Cell biological determinants underlying phenotypic severity of holoprosencephaly

前脑无裂畸形表型严重程度的细胞生物学决定因素

• 批准号: 9312664
• 负责人: Alexis Lainoff
• 金额: $ 4.08万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9312664
• 关键词: Alleles; Apoptosis; Binding; Biological; Biological Assay; Birds; Brain; CRISPR/Cas technology; Cell Culture Techniques; Cell Death; Cell Line; Cell Proliferation; Cells; Chick Embryo; Coculture Techniques; Complex; Conceptions; Congenital Abnormality; Coupled; Craniofacial Abnormalities; Defect; Development; Disease; Dominant-Negative Mutation; Embryo; Endocytosis; Etiology; Excision; Exhibits; Face; Family member; Genetic; Genotype; Heterogeneity; Heterozygote; Holoprosencephaly; Human; In Situ Hybridization; In Vitro; Infection; Ligands; Link; Live Birth; Luciferases; Measures; Mediating; Microforms; Minor; Modeling; Modification; Morphology; Mus; Mutation; N-terminal; Null Lymphocytes; Outcome; Pathway interactions; Patients; Pattern; Phenotype; Play; Population; Production; Prosencephalon; Proteins; Public Health; Publishing; Reporter; Research; Role; SHH gene; Series; Severities; Shapes; Signal Transduction; Sonic Hedgehog Pathway; Source; Testing; Time; Variant; Viral Envelope Gene; Virus; behavior change; cell behavior; craniofacial; craniofacial complex; genome editing; in vivo Model; innovation; mouse model; mutant; neuroepithelium; novel; overexpression; patient population; public health relevance; response; smoothened signaling pathway; tool

 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.

 描述（由申请人提供）：颅面复合体的许多结构性疾病的特征是高度的表型变异，但这种变异的根本原因在很大程度上是未知的。此类疾病的一个重要例子是前脑无裂畸形（HPE），它可以在人类身上产生从轻微的中面部狭窄到独眼的表型。 Sonic hidehog (SHH) 通路的突变是家族性 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如此异质的表型。 HPE 的发生率是每 250 例受孕中就有 1 例，但由于宫内致死率只有万分之一的活产率。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。本研究应用的目的是通过实验调节 SHH 通路激活来阐明 HPE 群体中线模式变化产生的机制。先前的研究表明，鸟类大脑中 SHH 信号的减少与模仿 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 SHH 分子浓度的微小范围可能是高度不同表型产生的基础。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。此应用旨在测试以下假设：SHH 通路激活的修改可以产生模仿 HPE 患者群体的连续面部中部表型。在目标 1、Subaim 1 中，我将确定是否可以通过 HPE 患者衍生的突变体 SHH 配体的存在在雏鸡群体中产生类似 HPE 的谱，该配体在体外以显性失活方式发挥作用。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 位点，并使用 qPCR 评估后续的 SHH 活性水平，从而确定五个 HPE 患者衍生的 SHH N 端突变是否会产生异质结果。在目标 2 中，我将测试在一系列等位基因小鼠胚胎中调节细胞对 SHH 的反应是否有助于产生可变的 HPE 表型。这项研究的结果可能为开发治疗颅面缺陷的新方法和创新方法提供必要的初步工具和/或信息。