全基因组单倍体特异性DNA甲基化鉴定和机理研究

As genetic effects on human health have been extensively studied, epigenetic effects, which represent factors such as the environment or dynamic cellular regulation, are still poorly understood, although they are believed to play an equally important role in human health. DNA methylation is repressive epigenetic marker that associates with normal development, aging process, and diseases such as cancer, mental disorders, and respiratory disorders. Mapping and understanding whole-genome DNA methylation (methylome) under the context of personal genomics adds an essential dimension of insights to personalized medicine. Rapid advances in DNA sequencing technologies have led to a detailed understanding of the human genome and epigenome. Yet, in spite of these advances, most genomics studies to date are based on a single reference of human genome and have given little consideration to a crucial aspect of human genetics: humans are diploid and differences between paternal and maternal chromosomes can affect many key biological mechanisms that are related to human health. In particular allele-specific DNA methylation is a key mechanism of allele-specific gene silencing, a phenomenon termed genomic imprinting. Allele-specific DNA methylation is shown to be important for normal embryonic development. However, its dynamics in human cells and its association with human health are poorly understood. .The goal of this project is to develop a new technology to completely phase human methylome and to study the dynamics of human methylome at chromosome level. The first two years of the project (K99 phase) will be carried out at Dr. Michael Snyder’s lab. During the K99 phase, Dr. Dan Xie will develop and test the phasing technology and apply this technology to phase the methylome of multiple cell types from one individual. This will reveal the dynamics of allele-specific DNA methylation in human cells and provide insights about the regulatory functions of DNA methylation. Having optimized the phasing technology and with the experience and results from the K99 study, as an independent researcher, Dr. Dan Xie will continue to phase the methylome in multiple lymphoblastoid cell lines from different individuals to understand the variation of allele-specific DNA methylation between individuals (R00 phase). The integration of existing large-scale genomics data, such as ENCODE and GWAS data, and the phased methylome data generated in this study will deepen our understanding of epigenetic regulation in much more detail.

人类是双倍体生物，单倍体间表观遗传和基因调控的差异有重要的生物功能，但是差异的形成机理和生物学功能并不清楚。本项目结合申请人先前参与开发的基因组单倍体测序技术(发表于Nature Biotechnology)和亚硝酸盐测序技术(BS-seq)来改进现有研究方法的缺点；通过设计新的干湿结合的生物技术达到识别全基因组单倍体特异性DNA甲基化的目的。利用这项新方法，申请人将研究全基因组单体型特异DNA甲基化在人类群体中的分布，并且通过利用已知染色体母本父本来源的基因组，区分单体型特异DNA甲基化是基于DNA序列(sequence dependent)还是基于上代来源(parent of origin)。通过和已有基因组数据整合分析，本项目数据将有助于理解单倍体特异性DNA甲基化和基因调控之间的关系。并回答为什么传统的基于DNA序列的遗传模型在某些基因组区域不能很好的解释性状显型。

人类是双倍体生物，单倍体间表观遗传和基因调控的差异有重要的生物功能，但是差异的形成机理和生物学功能并不清楚。结合申请人先前参与开发的基因组单倍体测序技术(发表于Nature Biotechnology)和亚硝酸盐测序技术(BS-seq)来改进现有研究方法的缺点；通过设计新的干湿结合的生物技术达到识别全基因组单倍体特异性DNA甲基化的目的。利用这项新方法，申请人研究了全基因组单体型特异DNA甲基化在人类群体中的分布，并且通过利用已知染色体母本父本来源的基因组，区分单体型特异DNA甲基化是基于DNA序列(sequence dependent)还是基于上代来源(parent of origin)。在本项目中，我们跟踪收集了一个54岁男性志愿者36个月共计54个时间点的外周血单个核细胞（PBMC），提取DNA和RNA数据分别进行全基因组甲基化和转录组测序。通过多组学数据分析，我们描述了一个历时1124天的外周血单个核细胞全基因组甲基化及转录组表达水平的动态变化过程。我们的研究揭示了外周血基因组甲基化及转录组的改变与个体健康状态之间的关联性。同时我们开发了一种基于化学基团修饰的低初始DNA羟甲基化建库测序方法。该方法可应用于细胞游离DNA（cfDNA）羟甲基化分子检测。利用此方法，我们从49例不同类型肿瘤病人外周血中提取并检验了cfDNA羟甲基化分子特征。我们在肺癌患者的cfDNA中发现了显著的DNA羟甲基化水平下降，而在肝癌和胰腺癌患者中分别找到了该类肿瘤特有的cfDNA羟甲基化分子模式。此项研究证实了cfDNA 羟甲基化测序手段可以用于肿瘤的早筛及一些肿瘤的分期预测。