"SHROOM3 is a Novel Cause of Congenital Heart Disease"

“SHROOM3 是先天性心脏病的新病因”

• 批准号: 10055378
• 负责人: Matthew D. Durbin
• 金额: $ 15.52万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-07 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10055378
• 关键词: Actomyosin; Antibodies; Apical; Automobile Driving; Award; Binding; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cardiac development; Cardiovascular system; Cause of Death; Cell Line; Cell Polarity; Characteristics; Cleft Palate; Co-Immunoprecipitations; Congenital Abnormality; Cytoskeleton; Data; Defect; Development; Developmental Biology; Disease; Disease model; Down-Regulation; Embryo; Environment; Etiology; Exhibits; Extramural Activities; F-Actin; Frequencies; Funding; Genes; Genetic; Genetic Transcription; Genomics; Goals; Grant; Heart; Heart Abnormalities; Heart Diseases; Human; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Indiana; Individual; Kidney; Lead; Left; Link; MAPK8 gene; Mentors; Molecular Biology; Movement; Mus; Names; Neural Crest; Neural Crest Cell; Neural tube; Pathogenesis; Pathway interactions; Patients; Pattern; Phenocopy; Phenotype; Physicians; ROCK1 gene; Research; Research Personnel; Rho-associated kinase; Risk; Scientist; Shapes; Signal Pathway; Signal Transduction; TNFSF5 gene; Testing; Time; United States National Institutes of Health; Universities; Variant; WNT Signaling Pathway; Western Blotting; Writing; Yang; body system; cardiogenesis; cell motility; comorbidity; congenital heart disorder; constriction; disease diagnosis; disease phenotype; exome sequencing; genome editing; improved; in vivo; individualized medicine; induced pluripotent stem cell; loss of function; migration; novel; outcome forecast; pediatric department; planar cell polarity; rare variant; statistics; supportive environment; targeted treatment; therapy development; transcriptome sequencing; translational research program; treatment strategy

Congenital heart disease (CHD) is the most common cause of death due to birth defects. Despite CHD frequency, the etiology remains mostly unknown. Understanding CHD pathogenesis will help establish prognosis, identify comorbidity risks, and develop targeted therapies. We have identified a novel CHD candidate, SHROOM3. SHROOM3 induces actomyosin constriction and is implicated in cleft palate, neural tube and kidney defects in humans, but is unexplored in the heart. A recent study demonstrates that SHROOM3 interacts with Dishevelled2 (DVL2), a component of the noncanonical Wnt/planar cell polarity (PCP) signaling pathway, suggesting that SHROOM3 may serve as an important link between actomyosin constriction and PCP signaling. PCP is required for cardiac development, driving neural crest and second heart field migration to the outflow tract. I utilized a Shroom3 gene-trap mouse (Shroom3gt/gt) to demonstrate SHROOM3-loss-of-function leads to cardiac defects, including outflow tract defects, phenocopying PCP disruption. Additionally, through whole exome sequencing in patients with PCP related CHD phenotypes, I identified rare and potentially damaging variants within SHROOM3's PCP-binding domain. I hypothesize SHROOM3 is a novel component of PCP signaling and disruption causes CHD. To test this hypothesis, Aim1a assays genetic interaction between SHROOM3 and PCP during cardiac development, by generating compound heterozygous Shroom3gt/+;Dvl2-/+ embryos and assaying for a cardiac defects. Aim1b assays Shroom3gt/gt embryos using western blot (WB) for phosphorylated, activated, PCP components and immunohistochemistry (IHC) analyses for neural crest cell and second heart field migration to the heart. Aim1c tests the hypothesis that SHROOM3 loss of function disrupts the PCP transcriptional profile by assaying Shroom3gt/gt hearts using RNA-Seq analysis. Aim 2 tests the hypothesis that specific SHROOM3 variants cause disrupted PCP signaling in vitro. Utilizing CRISPR/Cas9 genome editing, I will generate SHROOM3 variant induced pluripotent stem cell lines (iPSCs) and derive cardiomyocytes (iPSC-CMs). Aim 2a utilizes a coimmunoprecipitation WB to assay disrupted SHROOM3-DVL2 binding within the variant iPSC-CM lines. Aim 2b assays the variant iPSC-CMs for WB and IHC evidence of PCP disruption. Aim2c assays the variant iPSC-CMs for altered PCP transcription using RNA-seq. The research plan will demonstrate SHROOM3's mechanism as a novel component of the PCP pathway and a novel CHD candidate gene. Indiana University's top10 NIH funded Department of Pediatrics provides a highly supportive environment for physician-scientist development, and my unique panel of mentors each has world-class expertise in an important aspect of the project, including cardiovascular genetics, development, and disease modeling with mice and iPSCs. I will gain a skillset in statistics, molecular and developmental biology, mouse and iPSC disease models, CRISPR-Cas9 gene editing and genomics analysis. The project is a move towards my goal to become a physician-scientist with an extramurally-funded, translational research program that improves CHD diagnosis and treatment for patients.

