clone-based sequencing，CBS）的方法研究发现乙型肝炎病毒（hepatitis B virus, HBV）逆转录酶（reverse transcriptase, RT）区准种异质性可作为接受核苷类似物治疗的患者抗病毒疗效的一个预测指标。第二代测序技术可进行皮升（picoliter）规模的高通量平行反应以检测单个DNA分子，无需克隆这一费时繁琐的步骤。本研究首先通过比较超深焦磷酸测序（ultra-deeppyrosequencing UDPS），一种新的第二代测序技术，和CBS两种方法分析乙型肝炎病毒RT准种，结合生物信息学分析方法描述准种遗传异质性特征，探讨两种方法分析HBV生物多样性的表现力差异及其可能的临床意义。在此基础上，应用高通量UDPS方法检测拉米夫定抗病毒治疗早期阶段HBV RT区准种的动态变化，探讨病毒准种因素对抗病毒治疗应答的影响。 研究对象与方法：本研究的第一部分纳入了31例未接受过抗病毒药物治疗的慢性乙型肝炎患者，收集患者血清，抽提HBV基因组DNA，分别用CBS和UDPS方法平行分析RT区准种。用生物信息学方法对准种的异质性特征进行分析，准种的复杂度用下述公式计算（Sn=i(pi ln pi)/lnN）。本研究的第二部分共纳入了35例接受拉米夫定治疗至少48周的慢性乙型肝炎患者（16例为病毒学应答者，19例为部分病毒学应答者）。根据治疗48周时HBV DNA载量将患者分为应答组（HBVDNA载量低于检测下限）和部分应答组（HBV DNA载量高于检测下限，但较治疗基线水平下降1log10IU/mL）。收集治疗基线及治疗4周时的血清，抽提HBV基因组DNA，应用聚合酶链式（PCR）反应分别扩增HBV逆转录酶区三个相互重叠的约400bp的连续部分，将PCR产物进行超深焦磷酸测序。采用生物信息学方法分析准种的异质性特征，分析RT区准种基线和4周时的复杂度和离散度，并进一步分析两组患者的准种进化模式。 结果：第一部分：UDPS方法获得的有效准种株数远大于CBS方法（P0.001），相关分析表明UDPS和CBS两种方法的准种复杂度在核苷酸水平具有相关性（P0.05），UDPS方法准种的复杂度在核苷酸和氨基酸水平均高于CBS方法（P0.001）。 RT区变异分布研究发现，平均每个样本UDPS方法能检测到16.2±1.4个氨基酸变异位点，较CBS方法多9.7±1.1个位点。UDPS方法相关的进化分析较CBS方法显示了更多的遗传信息。第二部分：拉米夫定应答组RT区第1段和第2段的准种复杂度在治疗基线时高于部分应答组（P 0.05），应答组RT区第3段准种复杂度在治疗4周时高于部分应答组（P 0.05）。应答组的各个区段准种离散度相关参数（主要包括平均遗传距离和平均非同义替换数）在治疗基线时高于部分应答组（P 0.05），应答组的第1和第3区段准种离散度主要参数（主要包括平均遗传距离和平均非同义替换数）在治疗4周时亦高于部分应答组（P 0.05）。此外，，应答组RT区第1段的平均遗传距离（氨基酸水平）和平均非同义替换数的平均变化值和净变化值均高于部分应答组（P 0.05）。 结论：在低丰度变异的检测和准种模拟方面，UDPS方法描述准种异质性的表现力较CBS方法更敏感和高效，尽管UDPS方法尚存在不足，但其为HBV准种研究未来向临床推广应用指明了方向。拉米夫定抗病毒治疗初始及早期阶段时病毒学应答患者和部分病毒学应答患者HBV RT区准种的动态变化不同，治疗基线及治疗4周内HBV RT区准种的异质性及动态变化与拉米夫定抗病毒治疗的疗效相关。
[Abstract]:Background and purpose: by cloning and sequencing the research group before (clone-based sequencing CBS) method to study found that hepatitis B virus (hepatitis B virus HBV (reverse transcriptase) reverse transcriptase, RT) quasispecies heterogeneity can be used as a predictive index of patients receiving antiviral efficacy of nucleoside analogues therapy. Generation sequencing the technology can be second picoliter scale (picoliter) high-throughput parallel reactions to detect single DNA molecules, without cloning this time-consuming tedious steps. Firstly, through the comparison of ultra deep pyrosequencing (ultra-deeppyrosequencing UDPS), a new second generation sequencing technology, and CBS two methods of analysis of hepatitis B virus RT quasispecies, combined with bioinformatics analysis method to describe the quasispecies genetic heterogeneity, two methods of analysis of differences between HBV biodiversity expression and its clinical significance in May. Based on this, we applied high-throughput UDPS to detect the dynamic changes of quasispecies in HBV RT region at the early stage of lamivudine antiviral therapy, and to explore the influence of viral quasispecies on the response to viral therapy.
