Cell biological determinants underlying phenotypic severity of holoprosencephaly

 描述（由申请人提供）：颅面复合体的许多结构性疾病的特征在于高度的表型变异，但这种变异的潜在原因在很大程度上是未知的。这种疾病的一个关键例子是前脑无裂畸形（HPE），它可以在人类中产生从轻微的面中部狭窄到独眼畸形的表型。Sonic hedgehog（SHH）通路中的突变是家族性HPE的主要原因。尽管具有相同的突变，但家族成员通常表现出严重程度差异很大的表型;目前尚不清楚是什么产生了这种异质性表型。HPE发生率为1/250，但由于宫内致死性，每10，000例活产仅发生1例。因此，影响HPE严重程度的决定因素将对公共卫生产生重大影响。本研究应用的目的是阐明通过实验调节SHH通路激活在HPE人群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信号的减少与模拟HPE谱的颅面表型的连续分布相关。值得注意的是，SHH信号传导和面部形态之间的关系似乎是非线性的，这表明非常轻微的SHH分子浓度范围可以成为产生高度不同表型的基础。然而，SHH信号通路本身似乎是一个二元开关，其中非常低或高的配体浓度产生很少的表型变异，而非常接近中间的浓度产生更大的表型异质性。尽管该模型可以解释HPE患者中发现的表型谱，但迄今为止尚未在遗传体内模型中得到证实。本申请旨在检验以下假设：SHH通路激活的修饰可以产生模拟HPE患者人群的面中部表型的连续谱。在目标1，Subaim 1中，我将确定HPE样谱是否可以通过HPE患者衍生的突变SHH配体的存在在鸡群中产生，所述突变SHH配体显示在体外以显性阴性方式起作用。在目标1，Subaim 2中，我将通过使用CRISPR/Cas技术用每种突变替换细胞的内源性SHH基因座并使用qPCR评估后续SHH活性水平，确定五种HPE患者来源的SHH N末端突变是否产生异质性结果。在目标2中，我将测试是否在一个等位基因系列的小鼠胚胎中调节细胞对SHH的反应有助于产生可变的HPE表型。这项研究的结果可能会提供初步的工具和/或必要的信息，用于治疗颅面缺损的新的和创新的方法的发展。

 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.