Project I - Transcriptomic Analysis of Structural Birth Defects in Mouse Developmental Mutants

项目 I - 小鼠发育突变体结构性出生缺陷的转录组分析

• 批准号: 10327737
• 负责人: DAVID R. BEIER
• 金额: $ 81.43万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-11 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10327737
• 关键词: Anatomy; Biological; Cells; Complement; Computer Analysis; Congenital Abnormality; Data; Data Set; Development; Embryo; Embryonic Development; Frequencies; Gene Expression; Gene Expression Profiling; Generations; Genes; Grouping; Heterozygote; Human; Lethal Genes; Methods; Mus; Mutate; Mutation; Organogenesis; Pathway interactions; Phenotype; Play; Pregnancy; Proteins; RNA; RNA Sequences; Role; Severity of illness; Signal Pathway; Signal Transduction; Structural Congenital Anomalies; Techniques; Technology; Testing; base; bioinformatics tool; cell type; cohort; combinatorial; comparative; computerized tools; exome sequencing; gene discovery; human disease; indexing; insight; method development; mutant; novel; single cell analysis; single-cell RNA sequencing; transcriptome sequencing; transcriptomics

PROJECT SUMMARY In this project we plan to apply a powerful and scaleable technique of combinatorial indexing to characterize gene expression at the single-cell level in mice carrying mutations that are either known to or likely to result in structural birth defects. We propose to assess whether single-cell expression can be utilized as a phenotype; specifically, we aim to organize these data sets to assess whether there are signatures of single-cell gene expression that facilitate grouping mutant lines based on presumptive pathways of developmental signaling perturbation. This analysis will be complemented by the anatomical analysis that is proposed in Project 2. Given the novelty of this method, we will initially analyze 10 lines that are mutated for genes in the Shh signaling pathway, in order to correlate single-cell transcriptomic data with well-studied developmental phenotypes. To maximize the opportunity for new gene discovery, we will also examine novel genes that have not been previously annotated with respect to human structural birth defects. Specifically, using an analysis of human exome sequencing data, we have identified a large cohort of genes that are likely haploinsufficient; i.e., they are not compatible with survival when heterozygous null. We have furthermore developed a heterozygote selection (shet) statistic that correlates remarkably well with human disease severity. We aim to characterize 75 lines from the top quintile shet set that have limited functional annotation; these genes will be chosen either a) based on evidence from single-cell expression during embryogenesis (Cao et al. 2019) that they are novel cell- type-specific index genes or b) are known lethal genes (in mice) that have a high frequency of protein interactions. As part of this effort we will develop bioinformatic tools to facilitate comparisons across different datasets. These can identify mutant lines with common abnormalities of developmental signaling, as well as potentially serving as a means to understand the mechanistic basis for human congenital abnormalities.

项目概要 在这个项目中，我们计划应用一种强大且可扩展的组合索引技术来表征 携带已知或可能导致的突变的小鼠在单细胞水平上的基因表达 结构性出生缺陷。我们建议评估单细胞表达是否可以用作表型； 具体来说，我们的目标是组织这些数据集来评估是否存在单细胞基因的特征 促进基于发育信号的假定途径对突变系进行分组的表达 扰动。该分析将由项目 2 中提出的解剖分析进行补充。 鉴于这种方法的新颖性，我们将首先分析 10 个在 Shh 中基因发生突变的品系。 信号通路，以便将单细胞转录组数据与经过充分研究的发育相关联 表型。为了最大限度地增加新基因发现的机会，我们还将检查具有以下特征的新基因： 以前没有关于人类结构性出生缺陷的注释。具体来说，使用分析 人类外显子组测序数据，我们已经鉴定出一大群可能单倍体不足的基因； IE。， 当杂合子无效时，它们与生存不相容。我们还开发了杂合子 选择（Shet）统计数据与人类疾病的严重程度密切相关。我们的目标是描述 75 来自顶部五分位数图纸集的具有有限功能注释的行；这些基因将被选择a) 基于胚胎发生过程中单细胞表达的证据（Cao et al. 2019），它们是新型细胞- 类型特异性索引基因或 b) 是已知的致死基因（在小鼠中），具有高频率的蛋白质 互动。 作为这项工作的一部分，我们将开发生物信息学工具来促进不同数据集之间的比较。 这些可以识别具有发育信号传导常见异常的突变系，以及潜在的 作为了解人类先天异常机制基础的一种手段。