The Distinctive Genomic Landscape of Giant Congenital Melanocytic Nevi.

Congenital melanocytic nevi (CMN) are heterogeneous in their clinical appearance and implications and range in size from small to large and/or giant. Large–giant CMN (LGCMN) have an increased risk of malignant transformation, and the most severe complication manifests as melanoma of the CNS. Mutations in the oncogene NRAS are the most frequently observed; however, this postzygotic mutation is not present in all subtypes of CMN. In a study of LGCMN, Martins da Silva et al., 2017Martins da Silva V.P. Marghoob A. Pigem R. Carrera C. Aguilera P. Puig-Butillé J.A. et al.Patterns of distribution of giant congenital melanocytic nevi (GCMN): the 6B rule.J Am Acad Dermatol. 2017; 76: 689-694Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar performed RNA sequencing and multiregional sequencing (median coverage approximately ×19,000) using a targeted panel of genes (see Supplementary Materials) and discovered that ∼60% harbored an NRAS mutation, but other rare events such as gene fusions were identified. To date, there have been a limited number of CMN assessed using whole-genome (Colebatch et al., 2019Colebatch A.J. Ferguson P. Newell F. Kazakoff S.H. Witkowski T. Dobrovic A. et al.Molecular genomic profiling of melanocytic nevi.J Invest Dermatol. 2019; 139: 1762-1768Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) or whole-exome sequencing (Charbel et al., 2014Charbel C. Fontaine R.H. Malouf G.G. Picard A. Kadlub N. El-Murr N. et al.NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi.J Invest Dermatol. 2014; 134: 1067-1074Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar; Lim et al., 2020Lim Y. Shin H.T. Choi Y. Lee D.Y. Evolutionary processes of melanomas from giant congenital melanocytic nevi.Pigment Cell Melanoma Res. 2020; 33: 318-325Crossref PubMed Scopus (7) Google Scholar; Melamed et al., 2017Melamed R.D. Aydin I.T. Rajan G.S. Phelps R. Silvers D.N. Emmett K.J. et al.Genomic characterization of dysplastic nevi unveils implications for diagnosis of melanoma.J Invest Dermatol. 2017; 137: 905-909Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), with NRAS mutations reported at a high frequency in all studies (range, 80–100%). In consideration of our previous study, whereby ∼40% of LGCMN have no known driver mutation present, we sought to genomically characterize lesions from patients with LGCMN with known NRAS mutation status (mutant and wild type [WT]) to discover other potential somatic driver mutations and expand our knowledge of the mutational spectrum present in LGCMN. After institutional approval of experiments and written informed patient consent (see Supplementary Materials), we performed whole-exome sequencing of eight affected skin biopsies (lesional) from five patients with giant CMN (age range, 4–58 years) with matching unaffected skin (not available in one patient) along with germline DNA (Figure 1 and Supplementary Materials and Supplementary Table S1). In all the patients, we analyzed at least one biopsy of the largest CMN lesion, including a second biopsy of this lesion from two patients and an additional biopsy of a satellite lesion in another patient (Table 1). To ensure that high coverage was achieved across the exome region, sequencing was performed at a mean depth of ×135–204 for all CMN and matching unaffected skin and ×76–93 for germline DNA (Supplementary Table S2). The sequencing data were analyzed as previously described (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (1) Google Scholar, Stark et al., 2018Stark M.S. Tan J.M. Tom L. Jagirdar K. Lambie D. Schaider H. et al.Whole-exome sequencing of acquired nevi identifies mechanisms for development and maintenance of benign neoplasms [published correction appears in J Invest Dermatol 2018;138:2085].J Invest Dermatol. 