Subjects and methods: in the first part of the study included 31 cases of chronic hepatitis B patients had not received antiretroviral treatment, collect serum, extracting the genome of HBV DNA, CBS and UDPS respectively with the method of parallel analysis of RT quasispecies. By the methods of bioinformatics alignment heterogeneity for analysis of complex quasi use the following formula to calculate the degree of (Sn=i (PI ln PI) /lnN). The second part of this study included 35 cases of chronic hepatitis B patients treated with lamivudine for at least 48 weeks (16 cases of virological response, 19 cases with partial virological response patients). According to the treatment at 48 weeks HBV DNA the amount of patients will be divided into response group (load of HBVDNA below the detection limit) and partial response group (HBV DNA load is higher than the detection limit, but compared with the baseline 1log10IU/mL). Collected before treatment and after 4 weeks of treatment the serum, extracting the genome of HBV DN A, polymerase chain reaction (PCR) continuous part were amplified by HBV reverse transcriptase region three overlapping about 400bp, the PCR products were ultra deep pyrosequencing. Bioinformatics analysis of quasi heterogeneity for the analysis of RT quasispecies at baseline and after 4 weeks of complexity and from the divergence, and further analysis of the quasi evolutionary models of two groups of patients.
Results: the first part: a number of effective quasi UDPS method obtained more than CBS method (P0.001), correlation analysis showed that UDPS and CBS two methods of quasispecies complexity at the nucleotide level has correlation (P0.05) method, UDPS quasispecies complexity in nucleotide and amino acid levels were higher than the CBS method (P0.001). The RT variant distribution study found that the average sample UDPS method can detect 1.4 amino acid mutations was 16.2, compared with CBS 9.7 + 1.1.UDPS loci related methods of phylogenetic analysis showed that the genetic information is more than the CBS method. The second part: the response to lamivudine group of RT first and second segments of quasispecies the complexity in the baseline is higher than the partial response group (P 0.05), third groups of RT response section quasispecies complexity in the treatment of 4 weeks is higher than the partial response group (P 0.05). Each sector response group of quasispecies dispersion related parameters (mainly Including the average genetic distance and the average number of nonsynonymous substitutions) is higher than that of partial responders at baseline (P 0.05), the first and third section response group quasi discrete degree of main parameters (including the average genetic distance and the average number of nonsynonymous substitutions) in the treatment of 4 weeks before that of response group (P 0.05). In addition, the average genetic response group RT first distance (amino acid level) and the average change of the average value for the number of non synonymous substitutions and net change values were higher than the partial response group (P 0.05).
Conclusion: the detection of low abundance variation and quasi simulation, UDPS method to describe the quasispecies heterogeneity of expression than the CBS method is more sensitive and efficient, while the UDPS method is insufficient, but the HBV quasispecies study to future clinical application direction. Lamivudine antiviral therapy and early initial stage virological response patients and some patients HBV RT virological response zone quasi dynamic changes of different treatment at baseline and within 4 week of treatment HBV RT quasi heterogeneity and dynamic change and the kind of lamivudine therapy.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.62

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