Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ

Abstract

Background: The Sturge-Weber syndrome is a sporadic congenital neurocutaneous disorder characterized by a port-wine stain affecting the skin in the distribution of the ophthalmic branch of the trigeminal nerve, abnormal capillary venous vessels in the leptomeninges of the brain and choroid, glaucoma, seizures, stroke, and intellectual disability. It has been hypothesized that somatic mosaic mutations disrupting vascular development cause both the Sturge-Weber syndrome and port-wine stains, and the severity and extent of presentation are determined by the developmental time point at which the mutations occurred. To date, no such mutation has been identified.

Methods: We performed whole-genome sequencing of DNA from paired samples of visibly affected and normal tissue from 3 persons with the Sturge-Weber syndrome. We tested for the presence of a somatic mosaic mutation in 97 samples from 50 persons with the Sturge-Weber syndrome, a port-wine stain, or neither (controls), using amplicon sequencing and SNaPshot assays, and investigated the effects of the mutation on downstream signaling, using phosphorylation-specific antibodies for relevant effectors and a luciferase reporter assay.

Results: We identified a nonsynonymous single-nucleotide variant (c.548G→A, p.Arg183Gln) in GNAQ in samples of affected tissue from 88% of the participants (23 of 26) with the Sturge-Weber syndrome and from 92% of the participants (12 of 13) with apparently nonsyndromic port-wine stains, but not in any of the samples of affected tissue from 4 participants with an unrelated cerebrovascular malformation or in any of the samples from the 6 controls. The prevalence of the mutant allele in affected tissues ranged from 1.0 to 18.1%. Extracellular signal-regulated kinase activity was modestly increased during transgenic expression of mutant Gαq.

Conclusions: The Sturge-Weber syndrome and port-wine stains are caused by a somatic activating mutation in GNAQ. This finding confirms a long-standing hypothesis. (Funded by the National Institutes of Health and Hunter's Dream for a Cure Foundation.).

Figure 1. Representative Photographs and Magnetic Resonance Imaging (MRI) Scans from Study Participants with the Sturge–Weber Syndrome or Isolated Port-Wine Stains

Photographs of a participant with the Sturge–Weber syndrome (Patient 36), obtained at birth, show a facial port-wine stain with a left-sided V1 distribution (Panels A and B). The child began having seizures at 7 months of age. An isolated port-wine stain birthmark on the left shoulder from a participant without the Sturge–Weber syndrome (Patient 10) shows a birthmark that is flat and red without evidence of hypertrophy or cobblestoning or any other associated vascular or lymphatic anomaly (Panel C). Axial contrast-enhanced MRI scans of the brain in a participant with the Sturge–Weber syndrome (Panels D, E, and F; Patient 36 at 17 months of age) show left-sided hemispheric leptomeningeal enhancement (yellow arrows), an enlarged and enhancing left-sided choroid plexus (red arrow), and left hemispheric brain atrophy (white arrows).

Figure 2. Downstream Effectors of Gα_q

Plasmid encoding nonmutant Gα_q, p.Arg183Gln, and p.Gln209Leu were transfected into human embryonic kidney (HEK) 293T cells. Strongly increased phosphorylation of extracellular signal-regulated kinase (ERK) is seen with Gα_q p.Gln209Leu, and weaker but marked activation with Gα_q p.Arg183Gln (P<0.05 for both comparisons) (Panel A). Increased phosphorylation of p38 is seen with Gα_q p.Gln209Leu (P<0.05) but not with Gα_q p.Arg183Gln (Panel B). Increased phosphorylation of Jun N-terminal kinase (JNK) is seen with Gα_q p.Gln209Leu (P<0.05), and weaker activation with Gα_q p.Arg183Gln (P = 0.052) (Panel C). No change in phosphorylation of AKT is seen with either the Gα_q p.Arg183Gln or the p.Gln209Leu construct (Panel D). A control for transfection efficiency, transfected into HEK 293T cells, shows similar amounts of the three transfected, Flag-tagged proteins (Panel E). A serum-response- element (SRE) luciferase assay (Panel F) shows the relative luciferase activity expressed under the control of the SRE promoter, coexpressed with GNAQ encoding p.Arg183Gln and p.Gln209Leu, as compared with nonmutant Gα_q (P<0.05 for both comparisons). AU denotes arbitrary units, the prefix p antibody recognizing phosphorylated antigen, and the prefix t antibody recognizing total antigen.