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

描述（由申请人提供）：差异等位基因特异性表达（DASE）已经被很好地描述为基因组印迹和随机单等位基因表达（RME）的表观遗传现象。我们和其他小组最近的研究表明，DASE在非印迹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有关。由于多种遗传和表观遗传因素可参与DASE，因此DASE是顺式作用调控变异体和致病性突变的功能指标。这些发现清楚地支持癌症相关基因中的DASE可以作为癌症风险评估、早期检测和治疗的一类新的临床生物标志物。目前测量DASE的金标准平台，等位基因特异性定量PCR (AS-qPCR)，有几个主要的局限性，使得不可能将观察到的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）通常用于实验室研究和临床环境，但AS-RNA-ISH技术相当不发达。RNAscope(R)是一种新颖的RNA-ISH技术，最近开发了一种独特的探针设计策略，可以同时放大信号和抑制背景，在保持组织形态的同时实现单分子可视化。另一方面，近年来发展起来的“立足点”探针策略有助于规避传统单分子杂交产生的非特异性缺陷

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.