Heterozygosity for loss-of-function variants in LZTR1 is associated with isolated multiple café-au-lait macules.

PURPOSE: Pathogenic LZTR1 variants cause schwannomatosis and dominant/recessive Noonan syndrome (NS). We aim to establish an association between heterozygous loss-of-function (LoF) LZTR1 alleles and isolated multiple café-au-lait macules (CaLMs). METHODS: 849 unrelated participants with multiple CaLMs, lacking pathogenic/likely pathogenic NF1 and SPRED1 variants, underwent RASopathy gene panel sequencing. Data on 125 individuals with heterozygous LZTR1 variants were collected for characterizing their clinical features and the associated molecular spectrum. In vitro functional assessment was performed on a representative panel of missense variants and small in-frame deletions. RESULTS: Analysis revealed heterozygous LZTR1 variants in 6.0% (51/849) of participants, exceeding the general population prevalence. LZTR1-related CaLMs varied in number, displayed sharp or irregular borders, and were generally isolated, but occasionally associated with features recurring in RASopathies. In two families, CaLMs and schwannomas co-occurred. The molecular spectrum mainly consisted of truncating variants, indicating LoF. These variants substantially overlapped with those occurring in schwannomatosis and recessive NS. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate MAPK signaling. CONCLUSION: Our findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive NS, occur in individuals with isolated multiple CaLMs.

Revisiting the UK Genetic Severity Score for NF2: a proposal for the addition of a functional genetic component.

BACKGROUND: Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterised by the development of multiple schwannomas, especially on vestibular nerves, and meningiomas. The UK NF2 Genetic Severity Score (GSS) is useful to predict the progression of the disease from germline NF2 pathogenic variants, which allows the clinical follow-up and the genetic counselling offered to affected families to be optimised. METHODS: 52 Spanish patients were classified using the GSS, and patients' clinical severity was measured and compared between GSS groups. The GSS was reviewed with the addition of phenotype quantification, genetic variant classification and functional assays of Merlin and its downstream pathways. Principal component analysis and regression models were used to evaluate the differences between severity and the effect of NF2 germline variants. RESULTS: The GSS was validated in the Spanish NF2 cohort. However, for 25% of mosaic patients and patients harbouring variants associated with mild and moderate phenotypes, it did not perform as well for predicting clinical outcomes as it did for pathogenic variants associated with severe phenotypes. We studied the possibility of modifying the mutation classification in the GSS by adding the impact of pathogenic variants on the function of Merlin in 27 cases. This revision helped to reduce variability within NF2 mutation classes and moderately enhanced the correlation between patient phenotype and the different prognosis parameters analysed (R2=0.38 vs R2=0.32, p>0001). CONCLUSIONS: We validated the UK NF2 GSS in a Spanish NF2 cohort, despite the significant phenotypic variability identified within it. The revision of the GSS, named Functional Genetic Severity Score, could add value for the classification of mosaic patients and patients showing mild and moderate phenotypes once it has been validated in other cohorts.

Neurofibromatosis type 1 families with first-degree relatives harbouring distinct NF1 pathogenic variants. Genetic counselling and familial diagnosis: what should be offered?

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by pathogenic variants in NF1 Recently, NF1 testing has been included as a clinical criterion for NF1 diagnosis. Additionally, preconception genetic counselling in patients with NF1 focuses on a 50% risk of transmitting the familial variant as the risk of having a sporadic NF1 is considered the same as the general population. METHODS: 829 individuals, 583 NF1 sporadic cases and 246 patients with NF1 with documented family history, underwent genetic testing for NF1. Genotyping and segregation analysis of NF1 familial variants was determined by microsatellite analysis and NF1 sequencing. RESULTS: The mutational analysis of NF1 in 154 families with two or more affected cases studied showed the co-occurrence of two different NF1 germline pathogenic variants in four families. The estimated mutation rate in those families was 3.89×10-3, 20 times higher than the NF1 mutation rate (~2×10-4) (p=0.0008). Furthermore, the co-occurrence of two different NF1 germline pathogenic variants in these families was 1:39, 60 times the frequency of sporadic NF1 (1:2500) (p=0.003). In all cases, the de novo NF1 pathogenic variant was present in a descendant of an affected male. In two cases, variants were detected in the inherited paternal wild-type allele. CONCLUSIONS: Our results, together with previous cases reported, suggest that the offspring of male patients with NF1 could have an increased risk of experiencing de novo NF1 pathogenic variants. This observation, if confirmed in additional cohorts, could have relevant implications for NF1 genetic counselling, family planning and NF1 genetic testing.

