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: 7936085
• 负责人: CECILIA W. LO
• 金额: $ 165.42万
• 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/7936085
• 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）最多影响活产1％，但其遗传基础仍然不太了解。鉴于人类人口的遗传多样性，人类的研究是为了揭示CHD的遗传原因。鉴于小鼠的基因组是完全测序的，小鼠的遗传分析是有利的，并且近交小鼠提供了遗传上相同的动物。敲除小鼠研究已经确定了许多可能导致CHD的基因。但是，ftjnction冗余可能掩盖基因功能或早期胚胎致死性可能会排除对CHD的评估。我们提出了一种互补的方法，即通过乙酰硝基乙酰胺诱变进行正向遗传筛查，以恢复引起CHD的突变。我们先前显示非侵入性小鼠胎儿超声心动图对高通量心血管表型非常有效。我们的屏幕回收了许多编码纤毛或中心体中蛋白质的基因，这表明纤毛是CHD中的一种核心疾病途径。为了恢复导致冠心病的突变，我们计划使用非侵入性胎儿超声心动图将100,000小鼠胎儿从4000个谱系中筛选为估计的五倍基因组覆盖率（AIM 1）。我们将使用具有高吞吐量靶向和整个基因组DNA测序的两层方法来识别突变（AIM 2）。对于涉嫌在纤毛中起作用的基因，斑马鱼将用于快速地形敲低敲低的基因，以分析纤毛的动物/非运动功能，并可能破坏与纤毛缺陷相关的左右patter中的破坏（AIM 3）。衍生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中性功能（AIM 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，将心脏外细胞群体部署到心脏，流出道和腔室分隔。还可以检查纤毛介导的声波刺猬和非典型的Wnt信号传导 ^ili（AIM 5）。总而言之，拟议的研究将有助于阐明CHD的遗传基础。将生成许多新的CHD小鼠模型，并且在CHD中发挥重要作用的纤毛和其他途径的作用将出现，并鉴定出一组核心基因与CHD非常重要。相关性（请参阅说明）；鉴定一组参与先天性心脏病的核心基因可以为未来的翻译研究提供基础，以阐明人类先天性心脏病的复杂遗传学。这可能包括设计诊断性芯片，用于对先天性心脏病的基因分型患者进行研究，并检查基因型与疾病表型和长期结局之间的相关性。