HBV基本核心启动子和前C区变异的深度测序分析

Deep sequencing analysis of hepatitis B virus mutations in the basal core promoter and precore regions

目的利用Ion Torrent PGM深度测序技术,探讨乙型肝炎病毒e抗原（HBe Ag）阳性慢性乙型肝炎（CHB）患者乙型肝炎病毒（HBV）基本核心启动子和前C区突变特征。方法收集HBe Ag阳性CHB患者血清样本25例,提取HBV DNA,采用巢式PCR方法扩增HBV基本核心启动子和前C区片段,构建深度测序文库,基于Ion Torrent PGM平台深度测序,生物信息学分析突变位点及其突变率;构建HBV基本核心启动子和前C区突变型和野生型参考质粒,作为深度测序质控品。结果在25例HBe Ag阳性CHB患者HBV基本核心启动子和前C区检出流行率达20%的突变位点10个：G1746A、A1752G/T、T1753C/G、A1762T、G1764A、C1817G/A、T1825C/A、A1846T、G1896A、G1899A;G1746A和T1825C/A的流行率分别达92%和100%。突变位点在HBV B基因型和C基因型感染者的分布情况显示,A1752G/T和G1896A主要流行于B基因型感染者（63.6%vs 0.0%,χ^2=12.374、P=0.0007;72.7%vs 28.6%,χ^2=4.812、P=0.0472）,A1762T和G1764A主要流行于C基因型感染者（27.3%vs 78.6%,χ^2=6.579,P=0.0172）;C基因型感染者A1762T/G1764A突变率显著高于B基因型感染者,但差异无统计学意义（25.7%±28.4%vs 68.4%±42.7%,t=1.614、P=0.1326）。25例HBe Ag阳性CHB患者中,32.0%患者仅有A1762T/G1764A突变,24.0%患者仅有G1896A突变,24.0%患者同时携带A1762T/G1764A和G1896A突变。结论深度测序分析可用于定量检测HBV基本核心启动子和前C区突变,为HBV突变研究的临床应用提供了技术平台。

Objective To explore the characteristics of mutations in the basal core promoter（BCP） and precore（PC） regions of hepatitis B virus（HBV） genome in HBe Ag-positive patients with chronic hepatitis B（CHB） using Ion Torrent Personal Genome Machine（PGM） deep sequencing. Methods Serum samples from 25 HBe Ag-positive patients with CHB were collected, which were used for HBV DNA extraction. Nested PCR was used to amplify the BCP and PC regions of HBV genome. The PCR product was subjected to deep sequencing libraries preparation and subsequently Ion Torrent PGM sequencing. The variants and mutant percentages were detected through bioinformatics analysis. HBV BCP/PC wild type and mutant type reference plasmids were constructed, which were used as quality control in deep sequencing. Results Ten single nucleotide polymorphisms（SNPs） that had variant types with prevalence of greater than 20% were observed in the BCP and PC regions among the 25 HBe Ag-positive patients with CHB： G1746 A, A1752G/T, T1753C/G, A1762 T, G1764 A, C1817G/A, T1825C/A, A1846 T, G1896 A and G1899A; the prevalence of G1746 A and T1825C/A were 92% and 100%, respectively. The prevalence of these mutants was analyzed in patients with different HBV genotype infection. The A1752G/T and G1896 A were mainly prevalent in genotype B infection HCV patients（63.6% vs 0.0%, χ~2 = 12.374, P = 0.0007 and 72.7% vs 28.6%; χ~2 = 4.812, P = 0.0472）, A1762 T and G1764 A were mainly prevalent in genotype C HCV infection patients（27.3% vs 78.6%; χ~2 = 6.579, P = 0.0172）. Patients with genotype C HCV infection had higher A1762T/G1764 A mutant percentages than those with genotype B HCV infection, but without statistical significance（25.7% ± 28.4% vs 68.4% ± 42.7%; t = 1.614, P = 0.1326）. Among the 25 CHB patients with HBe Ag-positive, 32.0% cases had BCP mutants only, 24.0% had PC mutant only, and 24.0% had both BCP and PC mutants. Conclusions Deep sequencing could be used for quantification of HBV BCP and PC mutants, providing a suitable technology platform for the clinical application of HBV mutant research.