Discovery of aberrant enhancer activities during gut development that underlie genetic predisposition to pediatric Crohn's disease

项目概述：克罗恩病（CD）是一种特殊类型的炎症性肠病， 所有年龄段的患者，小肠远端区域（称为回肠）是最常见的 受影响的地点。它在美国折磨着近一百万人，并且在美国急剧增加。 在全球范围内流行。值得注意的是，相当一部分CD患者（约25%）是儿童或青少年， 发病，称为儿科CD，通常表现出高度侵袭性的症状和更复杂的临床 与晚发性成人CD相比。对人类群体的大规模遗传研究显示， 基因组中与CD相关的风险基因座，其中绝大多数发生在非编码区。 虽然人们普遍认为这些可能会影响监管因素，但这尚未得到明确的证实。 对大多数位点进行了验证。此外，风险位点可能影响肠道发育的程度尚不清楚 和探索不足，但值得调查的重要性，发展畸变的其他 肠道的炎症性疾病。值得注意的是，婴儿难治性先天性腹泻最近与 在出生前肠道发育过程中具有瞬时活性的调节元件的缺失。本研究旨在 （1）定义CD风险基因座，其包含在产前发育中短暂活跃的调节元件， (2)为特定CD风险等位基因影响早期肠道疾病中调节元件活性提供功能证据 发展，最终目标是提供一个新的角度对儿科CD的病因。上 目的研究CD相关单核苷酸多态性（SNP）在早期肠道发育中的作用， 我们将利用最新的人类多能干细胞定向分化技术， 将hPSC（hPSC）转化为人肠球状体，随后成熟为类器官（HIO）。该模型展示了 分子特征非常类似于原始人类胎儿肠道。定义活跃的监管元素 在全基因组范围内，我们将应用一种称为染色质连续测序（ChRO-seq）的尖端技术， hPSC-HIO模型以及儿科和成人CD和非CD回肠活组织检查。然后我们将定义这些 与CD风险基因座重叠且仅在hPSC-HIO模型的产前阶段有活性的调控元件 但不存在于出生后的儿童/成人组织中。在第二个目标中，我们将定义特定CD-SNP的作用， 在肠道发育早期，PRDM1基因座内含有调控元件。在小鼠中，Prdm 1调节 肠道成熟，包括潘氏细胞的发育，其在CD病理生物学中起重要作用。我们 已经鉴定了人PRDM1基因内的调控元件，其含有CD相关SNP， 仅在产前阶段有活性，而在成人CD或非CD回肠中无活性。我们将使用CRISPR/Cas9单核苷酸 碱基编辑技术，以检查该CD-SNP的不同等位基因在28天成熟期间的影响 回肠HIO的过程。该提案的成功完成将通过提供翻译来促进儿童健康 了解人类肠道发育和儿科CD病理学的基因调控机制。

PROJECT SUMMARY: Crohn’s disease (CD) is a specific type of inflammatory bowel disease that occurs in patients of all ages, with the distal region of the small intestine (referred to as ileum) as one of the most commonly affected locations. It afflicts nearly a million people in the United States and is dramatically increasing in prevalence worldwide. Notably, a substantial proportion of CD patients (~25%) are childhood- or adolescent- onset, referred to as pediatric CD, which often display highly aggressive symptoms and more complicated clinical course compared to late-onset, adult CD. Large-scale genetic studies of human populations have revealed >200 risk loci in the genome that are associated with CD, the vast majority of which occur in non-coding regions. Although it is widely conjectured that these likely impact regulatory elements, this has not yet been definitively demonstrated for most loci. Moreover, the extent to which the risk loci may affect gut development is unknown and poorly explored, but merits investigation given the importance of developmental aberrations to other inflammatory disorders of the gut. Notably, intractable congenital diarrhea in infants was very recently linked to the deletion of a regulatory element that is transiently active during prenatal gut development. This study aims to (1) define CD-risk loci harboring regulatory elements that are transiently active in pre-natal development and (2) provide functional evidence for a specific CD risk allele affecting regulatory element activity during early gut development, with the ultimate goal of offering a novel perspective on the etiology of pediatric CD. In the first Aim, to study the contribution of CD-associated single nucleotide polymorphisms (SNPs) to early gut development, we will leverage the recent technology of directed differentiation from human pluripotent stem cells (hPSCs) into human intestinal spheroids followed by maturation into organoids (HIOs). This model exhibits molecular features closely resembling the primary human fetal gut. To define active regulatory elements genome-wide, we will apply a cutting-edge technology called chromatin run-on sequencing (ChRO-seq) to the hPSC-HIO model as well as to pediatric and adult CD and non-CD ileal biopsies. We will then define those regulatory elements that overlap CD risk loci and are active only in the pre-natal stages of the hPSC-HIO model but not in the post-natal pediatric/adult tissue. In the second Aim, we will define the role of a specific CD-SNP containing regulatory element within the PRDM1 locus during early gut development. In mouse, Prdm1 regulates gut maturation, including the development of Paneth cells, which play important roles in CD pathobiology. We have identified a regulatory element within the human PRDM1 gene that contains a CD-associated SNP and is active only in pre-natal stages and not in adult CD or non-CD ileum. We will use CRISPR/Cas9 single-nucleotide base editing technology to examine the impact of different alleles of this CD-SNP during the 28-day maturation process of ileal HIOs. Successful completion of the proposal will promote child health by providing translational knowledge about the gene regulatory mechanisms of human gut development and pediatric CD pathobiology.