The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans.

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.

Verrucous epidermal nevus as a manifestation of a type 2 mosaic PTEN mutation in Cowden syndrome.

Linear Cowden nevus, also known as linear PTEN nevus, is a type of epidermal nevus, first described in 2007, which is seen in patients with PTEN hamartoma tumor syndrome. It is considered to be a type 2 form of segmental mosaicism, and we suggest that it has certain clinical features that distinguish it from epidermal nevi seen in similar conditions, such as Proteus syndrome. We present a case of linear Cowden nevus in a 4-year-old boy and review the literature.

Targeted massively parallel sequencing of candidate regions on chromosome 22q predisposing to multiple schwannomas: An analysis of 51 individuals in a single-center experience.

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.

CNVfilteR: an R/Bioconductor package to identify false positives produced by germline NGS CNV detection tools.

SUMMARY: Germline copy-number variants (CNVs) are relevant mutations for multiple genetics fields, such as the study of hereditary diseases. However, available benchmarks show that all next-generation sequencing (NGS) CNV calling tools produce false positives. We developed CNVfilteR, an R package that uses the single-nucleotide variant calls usually obtained in germline NGS pipelines to identify those false positives. The package can detect both false deletions and false duplications. We evaluated CNVfilteR performance on callsets generated by 13 CNV calling tools on three whole-genome sequencing and 541 panel samples, showing a decrease of up to 44.8% in false positives and consistent F1-score increase. Using CNVfilteR to detect false-positive calls can improve the overall performance of existing CNV calling pipelines. AVAILABILITY AND IMPLEMENTATION: CNVfilteR is released under Artistic-2.0 License. Source code and documentation are freely available at Bioconductor (http://www.bioconductor.org/packages/CNVfilteR). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mutational spectrum by phenotype: panel-based NGS testing of patients with clinical suspicion of RASopathy and children with multiple café-au-lait macules.

Children with neurofibromatosis type 1 (NF1) may exhibit an incomplete clinical presentation, making difficult to reach a clinical diagnosis. A phenotypic overlap may exist in children with other RASopathies or with other genetic conditions if only multiple café-au-lait macules (CALMs) are present. The syndromes that can converge in these inconclusive phenotypes have different clinical courses. In this context, an early genetic testing has been proposed to be clinically useful to manage these patients. We present the validation and implementation into diagnostics of a custom NGS panel (I2HCP, ICO-IMPPC Hereditary Cancer Panel) for testing patients with a clinical suspicion of a RASopathy (n = 48) and children presenting multiple CALMs (n = 102). We describe the mutational spectrum and the detection rates identified in these two groups of individuals. We identified pathogenic variants in 21 out of 48 patients with clinical suspicion of RASopathy, with mutations in NF1 accounting for 10% of cases. Furthermore, we identified pathogenic mutations mainly in the NF1 gene, but also in SPRED1, in more than 50% of children with multiple CALMs, exhibiting an NF1 mutational spectrum different from a group of clinically diagnosed NF1 patients (n = 80). An NGS panel strategy for the genetic testing of these two phenotype-defined groups outperforms previous strategies.

Analysis of intratumor heterogeneity in Neurofibromatosis type 1 plexiform neurofibromas and neurofibromas with atypical features: Correlating histological and genomic findings.

