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Division of Human Genetics, Medical University Innsbruck, Innsbruck, AustriaInstitute of Clinical Genetics, Technical University of Dresden, Dresden, Germany
Grupo de Carcinogénesis Epitelial, Programa de Patología Molecular, Centro Nacional de Investigaciones Oncológicas, Madrid, SpainDepartament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain
Phacomatosis pigmentokeratotica (PPK) is a rare epidermal nevus syndrome characterized by the co-occurrence of a sebaceous nevus and a speckled lentiginous nevus. The coexistence of an epidermal and a melanocytic nevus has been explained by two homozygous recessive mutations, according to the twin spot hypothesis, of which PPK has become a putative paradigm in humans. However, the underlying gene mutations remained unknown. Multiple tissues of six patients with PPK were analyzed for the presence of RAS, FGFR3, PIK3CA, and BRAF mutations using SNaPshot assays and Sanger sequencing. We identified a heterozygous HRAS c.37G>C (p.Gly13Arg) mutation in four patients and a heterozygous HRAS c.182A>G (p.Gln61Arg) mutation in two patients. In each case, the mutations were present in both the sebaceous and the melanocytic nevus. In the latter lesion, melanocytes were identified to carry the HRAS mutation. Analysis of various nonlesional tissues showed a wild-type sequence of HRAS, consistent with mosaicism. Our data provide no genetic evidence for the previously proposed twin spot hypothesis. In contrast, PPK is best explained by a postzygotic-activating HRAS mutation in a multipotent progenitor cell that gives rise to both a sebaceous and a melanocytic nevus. Therefore, PPK is a mosaic RASopathy.
Abbreviations
PPK
phacomatosis pigmentokeratotica
Introduction
Phacomatoses are a heterogeneous group of congenital neurocutaneous disorders showing different types of nevi and hamartomas. Phacomatosis pigmentokeratotica (PPK) was defined in 1996 as a distinct type of epidermal nevus syndrome (
). PPK is characterized by the coexistence of a sebaceous nevus and a speckled lentiginous nevus (Figure 1a and c). Further neurological, skeletal, or other extracutaneous abnormalities may be associated (
). The sebaceous nevus derives from an epithelial progenitor cell and shows epidermal hyperplasia, abundant sebaceous glands, and dilated apocrine glands. The speckled lentiginous nevus is characterized by a brownish café au lait macule, in which multiple dark papular speckles arise (
). Histopathologically, the nevus shows hyperpigmentation of the basal epidermis corresponding to the café au lait macule, as well as nests of melanocytes in the dermoepidermal junction and the dermal tissue corresponding to the speckles. The occurrence of two different nevus types in a single patient has been explained by the concept of twin spotting, also referred to as didymosis (
). The twin spot phenomenon is well known to occur in Drosophila and plants, but it has not been proven in vertebrates so far. PPK has become a putative paradigm for this genetic mechanism in humans. According to this hypothesis, somatic recombination of two nonallelic recessive mutations, each being heterozygous and localized on a different chromosome of a homologous pair, would result in two genetically different clones of neighboring daughter cells, each being homozygous for one recessive mutation and giving rise to two distinct nevi in close spatial proximity (Supplementary Figure 1a online;
Figure 1Clinical manifestations of phacomatosis pigmentokeratotica (patient 4). (a) Overview of a sebaceous nevus involving the right side of the scalp, neck, and back in close vicinity to a speckled lentiginous nevus on the left side of the back. It is noteworthy that a trichoblastoma (arrowhead) had developed on the sebaceous nevus of the scalp. (b) Detail of the sebaceous nevus (right) and the speckled lentiginous nevus (left). (c) A hairy congenital melanocytic nevus adjacent to the speckled lentiginous nevus was noted on the back.
). Therefore, we studied whether RAS gene mutations might contribute to PPK to test the proposed twin spot hypothesis.
