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.