Neurofibromatosis Type 1 Mutations Research Articles

Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study.

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.

SUMMARYNeurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.

RARE-03 CENTRAL NERVOUS SYSTEM TUMORS IN CHILDREN WITH NEUROFIBROMATOSIS TYPE 1, A COHORT FROM A NATIONAL PEDIATRIC ONCOLOGY CENTER

Approximately 15% of the children with Neurofibromatosis type 1 (NF1) develop a central nervous system (CNS) tumor, predominantly optic pathway glioma. However, other brain tumor types are also frequent, resulting in a heterogenous population regarding tumor type, symptoms and treatment. Even though many tumors are indolent, clinical deterioration or radiological progression requires surveillance and treatment. Pediatric oncology care in the Netherlands has been centralized into the Princess Máxima Center since June 2018, providing care for the majority of Dutch NF1 CNS tumor patients. This retrospective cohort study describes the course of disease in NF1 patients with a CNS tumor diagnosed between 2005–2020, based on data from patient files. The population consisted of 95 patients (48 males), median age at diagnosis 4.3 (range 0.8–17.2) and 12.6 (range 2.9–19.9) years at follow up, 27% familiar NF1, mostly nonsense or frameshift mutations. Neurological and/or visual symptoms were present in 64 (67.4%) patients. 63.6% of the tumors was in the optic pathway, 9.1% in the cerebellum. Biopsy in 19/95 revealed 1 ependymoma and 18 low grade glioma, with rarely additional molecular changes besides NF1. Kaplan Meier median PFS was 24 months (range 3–174.5, n=65) in symptomatic presenting patients, 34 months (range 5–165, n=20) in asymptomatic patients with abnormal ophthalmological and/or neurological exam and 67.3 months (range 7–130, n=10) in asymptomatic patients without abnormalities at exam. Treatment was indicated in 53 (55.8%), predominantly chemotherapy (n=49), with substantial toxicity. Carboplatin allergy was seen in 29/44 (65.9%) patients, peripheral neuropathy in 21/46 (45.7%) patients receiving vincristine, and 5/10 patients receiving irinotecan and bevacizumab ended treatment early due to toxicity. In conclusion: analysis of our extensive cohort of NF1 patients with a CNS tumor indicates the relevance of regular screening of NF1 patients without complaints and critical surveillance and systematical documentation of treatment toxicity.

Characterization of Two Loss-of-Function NF1 Variants in Chinese Patients and Potential Molecular Interpretations of Phenotypes.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by cafe’-au-lait spots, skinfold freckles, the formation of neurofibromas, skeletal dysplasia, vascular dysplasia, and an increased risk of malignant tumors. In this study, two Chinese NF1 children troubled with bone lesions or hypertension were reported. A de novo NF1 mutation (c.4925T > A/p.V1642E) and a maternally inherited NF1 mutation (c.4883T > A/p.L1628∗) were identified by molecular sequence. According to the ACMG/AMP guidelines, the c.4925T > A was classified as variants of uncertain significance (VOUS) while the c.4883T > A mutation was identified as likely Pathogenic. Further study found that these two NF1 mutants had lost their function to inhibit the Ras/Erk signaling and the proliferation of cells, which could interpretate some phenotypes of these two NF1 patients. We also observed these two NF1 mutants displayed decreased protein stability with increased ubiquitination levels compared with that of wild-type NF1.

Neurofibromatosis type 1: results of our own study (Republic of Bashkortostan)

