A system approach to the analysis of Heterotaxy Candidate Genes

项目摘要 先天性畸形是美国婴儿死亡的主要原因。然而，我们对 先天性畸形的遗传原因是严重有限的负面影响我们的能力，照顾这些 患者为了发现遗传原因，我们和其他人对患者进行了人类基因组学分析。 特别是，我们已经集中在Heterotaxy（Htx），一种左右模式的障碍。通常，我们的内部 器官沿着左右轴不对称分布，该过程的失败可导致严重的 疾病包括先天性心脏病、肠旋转不良和免疫缺陷。人类基因分析 这些患者中的许多人已经确定了许多候选基因，但功能相关性尚不清楚， 几乎所有候选基因的致病机制都是未知的。 在上一个资助期，我们提出了一个功能屏幕的增益，我们过度表达的HTX候选 非洲爪蟾的基因寻找有效的表型。虽然这种策略有局限性，但我们发现了多个 这些基因给出了有趣的表型，导致了对Wnt和纤毛信号通路的令人兴奋的新见解。 在该基金的资助下，我们还开发了新的技术，用于测定基于纤毛的细胞外液流 尤其是基于CRISPR的F0基因编辑。 这些新技术的发展已经改变了我们将采用的基因筛选策略 在随后的补助期内。重要的是，我们将使用基于F0 CRISPR的功能丧失筛查， 快速、廉价和高效。我们的"下一代"屏幕还将包括调查，不仅 但是LR信号级联中的额外步骤。最后，我们将研究患者变异 并使用GeneMatcher与世界各地的研究人员联系，以利用我们的高通量动物模型 以测试额外的患者变体并增加我们系统中实验测试的等位基因的数量。最后， 在随后的Aim中，我们将把Htx候选基因置于对LR模式化至关重要的四个关键途径中：纤毛， Wnt、Notch和TGF-β信号传导。我们有相当多的经验，在每一个这些途径转换小说 这些途径中的每一个的分子机制。因此，我们以往的经验 这表明，我们特别适合将患者驱动的基因发现转化为分子生物学， 机制等

PROJECT SUMMARY Congenital malformations are the major cause of infant mortality in the US. However, our understanding of the genetic causes of congenital malformations is severely limited negatively impacting our ability to care for these patients. To discover the genetic causes, we and others have employed human genomics analyses on patients. In particular, we have focused on Heterotaxy (Htx), a disorder of left-right patterning. Normally, our internal organs are asymmetrically distributed along the left-right axis and failure of this process can lead to severe disease including congenital heart disease, gut malrotation, and immune deficiencies. Human genetic analysis of these patients has identified many candidate genes, but the functional relevance is unclear since a plausible disease pathogenesis mechanism is unknown for nearly all of the candidate genes. In the previous grant period, we proposed a gain of function screen where we overexpressed Htx candidate genes in Xenopus looking for potent phenotypes. While this strategy has limitations, we discovered multiple genes that gave interesting phenotypes that led to exciting new insights into Wnt and Cilia signaling pathways. Funded by the grant proposal, we also developed new techniques for assaying cilia based extracellular fluid flow and notably CRISPR based F0 gene editing. The development of these new technologies has transformed the strategy that we will employ for gene screening in the subsequent grant period. Importantly, we will use F0 CRISPR based loss of function screening which is rapid, inexpensive, and highly effective. Our “next generation” screen will also include an investigation not only of cardiac looping but additional steps in the LR signaling cascade. Finally, we will investigate patient variants and connect with researchers across the world using GeneMatcher to exploit our high-throughput animal model to test additional patient variants and increase the number of experimentally tested alleles in our system. Finally, in a subsequent Aim, we will place Htx candidate genes into four key pathways critical for LR patterning: Cilia, Wnt, Notch, and TGF-β signaling. We have considerable experience in each of these pathways converting novel unexplored genes into molecular mechanisms in each of these pathways. Therefore, our previous experience demonstrates that we are especially well suited to translate patient driven gene discovery into molecular mechanisms.