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

• 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
• 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

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 结构性先天缺陷（SBD）涵盖了一系列先天性异常，影响了广泛的 人体器官系统。测序技术的进步已经在发现方面取得了重大进步 通过全外观测序进行SBD的编码突变。尽管如此，迄今为止大多数情况 继续保持“未解决”，从而为诊断解释和治疗发展带来了主要的障碍。 特别是，非编码序列中突变的识别和解释，占98％ 人类基因组提出了巨大的挑战。目前的提议解决了以下假设 非编码序列代表了因果突变的主要库，解释了许多未解决的SBD病例。 具体而言，我们将重点放在远处的转录增强子上，这是一类非编码基因组类别的类别 具有关键调节功能在胚胎发育中的元素。有孤立的sbd-例子 引起增强子突变，但三个主要障碍阻止了它们的标识：a）缺乏 来自未解决病例的整个基因组序列数据（WGS）； b）非编码基因组的注释不足 功能； c）缺乏测试管道来评估增强子突变的体内相关性并确定 他们的因果关系。在此提案中，我们通过为该挑战来解决这些挑战 识别，基于功能的优先级以及SBD增强子突变的休闲性的体内验证 案例。该建议将利用汇总的WGS数据，高级分析管道的优势 突变鉴定，是对体内增强子发育的优先预测的独特目录，以及 高级小鼠工程功能，用于体内验证增强剂和增强子突变。我们的 具体目的包括：1）优先考虑在生长中确定的从头非编码基因调节突变 SBD患者的WGS目录。利用已经存在的汇总WGS遗传数据和创新的优势 分析策略，我们将以前所未有的规模确定SBD中的非编码突变。非编码发现将 使用devcisreg进行解释和优先级，这是基因调节序列的全面目录 从对> 800个人类和小鼠表观基因组数据集的分析开发。 2）功能测试优先级的SBD 非编码突变对缩放转基因小鼠增强子测定中基因表达的影响。我们 将使用靶向CRISPR的转基因方法来表征小鼠的200个候选增强剂等位基因 并确定哪些突变对体内基因表达的影响。 3）功能模型优先考虑SBD 敲蛋白小鼠中的非编码突变。我们将与人类创建和表型40敲击鼠标线 等位基因测试活动物调节突变的体内影响。我们将专注于SBD的突变 可以通过简化小鼠的表型来建模和研究，以增加我们可以检测到的可能性 体内缺陷。这些努力将共同创建一个集成的突变与表型识别和测试 将在体内证据中确定增强子突变的偶然性的管道。