4C and Genome Editing for Causal SNP and Gene Discovery at Cancer Risk Loci

 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个连贯的战略，以发现复杂性状背后的因果基因和等位基因。虽然该提案关注的是癌症，但这些策略是通用的，可以应用于任何非蛋白质编码基因。中心假设是癌症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。因果基因和变异很难发现，因为科学界不太擅长对基因组的非蛋白质编码部分进行注释。这一建议寻求利用两个强大的工具，循环染色体构象捕获(4C)和基因组编辑来确定原因基因和等位基因。第一个目标是在已经识别出目标基因的危险基因座上发现因果等位基因。利用靶基因启动子作为4C的诱饵，我们将确定与启动子物理相互作用的基因组区域。这些相互作用的区域中的每一个都是候选的调控元素。同时，将对候选因果变异进行成簇的规则间隔短回文重复(CRISPR)评估。来自这些化验的信息将与遗传和表观遗传学数据相结合，以确定因果变异。因为我们知道目标基因，所以在因果等位基因位置上的修改将有望影响该基因的转录，并将为因果变异鉴定提供确凿的证据。AIM将专注于识别尚未识别目标基因的风险基因座上的因果基因和变异。研究中的基因座是乳腺癌、前列腺癌和卵巢癌的风险基因座。我们特别假设，这一区域对所有三种癌症都是通过一个共同的机制起作用的。同样，我们将使用4C和CRISPR技术来定义因果变体和基因。在这个项目完成时，我们完全预计我们将开始解开 启动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，还可能揭示机会 以更合理地介入治疗和预防。

 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 strategy to discover causal genes and alleles underlying complex traits. While the proposal focuses on cancer, the strategies are generic and can be applied to any non-protein coding locus. The central hypothesis is that 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. Causal genes and variants are difficult to discover because the scientific community is less adept at annotating the non-protein coding portion of the genome. This proposal seeks to utilize two powerful tools, circular chromosome conformation capture (4C) and genome editing to identify causal genes and alleles. The first aim will discover the causal alleles at risk loci where a target gene has already been identified. Using the target gene promoter as a bait for 4C, we will identify the genomic regions that are physically interacting with the promoter. Each of these interacting regions are candidate regulatory elements. In parallel, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) evaluation of the candidate causal variants will be performed. Information from these assays will be integrated with genetic and epigenetic data to define the causal variant. Since we know the target gene, modifications at the causal allele site will be expected to influence transcription of this gene and will provide definitive proof of causal variant identification. Aim will focus on identifying the causal gene and variant at a risk locus where a target gene has not been identified. The locus under study is a risk locus for breast, prostate, and ovarian cancers. We specifically hypothesize that this region is acting through a common mechanism for all three cancers. Again, we will employ 4C and CRISPR technologies to define the causal variant and gene. 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.