Introduction. Neurofibromatosis type 1 (NF1) is the most common hereditary tumor syndrome (frequency of its occurrence in the world is 1 : 3000 of the population). The main clinical manifestations of the disease are multiple café-au-lait macules on the skin and neurofibromas, skeletal abnormalities and cognitive deficits. The disease is based on mutations in the oncosuppressor gene NF1. This disease is characterized by significant clinical polymorphism of manifestations, even among members of the same family. No geno-phenotypic correlations were found for NF1. Therefore, it is assumed that modifier genes are the cause of the varying expressiveness of the disease. Materials and methods. Clinical-epidemiological and molecular-genetic research of patients with NF1 in the Republic of Bashkortostan (RB) was carried out. Sequencing was used to search for intragenic mutations in 57 exons of the NF1 gene. Microsatellite analysis was used to detect the deletion of the entire gene.Results. The frequency of occurrence of NF1 in RB was 1 : 10153 of the population. Analysis of the clinical manifestations of NF1 in RB patients showed a lower incidence of brain cysts in patients born in mixed marriages, which indicates the protective role of mestization. In patients with NF1 who inherited the disease from the mother, a more frequent development of skeletal anomalies and facial dysmorphism was determined. We identified 1 deletion of the entire NF1 gene in 1 patient and 14 intragenic mutations (c.205-1G>C, с.1278G>A, c.1369_1370insGGGTC, с.1570G>A, с.1973_1974delTC, c.2806A>T, с.2991-1G>C, c.3158C>G, с.3526_3528delAGA, с.3826delC, с.4514+5G>A, с.4537С>Т, c.5758_5761delTTGA, с.6792С>A) in 20 patients with NF1. We determined the random distribution of the types of mutations and did not reveal the specific features of the NF1 clinic depending on the type of mutations.Conclusions. The protective role of crossbreeding in relation to brain cysts, as well as the predominance of skeletal anomalies in patients with NF1 inheritance from the mother, indicate the role of modifier genes in the pathogenesis of the disease. The identified mutations in the NF1 gene will allow us to perform prenatal prevention of NF1 in RB patients.

Shared developmental gait disruptions across two mouse models of neurodevelopmental disorders

BackgroundMotor deficits such as abnormal gait are an underappreciated yet characteristic phenotype of many neurodevelopmental disorders (NDDs), including Williams Syndrome (WS) and Neurofibromatosis Type 1 (NF1). Compared to cognitive phenotypes, gait phenotypes are readily and comparably assessed in both humans and model organisms and are controlled by well-defined CNS circuits. Discovery of a common gait phenotype between NDDs might suggest shared cellular and molecular deficits and highlight simple outcome variables to potentially quantify longitudinal treatment efficacy in NDDs.MethodsWe characterized gait using the DigiGait assay in two different murine NDD models: the complete deletion (CD) mouse, which models hemizygous loss of the complete WS locus, and the Nf1+/R681X mouse, which models a NF1 patient-derived heterozygous germline NF1 mutation. Longitudinal data were collected across four developmental time points (postnatal days 21–30) and one early adulthood time point.ResultsCompared to wildtype littermate controls, both models displayed markedly similar spatial, temporal, and postural gait abnormalities during development. Developing CD mice also displayed significant decreases in variability metrics. Multiple gait abnormalities observed across development in the Nf1+/R681X mice persisted into early adulthood, including increased stride length and decreased stride frequency, while developmental abnormalities in the CD model largely resolved by adulthood.ConclusionsThese findings suggest that the subcomponents of gait affected in NDDs show overlap between disorders as well as some disorder-specific features, which may change over the course of development. Our incorporation of spatial, temporal, and postural gait measures also provides a template for gait characterization in other NDD models and a platform to examining circuits or longitudinal therapeutics.

Structural alteration of lung parenchyma in patients with NF1: a phenotyping study using multidetector computed tomography (MDCT)

