Evaluating the Impact of Mutations in Distant-Acting Enhancers in Structural Birth Defects

评估远效增强子突变对结构性出生缺陷的影响

基本信息

批准号：
10826564
负责人：
Len Alexander Pennacchio
金额：
$ 81.7万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-21 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10826564
关键词：
Address Affect Alleles Animals Biological Assay Brain Catalogs Categories Cells Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Code Complement 2 Congenital Abnormality Conserved Sequence DNA Sequence Data Data Set Defect Development Diagnosis Diagnostic Disease Distant Elements Embryonic Development Engineering Enhancers Etiology Gene Expression Generations Genes Genetic Genetic Enhancer Element Genetic Transcription Genome Genome engineering Genomics Histopathology Human Human Genome Human Resources International Knock-in Knock-in Mouse Manuals Maps Modeling Mus Mutate Mutation Organ Pathology Patients Perinatal Phenotype RNA analysis Regulator Genes Reporter Resolution Site Structural Congenital Anomalies Technology Testing Tissues Transgenic Mice Transgenic Organisms Untranslated RNA Validation Variant analysis pipeline analytical method body system brain malformation candidate selection causal variant cloud based cohort de novo mutation developmental disease epigenomics exome sequencing experimental study genetic variant genome annotation genome sequencing human data in vivo in vivo evaluation innovation mouse genome mouse model mutant prenatal prevent response risk variant screening therapeutic development transcriptome sequencing whole genome

项目摘要

SUMMARY Structural birth defects (SBDs) encompass a spectrum of congenital abnormalities affecting a wide range of human organ systems. Progress in sequencing technologies has enabled significant advances in the discovery of coding mutations underlying SBDs through whole-exome sequencing. Nonetheless, to date most cases continue to remain “unsolved”, creating a major barrier to diagnostic interpretation and therapeutic development. In particular, the identification and interpretation of mutations in noncoding sequence, which constitutes 98% of the human genome, has presented a formidable challenge. The present proposal addresses the hypothesis that noncoding sequence represents a major reservoir of causal mutations explaining many unsolved SBD cases. Specifically, we will focus on distant-acting transcriptional enhancers, a predominant class of noncoding genome elements with critical regulatory functions in embryonic development. There are isolated examples of SBD- causing enhancer mutations, but three principal hurdles have prevented their identification at scale: a) the lack of whole genome sequence data (WGS) from unsolved cases; b) inadequate annotations of noncoding genome functions; c) the lack of testing pipelines to assess the in vivo relevance of enhancer mutations and determine their causality. In this proposal, we address these challenges by creating an integrated pipeline for the identification, function-based prioritization, and in vivo validation of causality of enhancer mutations in SBD cases. This proposal will take advantage of growing aggregated WGS data, advanced analysis pipelines for mutation identification, a unique catalog of prioritized predictions of developmental in vivo enhancers, and advanced mouse engineering capabilities for in vivo validation of enhancers and enhancer mutations. Our specific aims include: 1) Prioritize de novo noncoding gene regulatory mutations identified in growing WGS catalogs in SBD patients. Taking advantage of preexisting aggregated WGS genetic data and innovative analysis strategies, we will identify noncoding mutations in SBD at unprecedented scale. Noncoding findings will be interpreted and prioritized using DevCisReg, a comprehensive catalog of gene regulatory sequences we developed from analysis of >800 human and mouse epigenomic data sets. 2) Functionally test prioritized SBD noncoding mutations for impacts on gene expression in scaled transgenic mouse enhancer assays. We will use a targeted CRISPR-enabled transgenic approach to characterize 200 candidate enhancer alleles in mice and determine which mutations impact on gene expression in vivo. 3) Functionally model prioritized SBD noncoding mutations in knockin mice. We will create and phenotype 40 knockin mouse lines with human alleles to test the in vivo impact of regulatory mutations in live animals. We will focus on mutations from SBDs that can be modeled and studied by streamlined phenotyping in mice to increase the likelihood we can detect a defect in vivo. Together, these efforts will create an integrated mutation-to-phenotype identification and testing pipeline that will provide conclusive in vivo evidence for establishing the causality of enhancer mutations in SBD.

概括结构性出生缺陷 (SBD) 涵盖一系列影响广泛的先天性异常人体器官系统的进步使这一发现取得了重大进展。尽管如此，迄今为止大多数病例都通过全外显子组测序发现了 SBD 的编码突变。继续保持“未解决”状态，为诊断解释和治疗发展造成主要障碍。特别是非编码序列突变的识别和解释，该序列占 98% 人类基因组，提出了一个巨大的挑战，目前的提议解决了这样的假设：非编码序列代表了因果突变的主要储存库，解释了许多未解决的 SBD 案例。具体来说，我们将重点关注远距离作用转录增强子，这是一类主要的非编码基因组在胚胎发育中具有关键调节功能的元件有 SBD- 的孤立例子。增强子突变，但三个主要障碍阻碍了它们的大规模识别：a）缺乏来自未解决案例的全基因组序列数据（WGS） b）非编码基因组注释不充分； c) 缺乏评估增强子突变的体内相关性并确定的测试管道在本提案中，我们通过创建一个集成的管道来解决这些挑战。 SBD 增强子突变因果关系的识别、基于功能的优先级排序和体内验证该提案将利用不断增长的 WGS 数据汇总和高级分析流程。突变识别，发育体内增强剂优先预测的独特目录，以及先进的小鼠工程能力，用于增强子和增强子突变的体内验证。具体目标包括： 1) 优先考虑在生长过程中发现的从头非编码基因调控突变 SBD 患者的 WGS 目录利用预先存在的汇总 WGS 遗传数据和创新。分析策略，我们将以前所未有的规模识别 SBD 中的非编码突变。使用 DevCisReg 进行解释和优先排序，DevCisReg 是我们的基因调控序列的综合目录根据对超过 800 个人类和小鼠表观基因组数据集的分析而开发。 2) 对优先 SBD 进行功能测试。非编码突变对大规模转基因小鼠增强子测定中基因表达的影响。将使用基于 CRISPR 的靶向转基因方法来表征小鼠中 200 个候选增强子等位基因并确定哪些突变影响体内基因表达 3) 功能模型优先 SBD。我们将用人类创建 40 个敲入小鼠品系并进行表型分析。我们将重点关注 SBD 的突变。可以通过简化的小鼠表型分析来建模和研究，以增加我们检测到的可能性这些努力将共同创建一个集成的突变到表型的识别和测试。该管道将为确定 SBD 增强子突变的因果关系提供决定性的体内证据。