Technology for detection and quantitation of telomeric DNA aberrations in cancer

描述(由申请人提供)：端粒DNA异常是癌症发生的关键和普遍方面。端粒的逐渐复制丢失最终导致端粒功能障碍，并在包括癌症在内的许多与年龄相关的疾病中发挥作用。散发性端粒缺失事件是一种普遍存在的细胞内源性突变机制，可导致端粒功能障碍和染色体不稳定；这些事件的发生频率被认为取决于一些因素，如DNA复制叉停滞和其他DNA复制应激，氧化损伤，以及同源重组事件，如不等姐妹染色单体交换(SCE)和T环重组。在正常细胞中，零星的端粒缺失事件发生的频率非常低；这些事件的频率增加(因此端粒功能障碍)可能是癌症发生中最早的突变事件之一。端粒功能障碍的修复可以导致端粒-端粒融合，端粒-染色体臂在内部双链DNA断裂部位的易位，以及反复融合-断裂-融合循环导致的额外DNA重排，从而导致基因组不稳定，并有助于推动癌症的进展。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。用目前的技术不可能准确地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，从细胞和器官癌症模型中对端粒功能的少数劳动密集型研究收集的端粒突变数据是不完整和有偏见的。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，我们建议利用这一知识开发一种通用的、高通量的检测和定量人类端粒DNA突变事件的方法，并获得其实用性的原则证明数据。该方法将端粒DNA的物理浓缩和纯化与通过高通量配对末端测序对端粒基因组部分进行定量分析相结合。它被设计用来检测和定量单等位基因分辨超短(TTAGGG)n轨迹轮廓、端粒融合和亚末端DNA断裂-重新连接事件。此外，未来还可以对其进行改进，以实现分析的小型化和多重化。对端粒长度和不稳定性的定量、单等位基因分辨率测量将使人们能够前所未有地深入了解端粒丢失和端粒融合在癌症发生中的作用(S)，包括对调节这些过程的分子事件的机械性见解，以及对该方法潜在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.