BackgroundDiffuse interstitial lung disease have been described in Neurofibromatosis type 1 (NF1), but its diversity and prevalence remain unknown. The aim of this study was to assess the prevalence and characteristics of (NF1)-associated lung manifestations in a large single-center study using multidetector computed tomography (MDCT) and to evaluate the smoking history, patients’ age, genetics, and the presence of malignant peripheral nerve sheath tumors (MPNST) as potential influencing factors for lung pathologies.MethodsIn this retrospective study, 71 patients with NF1 were evaluated for the presence of distinctive lung manifestations like reticulations, consolidations, type of emphysema, pulmonary nodules and cysts. All patients underwent F-18-FDG PET/CT scans, which were reviewed by two experienced radiologists in consensus. Patients’ subgroups were formed based on their smoking history (current smokers/previous smokers/never smokers), age (< 12 years, 12–18 years, > 18 years), and presence of MPNST (MPNST/no MPNST). In 57 patients (80%), genetic analysis of sequences coding for the neurofibromin on chromosome 17 was performed, which was correlated with different lung pathologies.ResultsAmong all NF1 patients (33 ± 14 years, 56% females), 17 patients (24%) were current smokers and 62 patients (87%) were > 18 years old. Pulmonary cysts, nodules, and paraseptal emphysema were the most common pulmonary findings (35%, 32%, 30%). The presence of pulmonary metastases, MPNST and centrilobular emphysema was associated with smoking. Cysts were observed only in adults, whereas no significant correlation between age and all other pulmonary findings was found (p > 0.05). Presence of MPNST was accompanied by higher rates of intrapulmonary nodules and pulmonary metastasis. Neither the presence nor absence of any of the specific gene mutations was associated with any particular lung pathology (p > 0.05).ConclusionsAll pulmonary findings in NF1 patients occurred independently from specific mutation subtypes, suggesting that many NF1 mutations can cause various pulmonary pathologies. The presence of pulmonary metastases, MPNST and centrilobular emphysema was associated with smoking, indicating the value of smoking secession or the advice not to start smoking in NF1 patients as preventive strategy for clinicians. For screening of pulmonary manifestations in NF1 patients, an MDCT besides medical history and physical examination is mandatory in clinical routine.

P2RY14-EGFR Crosstalk is Perturbed by Loss of the&amp;nbsp; &lt;i&gt;NF1&lt;/i&gt;&amp;nbsp;Tumor Suppressor and Modulates Schwann Cell Precursor Self-Renewal And&amp;nbsp;Neurofibroma&amp;nbsp;Initiation

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas. Expression profiling of human Schwann cells (SCs) and neurofibroma SC precursors (SCPs) identified enriched P2RY14 expression in neurofibroma SCPs. We show that genetic and pharmacological inhibition of P2RY14 in human and murine NF1 mutant SCPs reduces SCP self-renewal in vitro. We identified the mechanism of action being, P2RY14 forms a complex with the epidermal growth factor receptor (EGFR) to regulate cAMP and RAS signaling. In vivo, genetic deletion of P2RY14 in NF1 murine model delayed neurofibroma initiation, reduced cAMP and increased Caveolin 1 ( Cav1 ) expression. In a murine model where EGFR is overexpressed in SCs, genetic deletion of Cav1 increased neurofibroma initiation. Thus, P2RY14 regulates EGFR signaling, cAMP and Cav1 expression, facilitating SCP self-renewal and neurofibroma initiation. These gain and loss of function experiments suggest critical roles for P2RY14-EGFR crosstalk perturbed by NF1 tumor suppressor loss.

MicroRNA-155 contributes to plexiform neurofibroma growth downstream of MEK.

MicroRNAs (miRs) are small non-coding RNAs that can have large impacts on oncogenic pathways. Possible functions of dysregulated miRs have not been studied in neurofibromatosis type 1 (NF1) plexiform neurofibromas (PNFs). In PNFs, Schwann cells (SCs) have biallelic NF1 mutations necessary for tumorigenesis. We analyzed a miR-microarray comparing to normal and PNF SCs and identified differences in miR expression, and we validated in mouse PNFs versus normal mouse SCs by qRT-PCR. Among these, miR-155 was a top overexpressed miR, and its expression was regulated by RAS/MAPK signaling. Overexpression of miR-155 increased mature Nf1−/− mouse SC proliferation. In SC precursors, which model tumor initiating cells, pharmacological and genetic inhibition of miR-155 decreased PNF-derived sphere numbers in vitro and we identified Maf as a miR-155 target. In vivo, global deletion of miR-155 significantly decreased tumor number and volume, increasing mouse survival. Fluorescent nanoparticles entered PNFs, suggesting that an anti-miR might have therapeutic potential. However, treatment of established PNFs using anti-miR-155 peptide nucleic acid-loaded nanoparticles marginally decreased tumor numbers and did not reduce tumor growth. These results suggest that miR-155 plays a functional role in PNF growth and/or SC proliferation, and that targeting neurofibroma miRs is feasible, and might provide novel therapeutic opportunities.

