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

总结 结构性出生缺陷（SBD）包括一系列影响广泛的先天性异常， 人体器官系统测序技术的进步使发现的重大进展 通过全外显子组测序来分析SBD背后的编码突变。然而，迄今为止， 这些问题仍然“未解决”，对诊断解释和治疗发展造成了重大障碍。 特别是，非编码序列中突变的识别和解释，占98%的 人类基因组，已经提出了一个巨大的挑战。本提案针对的假设是， 非编码序列代表了解释许多未解决的SBD病例的因果突变的主要储存库。 具体来说，我们将集中在远距离作用的转录增强子，一类占主导地位的非编码基因组 在胚胎发育中具有重要调节功能的元素。有一些孤立的SBD的例子- 导致增强子突变，但三个主要障碍阻碍了它们的大规模鉴定：a）缺乏 未解决病例的全基因组序列数据（WGS）; B）非编码基因组注释不充分 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增强子突变的体内相关性; d）缺乏测试管道来评估增强子突变的体内相关性并确定增强子突变的体内相关性。 他们的因果关系。在本提案中，我们通过为以下项目创建一个综合管道来应对这些挑战： SBD中增强子突变因果关系的鉴定、基于功能的优先化和体内验证 例该提案将利用不断增长的聚合WGS数据，先进的分析管道， 突变鉴定，一个独特的目录优先预测的发展在体内增强，和 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。我们 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WGS基因数据和创新 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。非编码结果将 使用DevCisReg进行解释和优先排序，DevCisReg是一个全面的基因调控序列目录， 通过分析超过800个人类和小鼠表观基因组数据集开发。2)功能测试优先SBD 非编码突变对基因表达的影响，在缩放的转基因小鼠增强子测定。我们 将使用靶向CRISPR转基因方法来表征小鼠中的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)功能模型优先级SBD 基因敲入小鼠的非编码突变。我们将用人类基因组构建40个敲入小鼠品系， 等位基因来测试活动物中调节突变的体内影响。我们将重点关注SBD的突变 这可以通过在小鼠中进行简化的表型分析来建模和研究，以增加我们可以检测到 体内缺陷。总之，这些努力将创造一个综合的突变表型鉴定和测试 这将为建立SBD中增强子突变的因果关系提供决定性的体内证据。

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.