A novel somatic mutation in GNAQ in a capillary malformation provides insight into molecular pathogenesis

Abstract

Sturge-Weber syndrome (SWS) is a sporadic, congenital, neuro-cutaneous disorder characterized by a mosaic, capillary malformation. SWS and non-syndromic capillary malformations are both caused by a somatic activating mutation in GNAQ encoding the G protein subunit alpha-q protein. The missense mutation R183Q is the sole GNAQ mutation identified thus far in 90% of SWS-associated or isolated capillary malformations. In this study, we sequenced skin biopsies of capillary malformations from 9 patients. We identified the R183Q mutation in nearly all samples, but one sample exhibited a Q209R mutation. This new mutation occurs at the same residue as the constitutively-activating Q209L mutation, commonly seen in tumors. However, Q209R is a rare variant in this gene. To compare the effect of the Q209R mutation on downstream signaling, we performed reporter assays with a GNAQ-responsive reporter co-transfected with either GNAQ WT, R183Q, Q209L, Q209R, or C9X (representing a null allele). Q209L showed the highest reporter activation, with R183Q and Q209R showing significantly lower activation. To determine whether these mutations had similar or different downstream consequences we performed RNA-seq analysis in microvascular endothelial cells (HMEC-1) electroporated with the same GNAQ variants. The R183 and Q209 missense variants caused extensive dysregulation of a broad range of transcripts compared to the WT or null allele, confirming that these are all activating mutations. However, the missense variants exhibited very few differentially expressed genes (DEGs) when compared to each other. These data suggest that these activating GNAQ mutations differ in magnitude of activation but have similar downstream effects.

Keywords: GalphaQ; RNA sequencing; Signaling.

Fig. 1:

Images of a facial capillary malformation in a patient carrying the Q209R mutation. a) The clinical photo (patient 3225) shows the location of the lesion on the upper left lip. There is mild associated soft tissue hypertrophy of the involved area, at least in part due to dilation of the component vessels within the subcutis. b and c) Microscopic examination revealed dilated capillaries and small venules congested with erythrocytes (as indicated by the black arrows) distributed within the involved dermis (b, original mag 40x, scale bar 200 μm) and subcutis (c, original mag 100x, scale bar 100 μm) of the lip, characteristic of port-wine stain (hematoxylin & eosin).

Fig. 2

Q209R moderately activates downstream G-protein signaling. a For the luciferase reporter assays, we transfected human embryonic kidney cells (HEK293T) with a GNAQ-responsive luciferase reporter co-transfected with either GNAQ WT, R183Q, Q209L, Q209R, or C9X (premature termination codon). Compared to WT GNAQ or null alleles, expression of Q209R induced a 20-fold increase in the luciferase activity. Similarly, Q209L and R183Q induced increases of 50- and 10-fold, respectively (***p < 0.0001 each mutant vs. WT GNAQ, ###p ≤0.0001, ##p < 0.01 R183Q and Q209R vs. Q209L, respectively, n = 9). b Incubation with various concentrations of small molecule Gαq inhibitor YM-254890 in HEK293T cells, co-transfected with Q209R, resulted in dose-dependent decrease of luciferase activity; n = 6. c Representative blots indicate a increase of MAPK phosphorylation in cells transfected with GNAQ mutants (*p < 0.05 WT GNAQ vs. Q209L, n = 3). d Graphical representation of p-ERK/ERK signaling seen in panel c

Fig. 3

Activating mutations in GNAQ produce similar transcriptomic effects. (a) Principal component analysis of RNA-seq data produced from HMEC-1 cells transfected with WT or mutant GNAQ plasmids. (b, c) Top enriched terms for gene ontology (b) and KEGG signaling pathway (c) using the top 100 differentially expressed genes between WT and Q209R groups. Enriched terms are sorted by fold enrichment and all terms shown have p < 1E-5. (d) MA plots of RNA-seq data for pairwise comparisons between WT and mutant GNAQ (top) and comparisons between mutations (bottom). Blue circles are statistically significant DEGs, blue triangles are significant DEGs off the scale, grey circles are not significant