A system approach to the analysis of Heterotaxy Candidate Genes

分析异序候选基因的系统方法

基本信息

批准号：
10558564
负责人：
Mustafa K Khokha
金额：
$ 61.72万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-23 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10558564
关键词：
Aborted Fetus Adult Affect Alleles Animal Model Applications Grants Biological Assay Biological Process Breathing Candidate Disease Gene Cardiac Cause of Death Child Health Cilia Clustered Regularly Interspaced Short Palindromic Repeats Communities Congenital Abnormality Critical Pathways Data Development Developmental Gene Diagnostic Diploidy Disease Embryo Embryonic Development Environmental Exposure Etiology Europe Extracellular Fluid Failure Funding Genes Genetic Genetic Predisposition to Disease Genomics Goals Grant Heart Human Human Genetics Immune Incidence Infant Infant Mortality Inherited Investigation Left Life Live Birth Messenger RNA Modeling Molecular Operative Surgical Procedures Organ Output Pathogenesis Pathogenicity Pathway interactions Patient Care Patients Pattern Phenocopy Phenotype Population Process Publications Publishing Rana Research Personnel Role Signal Pathway Signal Transduction Situs Inversus Structure System Techniques Testing Therapeutic Transforming Growth Factor beta Translating Variant Work Xenopus cardiogenesis congenital heart disorder de novo mutation disease phenotype experience fascinate fluid flow gain of function gene discovery genetic analysis human genomics insight loss of function molecular marker mutant new technology next generation notch protein novel overexpression screening sharing platform statistics

项目摘要

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.

项目摘要先天性畸形是美国婴儿死亡率的主要原因。但是，我们对先天性畸形的遗传原因受到严重限制，对我们照顾这些的能力产生了负面影响患者。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，我们专注于异性恋（HTX），这是一种左右模式的疾病。通常，我们的内部器官沿左右轴不对称分布，此过程的故障可能导致严重包括先天性心脏病，肠道畸形和免疫缺陷的疾病。人类遗传分析这些患者已经确定了许多候选基因，但是功能相关性尚不清楚，因为合理几乎所有候选基因的疾病发病机理都是未知的。在上一个赠款期间，我们提出了一个函数屏幕的增益，在该屏幕上我们过表达HTX候选者爪蟾中寻找潜在表型的基因。尽管此策略有局限性，但我们发现了多个产生了有趣的表型的基因，从而引发了对Wnt和Cilia信号通路的激动人心的新见解。由赠款提案资助，我们还开发了用于测定基于纤毛的细胞外流体流量的新技术以及尤其是基于CRISPR的F0基因编辑。这些新技术的开发改变了我们将采用的基因筛查策略在随后的赠款期间。重要的是，我们将使用基于F0 CRISPR的功能筛选丢失快速，廉价且高效。我们的“下一代”屏幕还将不仅包括调查心脏循环的，但LR信号级联中的其他步骤。最后，我们将研究患者变体并使用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测试其他患者变体并增加系统中实验测试等位基因的数量。最后，在随后的目标中，我们将将HTX候选基因置于对LR模式至关重要的四个关键途径中：Cilia， Wnt，Notch和TGF-β信号传导。我们在这些途径中都有丰富的经验意外的基因进入这些途径的分子机制。因此，我们以前的经历证明我们特别适合将患者驱动基因发现转化为分子机制。