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Background: Neurofibromatosis type 1 (NF1) is one of the most common genodermatoses. It can affect every organ and is associated with an increased risk of benign and malignant tumors. Most common tumoral locations involve nervous system and soft tissues but a large variety of tumors have been described. So far, hepatoblastoma in a patient with NF1 has been reported twice in the literature., Case Presentation: A liver mass was discovered in a 11 year-old girl with NF1 leading to a diagnosis of epithelial hepatoblastoma with pulmonary metastasis. Targeted analysis on blood revealed a germline NF1 missense variant. Exome sequencing, RNA-seq and methylation analyses performed on tumoral and metastatic samples confirmed the germline NF1 variant and showed classical driver variants for hepatoblastoma., Conclusions: We present here the third case of hepatoblastoma in a patient with NF1 and discuss the possible link between this rare tumor and this neurocutaneous genetic condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

We report our 5-year experience in neurofibromatosis type 1 prenatal diagnosis (PND): 205 PNDs in 146 women (chorionic villus biopsies, 88% or amniocentesis, 12%). The NF1 variant was present in 85 (41%) and absent in 122 (59%) fetuses. Among 205 pregnancies (207 fetuses), 135 were carried to term (119 unaffected and 16 NF1 affected children), 69 pregnancy terminations (affected fetuses), 2 miscarriages, and 1 in utero death. The majority of PND requests came from parents with sporadic NF1. We describe two PNDs in women with mosaic NF1. In both families, direct PND showed the absence of the maternal NF1 variant in the fetus. However, microsatellite markers analysis showed that the risk haplotype had been transmitted. These rare cases of germline mosaicism illustrate the pitfall of indirect PND. Our study illustrates the crucial consequences of PND for medical and genetic counseling decisions. We also point to the challenges of germline mosaics., (© 2024. The Author(s).)

Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1 -deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1 -deficient SSPCs adopted a fibrotic fate, NF1 -deficient Schwann cells produced critical paracrine factors including transforming growth factor-β and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1 -deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.

Competing Interests: Competing interests: None declared.

