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Somatic Embryonic FGFR2 Mutations in Keratinocytic Epidermal Nevi

2016; Elsevier BV; Volume: 136; Issue: 8 Linguagem: Inglês

10.1016/j.jid.2016.03.040

1523-1747

Agustí Toll, Luis C. Fernández, Tirso Pons, Leopold Groesser, Ana Sagrera, Enrique Carrillo de Santa Pau, Asunción Vicente, Eulàlia Baselga, Miguél Vázquez, Sergi Beltrán, David G. Pisano, Daniel Rueda, Marta Gut, Ramón M. Pujol, Christian Hafner, Marta Gut, Alfonso Valencia, Francisco X. Real,

Skin and Cellular Biology Research

Keratinocytic epidermal nevi (KEN) frequently harbor somatic mutations in FGFR3, PIK3CA, HRAS, NRAS, and KRAS. We performed whole exome sequencing (WES) to discover additional causes of KEN in lesions that were wild type for hotspot codons of these genes. We now report somatic mutations in fibroblast growth factor receptor 2 (FGFR2) in 4 of 23 KEN. Existing evidence and computational analyses of the predicted mutations found in KEN strongly support their pathogenic role. We estimate that 5–10% of KEN are caused by embryonic FGFR2 mutations. Our findings emphasize the notion that multiple genes involved in cell signaling can contribute to KEN. Epidermal nevi (EN) are hamartomatous proliferations of the skin epithelium, including keratinocytes, sebocytes, pilosebaceous units, and eccrine or apocrine glands. EN includes nonorganoid KEN, nevus sebaceous, and nevus comedonicus. Approximately 40% of KEN harbor postzygotic activating mutations in FGFR3 and PIK3CA (Hafner et al., 2007Hafner C. Lopez-Knowles E. Luis N.M. Toll A. Baselga E. Fernandez-Casado A. et al.Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern.Proc Natl Acad Sci USA. 2007; 104: 13450-13454Crossref PubMed Scopus (172) Google Scholar, Hernandez et al., 2007Hernandez S. Toll A. Baselga E. Ribe A. Azua-Romeo J. Pujol R.M. et al.Fibroblast growth factor receptor 3 mutations in epidermal nevi and associated low grade bladder tumors.J Invest Dermatol. 2007; 127: 1664-1666Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and an additional 40% are caused by postzygotic activating RAS mutations, with a predominance of the HRAS G13R substitution (Hafner et al., 2012Hafner C. Toll A. Gantner S. Mauerer A. Lurkin I. Acquadro F. et al.Keratinocytic epidermal nevi are associated with mosaic RAS mutations.J Med Genet. 2012; 49: 249-253Crossref PubMed Scopus (75) Google Scholar). To identify additional genes causing KEN, we performed WES of the skin lesion and blood leukocytes from three patients whose KEN was wild type for mutational hotspots of FGFR3, PIK3CA, and RAS (Hafner et al., 2012Hafner C. Toll A. Gantner S. Mauerer A. Lurkin I. Acquadro F. et al.Keratinocytic epidermal nevi are associated with mosaic RAS mutations.J Med Genet. 2012; 49: 249-253Crossref PubMed Scopus (75) Google Scholar). The study was approved by the ethics committees of the participating institutions and performed according to the Declaration of Helsinki. All patients, or their legal representative if minor, gave written informed consent to the study and gave permission to publish images and case histories. WES disclosed two different FGFR2 mutations in the lesions of two of three patients that were undetectable in leukocyte DNA at the sequencing depth used. Patient 1 harbored a p.Y376C (c.1127A>G) (exon 9) mutation, whereas patient 2 harbored a p.S252W (c.755C>G) (exon 6) mutation (Figure 1). Additional details of the WES analyses are provided as Supplementary Materials online. Both mutations and their somatic nature were validated using Sanger sequencing of both strands of amplicons (Figure 1C and G, Supplementary Figure S1 online). The p.Y376C (c.1127A>G) and p.S252W (c.755C>G) mutations are predicted as damaging according to several bioinformatics algorithms (i.e., sorting intolerant from tolerant prediction tool, Polyphen-2, Mutation Assesor, and functional