Application of 454 Sequencing to Cancer Epigenomics

描述（由申请人提供）：尽管表观遗传组分在驱动许多人类癌症中的肿瘤进展中起主要作用，但癌症表观基因组中的甲基化景观在很大程度上仍未被探索。基于序列的系统性甲基化分析明显缺乏，因此，特定甲基化差异的潜在临床价值及其在癌症中的生物学影响在很大程度上仍未得到利用。通过鉴定癌症表观基因组中异常甲基化的“热点”，我们可以靶向这些基因进行治疗干预，并将其开发成DNA甲基化生物标志物，用于早期检测、诊断、预后和监测对治疗的反应。然而，为了充分理解甲基化和临床行为之间的相互作用，需要新的方法来确定整个基因组中单碱基水平的特异性甲基化模式。 作为我们工作的重要临床模型，我们将检查慢性淋巴细胞白血病（CLL）的子集，以发现区分CLL亚型的DNA甲基化改变，并提出差异临床行为和肿瘤进展的潜在机制。这项研究的成功完成将极大地影响CLL患者的临床管理，并允许表观遗传疗法的“前期”管理。为了实现这一目标，我们将开发一种高通量、大规模、基于测序的方法，为深入探索CLL甲基化组提供有效的方法。在我们的初步研究中，我们证明了亚硫酸氢盐测序可以使用创新的大规模平行测序系统（454-测序）进行，该系统能够在一次运行中分析数百万个DNA碱基。这种新一代亚硫酸氢盐测序将为多个CpG岛（CGI）中的特异性甲基化图谱提供高度定量的单个甲基胞嘧啶分辨率。 在这个R33应用中，我们建议优化和开发一种原型高通量亚硫酸氢盐测序方法，用于对来自CD 38+和CD 38- CLL B细胞的原发性CLL样本中的DNA甲基化模式进行超深度分析，并测试以下假设：CLL亚类的临床行为可以部分由其不同的DNA甲基化谱定义，这些DNA甲基化谱反过来影响多个基因和信号通路。具体而言，我们将：（1）开发用于高通量、超深度亚硫酸氢盐测序的多重扩增子制备方法;（2）开发用于甲基化富集的基因组DNA文库的亚硫酸氢盐测序的基因组规模方法;（3）将创新的高通量亚硫酸氢盐测序方法应用于CLL甲基化组的研究。我们相信，所开发的技术将彻底改变目前的DNA甲基化分析方法，为个体人类疾病提供异常DNA甲基化的数字图谱，并为疾病亚型的表观遗传分类提供深度测序，强大的方法。

DESCRIPTION (provided by applicant): Although epigenetic components play a major role in driving tumor progression in many human cancers, the methylation landscape in cancer epigenomes is still largely unexplored. Systematic sequence-based methylation analyses are notably absent and as a result, the potential clinical value of specific methylation differences and their biological impacts in cancers remain largely untapped. By identifying the aberrant methylation "hot spots" in the cancer epigenome, we can target these genes for therapeutic intervention and develop them into DNA methylation biomarkers for early detection, diagnosis, prognosis, and monitoring the response to therapy. However, to fully understand the interactions between methylation and clinical behaviors, new methods are needed to determine single-base-level specific methylation patterns across the genome. As an important clinical model for our work, we will examine subsets of chronic lymphocytic leukemia (CLL) to discover DNA methylation alterations that distinguish the sub-types of CLL and suggest underlying mechanisms for differential clinical behaviors and tumor progression. Successful completion of this study will substantially influence the clinical management of CLL patients and allow "up-front" administration of epigenetic therapies. To accomplish this, we will develop a high-throughput, large-scale, sequencing-based approach to provide efficient methods for deeply exploring the CLL methylome. In our preliminary study, we demonstrated that bisulfite sequencing can be carried out using an innovative massively parallel sequencing system (454-sequencing) that is capable of analyzing millions of DNA bases in a single run. This new generation of bisulfite sequencing will provide highly quantitative single methyl-cytosine resolution for specific methylation mapping in multiple CpG islands (CGIs). In this R33 application, we propose to optimize and develop a prototype high-throughput bisulfite sequencing method for ultra-deep analyses of DNA methylation patterns in primary CLL samples from CD38+ and CD38- CLL B-cells and test the hypothesis that the clinical behavior of subclasses of CLL can be defined in part by their distinct DNA methylation profiles that in turn affect multiple genes and signaling pathways. Specifically, we will: (1) develop a multiplexed amplicon preparation method for high-throughput, ultra-deep bisulfite sequencing; (2) develop a genome-scale approach for bisulfite sequencing of methylation-enriched genomic DNA libraries; (3) apply the innovative high-throughput bisulfite sequencing method to investigation of the CLL methylome. We believe that the technology developed will revolutionize the current analytical methods of DNA methylation, provide digital profiles of aberrant DNA methylation for individual human diseases and offer a deep-sequencing, robust method for epigenetic classification of disease subtypes.