Technology for detection and quantitation of telomeric DNA aberrations in cancer

描述（由申请人提供）：端粒DNA异常是致癌的一个关键和普遍的方面。端粒的逐渐复制丧失最终导致端粒功能障碍，并在许多与年龄相关的疾病中发挥作用，包括癌症。散发性端粒缺失事件是一种普遍的细胞内在突变机制，可导致端粒功能失调和染色体不稳定；这些事件的频率被认为取决于诸如DNA复制叉停滞和其他DNA复制应激、氧化损伤和同源重组事件（如不相等姐妹染色单体交换（SCE）和t环重组）等因素。散发性端粒缺失事件在正常细胞中发生的频率很低；这些事件的频率增加（因此端粒功能失调）可能是癌变过程中最早的突变事件之一。功能失调的端粒修复可导致端粒-端粒融合，端粒-染色体臂在内部双链DNA断裂位点易位，以及由于重复的融合-断裂-融合循环而导致的额外DNA重排，从而导致基因组不稳定并帮助推动癌症进展。最终，端粒维持机制的激活（基于端粒酶或基于alt）被认为有助于稳定功能失调的端粒并允许肿瘤细胞快速生长。目前的技术还无法准确测量散发性端粒缺失事件和端粒融合的全球频率。因此，作为癌症基因组图谱的一部分，从肿瘤样本中获得的高通量数据集中完全没有端粒突变数据，而从细胞和有机体癌症模型中端粒功能的一些劳动密集型研究中收集的端粒突变数据是不完整和有偏差的。我们的实验室专注于对人类端粒DNA结构和变异的详细分析；在这里，我们建议利用这些知识来开发一种通用的、高通量的检测和定量人类端粒DNA突变事件的方法，并为其实用性获得原理证明数据。该方法将端粒DNA的物理富集和纯化与端粒基因组片段的高通量配对端测序定量分析结合起来。它被设计用于检测和定量单等位基因分辨率超短（TTAGGG）n束谱、端粒融合和亚末端DNA断裂-重新连接事件。此外，它可以适应未来的改进，以允许分析的小型化和多路复用。端粒长度和不稳定性的定量、单等位基因分辨率测量将允许对端粒丢失和端粒融合在癌症发生中的作用进行前所未有的深入了解，包括对介导这些过程的分子事件的机制了解，以及对该方法潜在的预后和肿瘤分层适用性的翻译见解。

DESCRIPTION (provided by applicant): Telomeric DNA abnormalities are a critical and universal aspect of carcinogenesis. The gradual replicative loss of telomeres ultimately results in telomere dysfunction and plays a role in many age-related diseases, including cancer. Sporadic telomere deletion events are a universal cell-intrinsic mutational mechanism that can lead to dysfunctional telomeres and chromosome instability; the frequency of these events is believed to depend upon factors such as DNA replication fork stalling and other DNA replication stress, oxidative damage, and homologous recombination events such as unequal sister chromatid exchange (SCE) and T-loop recombination. Sporadic telomere deletion events occur at a very low frequency in normal cells; an increased frequency of these events (and hence dysfunctional telomeres) may be among the very first mutational events in carcinogenesis. Repair of dysfunctional telomeres can result in telomere-telomere fusions, telomere- chromosome arm translocations at sites of internal double-strand DNA breaks, and additional DNA rearrangements as a consequence of repeated fusion-breakage-fusion cycles that result in genome instability and help drive cancer progression. Eventually, activation of telomere maintenance mechanisms (either telomerase-based or ALT-based) are believed to help stabilize dysfunctional telomeres and permit rapid growth of tumor cells. It is impossible to measure accurately the global frequency of sporadic telomere deletion events and telomere fusions with current technology. As a consequence, telomere mutational data are totally absent from the high- throughput datasets being acquired from tumor samples as part of the Cancer Genome Atlas, and telomere mutational data gleaned from a few labor-intensive studies of telomere function in cellular and organismal cancer models are incomplete and biased. Our lab has focused upon detailed analyses of human telomeric DNA structure and variation; here, we propose to use this knowledge to develop a universal, high throughput assay for detection and quantitation of telomeric DNA mutational events in humans and to obtain proof-of-principle data for its utility. The method couples the physical enrichment and purification of telomeric DNA with quantitative analysis of the telomeric genome fraction by high-throughput paired-end sequencing. It is designed to detect and quantitate single-allele-resolution ultrashort (TTAGGG)n tract profiles, telomere fusions, and subterminal DNA breakage-rejoining events. In addition, it is amenable to future refinement to permit miniaturization and multiplexing of the assays. The quantitative, single-allele-resolution measurements of telomere length and instability will permit unprecedented insights into the role(s) telomere loss and telomere fusion play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the method.