Mechanism of LV Hypoplasia in Hypoplastic Left Heart Syndrome Supplement

左心发育不全综合征(HLHS)是一种以左小为特征的先天性心脏缺陷(CHD) 室壁(LV)和发育不良的主动脉和主动脉/二尖瓣。HLHS的遗传病因学被强烈提示 复发风险高，但HLHS的遗传基础尚不清楚。临床研究表明 HLHS具有多基因和遗传异质性。对HLHS遗传学的见解来自我们的 最近从大规模的小鼠诱变筛选中恢复了第一个HLHS小鼠模型。从8点开始 恢复了独立的HLHS小鼠系，鉴定出330个突变，没有共同的基因 在8行之间。这些发现表明，HLHS在基因上具有深刻的异质性，与 人体研究。对一种突变的小鼠品系奥伊亚的详细分析表明，HLSH是由两个突变引起的 基因：Sap130，染色质修饰组蛋白脱乙酰酶复合体(HDAC)中的Sin3a相关蛋白， 以及Pcdha9，一种介导细胞间黏附的原钙粘附素。左心室发育不全被证明是由 Sap130突变，在CRISPR产生的小脑室表型复制中证实了这一发现 SAP130A斑马鱼突变体。左心室发育不良与心肌细胞增殖缺陷有关。 和心肌细胞周期停滞。在这项研究中，我们将研究细胞和分子机制。 利用斑马鱼的独特优势，遗传交互作用推动了HLHS的LV发育不全 和老鼠模型。在目标1中，我们将使用斑马鱼的谱系追踪研究和CRE的实验 在小鼠中缺失Sap130以检验Sap130以细胞自主方式发挥作用的假设 调节心室/左心室的生长。这些研究将描绘Sap130调节LV的细胞背景 成长。在目标2中，我们将研究Sap1300Ha亚型突变导致LV的假设 通过调控心肌细胞周期和细胞增殖的靶基因实现发育不全。这些研究将 关注Meis1，一个Sap130的靶基因，也是已知的调节心肌细胞周期和出生后细胞的基因 周期拘禁。同时，通过Sap130芯片序列分析和rna-seq分析确定的其他候选基因将 通过CRISPR靶向胚胎的产生和分析来评估它们在左室发育不良中的作用 老鼠。在目标3中，我们将探讨染色质修饰物与Ras/MAPK信号通路的相互作用。 反义吗啉基因敲除致敏基因斑马鱼HLHS的发病机制 背景资料。积极的遗传相互作用将通过突变或CRISPR靶向斑马鱼或小鼠来验证。 这项研究的动机是染色质修饰物和RAS/MAPK突变的意外恢复 所有8个HLHS小鼠系中的途径成分，提示染色质修饰剂与 RAS/MAPK信号通路异常可能与LV发育不良和HLHS的复杂遗传学有关。 这些研究将有助于阐明驱动心室的细胞和分子机制。 HLHS的发育不全，这一发现可能为胎儿干预恢复LV生长提供新的治疗靶点。 好了！

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect (CHD) characterized by a small left ventricle (LV) and hypoplastic aorta and aortic/mitral valves. A genetic etiology for HLHS is strongly indicated by high recurrence risk, but the genetic underpinning for HLHS is poorly understood. Clinical studies suggest HLHS is multigenic and genetically heterogeneous. Insights into the genetics of HLHS has come from our recent recovery of the first mouse models of HLHS from a large-scale mouse mutagenesis screen. From 8 independent HLHS mouse lines recovered, 330 mutations were identified, with no genes shared in common between the 8 lines. These findings indicate HLHS is profoundly genetically heterogeneous, consistent with the human studies. Detailed analysis of one mutant mouse line, Ohia, showed HLSH is elicited by mutations in two genes: Sap130, a Sin3a associated protein in the chromatin modifying histone deacetylase complex (HDAC), and Pcdha9, a protocadherin mediating cell-cell adhesion. The LV hypoplasia was shown to be elicited by the Sap130 mutation, a finding confirmed with replication of a small ventricle phenotype in a CRISPR generated sap130a zebrafish mutant. The LV hypoplasia was associated with a cardiomyocyte cell proliferation defect and cardiomyocyte cell cycle arrest. In this study, we will investigate the cellular and molecular mechanisms and genetic interactions driving the LV hypoplasia in HLHS, leveraging the unique strengths of the zebrafish and mouse models. In Aim 1, we will employ lineage tracing studies in zebrafish and experiments with Cre deletion of Sap130 in mice to test the hypothesis that Sap130 functions in a cell autonomous manner to regulate ventricular/LV growth. These studies will delineate the cellular context in which Sap130 regulates LV growth. In Aim 2, we will investigate the hypothesis that the hypomorphic Sap130Ohia mutation causes LV hypoplasia via target genes that regulate cardiomyocyte cell cycle and cell proliferation. These studies will focus on Meis1, a Sap130 target gene, also known to regulate cardiomyocyte cell cycle and postnatal cell cycle arrest. In parallel, additional candidate genes identified via Sap130 ChIP-seq and RNA-seq analysis will be assessed for their role in LV hypoplasia with production and analysis of CRISPR targeted embryos and mice. In Aim 3, we will probe the interaction of chromatin modifiers with the Ras/MAPK signaling pathway in the pathogenesis of HLHS using antisense morpholino gene knockdown in zebrafish with a sensitized genetic background. Positive genetic interactions will be validated using mutant or CRISPR targeted zebrafish or mice. This study is motivated by the unexpected recovery of mutations in chromatin modifiers and Ras/MAPK pathway components in all 8 HLHS mouse lines, suggesting chromatin modifiers in combination with dysregulated Ras/MAPK signaling may contribute to the LV hypoplasia and complex genetics of HLHS. Together these studies will help to elucidate the cellular and molecular mechanisms driving the ventricular hypoplasia in HLHS, findings that may yield new therapeutic targets for fetal intervention to recover LV growth. !