Cell biological determinants underlying phenotypic severity of holoprosencephaly

 描述(申请人提供)：许多颅面复合体的结构性疾病的特征是高度的表型变异，但这种变异的潜在原因很大程度上是未知的。这种疾病的一个关键例子是无前脑畸形(HPE)，它可以在人类中产生从轻微的面中部狭窄到睫状眼的各种表型。Sonic hedgehog(SHH)途径的突变是家族性HPE的主要原因。尽管具有相同的突变，但家庭成员往往表现出严重程度大不相同的表型；目前尚不清楚是什么导致了这种不同的表型。每250个妊娠中就有1个发生HPE，但由于宫内死亡，每10,000个活产中只有1个。因此，确定影响HPE严重程度的因素将对公众健康产生重大影响。这项研究的目的是阐明通过实验调节SHH途径激活在HPE群体中产生中线图案变化的机制。先前的研究已经证明，鸟类大脑中SHH信号的减少与模仿HPE谱的头面部表型的连续分布相关。值得注意的是，SHH信号和面部形态之间的关系似乎是非线性的，这表明非常微小的SHH分子浓度范围可能是产生高度不同的表型的基础。然而，SHH信号通路本身似乎是一个二元开关，其中非常低或很高的配体浓度产生很小的表型变异，而非常接近中间的浓度产生更大的表型异质性。虽然这个模型可以解释在HPE患者中发现的表型谱，但到目前为止，它还没有在体内遗传模型中得到证实。这项应用旨在检验这样一个假设，即SHH途径激活的修饰可以产生模仿HPE患者群体的连续的面中部表型谱。在目标1，Subaim 1，我将确定是否可以通过存在HPE患者衍生的突变SHH配体在体外以显性-负向的方式在鸡群中产生HPE样谱。在目标1，Subaim 2中，我将使用CRISPR/Cas技术用每个突变替换细胞的内源性SHH基因座，并使用qPCR评估随后的SHH活性水平，以确定HPE患者衍生的五个SHH N端突变是否会产生不同的结果。在目标2中，我将测试在一系列等位基因的小鼠胚胎中调节细胞对SHH的反应是否有助于产生不同的HPE表型。这项研究的结果可能为开发治疗头面部缺陷的新的创新方法提供必要的初步工具和/或信息。

 DESCRIPTION (provided by applicant): Many structural diseases of the craniofacial complex are characterized by a high degree of phenotypic variation, but the underlying causation of such variation is largely unknown. A key example of such a disease is holoprosencephaly (HPE), which can produce phenotypes in humans ranging from minor midfacial narrowing to cyclopia. Mutations in the Sonic hedgehog (SHH) pathway are a predominant cause of familial forms of HPE. Family members often exhibit phenotypes of greatly varying severity despite possessing identical mutations; it remains unclear what produces such heterogeneous phenotypes. HPE occurs 1 in 250 conceptions, but only 1 in 10,000 live births due to intrauterine lethality. Determining factors that impact the severity of HPE would thus have a major impact on public health. The objective of this research application is to elucidate mechanisms by which variation in midline patterning is produced in the HPE population by modulating SHH pathway activation experimentally. Previous research has demonstrated that decreased SHH-signaling in the avian brain correlates with a continuous distribution of craniofacial phenotypes mimicking the HPE spectrum. Significantly, the relationship between SHH-signaling and facial morphology appears to be non-linear, suggesting that a very slight range of SHH molecule concentrations can underlie the production of highly dissimilar phenotypes. The SHH signaling pathway itself, however, appears to be a binary switch, wherein very low or high ligand concentrations produce little phenotypic variation, while concentrations very close to the middle produce greater phenotypic heterogeneity. Although this model could account for the phenotypic spectrum found in HPE patients, it has not been demonstrated in a genetic in vivo model to date. This application aims to test the hypothesis that modifications in SHH pathway activation can produce a continuous spectrum of midfacial phenotypes mimicking the HPE patient population. In Aim 1, Subaim 1, I will determine whether an HPE-like spectrum can be produced in a chick population by the presence of a HPE- patient derived mutant SHH ligand shown to act in a dominant-negative manner in vitro. In Aim 1, Subaim 2, I will determine whether five HPE patient-derived SHH N-terminal mutations produce heterogeneous outcomes by replacing the endogenous SHH locus of cells with each mutation using CRISPR/Cas technology and assessing subsequent SHH activity levels using qPCR. In Aim 2, I will test whether modulating the cellular response to SHH in an allelic series of mouse embryos contributes to the production of variable HPE phenotypes. Results from this study may provide the preliminary tools and/or information necessary for the development of new and innovative approaches for treating craniofacial defects.