Abstract 18196: Sh2 Domain-Containing 5-Inositol Phosphatase 2 (SHIP2) is an Effector of Lymphatic Dysfunction

Abstract

Background: Discovery of variants responsible for familial lymphatic abnormalities have been limited by candidate gene searches and inaccurate phenotyping that is restricted to overt symptomatic presentation. As a result, the majority of patients do not harbor any of the known genes associated with inherited lymphatic abnormalities. Methods: We employed near-infrared fluorescence lymphatic imaging (NIRFLI) to identify asymptomatic, but abnormal lymphatic phenotypes in a nucleus family with two daughters who were previously diagnosed with congenital and praecox lymphedema. Following DNA collection and whole exome sequencing, we first sought to identify causal variants known to be associated with lymphatic abnormalities and then secondly, to discover new gene variants, through unbiased analysis of rare, damaging SNPs with co-segregation analysis that was based upon NIRFLI phenotyping. Relevancy of variants was tested through functional cell culture studies following siRNA candidate gene knock down in lymphatic endothelial cells (LECs), and following overexpression of WT and mutant candidate gene variants. Results: A damaging mutation in hepatic growth factor (HGF), expressed by blood and lymphatic endothelial cells and ligand to its ubiquitously expressed receptor tyrosine kinase (RTK) cMET was passed to both daughters from the asymptomatic father who showed no abnormal NIRFLI phenotype. Yet the mother, who at time of study was found to have abnormal lymphatics through NIRFLI and minor swelling attributed to minor trauma several years prior, had passed a mutation in SHIP2, encoded by INPPL1 , to her daughters. Extended, asymptomatic family members, who agreed to the NIRFLI procedure and harbored the SHIP2 mutation, also possessed an abnormal lymphatic phenotype. Migration and tubulogenesis in LECs was arrested by SHIP2 knock down, and when compared to WT overexpression, was attenuated following HGF and VEGF-C stimulation with mutant SHIP2. Proximity ligation assay showed intracellular association of SHIP2 with cMET and with VEGFR3 upon HGF and VEGF-C stimulation respectively, consistent with SHIP2 functionality as an RTK adaptor protein. Conclusions: SHIP2 may be an effector of lymphatic dysfunction.