Defining the causality & biologic impact of genes within ulcerative colitis loci.

描述（申请人提供）：背景：全基因组关联研究（GWAS）已经确定了许多复杂疾病的基因座。这一成功对于炎症性肠病（IBD），克罗恩病（CD）和溃疡性结肠炎（UC）最为显着，其中已发现约100种遗传风险因素，其中一些是表型特异性的，另一些是一种以上疾病常见的。但是，疾病相关基因及其相应的等位基因如何对健康和疾病的生物学产生影响？遗传学数据表明，IBD可以从不同的信号转导途径的扰动中出现，这是由致病基因的失调引起的。假设和具体目标：虽然使用GWAS鉴定IBD遗传风险位点已经取得了巨大的成功，但这种方法主要导致鉴定常见的变异，其中大多数具有适度的遗传效应。我们推测，这些基因座内的罕见变异可能是观察到的效果的原因。为了解决这个问题，我们建议对经验证的IBD基因座进行靶向重新测序，以确定因果（罕见）变异。随着下一代测序技术的出现和获得良好表型的队列，我们现在能够使用这种创新方法，这是对我们以前使用GWAS的工作的补充，并对UC相关基因座进行测序，以识别具有更大遗传效应的罕见变异。这些罕见的，更渗透，等位基因预计有更显着的生物学影响，从而增加了可行性和相关性进行等位基因特异性功能研究，我们在这里提出。肠道炎症是一个复杂的过程，涉及遗传易感性和环境因素的相互作用，并且可能是涉及特定细胞类型的不同生物过程失调的结果。为了剖析这种相互作用， 通过研究遗传学和环境因素并了解UC发病机制的复杂性，我们将用系统的方法来补充我们的基因组学方法，以功能表征UC相关基因与不同的环境压力。鉴于我们团队的不同专业知识，我们将结合联合收割机几种功能方法，包括基因表达分析，shRNA和不同细胞类型中的等位基因特异性拯救实验，以将相关细胞类型和环境背景下的功能途径中的基因及其变体进行研究。我们预计，这些研究将指向UC中受干扰的关键途径的子集，以定义疾病机制簇，并将提供UC中涉及的复杂生物网络的更完整的图片。我们的具体目标是：目的1）为了鉴定由GWAS鉴定的UC相关区域内的风险和保护性等位基因，我们想要（1A）通过对来自患者和对照的样品进行深度重测序来鉴定新的不常见和罕见变体，并且（1B）通过对大群组进行基因分型来验证这些新变体。目的2）确定UC候选基因的功能我们建议采用系统的逐步方法将UC相关基因定位到生物学通路中。我们将：（2A）确定来自UC患者和对照个体的原代细胞和结肠活检中确认的UC基因的RNA和蛋白质表达;（2B）使用基于细胞的功能测定进行靶向过表达和RNA干扰（RNAi）筛选（2C）确定所选基因（及其变体）在细胞特异性背景下的生物学影响。影响：了解遗传变异导致UC的机制虽然具有挑战性，但通过提供对UC病理生理学的遗传和分子基础的新见解以及帮助改善诊断和治疗，在生物医学方面提供了令人兴奋和有前途的进展。与UC相关的致病基因（及其变体）的研究结合我们的高通量和细胞类型特异性功能分析，将提供对遗传因素在UC中作用的机制理解。确定与UC相关的致病性遗传变异及其功能意义可以为药物剂量调整、治疗方式的改变和预防策略提供新的指导。

DESCRIPTION (provided by applicant): Background: Genome-wide association studies (GWAS) have identified numerous loci for complex diseases. This success has been most notable for the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), where ~100 genetic risk factors have been discovered, some that are phenotype-specific and others that are common to more than one disease. But, how do disease-associated genes and their corresponding alleles exert their influence on the biology of health and disease? The genetic data indicates that IBD can emerge from the perturbations of distinct signal transduction pathways, resulting from the dysregulation of disease-causative genes. Hypothesis and specific aims: Although there has been tremendous success for identifying IBD genetic risk loci using GWAS, this approach primarily leads to the identification of common variants, most of which have modest genetic effect. We postulate that rare variants within these loci might be responsible for the effects observed. To tackle this issue, we propose to perform targeted re-sequencing of validated IBD loci to identify the causal (rare) variation. With the advent of next-generation sequencing technologies and access to well-phenotyped cohorts, we are now able to use this innovative approach, which is complementary to our previous work using GWAS, and sequence UC-associated loci to identify rare variants of greater genetic effect. These rarer, more penetrant, alleles are expected to have a more significant biological impact thus increasing the feasibility and relevance of performing allele-specific functional studies as we propose herein. Intestinal inflammation is a complex process involving an interplay genetic predisposition and environmental factors and can be the result of the dysregulation of different biological processes involving specific cell types. In an attempt to dissect this interplay between genetics and environmental factors and understand the complexity of UC pathogenesis, we will complement our genomics approach with a systematic approach to functionally characterize UC-associated genes in conjunction with different environmental stresses. Given the diverse expertise of our teams, we will combine several functional approaches, including gene-expression analysis, shRNA and allele-specific rescue experiments in different cell types to place the genes and their variants under study in functional pathways in the relevant cell type and environmental context. We anticipate that these studies will point to a subset of key pathways that are perturbed in UC to define disease-mechanism clusters and will provide a more complete picture of the complex biological networks involved in UC. Our specific aims are: Aim 1) To identify risk and protective alleles within UC-associated regions identified by GWAS We want to (1A) identify novel uncommon and rare variants by deep re-sequencing in samples from patients and controls and (1B) validate these novel variants by genotyping large cohorts. Aim 2) To determine the function of UC-candidate genes We propose to take a systematic stepwise approach to place UC-associated genes within biological pathways. We will: (2A) Determine the RNA and protein expression of confirmed UC genes in primary cells and colon biopsies from UC patients and control individuals; (2B) Perform targeted over- expression and RNA interference (RNAi) screens using cell-based functional assays (2C) Determine the biological impact of selected genes (and their variants) at a cell-specific context. Impact: Understanding the mechanisms through which genetic variation contributes to UC, although challenging, offers exciting and promising advances in biomedicine, both by providing new insights into the genetic and molecular basis of UC pathophysiology and by helping to improve diagnosis and therapy. The investigation of causal genes (and their variants) linked to UC combined with our high-throughput and cell-type specific functional analysis will provide a mechanistic understanding of the role of genetic factors in UC. The identification of causal genetic variants linked to UC and their functional implications could provide new guidelines for drug dose adjustment, for changes in treatment modalities and for prevention strategies.