LGG-27. TARGETED THERAPY FOR PEDIATRIC LOW-GRADE GLIOMAS AND PLEXIFORM NEUROFIBROMAS WITH TRAMETINIB

BACKGROUNDTargeted therapy aimed at modulating the RAS/RAF/MEK/ERK pathway is of increasing interest for patients with plexiform neurofibromas and low-grade gliomas. Trametinib is an FDA-approved MEK inhibitor that has little published pediatric experience to date.METHODSA retrospective chart review of patients treated with trametinib for low-grade gliomas (LGG) and/or plexiform neurofibromas (PN) between 2015–2018 was conducted at Children’s Hospital Colorado. Data collected included patient demographics, lesion location, Neurofibromatosis type 1 (NF1) status, best response of PN/LGG to trametinib, duration of trametinib therapy, and reported toxicities at least possibly attributed to trametinib.RESULTSThirty (57% male; 73% NF1) patients were identified. Sixteen (53%) patients had PN only, 12 (40%) had LGG only, and two (7%) patients had both PN and LGG. The most common LGG location was the optic pathway/hypothalamus (72%). The most common location of PN was the face (63%). Two-thirds (8/12) of patients with LGG had a BRAF alteration or NF1 mutation. The median age at start of trametinib therapy was 9.9 years (range, 2.0 – 18.8 years). The median duration of trametinib therapy was 0.8 years (range 0.1 – 2.9 years). The most commonly reported adverse event was rash. No patients developed retinal toxicity or cardiotoxicity. Only two (7%) patients discontinued for toxicity and one (3%) for progressive disease.CONCLUSIONSTrametinib can be administered without significant toxicity to children with PN or LGG. Clinical benefit is noted in this cohort; however, prospective clinical trials are necessary to characterize efficacy formally.

A novel RAB39B mutation and concurrent de novo NF1 mutation in a boy with neurofibromatosis type 1, intellectual disability, and autism: a case report

BackgroundMutations in RAB39B at Xq28 causes a rare form of X-linked intellectual disability (ID) and Parkinson’s disease. Neurofibromatosis type 1 (NF1) is caused by heterozygous mutations in NF1 occurring de novo in about 50% of cases, usually due to paternal gonadal mutations. This case report describes clinical and genetic findings in a boy with the occurrence of two distinct causative mutations in NF1 and RAB39B explaining the observed phenotype.Case presentationHere we report a 7-year-old boy with multiple café-au-lait macules (CALMs) and freckling, severe macrocephaly, peculiar facial gestalt, severe ID with absent speech, epilepsy, autistic traits, self-harming, and aggressiveness. Proband is an only child born to a father aged 47. Parents did not present signs of NF1, while a maternal uncle showed severe ID, epilepsy, and tremors.By RNA analysis of NF1, we identified a de novo splicing variant (NM_000267.3:c.6579+2T>C) in proband, which explained NF1 clinical features but not the severe ID, behavioral problems, and aggressiveness. Family history suggested an X-linked condition and massively parallel sequencing of X-exome identified a novel RAB39B mutation (NM_171998.2:c.436_447del) in proband, his mother, and affected maternal uncle, subsequently validated by Sanger sequencing in these and other family members.ConclusionsThe case presented here highlights how concurrent genetic defects should be considered in NF1 patients when NF1 mutations cannot reasonably explain all the observed clinical features.

