A system approach to the analysis of Heterotaxy Candidate Genes

分析异序候选基因的系统方法

• 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
• 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

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.

项目概要 先天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受到严重限制，这对我们照顾这些畸形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。 我们特别关注异向性（Htx），这是一种左右模式紊乱。通常情况下，我们的内部 器官沿着左右轴不对称分布，这个过程的失败可能会导致严重的后果 疾病包括先天性心脏病、肠道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鉴定出许多候选基因，但功能相关性尚不清楚，因为一个合理的 几乎所有候选基因的疾病发病机制都是未知的。 在之前的资助期内，我们提出了功能增益筛选，其中我们过度表达了 Htx 候选者 非洲爪蟾的基因寻找有效的表型。虽然这种策略有局限性，但我们发现了多种 这些基因赋予了有趣的表型，从而对 Wnt 和纤毛信号通路产生了令人兴奋的新见解。 在拨款提案的资助下，我们还开发了用于测定基于纤毛的细胞外液流动的新技术 尤其是基于 CRISPR 的 F0 基因编辑。 这些新技术的发展改变了我们用于基因筛选的策略 在随后的资助期内。重要的是，我们将使用基于 F0 CRISPR 的功能丧失筛选，即 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，但 LR 信号级联中的附加步骤。最后，我们将调查患者变异 并使用 GeneMatcher 与世界各地的研究人员联系，开发我们的高通量动物模型 测试其他患者变异并增加我们系统中经过实验测试的等位基因的数量。最后， 在后续目标中，我们将把 Htx 候选基因放入对 LR 模式至关重要的四个关键通路中：纤毛、 Wnt、Notch 和 TGF-β 信号传导。我们在每一个转化新颖的途径方面都有丰富的经验 将未探索的基因深入到这些途径的分子机制中。因此，我们之前的经验 表明我们特别适合将患者驱动的基因发现转化为分子 机制。