LETTER TO THE EDITOR See related commentary on pg 2493

Next-Generation Sequencing of Spilus–Type Congenital : Exquisite Genotype–Phenotype Correlation in Mosaic RASopathies

Journal of Investigative Dermatology (2014) 134, 2658–2660; doi:10.1038/jid.2014.195; published online 29 May 2014

TO THE EDITOR However, another nevus spilus–type on the basis of clustering of many CMN Nevus spilus is a descriptive term used phenotype has also been well described in one anatomical area. Smaller separate to denote any cutaneous lesion with in which large cafe´-au-lait macules have lesions in the same individual are often acafe´-au-lait macular background and superimposed lesions that are indistin- indistinguishable clinically from cafe´- superimposed on more pigmented areas. guishable both clinically and histopatho- au-lait macules, and are so faint that Small single nevus spilus are relatively logically from medium/large congenital they can be easily missed (Figure 1). common, and they have recently been melanocytic nevi (CMN), exhibit a wide Our primary aim in this study was to described to be due to somatic activating variety of colors and sizes (Schaffer et al., look for the genetic basis of this defined HRAS mutations (Sarin et al., 2014). 2001a, b), and continue to develop post- phenotypic subset of CMN, in the con- Larger superficial lesions with small natally in many cases. This is termed text of the recent discovery that NRAS superimposed junctional nevi in associ- nevus spilus–type CMN. In our experi- Q61K and Q61R mutations are the cause ation with phakomatosis pigmentokera- ence, there may be delayed appearance of most causes of multiple CMN (Kinsler totica have also been found to contain of the cafe´-au-lait background after birth, et al., 2013b). In particular, we were HRAS mutations (Groesser et al., 2013). but the lesion is still usually predictable interested in determining the mutation in

Figure 1. Clinical images of nevus spilus–type CMN in six different patients. The cafe´-au-lait macule background is often invisible at birth. Two separate lesions are indicated in one patient. CMN, congenital melanocytic nevi. Written consent was obtained for publication of all photographs.

Abbreviation: CMN, congenital melanocytic nevus or nevi Accepted article preview online 21 April 2014; published online 29 May 2014

2658 Journal of Investigative Dermatology (2014), Volume 134 & 2014 The Society for Investigative Dermatology VA Kinsler et al. Next-Generation Sequencing of Nevus Spilus–Type CMN

the background macular portion of the tary Table S1 online), and DNA was the sequencing files of 133 variants in nevus spilus–type CMN, working on the extracted directly by standard methods. 39 genes were reviewed manually. A hypothesis that this could be the ‘‘first Whole-exome sequencing using Nextera single mutation was found in the cafe´- hit’’inatwo-hitmodelofnevogenesis. library preparation and an ABI Hi-Seq au-lait macule and the superimposed These studies were approved by the bioanalyser was undertaken on all three CMN, with no further mutation con- appropriate Research Ethics Committee, samples from two patients, and data were firmed despite extensive analysis. These written consent was taken from partici- analyzed using an in-house pipeline mutations were missense activating pants, and the Declaration of Helsinki designed for somatic mosaicism. The mutations in NRAS in the skin, absent Principles protocols were followed. A principal governing analysis was to look from the blood, and this pattern of blood sample and skin biopsies of both for a mutation present in the cafe´-au-lait somatic mosaicism was confirmed in the cafe´-au-lait macule background and that was not present in the blood, and a the third patient by Sanger sequencing. a banal superimposed CMN were taken further mutation in the CMN not present The mutations, however, are undes- from three children with large nevus in the cafe´-au-lait or the blood. cribed so far in typical CMN, being spilus–type CMN, from a total of 17 In all, 1,991,478 variants were called 1:115256528 c.183A4C p.Q61H (two patients from our combined practices automatically in the overlying CMN, patients, Figure 2) and 1:115258745 (patients 5, 12, and 13 in Supplemen- affecting 20,693 genes. After filtering, c.37G4C p.G13R. Both mutations have

Figure 2. Sequencing results showing NRAS mutation. Next-generation sequencing reads of blood (upper left), cafe´-au-lait macule (upper centre), and overlying CMN (upper right) from the same patient showing mutation NRAS c.183A4C p.Q61H. Note the absence of mutation in the blood, and the much lower percentage of mosaicism in the cafe´-au-lait than the CMN, as would be expected from the number of affected cells in a biopsy sample. Sanger sequencing (below) confirmation of the heterozygous pQ61H mutation in CMN. CMN, congenital melanocytic nevi.

www.jidonline.org 2659 VA Kinsler et al. Next-Generation Sequencing of Nevus Spilus–Type CMN