Abstract 1087: NF1 regulates the RAS-related GTPases, RRAS and RRAS2, independent of RAS activity

Abstract Neurofibromin, which is encoded by the neurofibromatosis type 1 (NF1) gene, is a tumor suppressor that acts as a RAS-GTPase activating protein (RAS-GAP) to stimulate the intrinsic GTPase activity of RAS as well as the closely related RAS subfamily members, RRAS, RRAS2, and MRAS. This results in the conversion of the active GTP-bound form of RAS into the inactive GDP-bound state leading to the downregulation of several RAS downstream effector pathways, most notably MAPK signaling. While the region of NF1 that regulates RAS activity represents only a small fraction of the entire protein, a large extent of the NF1 structural domains and their corresponding mechanistic functions remain uncharacterized despite the fact there is a high frequency of NF1 mutations in several different types of cancer. Thus, we wanted to elucidate the underlying biochemical and signaling functions of NF1 that are unrelated to the regulation of RAS and how loss of these functions contributes to the pathogenesis of cancer. To accomplish this objective, we used CRISPR-Cas9 methods to knockout NF1 in an isogenic “RASless” MEF model system, which is devoid of the major oncogenic RAS isoforms (HRAS, KRAS, and NRAS) and reconstituted with the KRAS4b wild-type or mutant KRASG12C or KRASG12D isoform. Loss of NF1 led to elevated RAS-GTP levels, however, this increase was not as profound as the levels in KRAS-mutated cells or provided a proliferative advantage. Although ablation of NF1 resulted in sustained activation of MAPK signaling, it also unexpectedly, resulted in a robust increase in AKT phosphorylation compared to KRAS-mutated cells. Surprisingly, loss of NF1 in KRAS4b wild-type and KRAS-mutated cells potently suppressed the RAS-related GTPases, RRAS and RRAS2, with modest effects on MRAS, at both the transcript and protein levels. A Clariom™D transcriptome microarray analysis revealed a significant downregulation in the NF-κB target genes, insulin-like growth factor binding protein 2 (IGFBP2), argininosuccinate synthetase 1 (ASS1), and DUSP1, in both the NF1 knockout KRAS4b wild-type and KRAS-mutated cells. Moreover, NF1Null melanoma cells also displayed a potent suppression of RRAS and RRAS2 as well as these NF-κB transcription factors. Since RRAS and RRAS2 both contain the same NF-κB transcription factor binding sites, we hypothesize that IGFBP2, ASS1, and/or DUSP1 may contribute to the NF1-mediated regulation of these RAS-related GTPases. More importantly, this study provides the first evidence of at least one novel RAS-independent function of NF1 to regulate the RAS-related subfamily members, RRAS and RRAS2, in a manner exclusive of its RAS-GTPase activity and this may provide insight into new potential biomarkers and molecular targets for treating patients with mutations in NF1. Citation Format: Jillian M. Silva, Lizzeth Canche, Frank McCormick. NF1 regulates the RAS-related GTPases, RRAS and RRAS2, independent of RAS activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1087.

Nf1 heterozygous mice recapitulate the anthropometric and metabolic features of human neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a heritable cancer predisposition syndrome resulting from mutations in the NF1 tumor suppressor gene. Genotype-phenotype correlations for NF1 are rare due to the large number of NF1 mutations and role of modifier genes in manifestations of NF1; however, emerging reports suggest that persons with NF1 display a distinct anthropometric and metabolic phenotype featuring short stature, low body mass index, increased insulin sensitivity, and protection from diabetes. Nf1 heterozygous (Nf1+/-) mice accurately reflect the dominant inheritance of NF1 and are regularly employed as a model of NF1. Here, we sought to identify whether Nf1+/- mice recapitulate the anthropometric and metabolic features identified in persons with NF1. Littermate 16-20 week-old male wildtype (WT) and Nf1+/- C57B/6J mice underwent nuclear magnetic resonance (NMR), indirect calorimetry, and glucose/insulin/pyruvate tolerance testing. In some experiments, tissues were harvested for NMR and histologic characterization. Nf1+/- mice are leaner with significantly reduced visceral and subcutaneous fat mass, which corresponds with an increased density of small adipocytes and reduced leptin levels. Additionally, Nf1+/- mice are highly reliant on carbohydrates as an energy substrate and display increased glucose clearance and insulin sensitivity, but normal response to pyruvate suggesting enhanced glucose utilization and preserved gluconeogenesis. Finally, WT and Nf1+/- mice subjected to high glucose diet were protected from diet-induced obesity and hyperglycemia. Our data suggest that Nf1+/- mice closely recapitulate the anthropometric and metabolic phenotype identified in persons with NF1, which will impact the interpretation of previous and future translational studies of NF1.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Clinical characterization of children and adolescents with NF1 microdeletions

