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

摘要 结构性出生缺陷(SBD)包括一系列先天性异常，影响范围广泛 人体器官系统。测序技术的进步使这一发现取得了重大进展 通过全外显子组测序对SBD潜在的突变进行编码。尽管如此，到目前为止，大多数情况下 仍然“悬而未决”，对诊断解释和治疗发展造成了重大障碍。 尤其是非编码序列中突变的识别和解释，非编码序列占98% 人类基因组构成了一个巨大的挑战。本提案针对的假设是 非编码序列代表了原因突变的主要储存库，可以解释许多未解决的SBD病例。 具体地说，我们将专注于远距离作用的转录增强子，这是一类占主导地位的非编码基因组 在胚胎发育中具有关键调节功能的元件。SBD也有个别的例子- 导致增强子突变，但有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)对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 它们之间的因果关系。在本提案中，我们通过创建一条综合管道来应对这些挑战 SBD增强子突变的识别、基于功能的优先排序和体内验证的因果关系 案子。该提案将利用不断增长的聚合WGS数据、高级分析管道 突变鉴定，体内发育增强剂的唯一优先预测目录，以及 先进的小鼠工程能力，可在体内验证增强剂和增强子突变。我们的 具体目标包括：1)优先处理在生长中发现的从头开始的非编码基因调节性突变 SBD患者的WGS目录。利用先前存在的聚合WGS遗传数据和创新 分析策略，我们将在SBD中以前所未有的规模识别非编码突变。非编码结果将 使用DevCisReg进行解释和优先排序，这是我们对基因调控序列的全面目录 从分析&gt；800人和小鼠表观基因组数据集发展而来。2)功能测试优先级SBD 非编码突变对大规模转基因小鼠增强子分析中基因表达的影响。我们 将使用靶向CRISPR启用的转基因方法在小鼠身上表征200个候选增强子等位基因 并确定哪些突变会影响体内的基因表达。3)功能模型优先SBD 敲击小鼠的非编码突变。我们将创造和表型40敲击小鼠与人类 等位基因，以测试活体动物中调节性突变的影响。我们将专注于SBD的突变 这可以通过简化的小鼠表型来建模和研究，以增加我们可以检测到 体内缺陷。总之，这些努力将创建一个从突变到表型的综合鉴定和测试 这条管道将为确定SBD增强子突变的因果关系提供确凿的体内证据。

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.