Peng Yaqin, Liu Baoming, Hou Jinlin, Sun Jian, Hao Ran, Xiang Kuanhui, Yan Ling, Zhang Jiangbo, Zhuang Hui, Li Tong
Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
Antivir Ther. 2015;20(6):623-32. doi: 10.3851/IMP2955. Epub 2015 Apr 2.
Mutations in HBV core promoter (CP) are suggested to affect viral replication and disease progression. We investigated CP deletion/insertion mutations (Del/Ins) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients before and during antiviral treatment.
Direct and clone sequencings were used for detection of CP Del/Ins in 12 patients. The dynamic changes of CP Del/Ins were tracked in these cases until week 48 of treatment. The effects of Del/Ins on CP activities and hepatitis B X protein (HBx) were analysed using luciferase assay and sequence comparison, respectively. Furthermore, 292 untreated HBeAg-positive CHB cases were also analysed.
Twelve cases with multi-peak PCR direct sequencing electropherograms at baseline were confirmed to have CP Del/Ins by clone sequencing, with detection rates varying from 14.8% to 93.3% of clones analysed. Follow-up studies showed the detection rates of CP Del/Ins in patients decreased from 100% (12/12) at baseline to 16.7% (2/12) at week 48 of treatment (P<0.001), in parallel with a decline in HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotransferase (ALT) and aspartate transaminase (AST) levels along with an increase in HBeAg loss. Luciferase assay results showed distinct promoter activities among Del/Ins-harbouring CP sequences. Importantly, 71.8% (148/206) of Del/Ins sequences potentially resulted in HBx carboxy-terminal truncations. CP Del/Ins mutations were also found in 27.4% (80/292) of untreated cases.
Naturally occurring complex of CP Del/Ins mutants existed in untreated HBeAg-positive CHB patients. These mutations would affect HBV transcription activities and integrity of HBx, which might correlate with disease progression. Their prevalence decreases on antiviral therapy in parallel with the decline in HBV DNA, HBsAg and ALT and AST levels.
乙肝病毒核心启动子（CP）突变被认为会影响病毒复制和疾病进展。我们研究了乙肝e抗原（HBeAg）阳性慢性乙型肝炎（CHB）患者在抗病毒治疗前及治疗期间CP缺失/插入突变（Del/Ins）情况。
采用直接测序和克隆测序检测12例患者的CP Del/Ins。追踪这些病例中CP Del/Ins的动态变化直至治疗第48周。分别采用荧光素酶报告基因检测法和序列比较分析法分析Del/Ins对CP活性和乙肝X蛋白（HBx）的影响。此外，还分析了292例未经治疗的HBeAg阳性CHB病例。
12例基线时PCR直接测序电泳图谱呈多峰的病例经克隆测序证实存在CP Del/Ins，分析的克隆中检测率为14.8%至93.3%不等。随访研究显示，患者中CP Del/Ins的检测率从基线时的100%（12/12）降至治疗第48周时的16.7%（2/12）（P<0.001），同时乙肝病毒DNA（HBV DNA）、乙肝表面抗原（HBsAg）、丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平下降，HBeAg血清学转换率上升。荧光素酶报告基因检测结果显示，含有Del/Ins的CP序列之间启动子活性明显不同。重要的是，71.8%（148/206）的Del/Ins序列可能导致HBx羧基末端截短。在27.4%（80/292）的未经治疗病例中也发现了CP Del/Ins突变。
未经治疗的HBeAg阳性CHB患者中存在自然发生的CP Del/Ins突变复合体。这些突变会影响HBV转录活性和HBx完整性，可能与疾病进展相关。抗病毒治疗期间其发生率降低，同时HBV DNA、HBsAg以及ALT和AST水平下降。
