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

摘要 复杂的淋巴管异常，包括多种诊断：淋巴管扩张症、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)、 和戈勒姆斯图尔特病(GSD)，都是慢性衰弱的疾病，往往危及生命。不幸的是， 对于大多数患者，医生只能提供需要多次门诊和住院的姑息治疗 招生。缺乏分子病因学的数据，缺乏对潜在原因的了解 淋巴管异常的分子机制极大地阻碍了进一步的研究和精确医学 重点临床试验。我们的长期目标是寻找治疗复杂淋巴异常的有效方法。 这项应用的目标是发现新的致病基因/突变，并在体外和 我们在以前的研究中建立了活体模型，以确定最佳的治疗策略。我们的预赛 研究表明，多个基因在丝裂原活化蛋白激酶(MAPK)信号转导中汇聚 细胞和斑马鱼系统中的途径和建模突变概括了本质 淋巴异常患者的形态特征。我们发现有一小撮MEK/ERK 抑制剂显示了罗氏病基因突变的生物化学和形态逆转效应。 这项提案将检验这样一种假设，即对综合信息丰富的患者进行排序 多学科淋巴异常临床将公布新的类风湿疾病基因和突变，这些可能是 通过我们已建立的细胞和斑马鱼模型快速询问，以进一步研究突变 表型光谱效应与相关分子生物标记物。这项建议的具体目的是：1) 通过对复合体患者的外显子组测序发现其他类风湿病基因和突变 淋巴异常；2)描述细胞中新发现的基因和突变的分子机制 和斑马鱼模型；以及3)利用新的疾病模型进行治疗性救援，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 因为他们将回答有关遗传病因、分子机制、 和治疗选择，最重要的是，为分子实施的 用于临床试验的基于精确的疗法。这些知识将极大地促进我们对 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.