PurposeAn estimated 5–11% of patients with neurofibromatosis type 1 (NF1) harbour NF1 microdeletions encompassing the NF1 gene and its flanking regions. The purpose of this study was to evaluate the clinical phenotype in children and adolescents with NF1 microdeletions.MethodsWe retrospectively analysed 30 children and adolescents with NF1 microdeletions pertaining to externally visible neurofibromas. The internal tumour load was determined by volumetry of whole-body magnetic resonance imaging (MRI) in 20 children and adolescents with NF1 microdeletions. Furthermore, the prevalence of global developmental delay, autism spectrum disorder and attention deficit hyperactivity disorder (ADHD) were evaluated.ResultsChildren and adolescents with NF1 microdeletions had significantly more often cutaneous, subcutaneous and externally visible plexiform neurofibromas than age-matched patients with intragenic NF1 mutations. Internal neurofibromas were detected in all 20 children and adolescents with NF1 microdeletions analysed by whole-body MRI. By contrast, only 17 (61%) of 28 age-matched NF1 patients without microdeletions had internal tumours. The total internal tumour load was significantly higher in NF1 microdeletion patients than in NF1 patients without microdeletions. Global developmental delay was observed in 28 (93%) of 30 children with NF1 microdeletions investigated. The mean full-scale intelligence quotient in our patient group was 77.7 which is significantly lower than that of patients with intragenic NF1 mutations. ADHD was diagnosed in 15 (88%) of 17 children and adolescents with NF1 microdeletion. Furthermore, 17 (71%) of the 24 patients investigated had T-scores ≥ 60 up to 75, indicative of mild to moderate autistic symptoms, which are consequently significantly more frequent in patients with NF1 microdeletions than in the general NF1 population. Also, the mean total T-score was significantly higher in patients with NF1 microdeletions than in the general NF1 population.ConclusionOur findings indicate that already at a very young age, NF1 microdeletions patients frequently exhibit a severe disease manifestation which requires specialized long-term clinical care.

In Vitro Effect of Dovitinib (TKI258), a Multi-Target Angiokinase Inhibitor on Aggressive Meningioma Cells

Background: Meningiomas represent ∼30% of primary central nervous system (CNS) tumors. Although advances in surgery and radiotherapy have significantly improved survival, there remains an important subset of patients whose tumors have more aggressive behavior and are refractory to conventional therapy. Recent advances in molecular genetics and epigenetics suggest that this aggressive behavior may be due to the deletion of the DNA repair and tumor suppressor gene, CHEK2, neurofibromatosis Type 2 (NF2) mutation on chromosome 22q12, and genetic abnormalities in multiple RTKs including FGFRs. Management of higher-grade meningiomas, such as anaplastic meningiomas (AM: WHO grade III), is truly challenging and there isn’t an established chemotherapy option. We investigate the effect of active multi tyrosine receptor kinase inhibitor Dovitinib at stopping AM cell growth in in vitro with either frequent codeletion or mutated CHEK2 and NF2 gene.Methods: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot analysis, caspases assay, and DNA fragmentation assay.Results: Treatment of CH157MN and IOMM-Lee cells with Dovitinib suppressed multiple angiokinases—mainly FGFRs, leading to suppression of downstream signaling by RAS–RAF–MAPK molecules and PI3K–AKT molecules which are involved in cell proliferation, cell survival, and tumor invasion. Furthermore, Dovitinib induced apoptosis via downregulation of survival proteins (Bcl-XL), and over-expression of apoptotic factors (Bax and caspase-3) regardless of CHEK2 and NF2 mutation status.Conclusions: This study establishes the groundwork for the development of Dovitinib as a therapeutic agent for high-grade AM with either frequent codeletion or mutated CHEK2 and NF2, an avenue with high translational potential.