2018; 138: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar) and described briefly in the Supplementary Materials.Table 1Mutational Spectrum Detected in Lesions from Patients with Giant CMNPatientCMN Size (cm)Biopsy IDType of LesionBody SiteDriver Mutation (MAF)Total SNVsINDELsSBS MMR Signature, %1Giant (>60 cm)D16-0062CMNTrunk—right lower backNRAS p.Q61R (28%)3593—2Giant (40–60 cm)D16-0064CMNScalpTMEM2 c.3956-2->TT (15%)4346153Giant (>60 cm)D16-0090CMNTrunk—right scapulaLFNG p.T16M (30%)44369D16-0091SatelliteTrunk— inferior abdomenLFNG p.T16M (23%)66185—4Giant (>60 cm)D16-0105CMNTrunk—left scapulaNRAS p.Q61R (23%)5111418D16-0115CMNTruck—right hipNRAS p.Q61R (29%)52129125Giant (>60 cm)D16-0110CMNTrunk—right scapulaNRAS p.Q61R (1%)5110811D16-0111CMNGenital—right upper lipNRAS p.Q61R (22%)478210Abbreviations: CMN, congenital melanocytic nevi; ID, identification document; INDEL, insertion–deletion; MAF, mutant allele frequency; MMR, mismatch repair; SBS, single-base substitution signature; SNV, single-nucleotide variant.Summary of known and potential novel driver mutations in CMN biopsies and their associated MAF (%). The total number of SNVs were approximately two-fold less than the total number of small INDELS. SBS mutational signatures (proportion present, %) were commonly associated with defects in MMR. All somatic SNVs were detected in the coding and noncoding region, including splice sites and dinucleotide variants. Open table in a new tab Abbreviations: CMN, congenital melanocytic nevi; ID, identification document; INDEL, insertion–deletion; MAF, mutant allele frequency; MMR, mismatch repair; SBS, single-base substitution signature; SNV, single-nucleotide variant. Summary of known and potential novel driver mutations in CMN biopsies and their associated MAF (%). The total number of SNVs were approximately two-fold less than the total number of small INDELS. SBS mutational signatures (proportion present, %) were commonly associated with defects in MMR. All somatic SNVs were detected in the coding and noncoding region, including splice sites and dinucleotide variants. Somatic single-nucleotide variants were identified in all the lesional samples from the exome pull-down region with ranges from 35 to 66 mutations (median, 49) or 0 to 1 mutations per megabase (Supplementary Table S3). The total number of insertion–deletion mutations was 36–185 (median, 101), which was approximately two times higher than that of the single-nucleotide variants (Supplementary Table S3). This high proportion of insertion–deletion mutations (median, 69%) is consistent with our studies of acquired nevi located on the body sites with minimal sun-exposure (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (1) Google Scholar). We confirmed the presence of the common NRASQ61R mutation in all previously known NRAS mutated lesions (Table 1 and Supplementary Table S4). Along with the common NRAS driver mutation, other mutations were present at a high mutation frequency (Supplementary Tables S5 and S6). For example, in paired samples from patient 5, a nonsynonymous mutation in Keratin 81 (KRT81; NM_002281:exon2:c.C415A:p.Q139K) occurred at an equally high mutation frequency, indicating that it co-occurred with the NRASQ61R mutation. However, the NRASQ61R mutation in biopsies of patient 5 was not at an equally high frequency, which is consistent with our previous report (Martins da Silva et al., 2019Martins da Silva V. Martinez-Barrios E. Tell-Martí G. Dabad M. Carrera C. Aguilera P. et al.Genetic abnormalities in large to giant congenital nevi: beyond NRAS mutations.J Invest Dermatol. 2019; 139: 900-908Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) (Supplementary Table S4). KRT81 has recently been found to be associated with an invasive phenotype in breast cancer cell lines (Nanashima et al., 2017Nanashima N. Horie K. Yamada T. Shimizu T. Tsuchida S. Hair keratin KRT81 is expressed in normal and breast cancer cells and contributes to their invasiveness.Oncol Rep. 2017; 37: 2964-2970Crossref PubMed Scopus (12) Google Scholar); however, the p.Q139K is not predicted to be deleterious (Supplementary Table S5). It is interesting to speculate whether KRT81 is playing a more dominant role in the CMN development in this patient. Regarding the two patients with WT NRAS (patients 2 and 3), by determining the highest protein-altering mutation frequency, we identified potential novel driver mutations (Supplementary Tables S4 and S5). In patient 3, we found the p.T16M mutation (NM_002304:exon3:c.147C>T:p.T16M) in the LFNG gene. Importantly, the LFNGT16M mutation (in silico predicted to be deleterious [Supplementary Table S5]), was present in the largest CMN lesion (D16-0090) and in the satellite lesion (D16-0091) (Figure 1). LFNG is a member of the NOTCH-signaling pathway and plays a crucial role in somitogenesis during embryogenic development (Sparrow et al., 2006Sparrow D.B. Chapman G. Wouters M.A. Whittock N.V. Ellard S. Fatkin D. et al.Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype.Am J Hum Genet. 2006; 78: 28-37Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Interestingly, a loss of LFNG was recently found to be associated with promoting melanoma metastasis (Del Castillo Velasco-Herrera et al., 2018Del Castillo Velasco-Herrera M. van der Weyden L. Nsengimana J. Speak A.O. Sjöberg M.K. Bishop D.T. et al.Comparative genomics reveals that loss of lunatic fringe (LFNG) promotes melanoma metastasis.Mol Oncol. 2018; 12: 239-255Crossref PubMed Scopus (12) Google Scholar; Raimo et al., 2016Raimo M. Orso F. Grassi E. Cimino D. Penna E. De Pittà C. et al.miR-146a exerts differential effects on melanoma growth and metastatization.Mol Cancer Res. 2016; 14: 548-562Crossref PubMed Scopus (28) Google Scholar), possibly acting through the NOTCH/PTEN/Akt pathway (Raimo et al., 2016Raimo M. Orso F. Grassi E. Cimino D. Penna E. De Pittà C. et al.miR-146a exerts differential effects on melanoma growth and metastatization.Mol Cancer Res. 2016; 14: 548-562Crossref PubMed Scopus (28) Google Scholar). Because nevi are primarily driven by MAPK pathway activation through BRAF/NRAS mutations, conceivably, the LFNG mutation points to an alternative pathway to nevogenesis. In the other patient with WT NRAS (D16-0064), we found a splice-site mutation (NM_013390: exon24: c.3956-2->TT) in the cell migration inducing HYAL2 gene (CEMIP2/TMEM2) (Supplementary Tables S4 and S5). This mutation was found to have the highest mutation frequency in this sample (15%). Unfortunately, the presence of the TMEM2 mutation could not be assessed in any additional biopsy (Figure 1); however, copy number loss (loss of heterozygosity) was observed in D16-0090 (Supplementary Table S8). TMEM2 is known to promote metastasis in breast cancer (Lee et al., 2016Lee H. Goodarzi H. Tavazoie S.F. Alarcon C.R. TMEM2 is a SOX4-regulated gene that mediates metastatic migration and invasion in breast cancer.Cancer Res. 2016; 76: 4994-5005Crossref PubMed Scopus (41) Google Scholar), and more recently, TMEM2 has been tightly linked to the MAPK pathway, specifically mediated through extracellular signal–regulated kinase/p38 signaling (Schinzel et al., 2019Schinzel R.T. Higuchi-Sanabria R. Shalem O. Moehle E.A. Webster B.M. Joe L. et al.The hyaluronidase, TMEM2, promotes ER homeostasis and longevity independent of the UPRER.Cell. 2019; 179: 1306-1318.e18Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Because CMN are largely driven by MAPK signaling, the splice-site mutation in TMEM2 would be a strong candidate. The role of these potential driver mutations in LFNG and TMEM2 in CMN development is yet unknown and warrants further investigation both at the functional level and in an expanded cohort of NRAS WT CMN. Next, we performed a somatic mutation signature analysis using an updated mutation signature framework (Alexandrov et al., 2020Alexandrov L.B. Kim J. Haradhvala N.J. Huang M.N. Tian Ng A.W. Wu Y. et al.The repertoire of mutational signatures in human cancer.Nature. 2020; 578: 94-101Crossref PubMed Scopus (641) Google Scholar). In a previous study of acquired melanocytic nevi (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (1) Google Scholar), the UV-related single-base substitution (SBS) 7 signature was a dominant feature in predominantly sun-exposed nevi and was absent in the nevi with limited or no sun-exposure. In this study, there were no classical UVR signature mutations (SBS7) detected. The lack of SBS7 signatures reflects the congenital origin of these lesions, which are not induced by UVR exposure. Instead of UV-related SBS7 signatures, the signatures associated with defects in mismatch repair (MMR) (SBS15, SBS21, SBS44) were the most frequently observed (combined total, 6 of 8 or 75%; Table 