Derepression of Hedgehog Signaling in Congenital Heart Disease Patients

先天性心脏病（CHD）是最常见的出生缺陷之一，影响多达1%的活产婴儿。一 我们实验室先前进行的导致CHD的突变的小鼠正向遗传筛查回收了91 导致CHD的基因，其中超过50个与纤毛和纤毛信号相关。有趣的是， 在纤毛转导的细胞信号通路中，至少有5种与声波相关， Hedgehog（Shh）信号。事实上，已知Shh信号在神经元的发育中起重要作用。 通过心脏神经嵴和第二心野细胞的调节来调节流出道和半月瓣 血统虽然Shh在CHD发病机制中的作用现在在小鼠模型中得到了很好的描述，但其在人类中的作用仍然存在。 CHD仍然知之甚少。因此，为了研究Shh信号在人类CHD中的潜在重要性， 为了研究CHD的发病机制，我们使用CHD患者成纤维细胞进行了Shh信号传导缺陷的筛选。令人惊讶的是， 我们的分析显示，41例CHD患者中有17例表现出hedgehog信号的改变，但没有明显的纤毛发生 缺陷，表明hedgehog信号而不是纤毛结构受到影响。真实的PCR分析显示，9 17例患者中有异常的基底激活转录GLI，一个hegehog反应基因， 没有路径刺激。这表明Shh信号负调控的中断可能起作用， 在冠心病发病中起重要作用。有趣的是，9例基础GLI激活的患者中有6例表现出 包括左心室流出道梗阻的相似谱的CHD。由于Shh信号也起到了 在中枢神经系统的发育模式中起着重要作用，值得注意的是， 具有基底激活GLI转录的患者也具有脑发育不良。基于这些发现，我们 假设Shh信号负调节因子突变可能在CHD发病中起重要作用 和CHD相关的脑发育不良。为了验证这一假设，我们开展了3个目标的研究。在目标1中，我们 通过交叉患者成纤维细胞RNAseq鉴定导致CHD的负Shh调节的决定因素 以及具有在先前Shh CRISPR筛选中鉴定的Shh负调控子的全外显子组测序数据。在 目的2，我们将进行siRNA和CRISPR基因KO，以评估负调控因子对Shh的潜在作用。 发信号。在目标3中，我们将使用小鼠CRISPR基因编辑和F0创始人胚胎分析来评估小鼠CRISPR基因编辑。 负性Shh调节因子在CHD发病机制和神经发育异常中的作用表型分析 心脏和大脑异常将包括使用多普勒超声心动图的血液动力学评估， 随后使用microCT、MRI和连续组织病理学3D重建进行解剖学分析。这些 这些研究将为Shh信号的负调节如何有助于心脏提供新的见解。 冠心病的发生、发展及发病机制。这些研究也可能表明一种范式转变， Shh信号失调伴结构性心脏缺陷和神经发育异常，这些发现可能 具有长期的治疗意义。

Congenital heart disease (CHD) is one of the most prevalent birth defects, affecting up to 1% of live births. A previous mouse forward genetic screen for mutations causing CHD conducted in our laboratory recovered 91 genes causing CHD, with more than 50 being cilia and ciliary signaling related. Interestingly of the 15 genes recovered causing CHD in cilia transduced cell signaling pathways, at least 5 are associated with Sonic hedgehog (Shh) signaling. Shh signaling in fact is known to play an important role in development of the outflow tract and semilunar valve via the regulation of the cardiac neural crest and second heart field cell lineages. While the role of Shh in CHD pathogenesis is now well described in mouse models, its role in human CHD remains poorly understood. Hence, to examine the potential importance of Shh signalng in human CHD pathogenesis, we conducted a screen for defect in Shh signaling using CHD patient fibroblasts. Surprisingly, our analysis showed 17 of 41 CHD patients exhibited altered hedgehog signaling without overt ciliogenesis defects, suggesting hedgehog signaling but not ciliary structure is affected. Real time PCR analysis showed 9 of the 17 patients had abnormal basally activated transcription of GLI, a hegehog responsive gene, in the absence of pathway stimulation. This suggests disruption in the negative regulation of Shh signaling may play an important role in CHD pathogenesis. Interestingly, 6 of the 9 patients with basal GLI activation exhibited CHD of a similar spectrum comprising left ventricular outflow obstructions. As Shh signaling also plays an essential role in patterning development of the central nervous system, it is worth noting that 3 of the CHD patients with basally activated GLI transcription also had brain dysplasia. Based on these findings, we hypothesize mutations in negative regulators of Shh signaling may play a significant role in CHD pathogenesis and CHD associated brain dysplasia. To test this hypothesis, we developed studies in 3 aims. In Aim 1, we will identify determinants of negative Shh regulation contributing to CHD by intersecting patient fibroblast RNAseq and whole exome sequencing data with Shh negative regulators identified in previous Shh CRISPR screens. In Aim 2, we will conduct siRNA and CRISPR gene KO to assess the potential role of negative regulators on Shh signaling. In Aim 3, we will use mouse CRISPR gene editing and F0 founder embryo analysis to assess the role of negative Shh regulators on CHD pathogenesis and abnormal neurodevelopment. Phenotyping for cardiac and brain abnormalities will include hemodynamic assessments using Doppler echocardiography, followed by anatomical analyses using microCT, MRI, and serial histopathological 3D reconstruction. These studies will provide new insights into how negative regulation of Shh signaling may contribute to heart development and the pathogenesis of CHD. These studies also may suggest a paradigm shift linking dysregulated Shh signaling with structural heart defects and abnormal neurodevelopment, findings that may have therapeutic implications long term.