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Neurofibromatosis type 1 (NF1) is characterized by the highly variable and unpredictable development of benign peripheral nerve sheath tumours: cutaneous (cNFs), subcutaneous (scNFs) and plexiform (pNFs) neurofibromas., Objectives: To identify neurofibroma modifier genes, in order to develop a database of patients with NF1., Methods: All patients were phenotypically evaluated by a medical practitioner using a standardized questionnaire and the causal NF1 variant identified. We enrolled 1333 patients with NF1 who were genotyped for > 7 million common variants., Results: A genome-wide association case-only study identified a significant association with 9q21.33 in the pNF phenotype in the discovery cohort. Twelve, three and four regions suggestive of association at the P ≤ 1 × 10-6 threshold were identified for pNFs, cNFs and scNFs, respectively. Evidence of replication was observed for 4, 2 and 6 loci, including 168 candidate modifier protein-coding genes. Among the candidate modifier genes, some were implicated in the RAS-mitogen-activated protein kinase pathway, cell-cycle control and myelination. Using an original CRISPR/Cas9-based functional assay, we confirmed GAS1 and SPRED2 as pNF and scNF candidate modifiers, as their inactivation specifically affected NF1-mutant Schwann cell growth., Conclusions: Our study may shed new light on the pathogenesis of NF1-associated neurofibromas and will, hopefully, contribute to the development of personalized care for patients with this deleterious and life-threatening condition., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Differential diagnosis between constitutional mismatch repair deficiency (CMMRD) and neurofibromatosis type 1 (NF1 ) is crucial as treatment and surveillance differ. We report the case of a girl with a clinical diagnosis of sporadic NF1 who developed a glioblastoma. Immunohistochemistry for MMR proteins identified PMS2 loss in tumour and normal cells and WES showed the tumour had an ultra-hypermutated phenotype, supporting the diagnosis of CMMRD. Germline analyses identified two variants (one pathogenic variant and one classified as variant(s) of unknown significance) in the PMS2 gene and subsequent functional assays on blood lymphocytes confirmed the diagnosis of CMMRD. The large plexiform neurofibroma of the thigh and the freckling were however more compatible with NF1. Indeed, a NF1 PV (variant allele frequencies of 20%, 3% and 9% and in blood, skin and saliva samples, respectively) was identified confirming a mosaicism for NF1. Retrospective analysis of a French cohort identified NF1 mosaicism in blood DNA in 2 out of 22 patients with CMMRD, underlining the existence of early postzygotic PV of NF1 gene in patients with CMMRD whose tumours have been frequently reported to exhibit somatic NF1 mutations. It highlights the potential role of this pathway in the pathogenesis of CMMRD-associated gliomas and argues in favour of testing MEK inhibitors in this context., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Context: In patients with neurofibromatosis type 1 (NF1), guidelines suggest screening for pheochromocytoma by metanephrine measurement and abdominal imaging, which may lead to the discovery of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and their differential diagnosis, gastrointestinal stromal tumors (GISTs). Other endocrine manifestations such as follicular thyroid carcinoma and primary hyperparathyroidism have also been reported in a few cases., Objective: This study aimed to describe prevalence and clinical presentation of these manifestations through systematic screening in a large cohort of patients., Methods: In this monocentric retrospective study, 108 patients with NF1 were included and screened for endocrine manifestations and GISTs. Clinical, laboratory, molecular profile, pathology, and morphologic (abdominal computed tomography scan and/or magnetic resonance imaging) and functional imaging were collected., Results: Twenty-four patients (22.2% of the cohort, 16 female, mean age 42.6 years) presented with pheochromocytomas that were unilateral in 65.5%, benign in 89.7%, and with a ganglioneural component in 20.7%. Three female patients (2.8% of the cohort, aged 42-63 years) presented with well-differentiated GEP-NETs, and 4 (3.7%) with GISTs. One patient had primary hyperparathyroidism, 1 patient had medullary microcarcinoma, and 16 patients had goiter, multinodular in 10 cases. There was no correlation between pheochromocytoma and other NF1 tumoral manifestations, nor correlations between pheochromocytoma and NF1 genotype, despite a familial clustering in one-third of patients., Conclusion: The pheochromocytoma prevalence in this NF1 cohort was higher (>20%) than previously described, confirming the interest of systematic screening, especially in young women. The prevalence of GEP-NETs and GISTs was about 3%, respectively. No phenotype-genotype correlation was observed., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pacault et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but highly variable expressivity. In most patients, Next Generation Sequencing (NGS) technologies allow the identification of a loss-of-function pathogenic variant in the NF1 gene, a negative regulator of the RAS-MAPK pathway. We describe the 5-year diagnosis wandering of a patient with a clear NF1 clinical diagnosis, but no molecular diagnosis using standard molecular technologies. The patient presented with a typical NF1 phenotype but NF1 targeted NGS, NF1 transcript analysis, MLPA, and array comparative genomic hybridization failed to reveal a genetic aberration. After 5 years of unsuccessful investigations, trio WGS finally identified a de novo mosaic (VAF ~ 14%) 24.6 kb germline deletion encompassing the promoter and first exon of NF1. This case report illustrates the relevance of WGS to detect structural variants including copy number variants that would be missed by alternative approaches. The identification of the causal pathogenic variant allowed a tailored genetic counseling with a targeted non-invasive prenatal diagnosis by detecting the deletion in plasmatic cell-free DNA from the proband's pregnant partner. This report clearly highlights the need to make WGS a clinically accessible test, offering a tremendous opportunity to identify a molecular diagnosis for otherwise unsolved cases., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Objectives: Cell-free fetal DNA (cffDNA) analysis is performed routinely for aneuploidy screening, RhD genotyping or sex determination. Although applications to single gene disorders (SGD) are being rapidly developed worldwide, only a few laboratories offer cffDNA testing routinely as a diagnosis service for this indication. In a previous report, we described a standardised protocol for non-invasive exclusion of paternal variant in SGD. Three years later, we now report our clinical experience with the protocol., Design: Descriptive study., Setting: Multi-centre French., Population: Indications for referral included pregnancies at risk of 25% or 50% of paternally inherited SGD, and pregnancies associated with an increased risk of SGD due to a de novo variant, either from strongly suggestive ultrasound findings or from a possible parental germinal mosaicism in the context of a previously affected child., Methods: Non-invasive prenatal diagnosis was performed using custom assays for droplet digital PCR. Feasibility, diagnostic performance and turn-around time were evaluated., Results: Mean time for a new assay design and validation was evaluated at 14 days, and mean result reporting time was 6 days. All referred pathogenic variants could be targeted except one located in a complex genomic region. A result was obtained for every 198 referrals except two., Conclusion: This service was successfully implemented as a routine laboratory practice. It has been widely adopted by French clinicians and patients for paternal variant exclusion in various disorders., Tweetable Abstract: A robust approach to non-invasive prenatal exclusion of paternal pathogenic variant in a diagnosis setting., (© 2022 John Wiley & Sons Ltd.)

Individuals with the three base pair deletion NM_000267.3(NF1):c.2970_2972del p.(Met992del) have been recognised to present with a milder neurofibromatosis type 1 (NF1) phenotype characterised by café-au-lait macules (CALs) and intertriginous freckling, as well as a lack of cutaneous, subcutaneous and plexiform neurofibromas and other NF1-associated complications. Examining large cohorts of patients over time with this specific genotype is important to confirm the presentation and associated risks of this variant across the lifespan. Forty-one individuals with the in-frame NF1 deletion p.Met992del were identified from 31 families. Clinicians completed a standardised clinical questionnaire for each patient and the resulting data were collated and compared to published cohorts. Thirteen patients have been previously reported, and updated clinical information has been obtained for these individuals. Both CALs and intertriginous freckling were present in the majority of individuals (26/41, 63%) and the only confirmed features in 11 (27%). 34/41 (83%) of the cohort met NIH diagnostic criteria. There was a notable absence of all NF1-associated tumour types (neurofibroma and glioma). Neurofibroma were observed in only one individual-a subcutaneous lesion (confirmed histologically). Nineteen individuals were described as having a learning disability (46%). This study confirms that individuals with p.Met992del display a mild tumoural phenotype compared to those with 'classical', clinically diagnosed NF1, and this appears to be the case longitudinally through time as well as at presentation. Learning difficulties, however, appear to affect a significant proportion of NF1 subjects with this phenotype. Knowledge of this genotype-phenotype association is fundamental to accurate prognostication for families and caregivers., (© 2021. The Author(s).)