been described at a somatic level in non- nevus spilus–type CMN (Kinsler et al., REFERENCES CMN-related malignant 2009), and on current knowledge we Bastian BC, Xiong J, Frieden IJ et al. (2002) Genetic (Forbes et al., 2008). Independently, a would consider nevus spilus–type CMN changes in neoplasms arising in congenital fourth patient (patient 17, Supplementary patients at the same risk of malignancy melanocytic nevi: differences between nodu- lar proliferations and . Am J Pathol Table S1 online) had targeted exon as other genotypes. Similarly, although 161:1163–9 capture of two skin samples, and analy- interestingly none of the 17 patients Dadzie OE, Goerig R, Bhawan J (2008) Incidental sis also revealed only the same NRAS described here have neurological abnor- microscopic foci of nevic aggregates in skin. p.Q61H mutation in both the cafe´-au- malities on magnetic resonance imaging Am J Dermatopathol 30:45–50 lait macule and the superimposed CMN. scan (Supplementary Table S1 online), Forbes SA, Bhamra G, Bamford S et al. (2008) In conclusion, nevus spilus–type these numbers are too small to draw any The Catalogue of Somatic Mutations in Cancer (COSMIC). Curr Protoc Hum Genet CMN are a phenotypically and geno- conclusions about a possible low risk of Chapter 10:Unit 10 1 typically distinct variant of CMN, and neurological phenotype. Management Groesser L, Herschberger E, Sagrera A et al. (2013) are phenotypically and genotypically of nevus spilus–type CMN should there- Phacomatosis pigmentokeratotica is caused distinct from nevus spilus maculosus fore be the same as for other CMN. by a postzygotic HRAS mutation in a multi- and papulosus due to HRAS mutations potent progenitor cell. J Invest Dermatol 133:1998–2003 (Groesser et al., 2013; Sarin et al., 2013). CONFLICT OF INTEREST The authors state no conflict of interest. Kinsler VA, Anderson G, Latimer B et al. (2013a) This further extends the exquisite Immunohistochemical and ultrastructural fea- mutation specificity of the newly tures of congenital melanocytic naevus cells characterized mosaic RASopathies. We ACKNOWLEDGMENTS support a stem-cell phenotype. Br J Dermatol found no evidence of a second mutation We thank all the patients and families who parti- 169:374–83 to explain the superimposed nevi on the cipated in this research. This work was funded by a Kinsler VA, Birley J, Atherton DJ (2009) Great grant from Caring Matters Now charity and patient Ormond Street Hospital for Children Registry macular background. It is, however, support group. VAK is funded by the Livingstone for congenital melanocytic naevi: prospec- possible that there could be a mutation Skin Research Unit, UCL Institute of Child Health. tive study 1988-2007. Part 1-epidemiology, that does not appear pathogenic to us The laboratory work was done in London, UK, and phenotype and outcomes. Br J Dermatol and to the analysis pipelines at the in Dijon, France. 160:143–50 1,2,9 present time. Alternatively, there could Veronica A. Kinsler , Kinsler VA, Thomas AC, Ishida M et al. (2013b) 3,9 4 Multiple congenital melanocytic nevi and be either a translocation that does not Sven Krengel , Jean-Baptiste Riviere , 2 5 neurocutaneous melanosis are caused by disrupt exonic DNA sequence, or a Regula Waelchli , Carolina Chapusot , postzygotic mutations in codon 61 of NRAS. 1 6 growth-promoting copy number change, Lara Al-Olabi , Laurence Faivre , J Invest Dermatol 133:2229–36 7 8 although CMN have previously been Holger A. Haenssle ,LisaWeibel, Sarin KY, McNiff JM, Kwok S et al. (2014) Activat- 6 6 documented as having few such Ge´raldine Jeudy and Pierre Vabres ing HRAS mutation in nevus spilus. J Invest Dermatol (this issue) changes on array comparative genomic 1Clinical and Molecular Genetics Unit, UCL Sarin KY, Sun BK, Bangs CD et al. (2013) Activat- Institute of Child Health, London, UK; hybridization (Bastian et al., 2002). The ing HRAS mutation in agminated spitz nevi 2Paediatric Dermatology, Great Ormond St data at the moment suggest that only arising in a nevus spilus. JAMA Dermatol Hospital for Children, London, UK; 149:1077–81 one sequence-level mutation occurs, 3Department of Dermatology, University of and that these specific NRAS mutations Lu¨beck, Lu¨beck, Germany; 4Department of Schaffer JV, Orlow SJ, Lazova R et al. (2001a) are sufficient to cause cafe´-au-lait Molecular Genetics, Dijon University Hospital, Speckled lentiginous nevus—classic congenital melanocytic nevus hybrid not Dijon, France; 5Department of Clinical macular pigmentation, which can lead the result of ‘collision’. Arch Dermatol Genetics, Dijon University Hospital, Dijon, to macroscopic nevus formation over 6 137:1655 France; Department of Dermatology, Dijon time. Other experimental evidence University Hospital, Dijon, France; Schaffer JV, Orlow SJ, Lazova R et al. (2001b) supports that nevi can evolve in this 7Department of Dermatology, University Speckled lentiginous nevus: within the way, from a cell or collection of cells Medical Centre, Goettingen, Germany and spectrum of congenital melanocytic nevi. 8 Arch Dermatol 137:172–8 in the skin not visible to the naked eye, Department of Dermatology, University Children’s Hospital, Zurich, Switzerland as nevus cells have been found in normal E-mail: [email protected] or skin in patients with acquired melano- [email protected] This work is licensed under cytic nevi and typical CMN (Dadzie 9These authors contributed equally to this work. aCreativeCommons et al., 2008; Kinsler et al., 2013a). Attribution-NonCommercial-NoDerivs 3.0 It is important to note for clinical SUPPLEMENTARY MATERIAL Unported License. To view a copy of this management that malignant melanoma Supplementary material is linked to the online license, visit http://creativecommons.org/ has been described in patients with version of the paper at http://www.nature.com/jid licenses/by-nc-nd/3.0/

2660 Journal of Investigative Dermatology (2014), Volume 134