Modeling the genetic basis for human congenital heart disease in mice

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。考虑到人类群体的遗传多样性，人类研究揭开冠心病的遗传原因是具有挑战性的。小鼠的基因分析是有利的，因为小鼠的基因组是完全测序的，而近亲交配的小鼠提供了基因相同的动物。基因敲除小鼠的研究已经确定了许多可能导致CHD的基因。然而，基因功能冗余可能掩盖基因功能，或者早期胚胎死亡可能排除对CHD的评估。我们提出了一种互补的方法，通过乙基亚硝脲诱变进行正向遗传筛选，以恢复导致CHD的突变。我们先前的研究表明，无创小鼠胎儿超声心动图对于高通量心血管表型是非常有效的。我们的筛查在纤毛或中心体中发现了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，我们计划使用非侵入性胎儿超声心动图从4000个家系中筛选100,000个小鼠胚胎，以实现估计的五倍基因组覆盖(目标1)。我们将使用高通量、靶向和全基因组DNA测序的两级方法来识别突变(目标2)。对于被怀疑在纤毛中起作用的基因，斑马鱼将被用于快速吗啉敲除，以分析纤毛的动机/非运动功能，以及与纤毛缺陷相关的可能的左右模式中断(目标3)。来自突变胚胎的小鼠胚胎成纤维细胞和组织将被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环、心外细胞群部署到心脏、流出道和心腔分隔。纤毛介导的声波刺猬和非典型的Wnt信号也将被检测(目标5)。综上所述，建议的研究将有助于阐明冠心病的遗传基础。随着一组与CHD密切相关的核心基因的发现，许多新的CHD小鼠模型将被建立，纤毛和其他在CHD中起重要作用的通路的作用将显现出来。相关性(见说明)；确定一组涉及先天性心脏病的核心基因可以为未来与人类受试者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芯片，对先天性心脏病患者进行基因分型，并检查基因与疾病表型和长期结果之间的相关性。

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.