1 and Supplementary Table S7). MMR-related signatures are also common in acquired nevi and are often present in the absence of SBS7 (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (1) Google Scholar). In the satellite CMN of patient 3 (D16-0091; Figure 1), no detectable MMR signatures were found, whereas the larger lesion had MMR present, thus highlighting the heterogeneous nature of the giant CMN (Figure 1). The precise cause of the MMR signatures is unknown because there were no corresponding somatic mutations present, but patients carried rare germline variants in MMR pathway genes such as MSH2, MSH3, PMS1, and PMS2 (data not shown). In further supportive evidence, signatures relating to MMR have been previously detected in CMN (Colebatch et al., 2019Colebatch A.J. Ferguson P. Newell F. Kazakoff S.H. Witkowski T. Dobrovic A. et al.Molecular genomic profiling of melanocytic nevi.J Invest Dermatol. 2019; 139: 1762-1768Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), which suggests that the MMR pathway contributes to congenital nevogenesis. The next most frequent signatures were SBS39 (63%) and the ubiquitous SBS5 (50%; both have unknown etiology [Alexandrov et al., 2020Alexandrov L.B. Kim J. Haradhvala N.J. Huang M.N. Tian Ng A.W. Wu Y. et al.The repertoire of mutational signatures in human cancer.Nature. 2020; 578: 94-101Crossref PubMed Scopus (641) Google Scholar]) and SBS32 (50%; Table 1 and Supplementary Table S6). SBS32 is commonly found in squamous cell carcinomas derived from patients with transplantation that have been treated with the immune-suppressive drug azathioprine (Inman et al., 2018Inman G.J. Wang J. Nagano A. Alexandrov L.B. Purdie K.J. Taylor R.G. et al.The genomic landscape of cutaneous SCC reveals drivers and a novel azathioprine associated mutational signature.Nat Commun. 2018; 9: 3667Crossref PubMed Scopus (109) Google Scholar). However, because SBS32 has also been detected in acquired melanocytic nevi with no reported azathioprine therapy (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (1) Google Scholar), it can be suggested that this signature may also result from an unknown process that remains to be elucidated. Finally, we also determined the degree of copy number aberrations (CNAs) in the samples with matching normal skin available (Supplementary Tables S8 and S9). Atypical nodular proliferations, arising from congenital nevi, are known to contain CNAs (Bastian et al., 2002Bastian B.C. Xiong J. Frieden I.J. Williams M.L. Chou P. Busam K. et al.Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and melanomas.Am J Pathol. 2002; 161: 1163-1169Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The total CNAs ranged from 6 to 649 and were mostly in small regions of loss and/or gain (mean, 530 kilobases), which is in contrast to acquired nevi (mean, 8.3 megabases) (Stark et al., 2018Stark M.S. Tan J.M. Tom L. Jagirdar K. Lambie D. Schaider H. et al.Whole-exome sequencing of acquired nevi identifies mechanisms for development and maintenance of benign neoplasms [published correction appears in J Invest Dermatol 2018;138:2085].J Invest Dermatol. 2018; 138: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar). Heterogeneity of the CMN was also apparent in paired patient samples, with broad ranges of CNAs observed (Supplementary Tables S8 and S9). Copy number loss was observed in chromosomal regions encompassing known oncogenes (BRAF, SETDB1, MDM2, RAC1, PARP1, and DDX43) as well as tumor suppressor genes (TP53 and PPP6C) (Supplementary Tables S8 and S9). We have previously noted balanced CNA events in acquired melanocytic nevi (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (1) Google Scholar, Stark et al., 2018Stark M.S. Tan J.M. Tom L. Jagirdar K. Lambie D. Schaider H. et al.Whole-exome sequencing of acquired nevi identifies mechanisms for development and maintenance of benign neoplasms [published correction appears in J Invest Dermatol 2018;138:2085].J Invest Dermatol. 