HBV基本核心启动子和前C区变异的深度测序分析

发布时间：2018-07-25 06:34

【摘要】：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。
[Abstract]:Objective to investigate the characteristics of hepatitis B virus (HBV) (HBV) core promoter and precore region mutation in patients with hepatitis B virus (HBe Ag) positive chronic hepatitis B virus (CHB) by Ion Torrent PGM deep sequencing. Methods Serum samples of 25 patients with HBe Ag positive CHB were collected and HBV DNA was extracted. The HBV core promoter and precore region fragments were amplified by nested PCR method, and a deep sequencing library was constructed, which was based on Ion Torrent PGM platform. The mutation site and mutation rate were analyzed by bioinformatics, and the reference plasmids of the basic core promoter and precore promoter and wild type of HBV were constructed, which were used as quality control materials for deep sequencing. Results in 25 patients with HBe Ag positive CHB, 10 mutation sites with 20% prevalence rate were found in the basic core promoter and preC region of HBV. The prevalence rates of G1746A / A1752G / T / T1753C / G1764A / A1764A / A181764A / A181764A / A18176A / A1825C / A1846A / A1846A / G1896AG1899A G1746A and T1825C/A were 92% and 100%, respectively. The prevalence of G1746A and G1899A G1746A in 25 patients with HBe Ag positive CHB was 92% and 100%, respectively. The distribution of mutation locus in HBV B genotype and genotype C infection showed that A1762T and G1896A were mainly prevalent in patients with genotype B (63.6%vs 0.00, 蠂 ~ 2 12.374% P 0.00072.7 vs 28.6T, 蠂 ~ 2 + 4.812P 0.0472). A1762T and G1764A were mainly prevalent in C genotype carriers (27.3%vs 78.6p, 蠂 ~ 26.579P ~ (0.0172). Higher than genotype B, But there was no significant difference (25.7% 卤28.4%vs 68.4% 卤42.7%). Among 25 HBe Ag positive CHB patients, 32.0% had only A1762T/G1764A mutation 24.0%, only G1896A mutation 24.0% carried both A1762T/G1764A and G1896A mutations. Conclusion Deep-sequencing analysis can be used for quantitative detection of HBV core promoter and precore mutation, which provides a technical platform for clinical application of HBV mutation research.
【作者单位】：江苏大学医学院免疫学系;北京大学人民医院北京大学肝病研究所丙型肝炎及肝病免疫治疗北京市重点实验室;中国科学院微生物研究所生物信息中心;
【基金】：国家自然科学基金(No.81273202;No.31400773) 国家重大科技专项(No.2012ZX10002011-006) 江苏省临床医学科技专项(No.BL2013024)
【分类号】：R373.21;R392

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