Complete deletion of the NF1 gene is identified in 5-10% of patients with neurofibromatosis type 1 (NF1). Several studies have previously described particularly severe forms of the disease in NF1 patients with deletion of the NF1 locus, but comprehensive descriptions of large cohorts are still missing to fully characterize this contiguous gene syndrome. NF1 -deleted patients were enrolled and phenotypically characterized with a standardized questionnaire between 2005 and 2020 from a large French NF1 cohort. Statistical analyses for main NF1-associated symptoms were performed versus an NF1 reference population. A deletion of the NF1 gene was detected in 4% (139/3479) of molecularly confirmed NF1 index cases. The median age of the group at clinical investigations was 21 years old. A comprehensive clinical assessment showed that 93% (116/126) of NF1 -deleted patients fulfilled the NIH criteria for NF1. More than half had café-au-lait spots, skinfold freckling, Lisch nodules, neurofibromas, neurological abnormalities, and cognitive impairment or learning disabilities. Comparison with previously described "classic" NF1 cohorts showed a significantly higher proportion of symptomatic spinal neurofibromas, dysmorphism, learning disabilities, malignancies, and skeletal and cardiovascular abnormalities in the NF1 -deleted group. We described the largest NF1 -deleted cohort to date and clarified the more severe phenotype observed in these patients.

Background: New rescue regimens are needed for pediatric refractory/recurrent low-grade glioma. Nilotinib is a tyrosine kinase inhibitor that has potential synergistic effects with vinblastine on angiogenesis, tumor cell growth, and immunomodulation., Methods: This phase I trial aimed to determine the recommended doses of this combination for phase II trials (RP2D) using the dual-agent Bayesian continual reassessment method. Nilotinib was given orally twice daily (BID) in combination with once-weekly vinblastine injections for a maximum of 12 cycles of 28 days (clinicaltrials.gov, NCT01884922)., Results: Thirty-five pediatric patients were enrolled across 4 dose levels. The median age was 7 years and 10 had neurofibromatosis type 1. Patients had received a median of 3 prior treatment lines and 25% had received more than 4 previous treatment lines. Dose-limiting toxicity (DLT) during cycle 1 was hematologic, dermatologic, and cardiovascular. The RP2D was identified at 3 mg/m 2 weekly for vinblastine with 230 mg/m 2 BID for nilotinib (estimated probability of DLT = 18%; 95% credibility interval, 7-29%). Fifteen patients completed the 12 cycles; 2 stopped therapy prematurely due to toxicity and 18 due to disease progression. Three patients achieved a partial response leading to an objective response rate of 8.8% (95% confidence interval [CI], 1.9-23.7), and the disease control rate was 85.3% (95% CI, 68.9-95.1). The 12-month progression-free survival was 37.1% (95% CI, 23.2-53.67)., Conclusions: Vinblastine and nilotinib combination was mostly limited by myelosuppression and dermatologic toxicity. The efficacy of the combination at the RP2D is currently evaluated in a randomized phase II trial comparing this regimen to vinblastine alone., (© The Author(s) 2020. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Pediatric neurofibromatosis type 1 (NF1)-associated optic pathway gliomas (OPGs) exhibit different clinico-radiological features, treatment, and outcome compared with sporadic OPGs. While NF1-associated OPGs are caused by complete loss-of-function of the NF1 gene, other genetic alterations of the RAS-MAPK pathway are frequently described in the sporadic cases. We identified a group of patients who presented OPGs with typical radiological features of NF1-associated OPGs but without the NF1 diagnostic criteria. We aim to investigate into the possible molecular mechanisms underlying this "NF1-like" pediatric OPGs presentation., Methods: We analyzed clinico-radiological features of 16 children with NF1-like OPGs and without NF1 diagnostic criteria. We performed targeted sequencing of the NF1 gene in constitutional samples ( n  = 16). The RAS-MAPK pathway major genes were sequenced in OPG tumor samples ( n  = 11); BRAF FISH and IHC analyses were also performed., Results: In one patient's blood and tumor samples, we identified a NF1 nonsense mutation (exon 50: c.7285C>T, p.Arg2429*) with ~8% and ~70% VAFs, respectively, suggesting a mosaic NF1 mutation limited to the brain (segmental NF1). This patient presented signs of neurodevelopmental disorder. We identified a somatic alteration of the RAS-MAPK pathway in eight tumors: four BRAF activating p.Val600Glu mutations, three BRAF:KIAA oncogenic fusions, and one putative gain-of-function complex KRAS indel inframe mutation., Conclusions: NF1-like OPGs can rarely be associated with mosaic NF1 that needs specific constitutional DNA analyses for diagnosis. Further studies are warranted to explore unknown predisposition condition leading to the NF1-like OPG presentation, particularly in patients with the association of a neurodevelopmental disorder., (© The Author(s) 2019. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but high variable expressivity. NF1 is caused by loss-of-function mutations in the NF1 gene, a negative regulator of the RAS-MAPK pathway. The NF1 gene has one of the highest mutation rates in human disorders, which may explain the outbreak of independent de novo variants in the same family. Here, we report the co-occurrence of pathogenic variants in the NF1 and SPRED1 genes in six families with NF1 and Legius syndrome, using next-generation sequencing. In five of these families, we observed the co-occurrence of two independent NF1 variants. All NF1 variants were classified as pathogenic, according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. In the sixth family, one sibling inherited a complete deletion of the NF1 gene from her mother and carried a variant of unknown significance in the SPRED1 gene. This variant was also present in her brother, who was diagnosed with Legius syndrome, a differential diagnosis of NF1. This work illustrates the complexity of molecular diagnosis in a not-so-rare genetic disease.

