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）影响多达1%的活产婴儿，但其遗传基础仍不清楚。鉴于人类群体的遗传多样性，解开CHD遗传原因的人类研究具有挑战性。小鼠中的遗传分析是有利的，因为小鼠基因组被完全测序，并且近交系小鼠提供遗传上相同的动物。敲除小鼠研究已经确定了许多可能导致CHD的基因。然而，功能性冗余可能掩盖基因功能或早期胚胎致死可能妨碍CHD的评估。我们提出了一种补充的方法与正向遗传筛选与乙基亚硝基脲诱变恢复突变引起冠心病。我们以前显示，非侵入性小鼠胎儿超声心动图是非常有效的高通量心血管表型。我们的筛选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CHD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个小鼠胎儿，以达到估计的5倍基因组覆盖率（目标1）。我们将使用高通量靶向和全基因组DNA测序的双层方法来鉴定突变（目的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代敲除，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图案化的可能破坏（目的3）。小鼠胚胎成纤维细胞和突变胚胎组织将用于评价与纤毛和中心体相关的细胞内在功能（目的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成环、心外细胞群向心脏的部署、流出道和室分隔。还将检查纤毛介导的音刺猬和非经典Wnt信号传导（目的5）。总之，这些研究将有助于阐明冠心病的遗传基础。许多新的CHD小鼠模型将被产生，纤毛和其他途径在CHD中发挥重要作用的作用将随着一组关键参与CHD的核心基因的鉴定而出现。相关性（参见说明）;先天性心脏病相关核心基因的鉴定可以为未来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。这可能包括设计用于先天性心脏病患者基因分型的诊断芯片，并检查基因型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.