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

描述(申请人提供)：差异等位基因特异性表达(DASE)已经很好地描述了基因组印记和随机单等位基因表达(RME)的表观遗传现象。我们和其他研究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。我们以前的研究结果表明，BRCA1中的Dase表达与发生乳腺癌的风险增加有关。由于多种遗传和表观遗传因素可导致DASE，因此DASE是顺式作用的调节变异体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可以作为一类新的临床生物标记物用于癌症风险评估、早期发现和治疗。目前用于DASE检测的金标准平台--等位基因特异性定量聚合酶链式反应(AS-qPCR)存在几个主要的局限性，使得无法将所观察到的DASE信号定位于单个细胞并应用于常规临床实践。虽然原位杂交(ISH)经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)是一种新的RNA-ISH技术，最近开发了一种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，同时保持组织形态。另一方面，“脚趾”探针策略的最新发展有助于绕过传统单一杂交带来的非特异性杂交陷阱。 等位基因特异的探针设计。通过无缝结合这两种新兴技术，我们建议开发一种新的非放射性同位素AS-RNA-ISH分析，具有高灵敏度和特异性，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上评估“极端”和“中等”数据库。为了实现这一目标，我们将首先建立一种新的AS-RNA-ISH方法来检测BRCA1或BRCA2中截断突变相关的Dase。接下来，我们将使用乳腺癌患者的FFPE样本，用AS-qPCR验证这一新的AS-RNA-ISH方法。我们的研究结果将有助于建立一种新的AS-RNA-ISH方法，该方法将提供高灵敏度和高特异性分析存档临床标本中的DASE的能力，使这一新方法成为将DASE生物标记物转化为临床应用的一个有前景的平台。

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.