The tumor suppressor gene neurofibromin 1 (NF1) is a major regulator of the RAS-MAPK pathway. NF1 mutations occur in lung cancer but were not extensively explored. We hypothesized that NF1-mutated tumors could define a specific population with a distinct clinical and molecular profile. We performed NF1 sequencing using next generation sequencing (NGS) in 154 lung adenocarcinoma surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status, to evaluate the molecular and clinical specificities of NF1-mutated lung cancers. Clinical data were retrospectively collected, and their associations with molecular profiles assessed. In this series, 24 tumors were NF1 mutated (17.5%) and 11 were NF1 deleted (8%). There was no mutation hotspot. NF1 mutations were rarely associated with other RAS-MAPK pathway mutations. Most of patients with NF1 alterations were males (74.3%) and smokers (74.3%). Overall survival and disease-free survival were statistically better in patients with NF1 alterations (N = 34) than in patients with KRAS mutations (N = 30) in univariate analysis. Our results confirm that NF1 is frequently mutated and represents a distinct molecular and clinical subtype of lung adenocarcinoma., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background and Purpose Moyamoya angiopathy (MMA) is a rare cerebral vasculopathy outside of Asia. In Japanese patients, a vast majority of patients carry the founder p.R4810K variant in the RNF213 gene, and familial cases are around 10%. In European patients, data about familial occurrence are limited. The aim of this study was to characterize the clinical and molecular features of several European families with a parent-to-child transmission of MMA. Methods Out of 126 MMA probands referred, we identified 113 sporadic probands and 13 familial probands. Segregation analysis showed a vertical parent-to-child pattern of inheritance in the families of 5 of these probands. All 5 families were of German or Dutch ancestry. We investigated the clinical features of affected members and used whole-exome sequencing to screen RNF213 and 13 genes involved in Mendelian MMA and to identify genes recurrently mutated in these families. Results Twelve affected MMA patients were identified, including 9 females and 3 males. Age at clinical onset ranged from 11 to 65 years. In 3 of 5 families, associated livedo racemosa was found. We did not detect any deleterious variants in the 13 known MMA genes. RNF213 rare missense variants predicted to be pathogenic were detected in all affected members of 2 of these families, as well as 2 candidate variants of the PALD1 gene. Conclusions Nonsyndromic MMA was identified in 5 European families, including 2 to 3 clinically affected cases segregating with a parent-to-child pattern of inheritance in each family. Molecular screening detected rare deleterious variants within RNF213 and PALD1 in all affected members of 2 of these 5 families, as well as in some clinically unaffected members. Altogether these data raise the difficult and, to date unanswered, question of the medical indication of presymptomatic screening.

Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome in which glioma is one of the prevalent tumors. Gliomagenesis in NF1 results in a heterogeneous spectrum of low- to high-grade neoplasms occurring during the entire lifespan of patients. The pattern of genetic and epigenetic alterations of glioma that develops in NF1 patients and the similarities with sporadic glioma remain unknown. Here, we present the molecular landscape of low- and high-grade gliomas in patients affected by NF1 (NF1-glioma). We found that the predisposing germline mutation of the NF1 gene was frequently converted to homozygosity and the somatic mutational load of NF1-glioma was influenced by age and grade. High-grade tumors harbored genetic alterations of TP53 and CDKN2A, frequent mutations of ATRX associated with Alternative Lengthening of Telomere, and were enriched in genetic alterations of transcription/chromatin regulation and PI3 kinase pathways. Low-grade tumors exhibited fewer mutations that were over-represented in genes of the MAP kinase pathway. Approximately 50% of low-grade NF1-gliomas displayed an immune signature, T lymphocyte infiltrates, and increased neo-antigen load. DNA methylation assigned NF1-glioma to LGm6, a poorly defined Isocitrate Dehydrogenase 1 wild-type subgroup enriched with ATRX mutations. Thus, the profiling of NF1-glioma defined a distinct landscape that recapitulates a subset of sporadic tumors.