2018; 138: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar), which again was the norm in this collection of CMN. Conversely, there were no imbalanced CNA events, which are common in melanoma. In summary, we have discovered potential novel driver mutations in WT NRAS giant CMN, representing alternative mechanisms for the development of these lesions. In addition, we found that giant CMN show a distinctive molecular profile comprising mutational signatures associated with MMR genes and short CNAs. Further studies are necessary to explore the role of LFNG, TMEM2, and MMR genes in giant CMN development. Alignment files (.bam) generated in this study have been submitted to the European Genome-phenome Archive (https://ega-archive.org/) under accession number EGAS00001004541. Mitchell S. Stark: http://orcid.org/0000-0002-4510-2161 Gemma Tell-Martí: http://orcid.org/0000-0003-2728-6961 Vanessa Martins da Silva: http://orcid.org/0000-0002-0386-570X Estefania Martinez-Barrios: http://orcid.org/0000-0002-5856-1906 Neus Calbet-Llopart: http://orcid.org/0000-0003-0443-3025 Asunción Vicente: http://orcid.org/0000-0002-6077-728X Richard A. Sturm: http://orcid.org/0000-0003-1301-0294 H. Peter Soyer: http://orcid.org/0000-0002-4770-561X Susana Puig: http://orcid.org/0000-0003-1337-9745 Josep Malvehy: http://orcid.org/0000-0002-6998-914X Cristina Carrera: http://orcid.org/0000-0003-1608-8820 Joan A. Puig-Butillé: http://orcid.org/0000-0003-4345-9631 HPS is a shareholder of MoleMap New Zealand Limited and e-derm-consult Gesellschaft mit beschränkter Haftung and undertakes regular teledermatological reporting for both companies. HPS is a medical consultant for Canfield Scientific, MetaOptima Technology, and Revenio Research Oy and a medical advisor for First Derm. The remaining authors state no conflicts of interest. We thank the study participants. Part of this research was carried out at the Translational Research Institute, Woolloongabba, Queensland, Australia. The Translational Research Institute is supported by a grant from the Australian Government . The research at the Melanoma Unit from Hospital Clinic of Barcelona is partially funded by grants PI15/00716, PI15/00956, and PI18/00959 from Fondo de Investigaciones Sanitarias, Spain; by the Spanish Federation of Neuromuscular Disease; by the Spanish Federation of Rare Diseases; by Isabel Gemio Research Foundation of muscular dystrophy and other rare diseases through the Call for Research Projects on Rare Diseases 2014 through the initiative “We Are Rare, All Are Unique, by the CIBER de Enfermedades Raras of the Instituto de Salud Carlos III, Spain, co-funded by ISCIII-Subdirección General de Evaluación and European Regional Development Fund (ERDF), a way to build Europe”; by Agency for Management of University and Research Grants 2017_SGR_1134 of the Catalan Government, Spain; by the European Commission under the Seventh Framework Programme (DiagnOptics); by Centres de Recerca de Catalunya Programme/Generalitat de Catalunya, Spain; and by the Leo Messi Foundation. Part of the work was developed at the building Centro Esther Koplowitz, Barcelona, Spain. NCL is the recipient of a PhD Fellowship (FPU17/05453) from Ministerio de Educación, Cultura y Deportes, Spain. CC is the recipient of a research personal grant from Catalan Government through the Pla estratègic de recerca i innovació en salut 2018–2020, Ref. BDNS 357800. This work was partially funded by a grant from the Merchant Charitable Foundation and was supported by the National Health and Medical Research Council Centre of Research Excellence for the Study of Naevi ( APP1099021 ). MSS holds a National Health and Medical Research Council Fellowship (APP1106491), and HPS holds a National Health and Medical Research Council Medical Research Future Fund Next Generation Clinical Researchers Program Practitioner Fellowship (APP1137127). Conceptualization: MSS, JAPB; Data Curation: VMdS, AV, CC, GTM, EMB, NCL, MSS, JAPB, RAS, JM, SP; Formal Analysis: MSS, GTM, JAPB; Funding Acquisition: HPS, JAPB, CC, JM, SP; Writing - Original Draft Preparation: MSS; Writing - Review and Editing: MSS, GTM, VMdS, EMB, NCL, AV, RAS, HPS, SP, JM, CC, JAPB Patients diagnosed with large or giant congenital melanocytic nevi (CMN) who attended the Hospital Clinic of Barcelona, Spain, between 2013 and 2015 were included. Skin biopsy samples were taken from different phenotypic areas as indicated in Figure 1. Clinical information (sex, age at the time of biopsy, previous history of melanoma or neurocutaneous melanosis) was collected in all the patients. Patients were classified according to the Krengel classification (Krengel et al., 2013Krengel S. Scope A. Dusza S.W. Vonthein R. Marghoob A.A. New recommendations for the categorization of cutaneous features of congenital melanocytic nevi.J Am Acad Dermatol. 