Somatic GNAQ Mutation is Enriched in Brain Endothelial Cells in Sturge-Weber Syndrome

Abstract

Background: Sturge-Weber syndrome (SWS) is a rare congenital neurocutaneous disorder characterized by facial and extracraniofacial capillary malformations and capillary-venule malformations in the leptomeninges. A somatic mosaic mutation in GNAQ (c.548G>A; p.R183Q) was found in SWS brain and skin capillary malformations. Our laboratory showed endothelial cells in skin capillary malformations are enriched for the GNAQ mutation. The purpose of this study is to determine whether the GNAQ mutation is also enriched in endothelial cells in affected SWS brain.

Methods: Two human SWS brain specimens were fractionated by fluorescence-activated cell sorting into hematopoietic (CD45), endothelial (CD31, VE-Cadherin, and vascular endothelial growth factor receptor 2), and perivascular (platelet-derived growth factor receptor beta) cells and cells negative for all markers. The sorted cell populations were analyzed for GNAQ p.R183Q mutation by droplet digital polymerase chain reaction. SWS patient-derived brain endothelial cells were selected by anti-CD31-coated magnetic beads and cultured in endothelial growth medium in vitro.

Results: The GNAQ p.R183Q mutation was present in brain endothelial cells in two SWS specimens, with mutant allelic frequencies of 34.7% and 24.0%. Cells negative for all markers also harbored the GNAQ mutation. The mutant allelic frequencies in these unidentified cells were 9.2% and 8.4%. SWS patient-derived brain endothelial cells with mutant allelic frequencies of 14.7% and 21% survived and proliferated in vitro.

Conclusions: Our study provides evidence that GNAQ p.R183Q mutation is enriched in endothelial cells in SWS brain lesions and thereby reveals endothelial cells as a source of aberrant Gαq signaling. This will help to understand the pathophysiology of SWS, to discover biomarkers for predicting cerebral involvement, and to develop therapeutic targets to prevent neurological impairments in SWS.

Keywords: GNAQ; Sturge–Weber syndrome; capillary malformation; ddPCR; endothelial cell.

Figure 1. Brain specimens from two SWS patients

(A) Post-contrast axial T1-weighted MRI in patient 1 shows extensive left hemisphere leptomeningeal enhancement, left choroid plexus hypertrophy and marked left cerebral atrophy. (B) Axial susceptibility-weighted MRI in patient 1 shows enlarged medullary veins (arrows) and dilated deep veins (*). (C) Hematoxylin and eosin (H&E) staining of patient 1 brain tissue section shows abnormal clusters of leptomeningeal vascular channels of variable calibers. The underlying cortex demonstrates focal loss of neurons in layer 2 and subpial gliosis. Scale bar, 100μm. (D) H&E staining, viewed at higher magnification, shows some of these vascular walls are thick and rigid. Scale bar, 200μm. The histologic findings are consistent with leptomeningeal angiomatosis. (E–J) Anti human VE-cadherin, anti-human CD31 and anti-human VEGFR2 staining highlights enlarged vessels in leptomeningeal angiomas ( E,G and I, green) and shows positive staining predominately in the endothelium in the cortex (F,H and J, green) in patient 1. Nuclei counterstained with DAPI (blue). Scale bar, 100μm. (G–H) Fluorescence-activated cell sorting of freshly isolated brain cells from patient 2. Cells were sorted into hematopoietic and non-hematopoietic cells based on CD45/Glycophorin A expression (G). Non-hematopoietic cells were further fractioned into endothelial cells (using a set of anti-endothelial antibodies against CD31, VE-Cadherin and VEGFR2), PDGFRβ-positive cells and cells negative for all markers (H). Percentages of cells in each population are shown.

Figure 2. Cells isolated from SWS brain lesion

(A) Primary culture from patient 1 brain lesion. Dashed circle highlights cells with endothelial morphology. Scale bar, 50μm (B) Flow cytometric analysis of cells in the primary culture after selection using anti-CD31-coated magnetic beads: CD31-positive cells and CD31-negative cells. Black lines depict cells incubated with anti-CD31 antibody; shaded grey areas are cells incubated with isotype-matched control antibody.