Objective: Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy., Material and Methods: We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria., Results: A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2)., Conclusion: High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Background: Clinical overlap between neurofibromatosis type 2 (NF2), schwannomatosis, and meningiomatosis can make clinical diagnosis difficult. Hence, molecular investigation of germline and tumor tissues may improve the diagnosis., Methods: We present the targeted next-generation sequencing (NGS) of NF2, SMARCB1, LZTR1, SMARCE1, and SUFU tumor suppressor genes, using an amplicon-based approach. We analyzed blood DNA from a cohort of 196 patients, including patients with NF2 (N = 79), schwannomatosis (N = 40), meningiomatosis (N = 12), and no clearly established diagnosis (N = 65). Matched tumor DNA was analyzed when available. Forty-seven NF2-/SMARCB1-negative schwannomatosis patients and 27 NF2-negative meningiomatosis patients were also evaluated., Results: A NF2 variant was found in 41/79 (52%) NF2 patients. SMARCB1 or LZTR1 variants were identified in 5/40 (12.5%) and 13/40 (∼32%) patients in the schwannomatosis cohort. Potentially pathogenic variants were found in 12/65 (18.5%) patients with no clearly established diagnosis. A LZTR1 variant was identified in 16/47 (34%) NF2/SMARCB1-negative schwannomatosis patients. A SMARCE1 variant was found in 3/39 (∼8%) meningiomatosis patients. No SUFU variant was found in the cohort. NGS was an effective and sensitive method to detect mutant alleles in blood or tumor DNA of mosaic NF2 patients. Interestingly, we identified a 4-hit mechanism resulting in the complete NF2 loss-of-function combined with SMARCB1 and LZTR1 haploinsufficiency in two-thirds of tumors from NF2 patients., Conclusions: Simultaneous investigation of NF2, SMARCB1, LZTR1, and SMARCE1 is a key element in the differential diagnosis of NF2, schwannomatosis, and meningiomatosis. The targeted NGS strategy is suitable for the identification of NF2 mosaicism in blood and for the investigation of tumors from these patients.

Background: To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis practice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of paternally inherited mutations in maternal blood using droplet digital PCR (ddPCR)., Methods: This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting compound heterozygous CFTR mutations. NIPD was performed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Analytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample., Results: Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples., Conclusions: We report an NIPD protocol suitable for implementation in an experienced laboratory of molecular genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other monogenic diseases.

Neurofibromatosis type 1 (NF1) is a frequent autosomal dominant genetic disorder that predisposes to the development of benign and malignant tumors. Mutation of the NF1 gene affects the RAS-MAPK signaling pathway and leads to a dysfunction in cell proliferation and induces tumor development. Epidemiology of cancer in children with NF1 is very different from the general pediatric population, which requires regular and specific monitoring. Neurofibroma is the most frequent benign tumor. It can be very invalidating depending on the size and location of the tumor. Currently, there is no specific treatment for these tumors. The most frequent malignancies in children with NF1 are leukemias, rhabdomyosarcomas, malignant peripheral nerve sheath tumors and gliomas. The treatment of these tumors should consider the risk of second cancers induced by radio- and chemotherapy. We report on the case of a 5-year-old boy with NF1 developing two tumors., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Moyamoya syndrome (MMS) is the most common cerebral vasculopathy among children with neurofibromatosis type 1 (NF1). In this study, we clinically, radiologically, and genetically examined a cohort that was not previously described, comprising European children with NF1 and MMS. The NF1 genotyping had been registered. This study included 18 children. The mean age was 2.93 ± 3.03 years at the NF1 diagnosis and 7.43 ± 4.27 years at the MMS diagnosis. In seven patients, MMS was diagnosed before or at the same time as NF1. Neuroimaging was performed in 10 patients due to clinical symptoms, including headache (n = 6), cerebral infarction (n = 2), and complex partial seizures (n = 2). The remaining eight children (47%) had MMS diagnosed incidentally. Sixteen children were characterized molecularly. The features of MMS were similar between patients with and without NF1. Additionally, the NF1 phenotype and genotype were similar between children with and without MMS. Interestingly, three children experienced tumors with malignant histology or behavior. The presence of two first cousins in our cohort suggested that there may be potential genetic factors, not linked to NF1, with an additional role respect of NF1 might play a role in MMS pathogenesis. The incidental diagnosis of MMS, and the observation that, among children with NF1, those with MMS were clinically indistinguishable from those without MMS, suggested that it might be worthwhile to add an angiographic sequence to brain MRIs requested for children with NF1. A MMS diagnosis may assist in properly addressing an NF1 diagnosis in very young children who do not fulfill diagnostic criteria., (© 2017 Wiley Periodicals, Inc.)

The commonest tumors associated with neurofibromatosis type 1 (NF1) are benign peripheral nerve sheath tumors, called neurofibromas. Malignant transformation of neurofibromas into aggressive MPNSTs may occur with a poor patient prognosis. A cooperative role of SUZ12 or EED inactivation, along with NF1, TP53, and CDKN2A loss-of-function, has been proposed to drive progression to MPNSTs. An exome sequencing analysis of eight MPNSTs, one plexiform neurofibroma, and seven cutaneous neurofibromas was undertaken. Biallelic inactivation of the NF1 gene was observed in the plexiform neurofibroma and the MPNSTs, underlining that somatic biallelic NF1 inactivation is likely to be the initiating event for plexiform neurofibroma genesis, although it is unlikely to be sufficient for the subsequent MPNST development. The majority (5/8) of MPNSTs in our analyses demonstrated homozygous or heterozygous deletions of CDKN2A, which may represent an early event following NF1 LOH in the malignant transformation of Schwann cells from plexiform neurofibroma to MPNST. Biallelic somatic alterations of SUZ12 was also found in 4/8 MPNSTs. EED biallelic alterations were detected in 2 of the other four MPNSTs, with one tumor having a homozygous EED deletion. A missense mutation in the chromatin regulator KDM2B was also identified in one MPNST. No TP53 point mutations were found in this study, confirming previous data that TP53 mutations may be relatively rare in NF1-associated MPNSTs. Our study confirms the frequent biallelic inactivation of PRC2 subunits SUZ12 and EED in MPNSTs, and suggests the implication of KDM2B., (© 2017 Wiley Periodicals, Inc.)

