Functional Characterization of the GNAQ somatic mutation causing Sturge Weber syndrome

 描述（由申请人提供）：Sturge Weber综合征（SWS）是一种散发性、先天性、神经皮肤疾病，其特征为葡萄酒色斑（毛细血管畸形），影响皮肤和大脑和脉络膜软脑膜中的异常毛细血管静脉血管，导致青光眼、癫痫发作、中风和智力残疾。1987年，Rudolf Happle假设孤立的葡萄酒斑和SWS都是由于同一个未知基因的体细胞突变，其严重程度和表现程度由体细胞突变发生时的发育时间点决定。我们最近对SWS患者的受累和未受累组织进行了全基因组序列分析，以检验Happle的假设。我们在几乎所有的SWS和孤立的葡萄酒染色样本中发现了GNAQ中相同的体细胞突变，导致编码蛋白Gaq中的p.R183Q，Gaq是一种G蛋白亚基，调节广泛的下游信号通路。我们的初步研究表明，这是一个功能获得性突变，激活一个或多个下游途径。我们的数据还表明，这种体细胞突变存在于血管内皮细胞， SWS影响组织。在这个建议中，我们将功能性地描述这种体细胞的影响， 突变使用表达突变体Gaq的诱导型形式的内皮细胞系，我们将进行无偏信号筛选以检测适当细胞背景中突变的真实下游靶标。与我们的临床同事合作，这些信号通路将通过对来自SWS患者的受影响组织样本进行免疫染色来验证。使用建立在体外试验，我们将调查的影响，突变的离散内皮细胞功能相关的血管生成。最后，我们将通过在斑马鱼发育过程中表达突变体转录本来确定GNAQ体细胞突变在体内的表型效应。这项工作代表了SWS中GNAQ体细胞突变的第一次功能验证，为未来的研究奠定了基础，同时在短期内具有显着的翻译潜力。体细胞突变可以激活已经被小分子抑制剂靶向的下游信号传导途径。因此，这种探索性的R21可能会导致新的和可测试的治疗SWS。

 DESCRIPTION (provided by applicant): Sturge Weber Syndrome (SWS) is a sporadic, congenital, neuro-cutaneous disorder characterized by a port-wine stain (capillary vascular malformation) affecting the skin and abnormal capillary venous vessels in the leptomeninges of the brain and choroid, leading to glaucoma, seizures, stroke, and intellectual disability. In 1987 Rudolf Happle hypothesized that isolated port-wine stains and SWS are both due to somatic mutation of the same unidentified gene, with the severity and extent of presentation determined by the developmental time point when the somatic mutation occurred. We recently performed genome-wide sequence analysis of affected and unaffected tissue from SWS patients to test Happle's hypothesis. We discovered the identical somatic mutation in GNAQ in nearly all SWS and isolated port-wine stain samples, leading to p.R183Q in the encoded protein, Gaq, a G protein subunit modulating a wide spectrum of downstream signaling pathways. Our preliminary studies suggest this is a gain-of-function mutation, activating one or more downstream pathways. Our data also indicates that this somatic mutation is present in endothelial cells of the SWS affected tissue. In this proposal we will functionally characterize the effects of this somatic mutation. Using an endothelial cell line expressing an inducible form of the mutant Gaq, we will perform an unbiased signaling screen to detect the authentic downstream target(s) of the mutation in the appropriate cellular context. In collaborative with our clinical colleagues, these signaling pathways will be validated by immunostaining affected tissue samples from SWS patients. Using established in vitro assays we will investigate the effect of the mutation on discrete endothelial cell functions related to angiogenesis. Finally, we will determine the phenotypic effects of the GNAQ somatic mutation in vivo by expressing the mutant transcript during zebrafish development. This work represents the first functional validation of the GNAQ somatic mutation in SWS, laying the groundwork for future studies, while holding significant translational potential in the near term. The somatic mutation may activate downstream signaling pathways that have already been targeted with small molecule inhibitors. Thus, this exploratory R21 may lead to new and testable therapy for SWS.