Results
Material from six patients fulfilling diagnostic criteria of PPK was available for genetic analysis (Table 1). For each patient, DNA samples derived from multiple tissues were analyzed for the presence of RAS hotspot mutations. A heterozygous HRAS c.37G>C (p.Gly13Arg) mutation was identified in four of six PPK patients, and a heterozygous HRAS c.182A>G (p.Gln61Arg) mutation in two of six PPK patients by use of Sanger sequencing and the RAS SNaPshot multiplex assay. In each case, the same mutation was found in both the epidermal and the melanocytic nevus (Figure 2a and b). The different phenotype of the two nevi in the presence of the same mosaic mutation might be explained by distinct cell types that are affected by the genetic mosaicism. Epithelial cells (keratinocytes) have been previously identified to carry the mosaic p.Gly13Arg HRAS mutation in sebaceous nevi (
). We hypothesized that, in the speckled lentiginous nevus of PPK patients, the melanocytes instead of keratinocytes would carry the mosaic HRAS mutation. To test this assumption, we selectively microdissected melanocytic nests in the speckled lentiginous nevus of patient 1, and melanocytic nests, keratinocytes, and blood vessels in that of patient 4. In both subjects, the heterozygous HRAS c.37G>A mutation was found in the isolated melanocytes, whereas the epidermal keratinocytes and the dermal blood vessels derived from the speckled lentiginous nevus of patient 4 showed a wild-type sequence. This finding provides strong evidence that, in the speckled lentiginous nevi of PPK patients, the melanocytes carry the mosaic mutation and represent the relevant cell type for the development of this lesion. In patients 1, 2, 4, and 6, additional lesional tissue samples from keratinocytic epidermal nevi, secondary tumors which developed within the epidermal nevi, a congenital melanocytic nevus adjacent to the speckled lentiginous nevus (patient 4; Figures 1c and 2c), and multiple metachronous urothelial cell carcinomas (patient 6) were available for analysis (Table 1). All samples harbored the same HRAS mutation as the sebaceous nevus and the speckled lentiginous nevus of the respective patient. The two HRAS mutations detected in the PPK patients constitutively activate the Ras–Raf–MEK–ERK signaling pathway (
). Moreover, BRAF mutations and NRAS mutations were not detected by Sanger sequencing in the speckled lentiginous nevi and the congenital melanocytic nevus of patient 4, respectively. For each of the six patients, at least one sample of various nonlesional tissues was available comprising blood leukocytes (n=4), unaffected skin (n=3), hair roots (n=3), buccal swabs (n=4), an endometrial polyp (n=1), and a lymph node (n=1)(Table 1). All nonlesional tissues revealed an HRAS wild-type sequence at codon 13 and 61, indicating that the mutations occurred in mosaicism.
Table 1Genetic analysis of phacomatosis pigmentokeratotica
Hydrocephalus internus, abnormalities of right insular and temporo-parietal cortex, hearing loss, hypoglossus paresis, scoliosis, shortened left leg, equinus deformity of left foot, and dysplastic left pelvis.
Trichoblastomas with Merkel cell proliferation in nevi sebacei in Schimmelpenning-Feuerstein-Mims syndrome—histological differentiation between trichoblastomas and basal cell carcinomas.
1 Hydrocephalus internus, abnormalities of right insular and temporo-parietal cortex, hearing loss, hypoglossus paresis, scoliosis, shortened left leg, equinus deformity of left foot, and dysplastic left pelvis.
2 Seizures, right hand paresis, and hypoplastic left hip.
Figure 2Histological findings and corresponding RAS SNaPshot multiplex assay chromatograms (patient 4). (a) The sebaceous nevus shows abundant sebaceous glands and the HRAS c.37G>C mutation (blue peak; peaks indicate the DNA antisense strand of the HRAS gene). (b) The speckled lentiginous nevus showed the histopathology of a melanocytic compound nevus. The HRAS c.37G>C mutation was present in the melanocytic nevus cells in the dermis (left), whereas keratinocytes revealed a wild-type sequence at codon 13 of HRAS (right). (c) The congenital melanocytic nevus showed a deep infiltrate of melanocytic nevus cells in the dermis that revealed the HRAS c.37G>C mutation.
These results are not compatible with the hitherto widely accepted, yet unproven, hypothesis of nonallelic twin spotting as the cause of PPK, as this theory postulates that two different recessive mutations, each being present in a homozygous state, give rise to the (epidermal) sebaceous and the (melanocytic) speckled lentiginous nevus. Instead, we demonstrate that a single heterozygous activating HRAS mutation, acting dominantly, accounts for the two distinct nevus types. We hypothesize that the respective mutation occurs in a multipotent progenitor cell giving rise to the cutaneous and extracutaneous anomalies noted in PPK (Supplementary Figure 1b online). This alternative genetic concept highlights that the timing of the mutation during embryogenesis and the differentiation potential of the cell in which it occurs are crucial determinants for the resulting phenotype. As illustrated in our case series and in previous case reports of PPK, such a progenitor cell may comprise an ectodermal (e.g., epidermal nevi and central nervous system defects), mesodermal (e.g., vascular and skeletal anomalies), and endodermal (e.g., urothelial cell carcinoma) differentiation potential. Urothelial cell carcinomas have been reported previously in a patient with a keratinocytic epidermal nevus syndrome caused by an HRAS mosaic mutation (
). The finding that organoid sebaceous nevi, nonorganoid keratinocytic epidermal nevi, congenital melanocytic nevi, and speckled lentiginous nevi of the papular type can be caused by the same HRAS c.37G>C mutation, as demonstrated in this study, highlights the pleiotropy of HRAS mosaic mutations in the context of an ectodermal progenitor cell. According to this theory, the affected ectodermal progenitor cell may give rise to melanocytes and keratinocytes if the mutation occurs before neural crest differentiation, whereas it would be restricted to the latter if it occurs after neural crest formation. Similarly, this hypothesis can explain the divergent phenotypes of Schimmelpenning syndrome and PPK, which in fact can be considered as different variants of a broad clinical spectrum caused by a common HRAS mosaic mutation. What makes the PPK and the Schimmelpenning syndrome “look different” is that, in the case of PPK, the mutated progenitor cell has the ability to differentiate into epithelial cells and melanocytes, whereas in Schimmelpenning syndrome it has lost the latter. In PPK and Schimmelpenning syndrome, the sebaceous nevus is preferentially localized in the cranial parts of the body, whereas on the trunk and extremities the epidermal mosaicism more often results in a keratinocytic epidermal nevus. Sometimes, both a sebaceous and a keratinocytic component can be combined in the same lesion, with the sebaceous part at the center and the keratinocytic part at the margin. This peculiar lesion called nevus marginatus has been recently shown to be caused by an HRAS c.37G>C mosaic mutation (
). The preponderance of sebaceous nevi in the head and neck region may be explained by the fact that this area is rich with sebaceous glands, thus mutated progenitor cells in this region may be more often determined to differentiate into sebaceous cells, resulting in a sebaceous nevus.