Background: Heterozygous NSD1 mutations were identified in 60%-90% of patients with Sotos syndrome. Recently, mutations of the SETD2 and DNMT3A genes were identified in patients exhibiting only some Sotos syndrome features. Both NSD1 and SETD2 genes encode epigenetic 'writer' proteins that catalyse methylation of histone 3 lysine 36 (H3K36me). The DNMT3A gene encodes an epigenetic 'reader' protein of the H3K36me chromatin mark., Methods: We aimed at confirming the implication of DNMT3A and SETD2 mutations in an overgrowth phenotype, through a comprehensive targeted-next generation sequencing (NGS) screening in 210 well-phenotyped index cases with a Sotos-like phenotype and no NSD1 mutation, from a French cohort., Results: Six unreported heterozygous likely pathogenic variants in DNMT3A were identified in seven patients: two nonsense variants and four de novo missense variants. One de novo unreported heterozygous frameshift variant was identified in SETD2 in one patient. All the four DNMT3A missense variants affected DNMT3A functional domains, suggesting a potential deleterious impact. DNMT3A -mutated index cases shared similar clinical features including overgrowth phenotype characterised by postnatal tall stature (≥+2SD), macrocephaly (≥+2SD), overweight or obesity at older age, intellectual deficiency and minor facial features. The phenotype associated with SETD2 mutations remains to be described more precisely. The p.Arg882Cys missense de novo constitutional DNMT3A variant found in two patients is the most frequent DNMT3A somatic mutation in acute leukaemia., Conclusions: Our results illustrate the power of targeted NGS to identify rare disease-causing variants. These observations provided evidence for a unifying mechanism (disruption of apposition and reading of the epigenetic chromatin mark H3K36me) that causes an overgrowth syndrome phenotype. Further studies are needed in order to assess the role of SETD2 and DNMT3A in intellectual deficiency without overgrowth., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The discovery of free fetal DNA in the maternal circulation has inaugurated the era of non-invasive prenatal diagnosis. The latter has the advantage of avoiding the use of conventional obstetric procedures, such as chorionic villus sampling or aspiration of amniotic fluid, thus limiting the risks of miscarriage they induce. However, as free fetal DNA accounts for about 10% of cell-free DNA in maternal plasma, the presence of ambient maternal DNA can make it difficult to detect fetal alleles of paternal origin. Digital Droplet PCR (ddPCR) is a very sensitive method derived from quantitative real-time PCR (qPCR) for the detection of rare alleles and their absolute quantification by removing the necessity of standards. Here we show that this new technology can be applied in routine prenatal fetal RHD genotyping from maternal blood. In conclusion, the use of quantitative properties of digital PCR, in terms of accuracy, sensitivity and specificity, allows one to consider extending the applications of this new technology in non-invasive prenatal diagnosis of many diseases such as autosomal monogenic diseases, either dominant or recessive.

Neurofibromatosis-Noonan syndrome (NFNS) is a distinct entity which shows the features of both NF1 (neurofibromatosis 1) and Noonan syndrome (NS). While growth hormone deficiency (GHD) has been relatively frequently identified in NF1 and NS patients, there is limited experience in NFNS cases. The literature includes only one case report of a NFNS patient having GHD and that report primarily focuses on the dermatological lesions that accompany the syndrome and not on growth hormone (GH) treatment. Here, we present a 13-year-old girl who had clinical features of NFNS with a mutation in the NF1 gene. The case is the first NFNS patient reported in the literature who was diagnosed to have GHD and who received GH treatment until reaching final height. The findings in this patient show that short stature is a feature of NFNS and can be caused by GHD. Patients with NFNS who show poor growth should be evaluated for GHD.

Juvenile myelomonocytic leukemia (JMML) is a rare and severe myelodysplastic and myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations. Genetic profiling and whole-exome sequencing of a large JMML cohort (118 and 30 cases, respectively) uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38 events/Mb/case) and restricted to sporadic (49/78; 63%) or neurofibromatosis type 1 (NF1)-associated (8/8; 100%) JMML cases. Multiple concomitant genetic hits targeting the RAS pathway were identified in 13 of 78 cases (17%), disproving the concept of mutually exclusive RAS pathway mutations and defining new pathways activated in JMML involving phosphoinositide 3-kinase (PI3K) and the mTORC2 complex through RAC2 mutation. Furthermore, this study highlights PRC2 loss (26/78; 33% of sporadic JMML cases) that switches the methylation/acetylation status of lysine 27 of histone H3 in JMML cases with altered RAS and PRC2 pathways. Finally, the association between JMML outcome and mutational profile suggests a dose-dependent effect for RAS pathway activation, distinguishing very aggressive JMML rapidly progressing to acute myeloid leukemia.

