Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations

Abstract

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

Keywords: Connexin; Endothelial cell; Gap junction protein; Orbital disease; Vascular malformations; Whole-cell voltage clamp.

Fig. 1

Identification of GJA4 c.121G > T (p.Gly41Cys) in OCVM tissues. a Mutational profiles of vascular anomalies in the discovery cohort. Gene names on the left belong to genes in which somatic mutations were detected. Colored cells represent somatically mutated genes, and color denotes mutation type. b MAF of GJA4 c.121G > T (p.Gly41Cys) was determined using ddPCR analysis in tissue samples from the discovery and validation cohorts. c Position of GJA4 c.121G > T (p.Gly41Cys) in GJA4 (NP_002051.2). TM, transmembrane; EL extracellular loop, IL intracellular loop. d Conservation of Gly41 position in GJA4 orthologs. H. sapiens, human (NP_002051.2); M. musculus, mouse (NP_032146.1); R. norvegicus, rat (NP_067686.1); B. taurus, cattle (NP_001077207.1); G. gallus, chicken (NP_996867.3); X. tropicalis, western clawed frog (NP_001072904.1). Sequence alignments, CLUSTALW

Fig. 2

GJA4 expression and GJA4 c.121G > T (p.Gly41Cys) mutation allele frequency in OCVM endothelial cells. a Representative MRI images of OCVM lesions (images of P15). b Hematoxylin and eosin (HE) staining (left) and immunostaining images for GJA4, endothelial cell marker CD31, and nuclear marker TOTO®-3 (middle and right) of sections of OCVM tissue from P17 and P19. Asterisks indicate vascular channels. White dotted lines indicate the positions of magnified panels (right). Arrows indicate GJA4 expression in endothelial cells, and arrowheads indicate GJA4 expression in non-endothelial cells. Black, white, and yellow scale bars indicate 200, 100, and 50 μm respectively. c Results of the quantification of mutation allele abundance using ddPCR in bulk tissue, CD31-positive and negative cells, from prospectively collected samples from 2 OCVMs (P43 and P44). Each dot represents a droplet, with blue being mutation positive, green being wild-type positive, and orange being positive for both. The boxed number is the fractional abundance of mutation allele in each sample

Fig. 3

GJA4 p.Gly41Cys increases GJA4 hemichannel activity. a Representative whole-cell voltage clamp hemichannel-recording of Xenopus oocytes injected with 1 ng of GJA4 WT or GJA4 p.Gly41Cys cRNA. b Intensity of hemichannel currents at the steady state plotted as a function of membrane voltage. The number of oocytes analyzed is given in parentheses. Data are shown as means ± SEM. c Comparison of hemichannel currents at a steady state of 50 mV membrane voltage. Data are shown as means ± SEM. d Representative whole-cell voltage clamp gap junction-recording of Xenopus oocytes injected with 1 ng GJA4 WT or GJA4 p.Gly41Cys cRNA. e A plot of normalized steady state junctional conductance versus transjunctional voltage (Gj–Vj plot) for GJA4 WT. The number of oocytes analyzed is given in parentheses. Data are shown as means ± SEM. f Plots of normalized steady state junctional conductance versus transjunctional voltage (Gj–Vj plot) for GJA4 p.Gly41Cys. The number of oocytes analyzed is given in parentheses. Data are shown as means ± SEM. g Comparison of transjunctional current at a steady state of 100 mV membrane voltage. Data are shown as means ± SEM. P value was calculated using a two-tailed t test. h Images showing the deleterious effect of GJA4 on oocytes (arrows) injected with GJA4 c.121G > T (p.Gly41Cys) compared with GJA4 WT 36 h after cRNA injections. i Plots of survival rates of oocytes injected with GJA4 WT and c.121G > T (p.Gly41Cys). The number of oocytes analyzed is given in parentheses. The averages of two replicated experiments were plotted. P values were calculated using a two-tailed t test. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

GJA4 p.Gly41Cys dysregulates endothelial cell-function, which is rescued by a connexin inhibitor. a qPCR analyses of GJA4 mRNA in HUVECs transfected with GJA4 WT or GJA4 c.121G > T (p.Gly41Cys). b ddPCR analysis of GJA4 cDNA in HUVECs transfected with GJA4 WT or GJA4 c.121G > T (p.Gly41Cys). Each dot represents a droplet, with blue being mutation positive, and green being wild-type positive. c Immunostaining for FLAG and nuclear marker DAPI of HUVECs transfected with GJA4 WT-FLAG, GJA4 c.121G > T (p.Gly41Cys)-FLAG, or normal control. Scale bars, 15 μm. d Representative image of HUVECs transfected with GJA4 WT, GJA4 c.121G > T (p.Gly41Cys), or normal control, and efficacy of CBX. Scale bars, 50 μm. e Quantification of cell viability of HUVECs transfected with GJA4 WT, GJA4 c.121G > T (p.Gly41Cys), vector control, and normal control using the MTT assay. f Representative images of tube formation assay of HUVECs transfected with GJA4 WT, GJA4 c.121G > T (p.Gly41Cys), or normal control, and the efficacy of CBX. Scale bars, 200 μm. g Quantification of tube formation in HUVECs transfected with GJA4 WT, GJA4 c.121G > T (p.Gly41Cys), and normal control by total mesh area (left) and number master junctions (right). P values were calculated using a two-tailed t test. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001