Plexiform neurofibromas (PNFs) are benign peripheral nerve sheath tumors involving large nerves present in 30%-50% Neurofibromatosis type 1 (NF1) patients. Atypical neurofibromas (ANF) are distinct nodular lesions with atypical features on histology that arise from PNFs. The risk and timeline of malignant transformation in ANF is difficult to assess. A recent NIH workshop has stratified ANFs and separated a subgroup with multiple atypical features and higher risk of malignant transformation termed atypical neurofibromatous neoplasms with uncertain biological potential (ANNUBP). We performed an analysis of intratumor heterogeneity on eight PNFs to link histological and genomic findings. Tumors were homogeneous although histological and molecular heterogeneity was identified. All tumors were 2n, almost mutation-free and had a clonal NF1(-/-) origin. Two ANFs from the same patient showed atypical features on histology and deletions of CDKN2A/B. One of the ANFs exhibited different areas in which the degree of histological atypia correlated with the heterozygous or homozygous loss of the CDKN2A/B loci. CDKN2A/B deletions in different areas originated independently. Results may indicate that loss of a single CDKN2A/B copy in NF1(-/-) cells is sufficient to start ANF development and that total inactivation of both copies of CDKN2A/B is necessary to form an ANNUBP.

Unbalancing cAMP and Ras/MAPK pathways as a therapeutic strategy for cutaneous neurofibromas.

Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Individuals with neurofibromatosis type 1 (NF1) may develop thousands of cNFs, which greatly affect their quality of life. cNF growth is driven by the proliferation of NF1-/- SCs and their interaction with the NF1+/- microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to the SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by ogerin in combination with the MEK inhibitor (MEKi) selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an induced pluripotent stem cell-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with selumetinib. Interestingly, whereas primary SC cultures restarted their proliferation after treatment with selumetinib alone was stopped, the combination of ogerin-selumetinib elicited a permanent halt on SC expansion that persisted after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators could be used as a potential treatment for cNFs.

Deep genomic analysis of malignant peripheral nerve sheath tumor cell lines challenges current malignant peripheral nerve sheath tumor diagnosis.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1, CDKN2A, and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis.

Antisense oligonucleotides targeting exon 11 are able to partially rescue the NF2-related schwannomatosis phenotype in vitro.

NF2-related schwannomatosis (NF2-related SWN) is an autosomal dominant condition caused by loss of function variants in the NF2 gene, which codes for the protein Merlin and is characterized by the development of multiple tumors of the nervous system. The clinical presentation of the disease is variable and related to the type of the inherited germline variant. Here, we tested if phosphorodiamidate morpholino oligomers (PMOs) could be used to correct the splice signaling caused by variants at ±13 within the intron-exon boundary region and showed that the PMOs designed for these variants do not constitute a therapeutic approach. Furthermore, we evaluated the use of PMOs to decrease the severity of the effects of NF2 truncating variants with the aim of generating milder hypomorphic isoforms in vitro through the induction of the in-frame deletion of the exon-carrying variant. We were able to specifically induce the skipping of exons 4, 8, and 11 maintaining the NF2 gene reading frame at cDNA level. Only the skipping of exon 11 produced a hypomorphic Merlin (Merlin-e11), which is able to partially rescue the observed phenotype in primary fibroblast cultures from NF2-related SWN patients, being encouraging for the treatment of patients harboring truncating variants located in exon 11.

Modeling iPSC-derived human neurofibroma-like tumors in mice uncovers the heterogeneity of Schwann cells within plexiform neurofibromas.