Background & Aims: Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors in repetitive DNA sequences. We investigated whether these features could be used to identify patients with CMMRD., Methods: We examined MSI by PCR analysis and tolerance to methylating or thiopurine agents (functional characteristics of MMR-deficient tumor cells) in lymphoblastoid cells (LCs) from 3 patients with CMMRD and 5 individuals with MMR-proficient LCs (controls). Using these assays, we defined experimental parameters that allowed discrimination of a series of 14 patients with CMMRD from 52 controls (training set). We then used the same parameters to assess 23 patients with clinical but not genetic features of CMMRD., Results: In the training set, we identified parameters, based on MSI and LC tolerance to methylation, that detected patients with CMMRD vs controls with 100% sensitivity and 100% specificity. Among 23 patients suspected of having CMMRD, 6 had MSI and LC tolerance to methylation (CMMRD highly probable), 15 had neither MSI nor LC tolerance to methylation (unlikely to have CMMRD), and 2 were considered doubtful for CMMRD based on having only 1 of the 2 features., Conclusion: The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of ∼150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with ∼90[euro ] for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.

Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

Background: Neurofibromatosis 1 (NF1) belongs to the autosomal dominant neurocutaneous disorders' group, which mainly includes NF1 and NF2, tuberous sclerosis, von Hippel-Lindau disease and Cerebral Cavernous Malformations (CCMs). NF1 has a major impact on the nervous system, eye, skin, bone or cardiovascular system. Cerebrovascular lesions have been reported in NF1 including aneurysm, pseudoaneurysm, arteriovenous malformations, vascular stenosis or occlusion and Moya moya syndrome., Objective: To report a case of an NF1 patient with multiple CCMs., Observation: A 47-year-old man with café-au-lait skin lesions, countless cutaneous neurofibromas, short stature and scoliosis was admitted for progressive spinal cord compression due to histologically proven neurofibroma. Systematic cerebral MRI screening including gradient echo sequences showed multiple asymptomatic CCMs. Screening of CCM1, CCM2 and CCM3 genes was negative while a deleterious frameshift mutation was identified in NF1 gene., Conclusion: While single CCM can occur in NF1 patients following radiation exposure, they are only rarely reported in non-irradiated NF1 brain. Even if it could be a fortuitous association, plausible links and explanations exist. If cerebral MRI can be systematic in NF1 to detect asymptomatic gliomas, used protocols in neuroradiology do not usually include gradient echo sequences, the most sensitive test for CCM detection, leading possibly to failure to detect these vascular lesions. More reports having this combination and further investigations of NF1 families will certainly provide a better understanding of links between these 2 phakomatoses, as recently reported with "multiple meningiomas" phenotype associated with multiple CCMs in patients with CCM3 gene mutations or café-au-lait skin lesions in CCM1 mutation carriers., (Copyright © 2015. Published by Elsevier B.V.)

Constitutional dominant loss-of-function mutations in the SPRED1 gene cause a rare phenotype referred as neurofibromatosis type 1 (NF1)-like syndrome or Legius syndrome, consisted of multiple café-au-lait macules, axillary freckling, learning disabilities and macrocephaly. SPRED1 is a negative regulator of the RAS MAPK pathway and can interact with neurofibromin, the NF1 gene product. Individuals with NF1 have a higher risk of haematological malignancies. SPRED1 is highly expressed in haematopoietic cells and negatively regulates haematopoiesis. SPRED1 seemed to be a good candidate for leukaemia predisposition or transformation. We performed SPRED1 mutation screening and expression status in 230 paediatric lymphoblastic and acute myeloblastic leukaemias (AMLs). We found a loss-of-function frameshift SPRED1 mutation in a patient with Legius syndrome. In this patient, the leukaemia blasts karyotype showed a SPRED1 loss of heterozygosity, confirming SPRED1 as a tumour suppressor. Our observation confirmed that acute leukaemias are rare complications of the Legius syndrome. Moreover, SPRED1 was significantly decreased at RNA and protein levels in the majority of AMLs at diagnosis compared with normal or paired complete remission bone marrows. SPRED1 decreased expression correlated with genetic features of AML. Our study reveals a new mechanism which contributes to deregulate RAS MAPK pathway in the vast majority of paediatric AMLs.