先天性心脏病（CHD）是由于先天缺陷而导致的最常见死亡原因。尽管冠心病 频率，病因仍然是未知的。了解CHD发病机理将有助于建立 预后，确定合并症的风险并发展有针对性的疗法。我们已经确定了一个新颖的CHD候选者， Shroom3。 Shroom3诱导肌动蛋白收缩，并与left裂，神经管和肾脏有关 人类缺陷，但心脏中没有探索。最近的一项研究表明，Shroom3与 DISHEVELLED2（DVL2），非规范Wnt/Planar Cell极性（PCP）信号通路的组成部分， 表明Shroom3可能是Actomyosin收缩与PCP信号传导之间的重要联系。 PCP是心脏发育，驱动神经rest和第二心脏场迁移到流出道所必需的。 我利用shroom3基因陷阱鼠标（shroom3gt/gt）演示了shroom3功能的速度可导致心脏 缺陷，包括流出道缺陷，pCP的表现破坏。另外，通过整个外显子 在患有PCP相关的CHD表型患者的测序中，我确定了罕见且潜在的破坏变体 在Shroom3的PCP结合域中。我假设shroom3是PCP信号传导和 破坏会导致冠心。为了检验这一假设，AIM1A分析了Shroom3和PCP之间的遗传相互作用 在心脏发展过程中，通过产生复合杂合shroom3gt/+; dvl2 - /+胚胎和分析 对于心脏缺陷。 AIM1B使用Western印迹（WB）shroom3GT/GT胚胎进行磷酸化，激活， PCP成分和免疫组织化学（IHC）分析神经rest细胞和第二心脏场迁移 心脏。 AIM1C检验了shroom3功能损失的假设破坏了PCP转录概况 通过使用RNA-seq分析分析Shroom3GT/GT心脏。 AIM 2检验了特定扫描室的假设3 变体在体外引起PCP信号破坏。利用CRISPR/CAS9基因组编辑，我将生成 Shroom3变体诱导多能干细胞系（IPSC）和得出心肌细胞（IPSC-CMS）。目标2a 利用共免疫沉淀WB来测定变体IPSC-CM内的shroom3-dvl2绑定。 线。 AIM 2B分析为WB和IHC的PCP中断证据的变体IPSC-CMS。 AIM2C分析 使用RNA-seq更改PCP转录的变体IPSC-CM。该研究计划将展示Shroom3的 机制是PCP途径的新成分和新型CHD候选基因。印第安纳大学的 TOP10 NIH资助的儿科部为医师科学家提供了高度支持的环境 发展，我独特的导师小组都有世界一流的专业知识 项目，包括使用小鼠和IPSC进行的心血管遗传学，发育和疾病建模。我会收获 统计，分子和发育生物学，小鼠和IPSC疾病模型的技能，CRISPR-CAS9 基因编辑和基因组学分析。该项目是朝着我的目标迈进的迈进 一项由典型资助的转化研究计划，可改善患者的冠心病诊断和治疗。