Genetic susceptibility to Barrett's esophagus: From GWAS to biology

巴雷特食管（BE）是食管腺癌（EAC）的唯一已知的前体，其是一种高度致命的肿瘤。 癌症发病率上升，中位生存期<1年。大量的卫生保健资源用于BE 筛查、监测和治疗。胃食管反流引起的食管下段损伤和 慢性炎症是BE发展的关键驱动因素，但潜在风险的分子途径并不清楚， 定义了最近由我们团队成员领导的全基因组关联研究（GWAS）确定了>20种新的 BE/EAC的遗传易感性位点，为遗传风险的遗传组成部分提供了新的见解。 然而，在将协会与生物学联系起来方面进展甚微。与其他GWAS一致 复杂的疾病，所有BE指数变异体都定位于非编码区，缺乏明显的生物学功能， 与许多其他SNP的连锁不平衡，其中任何一个都可能是因果关系。绝大多数功能 GWAS信号的基础变体被认为映射到并改变调节元件的活性，包括 增强子，在等位基因特异性的方式，并反过来调节下游基因的表达参与风险。 重要的是，这种调节作用可能是组织和条件特异性的。要开始优先考虑候选人，请执行以下操作 功能变体的实验审讯，我们开发了一个定制的信息学评分管道 使用来自多个公共资源的全面的计算机注释。我们选择了四个得分高的BE 在食管细胞系中使用荧光素酶报告基因测定进行评估的风险位点，发现四分之二 区域表现出等位基因特异性增强子活性。CRISPR介导的增强子区域在两个位点的缺失 与下调几个候选风险基因相关的位点。在这些成功的激励下，我们力求 扩展我们的综合框架，阐明BE相关的遗传变异的功能后果。我们 假设这种变异通过转录调节改变而生物学表达， 下游基因表达。我们的目标是确定功能变异，风险增强子和靶基因 潜在的BE风险，利用独特的资源和互补的统计/实验方法。在 目标1，我们将通过贝叶斯精细映射定义候选因果变量，使用最大的BE GWAS世界- 广泛，并通过功能潜力分数进一步优先考虑领先的候选人。在目标2中，我们将执行新的 反流暴露的胃食管连接部组织和组成细胞的转录组谱分析，并鉴定 通过eQTL共定位和基于网络的分析，候选BE风险基因和途径。在目标3中，我们 使用荧光素酶报告增强子活性测定验证候选功能变体; 通过CRISPR介导的增强子缺失和RNA-Seq的风险增强子;和询问途径影响 通过CRISPR介导的基因敲减/过表达和RNA-聚合酶链反应（RNA-聚合酶链反应）， Seq.这项研究将推动非编码GWAS信号成为功能性生物特征，并支持未来的研究。 努力开发BE/EAC的新型预防/干预策略。

Barrett’s esophagus (BE) is the only known precursor for esophageal adenocarcinoma (EAC), a highly lethal cancer with rising incidence and median survival <1 year. Substantial health-care resources are devoted to BE screening, surveillance, and treatment. Gastroesophageal reflux-induced injury of the lower esophagus and chronic inflammation are key drivers of BE development, but molecular pathways underlying risk are not well defined. Recent genome-wide association studies (GWAS) led by members of our team identified >20 novel genetic susceptibility loci for BE/EAC, providing new insights into the inherited genetic component of risk. Nevertheless, little progress has been made in bridging associations to biology. Consistent with GWAS of other complex diseases, all BE index variants map to non-coding regions, lack obvious biologic function, and are in linkage disequlibrium with many other SNPs, any of which may be causal. The vast majority of functional variants underlying GWAS signals are believed to map to and alter activity of regulatory elements including enhancers, in an allele-specific manner, and in turn modulate expression of downstream genes involved in risk. Importantly, such regulatory effects may be tissue- and condition-specific. To begin prioritizing candidate functional variants for experimental interrogation, we developed a customized informatics scoring pipeline using comprehensive in-silico annotations from multiple public resources. We selected four high-scoring BE risk loci for evaluation using luciferase reporter assays in esophageal cell lines, and found that two of four regions exhibited allele-specific enhancer activity. CRISPR-mediated deletion of the enhancer region at both loci correlated with downregulation of several candidate risk genes. Motivated by these successes, we seek to expand our integrative framework for elucidating functional consequences of BE-related genetic variation. We hypothesize that such variation is biologically expressed through alterations in transcriptional regulation and downstream gene expression. Our goal is to identify functional variants, risk enhancers, and target genes underlying BE risk, leveraging unique resources and complementary statistical/experimental approaches. In Aim 1, we will define candidate causal variants via Bayesian fine-mapping, using the largest BE GWAS world- wide, and further prioritize leading candidates via functional-potential scores. In Aim 2, we will perform new transcriptome profiling of reflux-exposed gastroesophageal junction tissues and constituent cells, and identify candidate BE risk genes and pathways via eQTL colocalization and network-based analysis. In Aim 3, we will validate candidate functional variants using luciferase reporter enhancer activity assays; identify target genes of risk enhancers via CRISPR-mediated enhancer deletion and RNA-Seq; and interrogate pathways influenced by prioritized target genes in Aims 2 & 3 via CRISPR-mediated gene knockdown/overexpression and RNA- Seq. This study will advance noncoding GWAS signals into functional biological signatures and support future efforts to develop novel preventive/interventional strategies for BE/EAC.