Technology for detection and quantitation of telomeric DNA aberrations in cancer

癌症端粒 DNA 畸变的检测和定量技术

• 批准号: 7777979
• 负责人: Harold RIETHMAN
• 金额: $ 25.79万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777979
• 关键词: Affinity; Alleles; Atlases; Biological Assay; Cancer Model; Cancer cell line; Cells; Characteristics; Chromosomal Instability; Chromosome Arm; Complementary DNA; Couples; DNA; DNA Double Strand Break; DNA Sequence; DNA Sequence Rearrangement; DNA Structure; DNA biosynthesis; DNA replication fork; Data; Data Set; Databases; Detection; Development; Disease; Event; Frequencies; Functional disorder; Future; Gene Rearrangement; Genetic Recombination; Genome; Genomic Instability; Genomics; Glean; Human; Individual; Knowledge; Lead; Length; Libraries; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Miniaturization; Molecular; Mutation; Normal Cell; Oligonucleotides; Play; Preparation; Process; RNA Probes; Reading; Relative (related person); Resolution; Role; Sampling; Sequence Analysis; Sister Chromatid Exchange; Site; Source; Stratification; Stress; Stretching; Technology; Telomerase; Telomere Maintenance; Terminal Repeat Sequences; Testing; Variant; age related; anticancer research; base; cancer genome; cancer type; carcinogenesis; cost; design; genome-wide; high throughput analysis; high throughput screening; homologous recombination; insight; neoplastic cell; oxidative damage; prognostic; rapid growth; repaired; telomere; tumor; tumor progression

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.

描述（由申请人提供）：端粒DNA异常是致癌作用的关键和普遍方面。端粒的逐渐复制丧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有关的疾病中发挥作用。零星的端粒缺失事件是一种通用的细胞内部突变机制，可导致功能失调的端粒和染色体不稳定性。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复制应力，氧化损伤以及同源重组事件（例如不等姐妹染色单体交换（SCE）和T环重组）等因素。零星的端粒缺失事件发生在正常细胞中的频率很低。这些事件的频率增加（因此端粒功能失调）可能是致癌事件中的第一个突变事件之一。修复功能失调的端粒会导致端粒 - 凝聚液融合，内部双链DNA断裂部位的端粒 - 染色体臂易位，以及由于反复的融合融合融合周期的额外的DNA重排，导致基因组不稳定性并有助于促进癌症的进展。最终，据信端粒维持机理（基于端粒酶或基于ALT）的激活有助于稳定功能失调的端粒并允许肿瘤细胞的快速生长。不可能准确地衡量散发性端粒删除事件和与当前技术的端粒融合的全球频率。结果，作为癌症基因组地图集的一部分，从肿瘤样品中获取的高吞吐量数据集完全没有端粒突变数据，并且从端粒功能中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偏见的。我们的实验室重点是对人端粒DNA结构和变异的详细分析。在这里，我们建议使用这些知识来开发一种通用的高通量测定法，以检测和定量人类中的端粒DNA突变事件，并为其效用获得原理数据证明。该方法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，将端粒DNA的物理富集和纯化融合在一起。它旨在检测和量化单等位基因分辨率超舒比（ttaggg）n段曲线，端粒融合和亚末端DNA破裂持续的事件。此外，可以使未来的完善允许小型化和多重分析。端粒长度和不稳定性的定量，单层分辨率测量将允许对癌变中的端粒损失和端粒融合的作用前所未有的见解，包括对这些过程的机械见解，包括对这些过程的分子事件，以及对潜在的预后和tumor层层的转化洞察。