analysis through hidden Markov models) (Supplementary Table S1 online). We then assessed the prevalence of FGFR2 mutations in 20 additional KEN that were wild type for the genes known to cause EN. We used Sanger sequencing to interrogate exons 6, 9, 12, and 14, where the most common hotspot somatic mutations have been reported in human cancer (http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=FGFR2#histo). The clinical-pathological characteristics of these patients are summarized in Table 1. Two additional FGFR2-mutant KEN were identified. Patient 4 had a widespread KEN, with a phenotype consistent with macrocephaly-capillary malformation, and harbored a somatic p.Y376C (c.1127A>G) FGFR2 mutation (Figure 1G). Patient 5 had a conventional KEN, involving the head and neck region, harboring a p.P286S (c.857C>T) somatic mutation (Supplementary Figure S2 online). Altogether, we identified FGFR2 mutations in 4 of the 23 samples tested.Table 1Clinical characteristics of the KEN cases included in this study and mutation findingsPatientAgeGenderHistologyLocalizationExtracutaneous anomaliesExons sequencedSequence1 (ATN 10)5FHead, trunk, extremities (right > left)YES1Twisted hair, right eye bulging.All (WES)p.Y376C3The amino acid numbering refers to the FGFR2IIIb isoform, RefSeq ID: NM_022970.3, and Ensembl Transcript ID: ENST00000457416, which is the main transcript expressed in KEN.(c.1127A>G)2 (ATN 30)27FKEN with comedosBreastNOAll (WES)p.S252W(c.755C>G)3 (ATN 5)8FVerrucous KENLeft hemifaceNOAll (WES)WT4 (ATN 29)12FExternal area right leg, internal area left arm, right arm, chestYES2Macrocephaly + Arnold Chiari type II malformation + toe syndactyly + scoliosis + overgrowth of hands and feet + capillary malformation.6, 9p.Y376C(c.1127A>G)5 (EN 4120/96)16FKENTrunk (Blaschko)NO6, 9p.P286S(c.857C>T)6 (13084/07)22FKENTrunk (Blaschko)NO6, 9, 12, 14WT7 (EN 12003/08)16MPigmented KENNeck6, 9, 12, 14WT8 (EN 20162/01)17MPigmented KENHeadna6, 9, 12, 14WT9 (EN 1173/98)28FKENNeckna6, 9, 12, 14WT10 (EN 18563/00)7MKENHeadna6, 9, 12, 14WT11 (35901-08)7FKENHeadna6, 9, 12, 14WT12 (31753-08)11MKEN with lymphatic vesselTrunkna6, 9, 12, 14WT13 (256812-05)15MVerrucous KENHeadna6, 9, 12, 14WT14 (249388-05)13MVerrucous KENHeadna6, 9, 12, 14WT15 (242030-05)1FVerrucous KENArmna6, 9, 12, 14WT16 (20002-01)16FKENNeckna6, 9, 12, 14WT17 (180780-05)18FVerrucous KENLower legna6, 9, 12, 14WT18 (15012-99)9FIrritated KENHeadNO6, 9, 12, 14WT19 (13897-04)16FnaRight armNO6, 9, 12, 14WT20 (15190-99)1FKENHeadNO6, 9, 12, 14WT21 (567449-09)13FVerrucous KENHeadna6, 9, 12, 14WT22 (256812-05)15MVerrucous KENHeadna6, 9, 12, 14WTAbbreviations: KEN, keratinocytic epidermal nevus; na, no available information; WES, whole exome sequencing; WT, wild type.1 Twisted hair, right eye bulging.2 Macrocephaly + Arnold Chiari type II malformation + toe syndactyly + scoliosis + overgrowth of hands and feet + capillary malformation.3 The amino acid numbering refers to the FGFR2IIIb isoform, RefSeq ID: NM_022970.3, and Ensembl Transcript ID: ENST00000457416, which is the main transcript expressed in KEN. Open table in a new tab Abbreviations: KEN, keratinocytic epidermal nevus; na, no available information; WES, whole exome sequencing; WT, wild type. To assess whether the somatic mutations were present at lower allelic frequency in leukocyte DNA, we used targeted resequencing of FGFR2 in three of the mutant cases at >1,000 depth. In patients 2 and 4, no allelic variants were detected. By contrast, in patient 1 the c.1127A>G substitution was found at allelic frequencies of 35.85% and 3.37% in DNA from EN and leukocytes, respectively, indicating wider tissue mosaicism (Supplementary Table S2 and Supplementary Figure S3 online). The mutations identified, or their equivalent positions in other FGFR genes/isoforms, have been reported to occur in cancer (Supplementary Table S3 online), supporting their damaging