Effects of Thyroxine on the Craniosynostotic Phenotype

描述（由申请人提供）：颅缝早闭（CS）是颅骨的一个或多个纤维关节过早融合，通常允许扩张的脑颅生长。在美国，CS的发病率为每2500例活产中有1例。CS的病因是多因素的，并且对于大多数临床病例来说，遗传或环境因素通常是未知的。一般的基因-环境模型提出，如果遗传易感性与某些环境暴露相结合，其影响可能是累加的，甚至是倍增的，导致严重异常的表型。遗传变异和环境暴露之间的相互作用已经研究了几个颅面异常，包括口面裂。然而，目前，很少有人知道的作用，基因-环境相互作用在调制CS表型。美国疾病控制中心国家出生缺陷预防研究已经确定了与CS相关的几个环境因素，包括甲状腺激素的循环水平。虽然这些药物在流行病学上与CS相关，但其机制尚不清楚。我们建议探索细胞，分子和形态学的影响，这些外源性因素在CS小鼠模型与已知的突变。当与对照C57 BL小鼠交配时，Twist-1靶向突变小鼠产生类似于Saethre-Chotzen CS综合征的表型。与人类的情况类似，特质表达是高度可变的，这使得它有助于建模如何修改因素 可能与CS表型的范围有关。由于CS的时间和严重程度的表达范围，我们可以具体研究损伤对细胞形态、分子表达、组织中某些活性蛋白的存在和生物体发育的影响。在目标#1中，我们提出确定致畸挑战对Twist-1 CS小鼠模型原代细胞中细胞形态、蛋白质、表观遗传和分子表达的下游影响。具体来说，我们将用甲状腺素对细胞进行传统的液相刺激，并通过q-PCR评估骨生成的各种标志物的表达。此外，将分析全基因组微阵列中TWIST下游的靶点以及这些条件下与异常骨形成或CS相关的途径。在目标#2中，我们建议在Twist-1模型中确定子宫内暴露于甲状腺素的体内形态学效应。将通过骨和缝线微结构的3D成像、颅面形态测量学、免疫组织化学、缝线组织形态测量学和全颅骨分析的组合评估结局指标。通过确定基因的致畸作用的特定途径，拟议的研究将提供一个更好的理解CS的遗传-表观遗传-环境相互作用，并帮助颅面畸形的诊断和管理。然后，这种范例可以应用于已知与CS相互作用的其他致畸剂，例如选择性5-羟色胺再摄取抑制剂、缺氧或排卵刺激药物。

DESCRIPTION (provided by applicant): Craniosynostosis (CS) is the premature fusion of one or more of the fibrous joints of the skull, which normally allow for the growth of the expanding neurocranium. In the United States, the incidence of CS is one in every 2500 live births. The etiology of CS is multifactorial, and whether genetic or environmental is often unknown for the majority of clinical cases. The general gene-environment model proposes that if a genetic predisposition is coupled with certain environmental exposures, the effects can be additive or even multiplicative, resulting in severely abnormal phenotypes. The interaction between genetic variants and environmental exposures has been studied for several craniofacial anomalies, including orofacial clefting. However, at present, very little is known about the role of gene-environment interactions in modulating CS phenotypes. The Centers for Disease Control National Birth Defects Prevention Study has identified several environmental factors associated with CS, including circulating levels of thyroid hormone. Although these agents have been epidemiologically associated with CS, the mechanism is not understood. We propose to explore the cell, molecular, and morphological effects of these exogenous factors in a CS mouse model with a known mutation. The Twist-1 targeted mutation mouse when bred with control C57BL mice produces a phenotype similar to Saethre- Chotzen CS syndrome. Similar to the human condition, trait expression is highly variable, making it useful for modeling how modifying factors might relate to the range of CS phenotypes. Because of the range of expression in timing and severity of CS, we can specifically study the effects of the insults on cellular morphology, molecular expression, presence of certain active proteins in the tissues, and development of the organism. In Aim #1 we propose to determine the downstream effects of teratogenic challenges to cell morphology, protein, epigenetic and molecular expression in the Twist-1 CS mouse model primary cells. Specifically, we will subject cells to traditional liquid-phase stimulation wih thyroxine and various markers of osteogenesis will be evaluated for expression via q-PCR. In addition, genome wide microarrays will be analyzed for targets downstream of TWIST and for pathways linked to aberrant bone formation or CS under these conditions. In Aim #2 we propose to determine the in-vivo morphological effects of in utero exposure to thyroxine in the Twist-1 model. Outcome measures will be assessed by a combination of 3D imaging for bone and suture microarchitecture, craniofacial morphometrics, immunohistochemistry, suture histomorphometry and whole mount calvaria analysis. By determination of the specific pathway of teratogenic action upon the gene, the proposed studies will provide a better understanding of the genetic-epigenetic-environment interaction for CS and aid in the diagnosis and management of craniofacial anomalies. This paradigm can then be applied to other teratogens with known interaction with CS, e.g. selective serotonin reuptake inhibitors, hypoxia, or ovulation stimulatin drugs.