Application of 454 Sequencing to Cancer Epigenomics

描述（由申请人提供）：尽管表观遗传成分在许多人类癌症的肿瘤进展中起着重要作用，但癌症表观基因组的甲基化景观仍在很大程度上未被探索。系统的基于序列的甲基化分析明显缺失，因此，特异性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开发。通过识别癌症表观基因组中的异常甲基化“热点”，我们可以针对这些基因进行治疗干预，并将其发展成为DNA甲基化生物标志物，用于早期检测、诊断、预后和监测治疗反应。然而，为了充分了解甲基化与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定甲基化模式。

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.