Plexiform neurofibromas (pNFs) are developmental tumors that appear in neurofibromatosis type 1 individuals, constituting a major source of morbidity and potentially transforming into a highly metastatic sarcoma (MPNST). pNFs arise after NF1 inactivation in a cell of the neural crest (NC)-Schwann cell (SC) lineage. Here, we develop an iPSC-based NC-SC in vitro differentiation system and construct a lineage expression roadmap for the analysis of different 2D and 3D NF models. The best model consists of generating heterotypic spheroids (neurofibromaspheres) composed of iPSC-derived differentiating NF1(-/-) SCs and NF1(+/-) pNF-derived fibroblasts (Fbs). Neurofibromaspheres form by maintaining highly proliferative NF1(-/-) cells committed to the NC-SC axis due to SC-SC and SC-Fb interactions, resulting in SC linage cells at different maturation points. Upon engraftment on the mouse sciatic nerve, neurofibromaspheres consistently generate human NF-like tumors. Analysis of expression roadmap genes in human pNF single-cell RNA-seq data uncovers the presence of SC subpopulations at distinct differentiation states.

Centrosome dysfunction in Drosophila neural stem cells causes tumors that are not due to genome instability.

Genome instability (GI) and centrosomal alterations are common traits in human cancer [1, 2]. It is suspected that centrosome dysfunction may cause tumors by bringing about GI, but direct experimental proof is still lacking [3]. To explore the possible functional link between centrosome function and overgrowth, we have assayed the tumorigenic potential of a series of mutants that affect different centrosomal proteins in Drosophila. We have found that a significant number of such mutant conditions are tumorigenic in larval brain tissue, where self-renewing asymmetric division of neural stem cells is frequent, but not in symmetrically dividing epithelial cells. We have also found that mutations that increase GI without causing centrosome dysfunction are not tumorigenic in our assay. From these observations, we conclude that the tumors caused by centrosome dysfunction cannot be explained solely by the resulting genome instability. We propose that such tumors might be caused by impaired asymmetric division of neural stem cells [4]. These results show that centrosome loss, far from being innocuous, is a potentially dangerous condition in flies.

Clinical characteristics, imaging findings, and genetic results of a patient with CEP290-related cone-rod dystrophy.

PURPOSE: To describe the clinical characteristics, the imaging findings, and the genetic results of a patient with cone-rod dystrophy (CORD) related to mutations in CEP290. METHODS: A case report of atypical CEP290-related CORD. Ophthalmological examination was performed, including best-corrected visual acuity (BCVA), fundus photography, fundus autofluorescence (FAF) imaging, optical coherence tomography (OCT), a visual field test, and electroretinography testing. The genetic test was performed by next-generation sequencing (NGS)-based panel test containing 336 genes. RESULTS: A 57-year-old female who had reported a visual loss for 5 years. BCVA was 20/100 in both eyes. The fundus examination revealed a hypopigmented halo around the fovea, showing a paracentral hyperautofluorescent ring on FAF. OCT demonstrated the presence of atrophy in the outer retinal layers. The genetic test identified the probably pathogenic variants c.4028delA and c.5254C>T in compound heterozygosis in CEP290. CONCLUSIONS: This is the first report to present the clinical characteristics, imaging findings, and genetic test results of a patient with CEP290-related CORD. Our case contributes to expanding the clinical involvement of CEP290 pathogenic variants. This study indicates that CEP290-related CORD may have a mild phenotype with late-onset dystrophy, making these patients interesting candidates for innovative treatments such as genetic therapeutic approaches.

Using antisense oligonucleotides for the physiological modulation of the alternative splicing of NF1 exon 23a during PC12 neuronal differentiation.

Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide. The NF1 gene codes for neurofibromin protein, a GTPase activating protein (GAP) and a negative regulator of RAS. The NF1 gene undergoes alternative splicing of exon 23a (E23a) that codes for 21 amino acids placed at the center of the GAP related domain (GRD). E23a-containing type II neurofibromin exhibits a weaker Ras-GAP activity compared to E23a-less type I isoform. Exon E23a has been related with the cognitive impairment present in NF1 individuals. We designed antisense Phosphorodiamidate Morpholino Oligomers (PMOs) to modulate E23a alternative splicing at physiological conditions of gene expression and tested their impact during PC12 cell line neuronal differentiation. Results show that any dynamic modification of the natural ratio between type I and type II isoforms disturbed neuronal differentiation, altering the proper formation of neurites and deregulating both the MAPK/ERK and cAMP/PKA signaling pathways. Our results suggest an opposite regulation of these pathways by neurofibromin and the possible existence of a feedback loop sensing neurofibromin-related signaling. The present work illustrates the utility of PMOs to study alternative splicing that could be applied to other alternatively spliced genes in vitro and in vivo.

