Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

Abstract

Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Gα subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11(R183C) and GNA11(Q209L) in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11(R183C) under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities.

Figure 1

Clinical examples of dermatological, ophthalmological, and neurological aspects of phakomatosis pigmentovascularis. (a–c) Dermal melanocytosis and capillary malformation (port wine stain type) on the face, extensive dermal melanocytosis on the back and legs, and capillary malformation on the sole of the right foot. (d) Bilateral scleral melanocytosis with bilateral glaucoma. Capillary malformation and hemihypertrophy are just visible on right side of face. (e) Axial T1-weighed magnetic resonance image of the brain at the level of the lateral ventricles after administration of intravenous gadolinium contrast agent showing bilateral and asymmetrical thickening and enhancement of the pia mater. (Written consent for publication obtained in all cases.)

Figure 2

Sequencing results demonstrating mosaic GNA11 and GNAQ mutations. (a) Sanger sequencing of skin biopsy showing a very low peak in GNA11 at position c.547C>T (p.Arg183Cys) (asterisk). (b) Sanger sequencing of the same skin biopsy deoxyribonucleic acid after restriction enzyme digest of the normal allele, and hemi-nested amplification, revealing the mutation (asterisk). (c) Targeted next-generation sequencing showing low allele percentage mutations in skin but undetectable in blood, GNA11 c.547C>T (p.Arg183Cys) in 5% of reads. (d) GNAQ c.548G>A (p.Arg183Gln) in 6% of reads from skin but undetectable in blood.

Figure 3

Mutant GNA11 leads to activation of downstream signaling pathways. (a) Western blot for Flag, total GNA11, and the phosphorylated and total p38, JNK, ERK and AKT in total protein lysates from HEK293T cells transfected with either vector, wild-type GNA11 (WT), or one of two mutants R183C or Q209L mutant GNA11. (b) Ratio of phosphorylated to total p38, JNK, ERK, and AKT normalized by actin loading control. Bars indicate 1 standard deviation. ∗P < 0.05, unpaired t-test.

Figure 4

Mosaic expression of GNA11 promotes ectopic pigmentary lesions in zebrafish. (a) Images of adult zebrafish mosaic for GNA11, GNA11^R183C, or GNA11^Q209L expression. Large, ectopic pigmentary lesions are indicated next to white arrows. Dashed box indicates zoomed areas that show detail of pigmentary lesions. (b) Numbers of pigmentary lesions per fish expressing GNA11, GNA11^R183C, or GNA11^Q209L. Dark circles indicate ectopic pigmentary lesions. White circles indicate fish without pigmentary lesions. (c) Histology hematoxylin and eosin staining of a wild-type and GNA11^R183C zebrafish skin at ×100 and ×400 magnification. Melanocytes are clearly visible in the dermis by the black melanin (blue arrows), frequently also in the epidermis (not shown) and in a few cases within underlying muscle (yellow arrow).