Objective The involvement of HBV DNA integration in promoting hepatocarcinogenesis and the extent to which the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte transcriptome in hepatitis B ‘e’ antigen (HBeAg)-negative chronic hepatitis B (CHB). Design Liver tissue from 84 HBeAg-negative patients with CHB with low (n=12), moderate (n=25) and high (n=47) serum HBV DNA was analysed. Covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) were evaluated by quantitative PCR, whole exome and transcriptome sequencing was performed by Illumina, and the burden of HBV DNA integrations was evaluated by digital droplet PCR. Results Patients with low and moderate serum HBV DNA displayed comparable intrahepatic cccDNA and pgRNA, significantly lower than in patients with high HBV DNA, while hepatitis B core-related antigen correlated strongly with the intrahepatic HBV reservoir, reflecting cccDNA quantity. Whole exome integration was detected in a significant number of patients (55.6%, 14.3% and 25% in high, moderate and low viraemic patients, respectively), at a frequency ranging from 0.5 to 157 integrations/1000 hepatocytes. Hepatitis B surface antigen >5000 IU/mL predicted integration within the exome and these integrations localised in genes involved in hepatocarcinogenesis, regulation of lipid/drug metabolism and antiviral/inflammatory responses. Transcript levels of specific genes, including the proto-oncogene hRAS, were higher in patients with HBV DNA integration, supporting an underlying oncogenic risk in patients with low-level to moderate-level viraemia. Conclusions HBV DNA integration occurs across all HBeAg-negative patients with CHB, including those with a limited HBV reservoir; localising in genes involved in carcinogenesis and altering the hepatocyte transcriptome. Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. All clinical data have been collected in an anonymous database available at The Blizard Institute, Barts and The London SMD, QMUL. Responsible: PTFK

中文翻译：

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在 HBeAg 阴性慢性乙型肝炎中，整个外显子组 HBV DNA 整合与肝内 HBV 储存库无关

目的 HBV DNA 整合在促进肝癌发生中的作用以及肝内 HBV 储存库调节肝病进展的程度仍然知之甚少。我们检查了肝内 HBV 储存库、HBV DNA 整合的发生及其对乙型肝炎'e'抗原 (HBeAg) 阴性慢性乙型肝炎 (CHB) 肝细胞转录组的影响。设计分析了来自 84 名 HBeAg 阴性 CHB 患者的肝组织，这些患者具有低 (n=12)、中度 (n=25) 和高 (n=47) 血清 HBV DNA。通过定量 PCR 评估共价闭合环状 DNA (cccDNA)、前基因组 RNA (pgRNA)，由 Illumina 进行全外显子组和转录组测序，通过数字液滴 PCR 评估 HBV DNA 整合的负担。结果 低和中等血清 HBV DNA 患者的肝内 cccDNA 和 pgRNA 显着低于高 HBV DNA 患者，而乙型肝炎核心相关抗原与肝内 HBV 储库密切相关，反映了 cccDNA 的数量。在大量患者中检测到全外显子组整合（在高、中、低病毒血症患者中分别为 55.6%、14.3% 和 25%），频率范围为 0.5 至 157 个整合/1000 个肝细胞。乙型肝炎表面抗原 >5000 IU/mL 预示着外显子组内的整合，这些整合定位于与肝癌发生、脂质/药物代谢调节和抗病毒/炎症反应有关的基因中。特定基因的转录水平，包括原癌基因 hRAS，在 HBV DNA 整合的患者中更高，支持低水平至中度病毒血症患者潜在的致癌风险。结论 HBV DNA 整合发生在所有 HBeAg 阴性 CHB 患者中，包括那些 HBV 储库有限的患者；定位于参与致癌作用和改变肝细胞转录组的基因。可根据合理要求提供数据。所有与研究相关的数据都包含在文章中或作为补充信息上传。所有临床数据均收集在暴雪研究所、Barts 和伦敦 SMD、QMUL 的匿名数据库中。负责人：PTFK 包括那些乙肝病毒库有限的人；定位于参与致癌作用和改变肝细胞转录组的基因。可根据合理要求提供数据。所有与研究相关的数据都包含在文章中或作为补充信息上传。所有临床数据均收集在暴雪研究所、Barts 和伦敦 SMD、QMUL 的匿名数据库中。负责人：PTFK 包括那些乙肝病毒库有限的人；定位于参与致癌作用和改变肝细胞转录组的基因。可根据合理要求提供数据。所有与研究相关的数据都包含在文章中或作为补充信息上传。所有临床数据均收集在暴雪研究所、Barts 和伦敦 SMD、QMUL 的匿名数据库中。负责人：PTFK