Neurofibromatosis type 1 (NF1) is an autosomal dominant, multi-system, neurocutaneous disorder that predisposes to the development of benign and malignant tumors with a birth incidence rate of 1 in 2500-3000. 50% of cases are sporadic. The diagnosis is exclusively based on clinical assessment with clinical diagnostic criteria such as café-au-lait spots, neurofibromas, axillary or groin freckling, Lisch nodules, optic pathway glioma, bony dysplasia and first-degree relative with NF1. We report a family with NF1 in which two members presented atypical clinical features in addition to the classical diagnostic criteria. Three relatives affected by NF1, a father and two of his three sons, are described. The clinical diagnosis was originally worn in all three cases, with the association many spots café-au -lait over the entire body and some axillary freckling as well as first-degree relative. One case presented an Acute Myeloid Leukemia (AML) type 2 at 10 years of age diagnosed before the revelation of bicytopenia associated pallor and isolated asthenia. A second case presented a nephrotic syndrome at 4 years of age due to the association of hydrops with headache and asthenia. Direct sequencing of NF1 led to identify the familial mutation, a previously unreported heterozygous missense mutation c.3443C > A, p.Ala1148Glu in exon 20 which segregated with all three affected patients. The family described in this report confirms the high clinical variability of NF1, even intrafamilial, and raises the question as to whether rare features such as AML and nephrotic syndrome are associated with NF1. Some NF1 patients presenting glomerular diseases or AML have rarely been reported, but due to the small number of cases described the mechanisms underlying these associations are poorly understood. However, it seems important to be aware of the possible occurrence of nephritic syndrome and/or malignant blood diseases in NF1 patients., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Purpose: The hallmark of neurofibromatosis type 1 (NF1) is the onset of dermal or plexiform neurofibromas, mainly composed of Schwann cells. Plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST) that are resistant to therapies., Experimental Design: The aim of this study was to identify an additional pathway in the NF1 tumorigenesis. We focused our work on Wnt signaling that is highly implicated in cancer, mainly in regulating the proliferation of cancer stem cells. We quantified mRNAs of 89 Wnt pathway genes in 57 NF1-associated tumors including dermal and plexiform neurofibromas and MPNSTs. Expression of two major stem cell marker genes and five major epithelial-mesenchymal transition marker genes was also assessed. The expression of significantly deregulated Wnt genes was then studied in normal human Schwann cells, fibroblasts, endothelial cells, and mast cells and in seven MPNST cell lines., Results: The expression of nine Wnt genes was significantly deregulated in plexiform neurofibromas in comparison with dermal neurofibromas. Twenty Wnt genes showed altered expression in MPNST biopsies and cell lines. Immunohistochemical studies confirmed the Wnt pathway activation in NF1-associated MPNSTs. We then confirmed that the knockdown of NF1 in Schwann cells but not in epithelial cells provoked the activation of Wnt pathway by functional transfection assays. Furthermore, we showed that the protein expression of active β-catenin was increased in NF1-silenced cell lines. Wnt pathway activation was strongly associated to both cancer stem cell reservoir and Schwann-mesenchymal transition., Conclusion: We highlighted the implication of Wnt pathway in NF1-associated tumorigenesis., (©2013 AACR.)

Neurofibromatosis type 1 (NF1) affects about one in 3,500 people in all ethnic groups. Most NF1 patients have private loss-of-function mutations scattered along the NF1 gene. Here, we present an original NF1 investigation strategy and report a comprehensive mutation analysis of 565 unrelated patients from the NF-France Network. A NF1 mutation was identified in 546 of the 565 patients, giving a mutation detection rate of 97%. The combined cDNA/DNA approach showed that a significant proportion of NF1 missense mutations (30%) were deleterious by affecting pre-mRNA splicing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements that would have been missed if only sequencing or microsatellite analysis had been performed. In four unrelated families, we identified two distinct NF1 mutations within the same family. This fortuitous association points out the need to perform an exhaustive NF1 screening in the case of molecular discordant-related patients. A genotype-phenotype study was performed in patients harboring a truncating (N = 368), in-frame splicing (N = 36), or missense (N = 35) mutation. The association analysis of these mutation types with 12 common NF1 clinical features confirmed a weak contribution of the allelic heterogeneity of the NF1 mutation to the NF1 variable expressivity., (© 2013 WILEY PERIODICALS, INC.)

Background: Neurofibromatosis type 1 (NF1) is a common dominant tumor predisposition syndrome affecting 1 in 3,500 individuals. The hallmarks of NF1 are the development of peripheral nerve sheath tumors either benign (dermal and plexiform neurofibromas) or malignant (MPNSTs)., Results: To comprehensively characterize the role of microRNAs in NF1 tumorigenesis, we analyzed 377 miRNAs expression in a large panel of dermal and plexiform neurofibromas, and MPNSTs. The most significantly upregulated miRNA in plexiform neurofibromas was miR-486-3p that targets the major tumor suppressor gene, PTEN. We confirmed PTEN downregulation at mRNA level. In plexiform neurofibromas, we also report aberrant expression of four miRNAs involved in the RAS-MAPK pathway (miR-370, miR-143, miR-181a, and miR-145). In MPNSTs, significant deregulated miRNAs were involved in PTEN repression (miR-301a, miR-19a, and miR-106b), RAS-MAPK pathway regulation (Let-7b, miR-195, and miR-10b), mesenchymal transition (miR-200c, let-7b, miR-135a, miR-135b, and miR-9), HOX genes expression (miR-210, miR-196b, miR-10a, miR-10b, and miR-9), and cell cycle progression (miR-195, let-7b, miR-20a, miR-210, miR-129-3p, miR-449a, and miR-106b)., Conclusion: We confirmed the implication of PTEN in genesis of plexiform neurofibromas and MPNSTs in NF1. Markedly deregulated miRNAs might have potential diagnostic or prognostic value and could represent novel strategies for effective pharmacological therapies of NF1 tumors.