Modeling the genetic basis for human congenital heart disease in mice

在小鼠中模拟人类先天性心脏病的遗传基础

• 批准号: 8127905
• 负责人: CECILIA W. LO
• 金额: $ 165.04万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8127905
• 关键词: Affect; Animals; Biological Assay; Cardiac; Cardiovascular system; Cells; Centrosome; Cilia; Complex; DNA Sequence; Defect; Development; Diagnostic; Disease Pathway; Echocardiography; Embryo; Erinaceidae; Ethylnitrosourea; Event; Fetus; Fibroblasts; Future; Genes; Genetic; Genetic Models; Genetic Screening; Genetic Variation; Genome; Genotype; Heart; Human; Human Genetics; Inbred Mouse; Instruction; Knockout Mice; Left; Live Birth; Masks; Mediating; Morphogenesis; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Outcome; Pathway interactions; Patients; Phenotype; Play; Population; Proteins; Role; Signal Transduction; Tissues; Zebrafish; base; congenital heart disorder; design; disease phenotype; fetal; gene function; genetic analysis; genetic pedigree; genome sequencing; human subject; knock-down; mouse genome; mouse model; mutant; translational study

DESCRIPTION (provided by applicant): Congenital heart disease (CHD) affects up to 1% of live births, but its genetic basis is still not well understood. Human studies to unravel the genetic causes of CHD is challenging given genetic diversity of the human population. Genetic analysis in mice is advantageous given the mouse genome is completely sequenced, and inbred mice provide animals that are genetically identical. Knockout mouse studies have identified many genes that can cause CHD. However, ftjnctional redundancies may mask gene function or early embryonic lethality may preclude assessment of CHD. We propose a complementary approach with forward genetic screening with ethylnitrosourea mutagenesis to recover mutations causing CHD. We previously showed noninvasive mouse fetal echocardiography is highly effective for high throughput Cardiovascular phenotyping. Our screen recovered many genes encoding proteins in the cilia or centrosome, suggesting the cilium is a central disease pathway in CHD. To recover mutations causing CHD, we plan to use noninvasive fetal echocardiography to screen 100,000 mouse fetuses from 4000 pedigrees to achieve an estimated five-fold genome coverage (Aim 1). We will use a two-tier approach with high throughput targeted and whole genome DNA sequencing to identify the mutations (Aim 2). For genes suspected to have a role in the cilium, zebrafish will be used for rapid morpholino knock-down to analyze the motiie/nonmotile functions of the cilia and possible disruption of left-right patteming related to cilia defects (Aim 3). Mouse embryonic fibroblasts and tissues derived from mutant embryos will be used to evaluate cell-intrinsic function related to the cilium and centrosome (Aim 4). To elucidate the role of the cilia in cardiac morphogenesis, mutant embryos will be examined for heart looping, deployment of extracardiac cell populations to the heart, outflow tract and chamber septation. Cilia mediated sonic hedgehog and non-canonical Wnt signaling also ^ili be examined (Aim 5). In summary, the proposed studies will help elucidate the genetic basis for CHD. Many new CHD mouse models will be generated and the role of the cilium and other pathways playing important roles in CHD will emerge with the identification of a core set of genes critically involved in CHD. RELEVANCE (See instructions); The identification of a core set of genes involved in congenital heart disease can provide the basis for future translational studies with human subjects to elucidate the complex genetics of human congenital heart disease. This could include the design of diagnostic chips for genotyping patients with congenital heart disease and examining for correlation between genotype with disease phenotype and long term outcome.

描述（由申请人提供）： 先天性心脏病 (CHD) 影响高达 1% 的活产儿，但其遗传基础仍不清楚。鉴于人类遗传多样性，揭示冠心病遗传原因的人体研究具有挑战性。鉴于小鼠基因组已完全测序，并且近交小鼠提供了遗传相同的动物，因此对小鼠进行遗传分析是有利的。基因敲除小鼠研究已鉴定出许多可导致冠心病的基因。然而，功能冗余可能掩盖基因功能，或者早期胚胎致死性可能妨碍对 CHD 的评估。我们提出了一种利用乙基亚硝基脲诱变进行正向遗传筛选的补充方法，以恢复引起冠心病的突变。我们之前表明，无创小鼠胎儿超声心动图对于高通量心血管表型分析非常有效。我们的筛选恢复了纤毛或中心体中许多编码蛋白质的基因，表明纤毛是冠心病的中心疾病途径。为了恢复导致 CHD 的突变，我们计划使用无创胎儿超声心动图筛查来自 4000 个谱系的 100,000 个小鼠胎儿，以实现估计五倍的基因组覆盖率（目标 1）。我们将使用高通量靶向全基因组 DNA 测序的两层方法来识别突变（目标 2）。对于怀疑在纤毛中起作用的基因，将使用斑马鱼进行快速吗啉敲低，以分析纤毛的运动/非运动功能以及与纤毛缺陷相关的左右模式可能的破坏（目标 3）。源自突变胚胎的小鼠胚胎成纤维细胞和组织将用于评估与纤毛和中心体相关的细胞内在功能（目标 4）。为了阐明纤毛在心脏形态发生中的作用，将检查突变胚胎的心脏循环、心外细胞群向心脏的部署、流出道和心室分隔。纤毛介导的声波刺猬和非规范 Wnt 信号传导也值得研究（目标 5）。总之，拟议的研究将有助于阐明先心病的遗传基础。随着与 CHD 密切相关的一组核心基因的鉴定，将产生许多新的 CHD 小鼠模型，并且将出现纤毛和其他在 CHD 中发挥重要作用的途径的作用。相关性（参见说明）；鉴定与先天性心脏病有关的一组核心基因可以为未来以人类受试者为对象的转化研究奠定基础，以阐明人类先天性心脏病的复杂遗传学。这可能包括设计用于对先天性心脏病患者进行基因分型的诊断芯片，以及检查基因型与疾病表型和长期结果之间的相关性。