Smad6 deficient zebrafish as a model for multifactorial craniosynostosis

颅缝早闭（CS）是最常见的颅面出生缺陷之一，影响近1/2000 婴儿。大约20%的病例是由单基因突变引起的;最常见的是激活性的。 编码FGF受体的基因中的突变，以及在其它基因中鉴定的另外的突变， 既有传播的也有新生的现有的单基因CS小鼠模型已经产生了显著的 深入了解CS的分子和发展基础，并作为一个强有力的说明， 准确的动物模型。然而，大多数CS病例是非综合征性的， 潜在的遗传风险更为复杂，可能还涉及与环境的相互作用。 因素最近的人类遗传学研究暗示了其中一些特定基因 复杂的案件CS病例的全基因组关联研究（GWASs）确定了邻近 BMP 2和BMP 7基因。随后对个别CS患者进行全外显子组测序（WES）， 突变的高发生率使编码Smad6的基因的一个拷贝失活， BMP信号传导抑制剂。携带BMP 2附近风险基因座和一个SMAD 6突变等位基因的个体 据报道，CS的发病率大大增加，这表明一个简单的假设， BMP 2水平增加和抑制性Smad6降低的组合导致CS。虽然这 这一假设受到了最近研究的挑战，也有证据表明，其他基因突变也可能导致基因突变。 基因可以与SMAD6相互作用以增加CS风险。为了直接验证这些假设，我们建议 开发一个斑马鱼模型来研究CS背后复杂的遗传风险因素。我们已经生成 在两个斑马鱼smad6基因失活突变，并在目标1，我们将表征 缺乏Smad6A和Smad6B的一个或两个拷贝的鱼的表型。在目标2中，我们将采取两个- 评估斑马鱼遗传CS风险的方法。首先，我们将smad6突变体与 BMP通路抑制剂的现有突变体，以及与BMP通路相关的其他基因的突变体。 人类病人我们将分析所得的鱼CS和其他颅面和骨骼 异常，使用组织学染色和实时共聚焦成像。第二，我们将使用过表达 作为一种有效的检测方法，在斑马鱼胚胎中检测人SMAD6变体的功能， 在人类患者中鉴定的序列变异的后果。通过成功完成我们的 目的，我们将建立一个精确的模型系统，为复杂的遗传学基础的大多数 CS病例。斑马鱼在头骨的所有阶段都适合体内成像和直接操作 和缝线形成，提供了深入了解CS的病理生理学。同样重要的是，该模型可以 用于检测可能与smad6突变相互作用以增加CS风险的其他基因， 提供敏感的遗传背景，以评估潜在的环境因素。

Craniosynostosis (CS) is one of the most common craniofacial birth defects, affecting nearly 1/2000 infants. About 20% of cases are caused by mutations in single genes; most common are activating mutations in the genes encoding FGF receptors, with additional mutations identified in other genes, both transmitted and de novo. Existing mouse models for single gene CS have yielded significant insights into the molecular and developmental basis of CS and serves as a powerful illustration of the value of accurate animal models. However, the majority of CS cases are nonsyndromic, and the underlying genetic risk is more complex and probably also involves interactions with environmental factors. Recent human genetics studies have implicated several specific genes in some of these complex cases. Genome wide association studies (GWASs) of CS cases identified risk loci near the BMP2 and BMP7 genes. Subsequent whole exome sequencing (WES) of individual CS patients found a high incidence of mutations inactivating one copy of the gene encoding Smad6, a downstream inhibitor of BMP signaling. Individuals carrying the risk locus near BMP2 and one SMAD6 mutant allele reportedly had a greatly increased incidence of CS, suggesting a straightforward hypothesis that the combination of increased levels of BMP2 and reduced inhibitory Smad6 causes CS. Although this hypothesis has been challenged by more recent studies, there is also evidence that mutations in other genes can interact with SMAD6 to increase CS risk. To directly test these hypotheses, we propose to develop a zebrafish model to study the complex genetic risk factors underlying CS. We have generated inactivating mutations in the two zebrafish smad6 genes, and in Aim 1 we will characterize the phenotypes of fish lacking one or both copies of smad6a and smad6b. In Aim 2, we will take a two– pronged approach to assess genetic CS risk in zebrafish. First, we will cross the smad6 mutants with existing mutants for inhibitors of the BMP pathway, and with mutants for other genes implicated in human patients. We will assay the resulting fish for CS and other craniofacial and skeletal abnormalities, using histological staining and live confocal imaging. Second, we will use overexpression of human SMAD6 variants in zebrafish embryos as an efficient assay, to test the functional consequences of sequence variants identified in human patients. Through successful completion of our aims, we will establish an accurate model system for the complex genetics underlying the majority of CS cases. Zebrafish are amenable to in vivo imaging and direct manipulations during all stages of skull and suture formation, providing insight into the pathophysiology of CS. Also importantly, the model can be used to test other genes that may interact with smad6 mutations to increase CS risk, and will provide a sensitized genetic background to assess potentially contributing environmental factors.