Evaluation of CNV detection tools for NGS panel data in genetic diagnostics.

Although germline copy-number variants (CNVs) are the genetic cause of multiple hereditary diseases, detecting them from targeted next-generation sequencing data (NGS) remains a challenge. Existing tools perform well for large CNVs but struggle with single and multi-exon alterations. The aim of this work is to evaluate CNV calling tools working on gene panel NGS data and their suitability as a screening step before orthogonal confirmation in genetic diagnostics strategies. Five tools (DECoN, CoNVaDING, panelcn.MOPS, ExomeDepth, and CODEX2) were tested against four genetic diagnostics datasets (two in-house and two external) for a total of 495 samples with 231 single and multi-exon validated CNVs. The evaluation was performed using the default and sensitivity-optimized parameters. Results showed that most tools were highly sensitive and specific, but the performance was dataset dependant. When evaluating them in our diagnostics scenario, DECoN and panelcn.MOPS detected all CNVs with the exception of one mosaic CNV missed by DECoN. However, DECoN outperformed panelcn.MOPS specificity achieving values greater than 0.90 when using the optimized parameters. In our in-house datasets, DECoN and panelcn.MOPS showed the highest performance for CNV screening before orthogonal confirmation. Benchmarking and optimization code is freely available at https://github.com/TranslationalBioinformaticsIGTP/CNVbenchmarkeR .

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(-/-) cells, descendants from the tumor originating cell. These NF1(-/-)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(-/-) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

PTOV1 enables the nuclear translocation and mitogenic activity of flotillin-1, a major protein of lipid rafts.

PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.

Early Genetic Diagnosis of Neurofibromatosis Type 2 From Skin Plaque Plexiform Schwannomas in Childhood.

Importance: Neurofibromatosis type 2 (NF2) is a devastating genetic condition characterized by the development of multiple tumors of the nervous system. An early diagnosis of individuals with NF2 would facilitate treatment and reduction of disease impact because most severe effects of the disease do not usually develop before adolescence. Little attention has traditionally been paid to dermatological signs in NF2. However, skin plaques are commonly seen in patients with NF2, normally appearing either at birth or early childhood, providing an opportunity for early NF2 detection and testing. Objective: To determine the clinical utility of skin plaque identification and characterization in children for reaching an early diagnosis of patients with NF2 and to evaluate their molecular pathogenesis and their use in the genetic diagnostics of NF2. Design, Setting, and Participants: Diagnostic test study by the histological and genetic characterization of skin plaques from patients with NF2. Patients were 7 individuals with NF2 or clinical suspicion of NF2 treated at the Spanish Reference Center on Phakomatoses. Main Outcomes and Measures: Histological evaluation of all skin plaques was performed. Fresh skin plaques were cultured to obtain Schwann cells and the NF2 gene was genetically analyzed. For all 7 patients, NF2 clinical history was reviewed. Results: In all 7 patients (4 male and 3 female), all skin plaques analyzed were histologically characterized as plexiform schwannomas. Genetic analysis of primary Schwann cell cultures derived from them allowed the identification of a constitutional and a somatic NF2 mutation. Genetic testing allowed the early diagnosis of NF2 in a child only exhibiting the presence of skin plaques. Most of the patients with NF2 analyzed had an early presentation of skin plaques and a severe NF2 phenotype. Conclusions and Relevance: This work emphasizes the clinical utility of a careful dermatological inspection and the correct identification of skin plaques in children for an early diagnosis of NF2. We show for the first time that Schwann cells derived from skin plaque plexiform schwannomas bear the double inactivation of the NF2 gene and thus constitute an excellent source of tissue for genetic testing, especially in the context of mosaicism.