Interestingly, Spitz nevi may arise within speckled lentiginous nevi of PPK patients. Both the HRAS c.37G>C and the HRAS c.182A>G mutation have already been reported in sporadic Spitz nevi (
), which further substantiates our data. Alternatively, the sebaceous and melanocytic nevi in PPK may be caused by two independent mutations occurring in two different progenitor cells at the same hotspot gene locus, but this possibility appears to be very unlikely.
In an analogy to a group of phenotypically overlapping developmental syndromes caused by germline mutations in components of the Ras–Raf–MEK–ERK signaling pathway that have been designated as “RASopathies” (
). Our findings in patients with PPK incorporate this syndrome to the group of mosaic RASopathies. The possible contribution of modifier genes to the clinical presentation and the risk of cancer development in patients with mosaic RASopathies merit further study.
In conclusion, our findings do not support the previously proposed twin spot hypothesis in PPK. Alternatively, we show that a postzygotic-activating HRAS mutation in a multipotent progenitor cell represents the underlying pathogenetic mechanism. Moreover, it is possible or even likely that this genetic mechanism applies to some other phacomatoses and related syndromes that have been previously explained by the twin spot hypothesis.
Materials and Methods
Sample acquisition
The study was approved by the local ethics committee of the University of Regensburg and performed according to the guidelines of the declaration of Helsinki.
Multiple archived lesional tissues (sebaceous nevi, speckled lentiginous/melanocytic nevi, keratinocytic epidermal nevi, and urothelial carcinomas) were available from one unpublished and five previously published individuals with PPK (
Trichoblastomas with Merkel cell proliferation in nevi sebacei in Schimmelpenning-Feuerstein-Mims syndrome—histological differentiation between trichoblastomas and basal cell carcinomas.
). In addition, various nonlesional tissues of the patients were investigated including blood leukocytes (four patients), unaffected skin (three patients), hair roots (three patients), buccal swabs (four patients), an endometrial polyp (one patient), and a lymph node (1 patient). A detailed list of the analyzed samples and clinical characteristics of the respective patients are displayed in Table 1. Written informed consent had been obtained from patients 1, 2, 3, 4, and 6 before sample acquisition. Samples from patient 5 were derived from an autopsy. Formalin-fixed paraffin-embedded tissue was manually microdissected with a needle under an inverted microscope. Selective microdissection of melanocytic nests was carried out within the speckled lentiginous nevus of patient 1 and 4. In addition, in patient 4, keratinocytes and blood vessels from the speckled lentiginous nevus were microdissected separately. DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Mutation analyses
A highly sensitive SNaPshot multiplex assay was used for the detection of RAS hotspot mutations as described previously (
). The assay (set I) covers the following nucleotide positions: 34, 35, 37, 181, 182 of HRAS; 34, 35, 181 of KRAS; and 34, 182 of NRAS. Each mutation was confirmed by a second independent PCR. In addition, PCR products of exons 1 and 2 of HRAS were directly sequenced in sebaceous and speckled lentiginous nevi samples of patients 1–5 by the Sanger method. Furthermore, PCR products of exon 15 of BRAF and exons 1 and 2 of NRAS were directly sequenced in the melanocytic lesions of patient 4. Primer sequences are provided in the supplement (Supplementary Table S1 online), and PCR conditions can be obtained from the authors. FGFR3 and PIK3CA hotspot mutations were analyzed by SNaPshot multiplex assays as described previously (
We thank all subjects who participated in this study, and L. Kuenzel, P. Jaén, X. Langa, G. Morata, C. Moreno, and M. Marqués for their valuable contributions. This work was supported by the DFG grant HA 5531/1-1 (www.dfg.de).
Trichoblastomas with Merkel cell proliferation in nevi sebacei in Schimmelpenning-Feuerstein-Mims syndrome—histological differentiation between trichoblastomas and basal cell carcinomas.
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