A Novel Allele-Specific RNA-ISH for Differential Allele-specific Expression

用于差异等位基因特异性表达的新型等位基因特异性 RNA-ISH

• 批准号: 8739018
• 负责人: XIAOWEI CHEN
• 金额: $ 23.29万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8739018
• 关键词: Alleles; Archives; BRCA1 gene; BRCA2 gene; Binding; Biological Assay; Biological Markers; Breast; Cancer Patient; Cells; Clinical; Cultured Cells; DNA; Development; Duct (organ) structure; Early Diagnosis; Emerging Technologies; Epigenetic Process; Event; Family; Formalin; Genes; Genetic; Genomic Imprinting; Goals; Gold; Hereditary Breast Carcinoma; Histopathology; Human; Imagery; In Situ; In Situ Hybridization; Individual; Laboratory Research; Lead; Malignant Neoplasms; Mammary gland; Maps; Measurement; Mediating; Methods; Molecular; Molecular Analysis; Morphology; Mutation; Paraffin Embedding; Pathogenesis; Patients; Pattern; Process; RNA; Resources; Risk; Risk Assessment; Sampling; Sensitivity and Specificity; Signal Transduction; Specificity; Specimen; Technology; Tissue Microarray; Tissues; Transcript; Translating; Variant; cancer initiation; cancer risk; cancer therapy; clinical practice; cohort; design; imprint; indexing; innovation; mRNA Decay; malignant breast neoplasm; novel; novel strategies; programs; public health relevance; single molecule; tumor progression

DESCRIPTION (provided by applicant): Differential allele-specific expression (DASE) has been well described with the epigenetic phenomena of genomic imprinting and random monoallelic expression (RME). Recent studies from us and other groups have demonstrated that DASE is also relatively common among non-imprinted autosomal genes. Results from our previous studies have shown that DASE in BRCA1 expression was associated with an increased risk of developing breast cancer. As multiple genetic and epigenetic factors can contribute to DASE, DASE is a functional index for cis-acting regulatory variants and pathogenic mutations. These findings clearly support that DASE in cancer-associated genes could serve as a novel class of clinical biomarkers for cancer risk assessment, early detection, and treatment. The current gold standard platform for DASE measurement, allele-specific quantitative PCR (AS-qPCR), has several major limitations to making it impossible to map the observed DASE signals to individual cells and to apply for routine clinical practice. Although in situ hybridizaton (ISH) is routinely used in laboratory research and clinical settings, AS-RNA-ISH technology is considerably underdeveloped. RNAscope(R) is a novel RNA-ISH technology, which was recently developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. On the other hand, the recent development of "toehold" probe strategy helps to circumvent the pitfall of nonspecific hybridization created by traditional single allele-specific probe design. By seamlessly combining these two emerging technologies, we propose to develop a novel non-radioisotopic AS-RNA-ISH assay with high sensitivity and specificity, which is capable of evaluating both "extreme" and "moderate" DASE at individual cell levels using archived Formalin- Fixed Paraffin-Embedded (FFPE) tissues. To achieve this goal, we will first establish a new AS-RNA-ISH assay to detect truncating mutation-associated DASE in BRCA1 or BRCA2. Next we will validate this novel AS-RNA-ISH method with AS-qPCR using FFPE specimens from breast cancer patients. Results from our proposed study will help to establish a new AS-RNA-ISH method which will provide the capability of analyzing DASE in archived clinical specimens with high sensitivity and specificity, making this new approach a promising platform for translating DASE biomarkers into clinical use.

描述（由申请人提供）：差异等位基因特异性表达（DASE）已经通过基因组印记和随机单等位基因表达（RME）的表观遗传现象得到了很好的描述。我们和其他小组最近的研究表明，DASE 在非印记常染色体基因中也相对常见。我们之前的研究结果表明，BRCA1 表达中的 DASE 与患乳腺癌的风险增加相关。由于多种遗传和表观遗传因素可促成 DASE，因此 DASE 是顺式作用调控变异和致病突变的功能指标。这些发现清楚地支持癌症相关基因中的 DASE 可以作为一类新型临床生物标志物，用于癌症风险评估、早期检测和治疗。当前 DASE 测量的黄金标准平台，等位基因特异性定量 PCR (AS-qPCR)，有几个主要限制，无法将观察到的 DASE 信号映射到单个细胞并应用于常规临床实践。尽管原位杂交 (ISH) 常用于实验室研究和临床环境，但 AS-RNA-ISH 技术还相当不发达。 RNAscope(R) 是一种新颖的RNA-ISH技术，最近开发出独特的探针设计策略，允许同时进行信号放大和背景抑制，以实现单分子可视化，同时保留组织形态。另一方面，最近发展的“立足点”探针策略有助于规避传统单一杂交所造成的非特异性杂交的陷阱。 等位基因特异性探针设计。通过无缝结合这两种新兴技术，我们建议开发一种具有高灵敏度和特异性的新型非放射性同位素 AS-RNA-ISH 测定，能够使用存档的福尔马林固定石蜡包埋 (FFPE) 组织在单个细胞水平评估“极端”和“中等”DASE。为了实现这一目标，我们将首先建立一种新的 AS-RNA-ISH 检测方法来检测 BRCA1 或 BRCA2 中截短突变相关的 DASE。接下来，我们将使用乳腺癌患者的 FFPE 标本通过 AS-qPCR 验证这种新型 AS-RNA-ISH 方法。我们提出的研究结果将有助于建立一种新的 AS-RNA-ISH 方法，该方法将提供以高灵敏度和特异性分析存档临床标本中的 DASE 的能力，使这种新方法成为将 DASE 生物标志物转化为临床应用的有前景的平台。