Utilizing genetic and functional strategies to identify causal genes and alleles

描述(由申请人提供)： 与孟德尔疾病形成鲜明对比的是，大多数复杂的与特征相关的常见变异映射到非蛋白质编码区。由于基因组中较大的非蛋白质编码部分的遗传密码不太发达，识别该基因(S)和潜在的非孟德尔/复杂性状的因果等位基因是一个挑战。鉴于全基因组关联研究(GWAS)发现与复杂性状相关的区域的速度之快，基因和因果等位基因识别已成为严重的瓶颈。这项提议的总体目标是勾勒出一套连贯的战略，以发现复杂特征背后的因果基因和等位基因。虽然该提案关注的是前列腺癌，但这些策略是通用的，可以应用于任何非蛋白质编码基因。核心假设是前列腺癌风险基因是调控因素。最近的数据令人信服地证明，Gwas基因座富含调控元件。调控元件可以控制基因的表达水平。可以研究个体携带的等位基因数量(0、1或2)与转录水平之间的相关性。控制RN水平的变异通常被称为表达数量性状基因座(EQTL)。EQTL-靶基因关系的存在为进行基因和因果等位基因鉴定提供了坚实的基础。第一个目标是在500名男性的前列腺组织中发现所有已知前列腺癌风险等位基因的eQTL/转录对。高度定量的纳米串平台将被用来测量转录水平。AIM 2将使用功能分析来确保在AIM 1中发现的基因与前列腺癌生物学相关。功能分析将使用核酸酶技术，这是一种上调和下调基因的新策略。转录激活子样效应子核酸酶(TALEN)技术能够直接在基因组选择的位置以定向的方式精确地产生DNA序列修饰。这项技术与更传统的方法有根本的不同，因为它在调查人员所针对的位置产生了稳定的、可遗传的变化。 目标3将侧重于对eQTL/转录本关联的基因座进行因果等位基因鉴定。使用遗传和表观遗传学方法的综合策略将被用来识别候选因果等位基因集。然后将使用以下工具对这些候选人进行功能测试 TALEN在适当的细胞系中设计特定的基因修改。因果等位基因位点的改变预计会影响转录。在这个项目完成时，我们完全预计我们将开始解开引发人类前列腺癌的基因/途径。前列腺癌潜在机制的发现不仅将为这种疾病的生物学研究提供信息，还可能揭示更合理地干预治疗和预防的机会。

DESCRIPTION (provided by applicant): In stark contrast to Mendelian disorders, the majority of complex trait-associated common variants map to non-protein coding regions. Since there is a less well-developed genetic code for the much larger non- protein coding portion of the genome, identifying the gene(s) and causal alleles underlying non- Mendelian/complex traits presents a challenge. Given the rapidity with which genome wide association studies (GWAS) are discovering regions associated with complex traits, gene and causal allele identification have become severe bottlenecks. The overall goal of this proposal is to outline a coherent set of strategies to discover causal genes and alleles underlying complex traits. While the proposal focuses on prostate cancer, the strategies are generic and can be applied to any non-protein coding locus. The central hypothesis is that prostate cancer risk loci are regulatory elements. Recent data convincingly demonstrate that GWAS loci are enriched for regulatory elements. Regulatory elements can control the level of expression of genes. The correlation between the number of alleles an individual carries (0, 1, or 2) and transcript levels can be investigated. Variants that control RN levels are often referred to as expression quantitative trait loci (eQTLs). The existence of an eQTL-target gene relationship provides a strong foundation upon which to pursue gene and causal allele identification. The first aim will discover eQTL/transcript pairs for all known prostate cancer risk alleles in prostate tissue from 500 men. The highly quantitative Nanostring platform will be used to measure transcript levels. Aim 2 will employ functional assays to ensure that the genes discovered in Aim 1 are relevant to prostate cancer biology. The functional assays will be performed using nuclease technology, a novel strategy to upregulate and downregulate genes. Transcription activator-like effector nuclease (TALEN) technology has the ability to create DNA sequence modifications in a directed manner and with exquisite precision directly in a genomic location of choice. This technology radically differs from more traditional methods in that it creates stable and heritable changes in a location targeted by the investigator. Aim 3 will focus on causal allele identification for loci demonstrating an eQTL/transcript association. An integrative strategy using genetic and epigenetic approaches will be used to identify a candidate set of causal alleles. These candidates will then be functionally tested using TALENs to engineer specific genetic modifications in appropriate cell lines. Modifications at the causal allele site will be expected to influence transcription. At the completion of this project,we fully anticipate that we will have begun to unravel the genes/pathways that initiate human prostate cancer. Discovering the mechanisms underlying prostate cancer will not only inform the biology of this disease, but may also reveal opportunities to more rationally intervene in treatment and prevention.