2013; 68: 441-451Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and those with giant CMN were classified by the B6 classification (Martins da Silva et al., 2017Martins da Silva V.P. Marghoob A. Pigem R. Carrera C. Aguilera P. Puig-Butillé J.A. et al.Patterns of distribution of giant congenital melanocytic nevi (GCMN): the 6B rule.J Am Acad Dermatol. 2017; 76: 689-694Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). All CMN were classified as classic CMN. Written informed consent was obtained from patients, or from the patients’ parents and/or guardians, and the study was approved by the medical ethics committee of the Hospital Clinic of Barcelona and complied with the Declaration of Helsinki Principles. Patients, or the patients’ parent and/or guardian, consented for the publication of their images where presented in this study (except patient 4). DNA was extracted from fresh tissue using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was quantified using a Qubit dsDNA BR Assay Kit (Invitrogen, Waltham, MA). Exome capture was performed using the Agilent SureSelect Target Enrichment System, Human All Exon V5+UTRs kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer’s instructions. Sequencing of germline DNA from the five study participants along with normal skin (4 of the 5 participants) and congenital nevi biopsies (seven in total, with two paired specimens) was carried out on an Illumina NovaSeq6000 platform (Illumina, CA) with paired-end 100 basepair reads following Illumina-provided protocol by a fee-for-service provider (Macrogen, Seoul, Republic of Korea). Coverage statistics for the capture regions were generated with Genome Analysis Toolkit, version 3.1-1 (McKenna et al., 2010McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. et al.The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (12687) Google Scholar). The analysis pipeline has been previously described (Stark et al., 2018Stark M.S. Tan J.M. Tom L. Jagirdar K. Lambie D. Schaider H. et al.Whole-exome sequencing of acquired nevi identifies mechanisms for development and maintenance of benign neoplasms [published correction appears in J Invest Dermatol 2018;138:2085].J Invest Dermatol. 2018; 138: 1636-1644Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Somatic variants present in the CMN and normal skin biopsies were determined by filtering out all the variants that were present in the matching blood‒derived germline DNA, followed by all variants that were present in the European population in the 1,000 genomes project (version 1000g2014oct_eur) and Exome Aggregation Consortium Non-Finnish European databases. Variants present in Single Nucleotide Polymorphism Database (version 138) were not used as a filter owing to the presence of somatic mutations (e.g., BRAFV600E). Accordingly, a proportion of the variants presented in Supplementary Table S6 may be polymorphisms. Next, to generate a list of somatic mutations that were present only in the CMN biopsy, we pooled all the somatic mutations that were found in the four normal skin biopsies and removed these from the CMN data. To minimize the chance of a rare variant or commonly occurring somatic variant (potentially sequencing artifact), any variant present in a pool of 30 normal skin samples (Stark et al., 2018Stark M.S. Tan J.M. Tom L. Jagirdar K. Lambie D. Schaider H. et al.Whole-exome sequencing of acquired nevi identifies mechanisms for development and maintenance of benign neoplasms [published correction appears in J Invest Dermatol 2018;138:2085].J Invest Dermatol. 