Characterization of RASopathy Mutations Underlying Lymphatic Anomalies and Preparation for Clinical Development

摘要 复杂的淋巴异常，包括各种诊断：淋巴管扩张，中枢传导 淋巴异常（CCLA）、全身性淋巴异常（GLA）、卡波西样淋巴管瘤病（KLA）， 和戈勒姆斯托特病（GSD）是慢性衰弱性疾病，并且常常危及生命。不幸的是， 对于大多数病人，医生只能提供姑息治疗，需要多次门诊和住院治疗， 招生。缺乏分子病因学方面的数据，以及缺乏对潜在病因的了解， 淋巴异常的分子机制极大地阻碍了进一步的研究和精确医学 集中的临床试验。我们的长期目标是确定复杂淋巴异常的有效疗法。 本申请的目的是发现新的致病基因/突变，并在体外和体内使用。 在我们以前的研究中建立的体内模型，以确定最佳的治疗策略。我们的初步 研究揭示了多个基因会聚在促分裂原活化蛋白激酶（MAPK）信号传导上 细胞和斑马鱼系统中的途径和建模突变已经概括了 淋巴管畸形患者的形态学特征。我们已经发现少数MEK/ERK 抑制剂显示了RAS病基因突变效应的生物化学和形态学逆转。 该提议将检验以下假设： 多学科淋巴异常诊所将揭示新的RAS病基因和突变，这些可以 通过我们建立的细胞和斑马鱼模型快速询问，以进一步研究突变 表型谱效应和相关的分子生物标志物。这项建议的具体目标是：1） 通过复杂RAS患者的外显子组测序发现其他RAS病基因和突变 淋巴管异常; 2）描述新发现的基因和细胞内突变的分子机制， 和斑马鱼模型; 3）利用新的疾病模型进行治疗拯救，以探索潜在的未来 人类疾病的治疗目标。这些实验的结果将对未来产生重大影响。 领域，因为他们将回答有关遗传病因学，分子机制， 和治疗方案，最重要的是，提供有效的临床前数据， 用于临床试验的精准疗法。这些知识将大大促进我们对 不同类型的淋巴异常

ABSTRACT Complex lymphatic anomalies, which include a variety of diagnoses: lymphangiectasia, Central Conducting Lymphatic Anomaly (CCLA), Generalized Lymphatic Anomaly (GLA), Kaposiform Lymphangiomatosis (KLA), and Gorham Stout Disease (GSD), are chronically debilitating and often life-threatening diseases. Unfortunately, for most patients, physicians can offer only palliative care that requires multiple outpatient visits and hospital admissions. The absence of data on the molecular etiology, and lack of understanding of the underlying molecular mechanisms in lymphatic anomalies have greatly hampered further research and precision medicine focused clinical trials. Our long-term goal is to identify efficacious therapies for complex lymphatic anomalies. The objective of this application is to uncover novel disease-causing genes/mutations and use in vitro and in vivo models established in our previous studies to determine optimal treatment strategies. Our preliminary studies have revealed multiple genes converging on the Mitogen-Activated Protein Kinase (MAPK) signaling pathway and modeling mutations in cellular and zebrafish systems have recapitulated the essential morphological features seen in the lymphatic anomaly patients. We have found that a handful of MEK/ERK inhibitors showed the biochemistry and morphological reversal of the effects of mutations in RASopathy genes. This proposal will test the hypothesis that sequencing of highly informative patients referred by an integrated multidisciplinary lymphatic anomalies clinic will unveil novel RASopathy genes and mutations, and these can be rapidly interrogated through our established cellular and zebrafish models to further investigate the mutation phenotype spectrum effect and correlating molecular biomarkers. The specific aims of this proposal are to: 1) Discover additional RASopathy genes and mutations through exome sequencing of patients with complex lymphatic anomalies; 2) Delineate the molecular mechanisms of newly identified genes and mutations in cellular and zebrafish models; and 3) Leverage novel disease models for therapeutic rescue to explore potential future therapeutic targets for human disease. The results from these experiments will have a significant impact on the field because they will answer fundamental questions regarding the genetic etiology, molecular mechanisms, and treatment options, and most importantly, provide validated pre-clinical data for molecularly implemented precision-based therapies for clinical trials. This knowledge will significantly advance our understanding of different types of lymphatic anomalies.