乙型肝炎病毒感染后表型不一致的单卵孪生子基因组CpG岛甲基化谱差异分析

Difference analysis of genome-wide DNA methylation status of CpG islands between the monozygotic twins with disconcordant phenotypes of chronic hepatitis B virus infection

目的进行乙型肝炎病毒感染后表型不一致的单卵孪生子基因组CpG岛甲基化谱差异分析,以发现可能的差异甲基化基因。方法采用改良甲基化间区位点扩增技术,根据人基因组CpG岛甲基化位点的两种主要碱基组合,CGCG和CCGG,用3种不同的酶切组合(SmaⅠXmaⅠ、HpaⅡMspⅠ、BssHⅡPauⅠ/BsePⅠ),同时采用PersonalMolecularImagerFX分子成像系统及放射自显影相结合的方法,经QuantityOne4.4.0凝胶图像分析软件进行差异条带分析,分离回收差异甲基化条带并克隆入T载体,阳性克隆进行测序,将测序结果进行BLAST分析,初步了解与乙型肝炎病毒感染后表型不一致的单卵孪生子相关的差异甲基化基因情况。结果在一对表型一致的单卵孪生子间基因组CpG岛甲基化分析显示条带几乎相同,而在另一对表型一致以及一对表型不一致的单卵孪生子之间均存在差异甲基化条带,前者差异甲基化条带数目少于后者,将得到的差异甲基化条带克隆入T载体,目前测序分析得到4个可能与乙型肝炎病毒感染后表型不一致的单卵孪生子相关的差异甲基化基因。结论乙型肝炎病毒感染后表型不一致的单卵孪生子间基因组CpG岛甲基化水平有差异,其对疾病表型的影响及机制尚需后续深入研究。

Objective To analyse the difference of genome-wide DNA methylation status of CpG island between the monozygotic twins with disconcordant phenotype of HBV infection and to discover possible differentially methylated genes. Methods Modified AIMS （amplification of inter-methylated sites） method was adopted. According to the frequent sites of CpG islands （-CGCG- and -CCGG-）, three groups of isochizomers with distinct methylation sensitivity （ Sma Ⅰ -Xma Ⅰ , Hpa Ⅱ -Msp Ⅰ and BssH Ⅱ -Pau Ⅰ/BseP Ⅰ ）were used to modify the AIMS method. The modified AIMS method combined with Personal Molecular Imager FX system analysis and radioautographic analysis was used to make the global detection of the methylome of CpG islands. Multiple anonymous bands were compared between the twins with the Quantity One bio-soft. The different bands were cloned into T vectors and positive clones were sequenced. BLAST analysis of positive clone sequences was conducted to give the clues for the differential methylated genes between monozogotic twins with discordant phenotype of HBV infection. Results Nearly the same bands were found between one pair of twins with concordant phenotype of HBV infection. Different methylated bands were found not only between monozygotic twins with concordant phenotype but also between those with disconcordant phenotype. More differential methylated bands were found in the latter groups. By BLAST analysis with sequences of differential methylated bands, four possible genes were got. These genes might relate to the monozygotic twins with disconcordant phenotype of chronic HBV infection. Conclusion Differential methylation of genes occurs in monozygotic twins with discordant phenotype of HBV infection. Whether these changes are involved in the pathogenesis of different phenotypes needs further elucidation.