2018; 138: 1636-1644Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) were also removed from the CMN samples, resulting in a list of somatic variants present only in the CMN. Next, all the somatic variants present in the nevi were filtered further according to the criteria previously described (Stark et al., 2018Stark M.S. Tan J.M. Tom L. Jagirdar K. Lambie D. Schaider H. et al.Whole-exome sequencing of acquired nevi identifies mechanisms for development and maintenance of benign neoplasms [published correction appears in J Invest Dermatol 2018;138:2085].J Invest Dermatol. 2018; 138: 1636-1644Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and are presented in Supplementary Table S6. The common NRAS Q61R somatic mutation was known to be present in the CMN samples D16-0062, D16-0105, D16-0115, D16-0110, and D16-0111 and confirmed to be absent (NRAS wild type) in D16-0064, D16-0090, and D16-0091. The NRAS wild-type samples were also known to be the wild type for somatic mutations in BRAF, AKT1, EGFR, GNAS, ALK, ERBB2, KIT, APC, FBXW7, KRAS, STK11, PDGFRA, TP53, PIK3CA, BRAF, FGFR2, MAP2K1, PTEN, CDH1, FOXL2, MET, CTNNB1, GNAQ, MSH6, SRC, and SMAD4 (Martins da Silva et al., 2019Martins da Silva V. Martinez-Barrios E. Tell-Martí G. Dabad M. Carrera C. Aguilera P. et al.Genetic abnormalities in large to giant congenital nevi: beyond NRAS mutations.J Invest Dermatol. 2019; 139: 900-908Abstract Full Text Full Text PDF PubMed Google Scholar). We confirmed the NRAS Q61R mutation in 5 of the 5 CMN; however, because D16-0110 was known to have a low mutant frequency (Martins da Silva et al., 2019Martins da Silva V. Martinez-Barrios E. Tell-Martí G. Dabad M. Carrera C. Aguilera P. et al.Genetic abnormalities in large to giant congenital nevi: beyond NRAS mutations.J Invest Dermatol. 2019; 139: 900-908Abstract Full Text Full Text PDF PubMed Google Scholar), it was not present in the stringently filtered list of mutations (Supplementary Table S6) and was only detectable in the Binary Alignment Map file (visualized using the Integrative Genome Viewer [Broad Institute, Cambridge, CA]) (Supplementary Table S4) Filtered somatic single-nucleotide variants present in the CMN were imported into the deconstructSigs (Rosenthal et al., 2016Rosenthal R. McGranahan N. Herrero J. Taylor B.S. Swanton C. DeconstructSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and patterns of carcinoma evolution.Genome Biol. 2016; 17: 31Crossref PubMed Scopus (494) Google Scholar) package using R 3.4.0 for Windows (https://github.com/raerose01/deconstructSigs). Mutation signatures were determined as previously described (Stark et al., 2020Stark M.S. Denisova E. Kays T.A. Heidenreich B. Rachakonda S. Requena C. et al.Mutation signatures in melanocytic nevi reveal characteristics of defective DNA repair [e-pub ahead of print].J Invest Dermatol. 2020; (accessed 6 March 2020)https://doi.org/10.1016/j.jid.2020.02.021Abstract Full Text Full Text PDF Scopus (2) Google Scholar). Copy number aberrations were determined through the CNVkit (Talevich et al., 2016Talevich E. Shain A.H. Botton T. Bastian B.C. CNVkit: Genome-wide copy number detection and visualization from targeted DNA sequencing.PLoS Comput Biol. 2016; 12e1004873Crossref PubMed Scopus (573) Google Scholar) package (https://github.com/etal/cnvkit) and run using Python 2.7. Only the CMN that had matching normal skin were able to be analyzed for copy number aberrations. Briefly, the CMN (D16-0062, D16-0064, D16-0090, D16-0091, D16-0105, and D16-0115) and matching normal skin Binary Alignment Map files, with duplicates marked and sorted (see methods above) were analyzed within the CNVkit according to the standard methods (Talevich et al., 2016Talevich E. Shain A.H. Botton T. Bastian B.C. CNVkit: Genome-wide copy number detection and visualization from targeted DNA sequencing.PLoS Comput Biol. 2016; 12e1004873Crossref PubMed Scopus (573) Google Scholar). A segmentation file (Supplementary Table S8) was compiled from all the lesions. Genes involved in regions of gain (copy number of ≥3) and loss (copy number of ≤1), summarized in Supplementary Table S9, were those commonly mutated in melanoma (Cancer Genome Atlas Network, 2015Cancer Genome Atlas NetworkGenomic classification of cutaneous melanoma.Cell. 2015; 161: 1681-1696Abstract Full Text Full Text PDF PubMed Scopus (1635) Google Scholar). Download .xlsx (.47 MB) Help with xlsx files Supplementary Data