potential. To gain further insight into their putative functional effects, we mapped the predicted amino acid substitutions onto a structural model of the extracellular ligand-binding domains of a dimeric 2:2:2 FGFR2IIIb:FGF10:heparin ternary complex (Supplementary Materials for details). FGF ligands interact with the second and third Ig domains (hereafter referred to as D2 and D3) and with the interconnecting D2-D3 linker region of FGFR. The p.S252W (c.755C>G) mutation is localized in the D2-D3 linker and can alter the ligand-binding specificity, being classified as an activating damaging mutation. The p.P286S (c.857C>T) mutation—which has not been reported previously—is also positioned in D3 in the ligand-binding site and is in a position equivalent to that of the p.P283S mutation in FGFR3b. The structure-PPi system (http://structureppi.bioinfo.cnio.es/Structure) suggests that this mutation could also have an activating effect through changes in ligand-binding specificity. Pro286 is a strictly conserved residue among members of the human FGFR family and the p.P286S (c.857C>T) mutation is predicted to destabilize the structure of the D3 domain in FGFR2 and to increase the binding affinity of FGF10 (Supplementary Figure S4 online). The p.Y376C (c.1127A>G) mutation localizes to a segment between D3 and the transmembrane region that is not included in the structural model. Bioinformatics analysis classified this mutation as harmful (Supplementary Table S1). The experimental evidence, in silico modeling, and genotype/phenotype correlations strongly support the notion that the three FGFR2 mutations that we have found in KEN are pathogenic (Supplementary Table S1, Supplementary Figure S4). Moreover, these—or equivalently positioned—mutations are recurrently found in several cancers, such as endometrial, breast, and bladder cancers (Byron et al., 2012Byron S.A. Gartside M. Powell M.A. Wellens C.L. Gao F. Mutch D.G. et al.FGFR2 point mutations in 466 endometrioid endometrial tumors: relationship with MSI, KRAS, PIK3CA, CTNNB1 mutations and clinicopathological features.PloS One. 2012; 7: e30801Crossref PubMed Scopus (132) Google Scholar) (Supplementary Table S3). Patient 4 fulfilled criteria of macrocephaly-capillary malformation [OMIM 602501]. This syndrome consists of megalencephaly, neonatal hypotonia, growth dysregulation with asymmetry, vascular anomalies, distal limb malformations (syndactyly and polydactyly), cortical malformation, and a mild connective tissue dysplasia. Although infrequent, KEN may occur in patients with macrocephaly-capillary malformation (Katugampola et al., 2008Katugampola R. Moss C. Mills C. Macrocephaly-cutis marmorata telangiectatica congenita: A case report and review of salient features.J Am Acad Dermatol. 2008; 58: 697-702Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Postzygotic mosaic mutations in several genes involved in the PI3K pathway (AKT3, PIK3CA, and PIK3R2) have recently been reported in overgrowth syndromes (Riviere et al., 2012Riviere J.B. Mirzaa G.M. O'Roak B.J. Beddaoui M. Alcantara D. Conway R.L. et al.De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes.Nat Genet. 2012; 44: 934-940Crossref PubMed Scopus (520) Google Scholar). However, 25% of patients with macrocephaly-capillary malformation lack mutations in these genes (Riviere et al., 2012Riviere J.B. Mirzaa G.M. O'Roak B.J. Beddaoui M. Alcantara D. Conway R.L. et al.De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes.Nat Genet. 