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）是一种特定类型的炎症性肠病，发生在 各个年龄段的患者，小肠的远端区域（称为回肠）是最常见的 受影响的位置。它影响了美国近一百万的人，并且正在急剧增加 全球流行。值得注意的是，很大一部分CD患者（约25％）是儿童或青少年 发作，称为儿科CD，通常表现出高度侵略性的症状和更复杂的临床 与迟到的成人CD相比。人类种群的大规模遗传研究表明> 200 与CD相关的基因组中的风险基因座，其中绝大多数发生在非编码区域。 尽管人们广泛认为这些可能会影响调节元素，但尚未确定 为大多数基因座展示。而且，风险基因座可能影响肠道发展的程度尚不清楚 而且探索不佳，但鉴于发展畸变的重要性，因此值得投资 肠道疾病。值得注意的是，婴儿最近与 在产前肠道发育过程中暂时活跃的调节元件的删除。这项研究的目的是 到（1）定义具有瞬时活跃在产前发育和 （2）提供了特定CD风险等位基因的功能证据 发展，最终的目标是提供有关儿科CD病因的新颖观点。在第一个 目的，研究CD相关的单个核苷酸多态性（SNP）对早期肠道的贡献 开发，我们将利用最新的指导分化与人类多能干细胞的技术 （HPSC）进入人肠球体，然后成熟到类器官（HIO）。该模型展出 分子特征与原代人胎儿肠道非常相似。定义主动调节元素 全基因组，我们将应用一种称为染色质跑步测序（CHRO-SEQ）的尖端技术 HPSC-HIO模型以及儿科和成人CD以及非CD回肠活检。然后，我们将定义那些 重叠CD的调节元素风险位点，并且仅在HPSC-HIO模型的产前阶段处于活动状态 但不在产后小儿/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包含PRDM1基因座中的调节元件。在鼠标中，PRDM1调节 肠道成熟，包括Paneth细胞的发展，在CD病理生物学中起重要作用。我们 已经确定了包含CD相关SNP的人PRDM1基因中的调节元件，IS 仅在产前阶段活跃，而不在成人CD或非CD回肠中活跃。我们将使用CRISPR/CAS9单核苷酸 基础编辑技术以检查28天成熟期间该CD-SNP不同等位基因的影响 回肠hios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 关于人肠发展和小儿CD病理生物学基因调节机制的知识。