Dissecting the Cellular and Molecular Mechanisms Contributing to Craniofacial Fibrous Dysplasia

项目总结/摘要 颅面FD是最常见的和使人衰弱的骨骼发育不良之一。膨胀的纤维状骨质 在面骨和颅底中形成的损伤导致严重的残疾，包括畸形的面容， 骨质脆弱、疼痛、视力和听力丧失。FD没有有效的治疗方法， 这是一个急需医疗的领域。FD可以发生在一个骨骼（单骨型）或多个骨骼（多骨型） 并且可以与McCune-Albright综合征（MAS）相关地发生，这是一种体细胞嵌合遗传病 特征为FD、性早熟、咖啡牛奶皮肤病变、各种内分泌疾病和实体器官 恶性肿瘤。FD可以说是MAS最具临床意义的特征。FD可以在任何地点形成， 骨骼，但最常见的涉及网站是神经嵴衍生颅面骨。尽管如此， 关于驱动颅面FD的细胞和分子机制知之甚少，以及为什么会有一个 神经嵴衍生骨中FD形成的偏好。除了颅面FD，MAS患者还 通常有其他神经嵴衍生组织的马赛克参与，包括黑色素细胞皮肤病变， 垂体和肾上腺肿瘤该提案的目标是更好地了解细胞和分子 驱动颅面FD的机制，以便我们能够努力确定潜在的治疗目标。我们 将通过以下3个目标追求这一目标：目标1：测试WNT抑制是否逆转颅面FD， Col 1（2.3）+/Rs 1+小鼠。我们先前已经通过激活成骨细胞中的Gs-GPCR信号在小鼠中建立了FD模型。 细胞通过工程GPCR“Rs 1”。Col 1（2.3）+/Rs 1+小鼠出现了一种引人注目的骨表型， 人FD。我们已经表明，停止异常Gs-GPCR信号转导逆转FD病变，表明 FD是可以逆转的。我们还发现从这些组织中获得的骨骼中WNT表达水平增加， 小鼠，表明WNT信号传导在FD中很重要。我们将使用该模型来测试WNT的抑制是否 信号传导可以逆转颅面FD病变。目的2：确定人类中的GNASR 201 H突变负荷 颅面FD骨，并确定携带这种突变的细胞类型。尽管知道因果关系 尽管我们在FD/MAS中发现了突变，但我们仍然不知道哪种细胞类型在人FD骨中携带突变以及突变水平如何， 才能导致疾病我们将使用创新的方法来分离和基因分型 来自人颅面FD病变的单细胞。这将使我们能够了解人类基因突变的负担， FD病变并鉴定携带突变的细胞类型。目标3：阐明镶嵌GNASR 201 H 使用FD/MAS的人iPSC模型，突变影响细胞多能性和分化能力。 我们将使用我们新开发的MAS人iPSC模型，该模型在其基因组中含有致病性GNAS突变。 内源性位点，以探索GNAS对早期细胞命运的影响，特别是神经嵴和成骨细胞 血统总之，这些目标旨在解决我们理解中存在的关键知识差距， 颅面FD的发病机制

PROJECT SUMMARY/ABSTRACT Craniofacial FD is one of the most common and debilitating skeletal dysplasias. The expansile, fibrous bony lesions that form in the facial bones and skull base cause significant disability, including dysmorphic facies, bone fragility, pain, and vision and hearing loss. There are no effective medical treatments for FD, making this an area of critical medical need. FD can develop in one bone (monoostotic) or multiple bones (polyostotic) and can occur in association with McCune-Albright Syndrome (MAS), which is a somatic mosaic genetic disease characterized by FD, precocious puberty, café-au-lait skin lesions, various endocrinopathies, and solid organ malignancies. FD is arguably the most clinically significant feature of MAS. FD can form at any site within the skeleton, but the most commonly involved sites are the neural crest-derived craniofacial bones. Despite this, little is known about the cellular and molecular mechanisms that drive craniofacial FD, and why there is a predilection for FD formation in neural crest-derived bone. In addition to craniofacial FD, patients with MAS also commonly have mosaic involvement of other neural crest-derived tissues, including melanocytic skin lesions and pituitary and adrenal tumors. The goal of this proposal is to better understand the cellular and molecular mechanisms that drive craniofacial FD so that we can work towards identifying potential treatment targets. We will pursue this goal through the following 3 aims: Aim 1: Test if WNT inhibition reverses craniofacial FD in Col1(2.3)+/Rs1+ mice. We have previously modeled FD in mice by activating Gs-GPCR signaling in osteoblastic cells via the engineered GPCR “Rs1.” Col1(2.3)+/Rs1+ mice develop a dramatic bone phenotype that resembles human FD. We have shown that stopping the abnormal Gs-GPCR signaling reverses the FD lesions, suggesting that FD can be reversed. We also found increased levels of WNT expression in bones harvested from these mice, suggesting that WNT signaling is important in FD. We will use this model to test if inhibition of WNT signaling can reverse craniofacial FD lesions. Aim 2: Determine the GNASR201H mutational burden in human craniofacial FD bone and identify the cell type(s) that harbor this mutation. Despite knowing the causative mutation in FD/MAS, we still do not know which cell type(s) carry the mutation in human FD bone and what level of mosaicism is needed in order to cause disease. We will use an innovative approach to isolate and genotype single cells from human craniofacial FD lesions. This will allow us to understand the mutational burden of human FD lesions and identify the cell type(s) that carry the mutation. Aim 3: Elucidate how the mosaic GNASR201H mutation affects cell multipotency and differentiation capacity using a human iPSC model of FD/MAS. We will use our newly-developed human iPSC model of MAS that contains the causative GNAS mutation in its endogenous locus to explore the effects of GNAS on early cell fate, particularly the neural crest and osteoblast lineages. Together, these aims seek to address the critical knowledge gaps that exist in our understanding of the pathogenesis of craniofacial FD.