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，发生在 所有年龄段的患者，以小肠远端区域(简称回肠)为最常见的 受影响的地点。它困扰着美国近100万人，并在#年急剧增加。 在全球流行。值得注意的是，相当一部分CD患者(约25%)是儿童--或青少年-- 起病，称为儿科CD，通常表现为高度侵袭性症状和更复杂的临床 病程与晚发性成人CD相比。对人类群体的大规模遗传研究揭示了&gt；200 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。 尽管人们普遍猜测，这些因素可能会影响监管要素，但这一点尚未确定。 对大多数基因座进行了验证。此外，风险基因对肠道发育的影响程度尚不清楚。 研究很少，但考虑到发育异常对其他 肠道炎症性疾病。值得注意的是，婴儿难治性先天性腹泻最近与 缺失在胎儿期肠道发育过程中短暂活跃的调控元件。本研究旨在 为了(1)确定CD风险基因座，该基因座含有在出生前发育中短暂活跃的调控元件，并且 (2)为特定的镉风险等位基因在肠道早期影响调节元件活性提供功能证据 发展，最终目的是为儿童CD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(SNPs)在早期肠道疾病中的作用 发展，我们将利用最新的人类多能干细胞定向分化技术 (HPSCs)进入人体肠道球体，然后成熟为类器官(HIO)。这个模型展示了 分子特征与人类主要的胎儿肠道非常相似。定义有效的法规要素 在全基因组范围内，我们将把一种名为染色质连续测序(CHRO-SEQ)的尖端技术应用于 HPSC-HIO模型以及儿童和成人CD和非CD回肠活检。然后我们将定义这些 与CD风险基因重叠且仅在hPSC-HIO模型的出生前阶段活跃的调控元件 但在出生后的儿童/成人组织中不存在。在第二个目标中，我们将确定一个具体的CD-SNP的作用 在肠道发育早期，在PRDM1基因座内含有调控元件。在小鼠中，Prdm1调节 肠道成熟，包括潘氏细胞的发育，在CD的病理生物学中起着重要作用。我们 已经在人类PRDM1基因中发现了一个调控元件，它包含与CD相关的SNP，并且是 仅在出生前阶段活跃，在成人CD或非CD回肠中不活跃。我们将使用CRISPR/Cas9单核苷酸 碱基编辑技术检测该CD-SNP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为儿童提供 了解人类肠道发育的基因调控机制和儿童CD病理生物学。