ORIGINAL ARTICLE Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis Anna C. Thomas1,18, Zhiqiang Zeng2,18, Jean-Baptiste Rivie`re3,18, Ryan O’Shaughnessy4, Lara Al-Olabi1, Judith St.-Onge3, David J. Atherton5,He´le`ne Aubert6, Lorea Bagazgoitia7, Se´bastien Barbarot6, Emmanuelle Bourrat8,9, Christine Chiaverini10, W. Kling Chong11, Yannis Duffourd3, Mary Glover5, Leopold Groesser12, Smail Hadj-Rabia13, Henning Hamm14, Rudolf Happle15, Imran Mushtaq16, Jean-Philippe Lacour10, Regula Waelchli5, Marion Wobser14, Pierre Vabres3,17,19, E. Elizabeth Patton2,19 and Veronica A. Kinsler1,5,19 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 pha- komatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Ga 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. R183C Q209L In vitro expression of mutant GNA11 and GNA11 in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic R183C mosaic zebrafish models expressing mutant GNA11 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. Journal of Investigative Dermatology (2016) 136, 770e778; doi:10.1016/j.jid.2015.11.027 INTRODUCTION flat, dark-blue lesions with indistinct edges overlying the but- Mongolian blue spots (or, more appropriately, dermal mela- tocks and lower back, which undergo spontaneous resolution nocytosis) are common birthmarks, seen in up to 95% of over a period of years. However, the appearances and behavior African neonates and approximately 10% of white Caucasians are not always classic (Gupta and Thappa, 2013). Some dermal (Cordova, 1981). As a result they are easily overlooked as a melanocytosis involve unusual sites, cover large areas of the possible sign of underlying genetic disease. Classically they are body surface, are more deeply pigmented, are better defined, 1Genetics and Genomic Medicine, UCL Institute of Child Health, London, Urology, Great Ormond Street Hospital for Children, London, UK; and UK; 2MRC Institute of Genetics and Molecular Medicine, MRC Human 17Dermatology, Dijon University Hospital, Dijon, France Genetics Unit & Edinburgh Cancer Research UK Centre, Edinburgh, UK; 18 These authors contributed equally to this work. 3Equipe d’Accueil 4271, Ge´ne´tique des Anomalies du De´veloppement, 19 University of Burgundy, Dijon, France; 4Livingstone Skin Research Unit, These authors contributed equally to this work. 5 UCL Institute of Child Health, London, UK; Paediatric Dermatology, Correspondence: Veronica A. Kinsler, Genetics and Genomic Medicine, UCL 6 Great Ormond Street Hospital for Children, London, UK; Department of Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. E-mail: 7 Dermatology, Nantes University Hospital, Nantes, France; Dermatology, [email protected] or Elizabeth Patton, MRC Institute of Genetics and 8 Hospital Universitario Ramo´n y Cajal, Madrid, Spain; Dermatology, Molecular Medicine, MRC Human Genetics Unit & Edinburgh Cancer 9 Saint-Louis Hospital, Paris, France; General Paediatrics, Robert-Debre´ Research UK Centre, Edinburgh, UK. E-mail: [email protected] Hospital, Paris, France; 10Dermatology, University Hospital of Nice, Abbreviations: DNA, deoxyribonucleic acid; PPV, phakomatosis pigmento- Nice, France; 11Neuroradiology, Great Ormond Street Hospital for vascularis; SWS, Sturge-Weber syndrome; WT, wild type Children, London, UK; 12Dermatology, Regensburg University Clinic, Regensburg, Germany; 13Paediatric Dermatology, Necker Enfants- Received 29 June 2015; revised 31 October 2015; accepted 2 November Malades Hospital, Paris, France; 14Dermatology, University Hospital 2015; accepted manuscript published online 14 January 2016; corrected Wuerzburg, Wuerzburg, Germany; 15Dermatology, Freiburg University proof published online 26 February 2016 Medical Center, University of Freiburg, Freiburg, Germany; 16Paediatric ª 2016 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. This is 770 Journal of Investigative Dermatology (2016), Volume 136 an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). AC Thomas et al. GNA11 and GNAQ Mosaicism and persist rather than resolve. These more atypical patterns of limbs (Chhajed et al., 2010; Jeon et al., 2013; Vidaurri-de la dermal melanocytosis can be associated with anomalies such Cruz et al., 2003), and melanoma. The latter can arise in as colocalized cleft lip (Igawa et al., 1994), ocular melanocy- choroid or conjunctiva, with melanocytoma of the optic disk tosis (Teekhasaenee et al., 1990), ocular melanoma (Shields also described (Krema et al., 2013; Shields et al., 2011; et al., 2013), lysosomal storage disorders (Hanson et al., Teekhasaenee and Ritch, 1997; Tran and Zografos, 2005). 2003), or vascular birthmarks. This latter association then Without an understanding of the genetic pathogenesis of the falls under the diagnostic group of phakomatosis pigmento- disease, targeted therapies for the congenital and neoplastic vascularis (PPV) (for clinical examples see Figure 1). Phako- aspects of the disease have been impossible. matosis pigmentovascularis is a group of sporadic disorders of When considering the genetic basis of extensive dermal unknown frequency, defined by the co-occurrence of melanocytosis and of PPV, we hypothesized that these con- pigmentary and vascular birthmarks and subclassified clini- ditions could be the result of a postzygotic mutation in a cally by the exact cutaneous phenotypes (Supplementary member of the G-protein nucleotide binding protein alpha Table S1 online) (Happle, 2013; Hasegawa and Yasuhara, subunit family. This hypothesis was generated by the rare 1979; Ota et al., 1947). Whether the subtypes are distinct concurrent description of PPV with Sturge-Weber syndrome disorders or variable expressions of a single disorder has not (SWS) (Chhajed et al., 2010; Vidaurri-de la Cruz et al., 2003), been clear as the molecular and developmental pathogenesis a vascular disorder with no pigmentary phenotype recently of PPV has been unknown. The hypothesis of non-allelic twin found to be the result of postzygotic mosaicism for activating spotting was previously expounded to explain the co- mutations in GNAQ (Shirley et al., 2013). We also hypoth- occurrence of two disparate birthmarks (Danarti and Happle, esized that an identical mutation could be present in both 2003) but was recently retracted by the author because of types of birthmarks in PPV, secondary to a single mutation in lack of supporting molecular evidence (Happle, 2013). a pluripotent progenitor cell. Extracutaneous associations of PPV can be severe, including scleral or intraocular melanocytosis (Shields et al., 2011), RESULTS glaucoma (Henry et al., 2013; Teekhasaenee and Ritch, 1997; Missense GNA11 or GNAQ mutations were found in 8 of 11 Vidaurri-de la Cruz et al., 2003), intracerebral vascular or other patients tested. In each case, these mutations were detected malformations leading to seizures and cognitive delay (Hall in affected skin (and in one case ocular tissue) at very low et al., 2007; Shields et al., 2011; Tsuruta et al., 1999; levels and were undetectable in blood, indicating postzygotic Vidaurri-de la Cruz et al., 2003), overgrowth of soft tissues or mosaicism (Table 1). When levels of mutant allele <1% were Figure 1. Clinical examples of dermatological, ophthalmological, and neurological aspects of phakomatosis pigmentovascularis. (aec) 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.) www.jidonline.org 771 AC Thomas et al. GNA11 and GNAQ Mosaicism Table 1. Clinical phenotype and GNA11 and GNAQ genotypes with percentage mosaicism (where available) of different samples from eight patients with PPV (patients 1e8) and three