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

肠道发育过程中异常增强子活性的发现是儿童克罗恩病遗传易感性的基础

• 批准号: 10494257
• 负责人: Praveen Sethupathy
• 金额: $ 23.55万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10494257
• 关键词: Adolescent; Adult; Affect; Age; Alleles; Applications Grants; Binding Sites; Bioinformatics; Biopsy; CRISPR/Cas technology; Cell Lineage; Child Health; Childhood; Chromatin; Clinical; Crohn' s disease; Data; Defect; Development; Diarrhea; Disease; Distal; Enhancers; Ensure; Epithelial; Etiology; Exhibits; Fetal Tissues; Gastrointestinal tract structure; Genes; Genetic Predisposition to Disease; Genetic study; Genome; Genomics; Goals; Human; Ileal Diseases; Individual; Infant; Inflammatory; Inflammatory Bowel Diseases; Interdisciplinary Study; Intestines; Investigation; Knowledge; Link; Linkage Disequilibrium; Location; Modeling; Molecular; Mus; Necrotizing Enterocolitis; Nucleic Acid Regulatory Sequences; Nucleotides; Organoids; Paneth Cells; Patients; Pediatric Crohn' s disease; Persons; Play; Population; Predisposition; Prevalence; Process; Protein Isoforms; Regulator Genes; Regulatory Element; Research; Resources; Rest; Role; Running; SET Domain; Single Nucleotide Polymorphism; Small Intestines; Stains; Symptoms; Technology; Testing; Tissues; United States; United States National Institutes of Health; Untranslated RNA; Variant; base; base editing; directed differentiation; disorder risk; early onset; fetal; genome wide association study; genome-wide; genomic locus; human data; human pluripotent stem cell; human population study; ileum; novel; novel strategies; premature; prenatal; promoter; risk variant; transcription factor; transcriptome sequencing

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.

项目摘要：克罗恩病 (CD) 是一种特殊类型的炎症性肠病，发生于 所有年龄段的患者，其中小肠远端区域（称为回肠）是最常见的区域之一 受影响的地点。它在美国困扰着近一百万人，并且发病率正在急剧增加 全世界流行。值得注意的是，很大一部分 CD 患者 (~25%) 是儿童或青少年患者 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 病程与晚发成人 CD 相比。对人类群体的大规模遗传学研究表明>200 基因组中与 CD 相关的风险位点，其中绝大多数发生在非编码区。 尽管人们普遍猜测这些可能会影响监管因素，但这尚未得到明确的证实 大多数基因座都得到了证明。此外，风险位点可能在多大程度上影响肠道发育尚不清楚 且探索不足，但考虑到发育畸变对其他方面的重要性，值得研究 肠道炎症性疾病。值得注意的是，婴儿顽固性先天性腹泻最近与 删除在产前肠道发育过程中短暂活跃的调节元件。本研究目的 (1) 定义包含在产前发育中短暂活跃的调节元件的 CD 风险基因座，以及 (2) 为影响早期肠道调节元件活性的特定 CD 风险等位基因提供功能证据 开发，最终目标是为儿科克罗恩病的病因学提供一个新的视角。在第一个 目的，研究 CD 相关单核苷酸多态性 (SNP) 对早期肠道的贡献 开发中，我们将利用最新的人类多能干细胞定向分化技术 （hPSC）转化为人肠球体，然后成熟为类器官（HIO）。该模型展示了 分子特征与人类胎儿的原始肠道非常相似。定义积极的监管要素 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 模型以及儿童和成人 CD 和非 CD 回肠活检。然后我们将定义那些 与 CD 风险位点重叠且仅在 hPSC-HIO 模型的产前阶段活跃的调控元件 但不在产后儿科/成人组织中。在第二个目标中，我们将定义特定 CD-SNP 的作用 在早期肠道发育过程中，PRDM1 位点内含有调节元件。在小鼠中，Prdm1 调节 肠道成熟，包括潘氏细胞的发育，潘氏细胞在 CD 病理学中发挥重要作用。我们 已鉴定出人类 PRDM1 基因内的一个调控元件，该元件包含 CD 相关的 SNP，并且 仅在产前阶段活跃，在成人 CD 或非 CD 回肠中不活跃。我们将使用 CRISPR/Cas9 单核苷酸 碱基编辑技术，用于检查该 CD-SNP 的不同等位基因在 28 天成熟过程中的影响 回肠 HIO 的过程。该提案的成功完成将通过提供转化来促进儿童健康 有关人类肠道发育的基因调控机制和儿科 CD 病理学的知识。