Abstract 851: Functional dissection of GNAQ and GNA11 oncogenic mutations identifies potential targeted therapy

Background: Oncogenic variants of GNAQ and GNA11 arising in embryonic or fetal development lead to a spectrum of congenital mosaic disorders including Sturge-Weber syndrome (SWS) and Phakomatosis Pigmentovascularis (PPV). Both SWS and PPV are characterised by vascular malformations in brain, skin and eye, with worsening neurological sequelae after birth suggesting a potential treatment window. Although the genetic causes are understood, and previous data in non-disease-specific cell lines have suggested MAPK activation, the functional effects of the mutations in vascular endothelium have not been studied. The characteristic finding of intracerebral intravascular calcification led us to hypothesise that intra-cellular calcium-signalling disturbances leading to localised imbalances in calcium homeostasis may be involved in disease pathogenesis. Methods: Stable cell lines were established to study cell signalling downstream of GNAQ c.548G>A, p.(R183Q) or GNA11 c.547C>T, p.(R183C) variants in endothelial cells. We re-expressed mutant GNAQ or GNA11 alleles in HEK293 cells in which both endogenous genes had been knocked out for validation purposes. Constitutive calcium signaling was evaluated by measuring inositol-monophosphate accumulation and by a NFAT-luciferase reporter assay, while ligand-stimulated calcium signaling was monitored over time following incubation with intra-cellular calcium probe Fluo-8. In a parallel complementary study, serum calcium and related indices were assayed in 35 patients with SWS or PPV, consented for research under appropriate approvals. Results: GNAQ and GNA11 variants led to marked constitutive calcium signalling in vascular endothelial cells, but not to MAPK activation. GNAQ-mutant endothelial cells also showed aberrant calcium signalling responses to GPCR ligand, which led in turn to sustained replenishment of intracellular calcium stores from the extracellular space. These calcium signalling defects could be rescued by a specific calcium channel inhibitor. Strikingly, and previously undescribed in these diseases, 43% of patients were found to have hypocalcaemia, and 20% hyperparathyroidism, currently presumed secondary but under further investigation. Conclusions: GNAQ mosaicism leads to constitutive and ligand-induced over-activation of intracellular calcium signalling, which increases influx of calcium from the extracellular to the intracellular space and could be the explanation for systemic hypocalcaemia in a substantial