Smad6 deficient zebrafish as a model for multifactorial craniosynostosis

颅缝早闭(CS)是头面部最常见的出生缺陷之一，其发病率接近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 编码成纤维细胞生长因子受体的基因突变，以及在其他基因中发现的额外突变， 既有传播性的也有从头开始的。现有的单基因CS小鼠模型已经取得了显著的成果 对CS的分子和发育基础的洞察，并有力地说明了 准确的动物模型的价值。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 各种因素。最近的人类遗传学研究表明，其中一些基因与某些特定基因有关 复杂的案子。CS病例的全基因组关联研究(GWASs)确定了靠近 BMP2和BMP7基因。随后发现单个CS患者的完整外显子组测序(WES) 编码Smad6的下游基因Smad6的一个拷贝失活的高发突变 骨形态发生蛋白信号的抑制物。携带BMP2和一个SMAD6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是 BMP2水平升高和抑制Smad6水平降低共同导致CS。虽然这件事 假说受到了最近的研究的挑战，也有证据表明，其他基因的突变 基因可以与SMAD6相互作用，增加CS的风险。为了直接检验这些假设，我们建议 建立斑马鱼模型以研究CS潜在的复杂遗传风险因素。我们已经产生了 两个斑马鱼smad6基因的失活突变，在目标1中，我们将描述 缺少一个或两个smad6a和smad6b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风险的综合方法。首先，我们将让smad6突变体与 BMP途径抑制物的现有突变，以及与 人类病人。我们将对得到的鱼进行CS和其他头面部和骨骼的检测 组织学染色和活体共聚焦成像。第二，我们将使用过度表达 在斑马鱼胚胎中检测人类SMAD6变异体作为一种有效的测试方法，以测试其功能 在人类患者中发现的序列变异的后果。通过成功完成我们的 目标，我们将建立一个准确的模型系统的复杂遗传学基础上的大多数 CS病例。斑马鱼在头骨的所有阶段都可以进行活体成像和直接操作 和缝线的形成，提供了对CS的病理生理学的洞察。同样重要的是，该模型可以 被用来测试其他可能与smad6突变相互作用的基因，从而增加CS的风险，并将 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.