Integration of genomics and transcriptomics to investigate biological pathways in very early onset IBD

摘要 基因组对极早发性炎症性肠病（VEO-IBD）发展的贡献 在<6岁时被诊断出来，仍然没有得到充分的研究，但遗传风险因素的阐明无疑将 提高我们对发病机制的理解，并提出新的治疗方法。咄咄逼人的 表型，通常对常规治疗难治，早期发病，和强家族史点富集 单基因缺陷事实上，我们和其他人已经确定了VEO-IBD的因果变异， 为了这些孩子全外显子组测序从根本上改变了我们对VEO-IBD的方法，然而，它 仅在~18%的病例中成功，尽管有证据高度提示潜在的遗传缺陷。在 此外，将鉴定的变异体与VEO-IBD表型的发展联系起来仍然很困难。 该提案的目标是通过全基因组测序（WGS）扩大我们的遗传分析， 利用转录组修饰来增强我们识别致病变异的能力。我们的中央 假设一部分VEO-IBD是单基因疾病，其子集将改变基因转录， 和转录组分析，包括单细胞分析，将使我们能够更快速，更准确地识别 在检测到的候选变体中的这种突变。使用一种新的创新方法，基于WGS，我们 将扩大我们在VEO-IBD中的因果缺陷库。我们将这些发现与RNA-seq整合， scRNA-seq数据，它可以产生一种更敏感和特异的方法来检测致病变异， 描述其作用机制。此外，当WGS无法确定明确的致病变异时，RNA-seq scRNA-seq可以提供对潜在疾病机制的深入了解，支持 候选突变 为了检验我们的中心假设，在具体目标1中，我们将扩展VEO中潜在因果突变的数据集- IBD通过WGS。我们将在其他VEO-IBD病例中验证候选基因。在第二阶段，我们将测试 结肠组织和PBMC的转录谱分析能够增强新的致病基因的鉴定， VEO-IBD的变体。最后，在具体目标3中，使用scRNA测序，我们将评估结肠和免疫 细胞异质性和表征基因和途径与婴儿发病IBD使用转录 分析来自结肠组织和PBMC的单个细胞。 该项目的完成将导致VEO-IBD的新遗传原因的鉴定， 生物过程的来源，以更好地了解疾病的基本机制，最终 我们的目标是通过个性化和个性化的方式， 靶向治疗

ABSTRACT The genomic contribution to the development of very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at <6 years of age, remains understudied, yet elucidation of genetic risk factors would undoubtedly enhance our understanding of pathogenesis and suggest novel therapeutic approaches. The aggressive phenotype, often refractory to conventional therapy, early onset, and strong family history points to an enrichment of monogenic defects. Indeed, we and others have identified causal variants in VEO-IBD that has changed care for these children. Whole exome sequencing has radically changed our approach to VEO-IBD, however, it has only been successful in ~18% of cases, despite evidence highly suggestive of an underlying genetic defect. In addition, linking the identified variant to the development of the VEO-IBD phenotype remains difficult. The goal of this proposal is to widen our genetic analysis through whole genome sequencing (WGS) and leverage transcriptome modifications to enhance our capacity in identifying causal variants. Our central hypothesis is that a proportion of VEO-IBD is a monogenic disease, a subset of which will alter gene transcription, and transcriptome analysis, including single cell analysis, will allow us to more rapidly and accurately identify such mutations among the detected candidate variants. Using a novel innovative method, based on WGS, we will expand our repertoire of causal defects in VEO-IBD. We will integrate these findings with RNA-seq and scRNA-seq data, which can generate a more sensitive and specific approach to detect causal variants and characterize their mechanism of action. In addition, when WGS cannot identify a clear causal variant, RNA-seq and scRNA-seq can provide insight into the underlying disease mechanism, supporting the implication of candidate mutations. To test our central hypothesis, in Specific Aim 1 we will expand our dataset of potential causal mutations in VEO- IBD through WGS. We will validate candidate genes in additional VEO-IBD cases. In Specific Aim 2, we will test the ability of transcriptional profiling of colonic tissue and PBMCs to enhance the identification of novel causal variants of VEO-IBD. Finally, in Specific Aim 3, using scRNA sequencing, we will assess the colonic and immune cell heterogeneity and characterize genes and pathways associated with infantile onset IBD using transcriptional profiling of individual cells from colonic tissue and PBMCs. Completion of this project will result in the identification of novel genetic causes of VEO-IBD, providing a fertile source of biologic processes to pursue to better understand the basic mechanisms of disease, with the ultimate goal of translating this knowledge into improved care for children with VEO-IBD through individualized and targeted therapy.