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，大多数复杂的性状相关的常见变体映射到非蛋白质编码区。由于对于基因组的大得多的非蛋白质编码部分存在发育不太完善的遗传密码，因此鉴定非孟德尔/复杂性状的基因和致病等位基因存在挑战。鉴于全基因组关联研究（GWAS）发现与复杂性状相关的区域的快速性，基因和致病等位基因鉴定已成为严重的瓶颈。这个建议的总体目标是概述一套连贯的策略，以发现复杂性状的致病基因和等位基因。虽然该提案的重点是前列腺癌，但这些策略是通用的，可以应用于任何非蛋白质编码位点。 核心假设是前列腺癌风险基因座是调节元件。最近的数据令人信服地表明，GWAS基因座富集的调控元件。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水平的变体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基因和致病等位基因鉴定提供了坚实的基础。 第一个目标是发现来自500名男性的前列腺组织中所有已知前列腺癌风险等位基因的eQTL/转录本对。高定量Nanostring平台将用于测量转录水平。目标2将采用功能性测定来确保目标1中发现的基因与前列腺癌生物学相关。功能测定将使用核酸酶技术进行，这是一种上调和下调基因的新策略。转录激活因子样效应物核酸酶（TALEN）技术具有以定向方式和直接在选择的基因组位置中精确地产生DNA序列修饰的能力。这项技术与传统方法的根本不同之处在于，它在研究人员的目标位置产生了稳定和可遗传的变化。 目标3将集中于证明eQTL/转录本关联的基因座的因果等位基因鉴定。使用遗传和表观遗传方法的综合策略将用于鉴定一组候选的致病等位基因。这些候选人将接受功能测试， TALEN在适当的细胞系中设计特定的遗传修饰。预期致病等位基因位点的修饰会影响转录。 在这个项目完成后，我们完全预期我们将开始解开引发人类前列腺癌的基因/途径。发现前列腺癌的潜在机制不仅可以为这种疾病的生物学提供信息，还可以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.