2012; 44: 934-940Crossref PubMed Scopus (520) Google Scholar) and our findings support the concept that somatic mutations in genes coding for upstream PI3K activators may lead to a similar clinical phenotype. Patient 1 had a KEN, unilateral exophthalmia, and a p.Y376C (c.1127A>G) mutation in mosaicism in the skin and blood (Supplementary Table S4 online). This mutation has been reported in the germline in Beare-Stevenson syndrome [OMIM 123790] that associates craniosynostosis, ocular proptosis, cutis gyrata, acanthosis nigricans, prominent umbilical stump, furrowed palms and soles, and anogenital anomalies. Thus, the patient may be considered a mosaic form of the syndrome. Patient 2 had a KEN carrying the p.S252W (c.755C>G) mutation (Figure 1h–j). This mutation causes Apert syndrome [OMIM 101200] when present in the germline and can also be found in its mosaic state, unilateral/segmental acneiform nevus (Munro's acne nevus) (Melnik et al., 2008Melnik B.C. Vakilzadeh F. Aslanidis C. Schmitz G. Unilateral segmental acneiform naevus: a model disorder towards understanding fibroblast growth factor receptor 2 function in acne?.Br J Dermatol. 2008; 158: 1397-1399Crossref PubMed Scopus (33) Google Scholar, Munro and Wilkie, 1998Munro C.S. Wilkie A.O. Epidermal mosaicism producing localised acne: somatic mutation in FGFR2.Lancet. 1998; 352: 704-705Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Both conditions show acneiform lesions. Intriguingly, the lesion in patient 2 showed histological features reminiscent of acne, with dilated follicular infundibula (Melnik et al., 2008Melnik B.C. Vakilzadeh F. Aslanidis C. Schmitz G. Unilateral segmental acneiform naevus: a model disorder towards understanding fibroblast growth factor receptor 2 function in acne?.Br J Dermatol. 2008; 158: 1397-1399Crossref PubMed Scopus (33) Google Scholar). A 13-year-old boy with acne within a pre-existing KEN, with no genotype studies, has been reported (Hivnor et al., 2007Hivnor C.M. Yan A.C. Honig P.J. Acne arising in an epidermal nevus.Pediatr Dermatol. 2007; 24: 534-535Crossref PubMed Scopus (6) Google Scholar). These observations suggest that some KEN share histological and genetic changes with mosaic acne and Apert syndrome. The phenotypic pleiotropy described above for the FGFR2 mutations parallels previous observations regarding HRAS mutations that can occur both in KEN and in Costello syndrome (frequently showing acanthosis nigricans) (Gripp et al., 2006Gripp K.W. Lin A.E. Stabley D.L. Nicholson L. Scott Jr., C.I. Doyle D. et al.HRAS mutation analysis in Costello syndrome: genotype and phenotype correlation.Am J Med Genet A. 2006; 140: 1-7Crossref PubMed Scopus (153) Google Scholar, Hafner et al., 2011Hafner C. Toll A. Real F.X. HRAS mutation mosaicism causing urothelial cancer and epidermal nevus.N Engl J Med. 2011; 365: 1940-1942Crossref PubMed Scopus (34) Google Scholar). Altogether, these findings suggest that the cellular compartments (i.e., progenitor vs. stem cells) and cell types (epithelial and/or mesenchymal) affected by the mutation may determine, among others, the clinical presentation. In conclusion, we report that 5–10% of EN harbor embryonic postzygotic FGFR2 activating mutations. Overall, the genetic cause of 80–90% of KEN is thus identified as an activating mutation in an oncogene. The products of these genes are positioned from the cell membrane to various tiers of intracellular signaling. Therefore, we propose to extend the concept of "mosaic RASopathy" to one of "mosaic signalopathy" as the general cause of KEN and related conditions. Tirso Pons: http://orcid.org/0000-0002-7616-7750 The authors state no conflict of interest. We thank M. Bayés for valuable contributions. This work was supported, in part, by grants from Comunidad Autónoma de Madrid (S2010/BMD-2315), Instituto de Salud Carlos III (RTICC RD12/0036/0034, cofinanced by the European Regional Development Fund), and Consolíder ONCOBIO to FXR; from Instituto de Salud Carlos III, Ministerio de Sanidad (PI10/00785), to AT; from EU FP7 project ASSET (agreement 259348) to AV, and from Deutsche Forschungsgemeinschaft (GR4610/1-1) to LG. LCF was supported by a Marie Curie Training Grant (FP7-PEOPLE-2010-IEF, project 274946). Download .pdf (8.22 MB) Help with pdf files Supplementary Data