High Throughput Method to Assess SNP Functionality in Prostate Cancer

描述（由申请人提供）：实施个性化医疗的一个关键的未满足的需求是能够对人类基因组中存在的数百万个单核苷酸多态性（SNP）进行分类，并查明这些DNA变异中哪些是疾病的病因。SNPs的一个关键的未被研究的功能是它们产生或破坏与癌症有关的转录因子的基因组结合位点的能力。为了实现这一目标，我们发明了SNP-SNAP（蛋白质特异性和亲和力）微阵列作为原型高通量设备，以评估SNP功能。SNP-SNAP阵列将用于显示25万个前列腺癌相关的SNP作为双链DNA分子，并测定转录因子（即，药物靶点）用于它们与这些SNP DNA序列的结合。所得数据将与前列腺癌发病率相关。来自SNP-SNAP阵列的数百万个数据点将使用SNP-Sequence Specificity Landscapes进行分析，创建一个前列腺癌“分子签名”，该签名与转录因子结合，SNP偏好和最近基因的染色体位置有关。我们的研究结果还将前列腺癌相关SNP功能与癌症分期和侵袭性联系起来。了解SNP功能将对个性化医疗产生重大影响，通过提供个性化疾病风险评估，识别新的个性化治疗靶点，预测常用疗法的疗效和潜在脱靶副作用。第一阶段项目的目标是：1。设计并合成定制的SNP-SNAP DNA微阵列，以覆盖与前列腺癌相关的25万个SNPs。2.在SNP-SNAP阵列上检查5种前列腺癌相关转录因子（作为纯化蛋白和来自细胞裂解物）的DNA结合特异性和亲和力，并用SNP引起的转录因子结合差异注释人类基因组。验证前列腺癌细胞染色质免疫沉淀的结果。3.从前列腺癌患者中获得SNP数据，并确定功能性SNP是否与前列腺癌发病率存在统计学显著相关性，这些功能性SNP在SNP-SNAP阵列上产生前列腺癌特异性转录因子的差异结合。该技术可以同时测定数百万个SNP和多种转录因子，因此代表了以高通量方式评估SNP功能的首批方法之一。我们的SNP-SNAP技术，凭借阵列定制设计和检查数百万DNA排列的能力，也广泛适用于任何癌症类型和疾病模型。 公共卫生相关性：实施个性化医疗的一个关键的未满足的需求是能够对人类基因组中存在的数百万个单核苷酸多态性（SNP）进行分类，并确定这些DNA变异中哪些是疾病的病因。SNPs的一个关键的未被研究的功能是它们产生或破坏转录因子的基因组结合位点的能力，这些转录因子调节与癌症有关的基因。为了实现这一目标，我们发明了SNP-SNAP（蛋白质特异性和亲和力）微阵列作为原型高通量装置，通过将25万个前列腺癌相关SNP显示为双链DNA分子，测定转录因子（即药物靶点）与这些SNP DNA序列的结合，并将这些发现与前列腺癌发病率相关联，来评估SNP功能。

DESCRIPTION (provided by applicant): A critical unmet need in implementing personalized medicine is the ability to sort through the millions of single nucleotide polymorphisms (SNPs) present in the human genome and to pinpoint which of these DNA variations are causative in disease. A key under-studied function of SNPs is their ability to generate or disrupt genomic binding sites for transcription factors involved in cancer. Toward this goal, we are inventing the SNP-SNAP (Specificity and Affinity for Proteins) microarray as a prototype high throughput device to evaluate SNP function. The SNP-SNAP arrays will be used to display a quarter-million prostate cancer- related SNPs as double-stranded DNA molecules and to assay transcription factors (i.e., drug targets) for their binding to these SNP DNA sequences. The resulting data will be correlated with prostate cancer incidence. The million-plus data points from the SNP-SNAP arrays will be analyzed using SNP-Sequence Specificity Landscapes, creating a prostate cancer "molecular signature" that relates transcription factor binding, SNP preferences, and chromosomal position of the nearest genes. Our findings will also relate prostate cancer-associated SNP function with cancer stage and aggressiveness. Understanding SNP function will have a major impact on personalized medicine, by providing individualized disease risk assessment, identifying new personalized therapeutic targets, and predicting efficacy and potential off- target side effects of common therapeutics. The goals of this Phase I project are to: 1. Design and synthesize a customized SNP-SNAP DNA microarray to tile across a quarter-million SNPs that are associated with prostate cancer. 2. Examine the DNA binding specificity and affinity of 5 prostate cancer-related transcription factors, as purified proteins and from cell lysates, on the SNP-SNAP array and annotate the human genome with the transcription factor binding differences due to SNPs. Verify results with chromatin immunoprecipitation in prostate cancer cells. 3. Obtain SNP data from patients with prostate cancer and determine if there is a statistically significant association of functional SNPs, which yielded differential binding of prostate cancer specific transcription factors on the SNP-SNAP array, with prostate cancer incidence. This technology can assay millions of SNPs and multiple transcription factors simultaneously, thus representing one of the first methods to evaluate SNP functionality in a high throughput manner. Our SNP- SNAP technology, by virtue of the array custom design and ability to examine millions of DNA permutations, is also broadly applicable to any cancer type and disease model. PUBLIC HEALTH RELEVANCE: A critical unmet need in implementing personalized medicine is the ability to sort through the millions of single nucleotide polymorphisms (SNPs) present in the human genome and to pinpoint which of these DNA variations are causative in disease. A key under-studied function of SNPs is their ability to generate or disrupt genomic binding sites for transcription factors which regulate genes involved in cancer. Toward this goal, we are inventing the SNP-SNAP (Specificity and Affinity for Proteins) microarray as a prototype high throughput device to evaluate SNP function by displaying a quarter-million prostate cancer-related SNPs as double-stranded DNA molecules, assaying transcription factors (i.e. drug targets) for their binding to these SNP DNA sequences, and correlating these findings with prostate cancer incidence.