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

摘要 基因组在极早发型炎症性肠病(VEO-IBD)发病中的作用 在6岁时被诊断出来，目前仍未得到研究，但阐明遗传风险因素无疑将是 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 表现型，通常对常规治疗无效，起病早，有很强的家族史，表明有丰富的 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，这些变异改变了人们的治疗 为了这些孩子。整个外显子组测序已经从根本上改变了我们对VEO-IBD的方法，然而，它 尽管有证据表明存在潜在的遗传缺陷，但只有18%的病例成功。在……里面 此外，将已识别的变异与VEO-IBD表型的发展联系起来仍然是困难的。 这项建议的目标是通过全基因组测序(WGS)和 利用转录组修改来增强我们识别因果变异的能力。我们的中央 假设一部分VEO-IBD是单基因疾病，其中一部分会改变基因转录， 转录组分析，包括单细胞分析，将使我们能够更快、更准确地识别 在检测到的候选变异体中存在这种突变。使用一种基于WGS的创新方法，我们 将扩大我们的VEO-IBD原因缺陷曲目。我们将把这些发现与RNA-seq和 ScRNA-seq数据，这可以产生一种更敏感和更具体的方法来检测因果变异和 描述它们的作用机制。此外，当WGS不能确定一个明确的因果变量时，RNA-seq 而scRNA-seq可以提供对潜在疾病机制的洞察，支持 候选突变。 为了测试我们的中心假设，在特定的目标1中，我们将扩展我们的VEO-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。在具体目标2中，我们将测试 结肠组织和外周血单核细胞的转录图谱增强新病因鉴定的能力 VEO-IBD的变种。最后，在特定的目标3中，使用scrna测序，我们将评估结肠和免疫 细胞异质性及其与婴儿期IBD相关基因和途径的转录研究 结肠组织和外周血单核细胞的单个细胞图谱。 该项目的完成将导致确定VEO-IBD的新遗传原因，为 追求源头生物过程，以更好地了解疾病的基本机制，具有终极意义 将这些知识转化为改善对VEO-IBD儿童的护理的目标是通过个性化和 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.