Mosaic Neurofibromatosis Type 1 With Multiple Cutaneous Diffuse and Plexiform Neurofibromas of the Lower Leg

Neurofibromatosis type 1 (NF1) is an autosomal dominant hereditary disease with complete penetrance and a very variable phenotype. Recent research has shown that postzygotic NF1 gene mutations occur to a far greater extent than previously thought. The phenotype of affected individuals reflects the time of somatic mutation and the phenotype is correspondingly diverse. This report describes histological and genetic findings in a case of mosaic NF1, the clinical control of which documents almost stationary skin findings over a period of 9 years. The 55-year-old female first presented for advice on a strip of nodular skin tumours of the calf skin. She had no hallmarks of NF1. It was only 9 years later that she had the skin tumours removed, all of which were partially diffuse and partially plexiform neurofibroma. The genetic examination showed an atypical large deletion of the NF1 gene in the skin tumours, but not in overlying skin or blood. Segmental NF1 is a distinct type of mosaic/somatic NF1 mutation. The phenotype of diffuse and plexiform skin neurofibromas can resemble cutaneous neurofibroma. Surgical therapy for segmental neurofibromatosis does not differ from the concepts for treating nerve sheath tumours in NF1 patients with a germline NF1 mutation.

SAT-222 Aggressive Phenotype Pheochromocytoma Associated with NF-1 and BRCA Mutation

IntroductionPheochromocytomas are catecholamine-secreting tumors of the adrenal medulla that occur sporadically or with familial association. Familial predilection occurs in those with multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis Type 1 (NF-1). To date, more than 21 gene mutations have been identified that are involved in the development of pheochromocytomas. However, co-existence of two different mutations such as NF-1 and BRCA gene mutations with pheochromocytoma has not been well described.Clinical CaseA 40 year-old woman with NF-1 and recently diagnosed left breast invasive ductal carcinoma, estrogen positive, progesterone positive, HER-2 negative BRCA2 positive breast cancer and right breast lobular carcinoma in situ underwent staging CT scan. A 2.7 cm left adrenal incidentaloma with heterogeneous texture and 69–100 HU was discovered. Symptom review revealed chronic headaches and increasing frequency of anxiousness and irritability. She had no personal or family history of hypertension. Laboratory analysis showed elevated catecholamine levels included plasma normetanephrine of 2.8 nmol/L (normal <0.9), metanephrine of 0.9 nmol/L (normal <0.5), 24 hour urine metanephrine 498 mcg/24 hr (normal <180) and 24 hour urine normetanephrine of 1,152 mcg/24 hr (normal <451). Findings were consistent with adrenal pheochromocytoma.She was immediately started on phenoxybenzamine and encouraged to increase fluid intake to expand intravascular volume before surgery. She underwent laparoscopic left partial adrenalectomy fourteen days later. Surgical pathology revealed a 3.0 cm pheochromocytoma in a background of nodular hyperplasia of the medulla, described as an aggressive tumor phenotype based on morphological features.ConclusionsIt has been estimated that 1.5–14% of adrenal incidentalomas are pheochromocytomas. While only 3% of NF-1 patients have pheochromocytomas, 20–50% of NF-1 patients with concurrent hypertension have underlying chromaffin tumors. This association is significant when compared to the pheochromocytoma incidence of 0.1% in all hypertensive individuals. Interestingly, germline mutations of BRCA-1 associated protein-1 gene have been reported in patients with paragangliomas, therefore it may be plausible to consider interactions between NF-1 and breast cancer BRCA-2 gene mutations, resulting in an unusual and more aggressive pheochromocytoma phenotype, even when detected at an early stage, as in this case. It is also important to recognize and adequately treat pheochromocytoma prior to having surgery to avoid possible intraoperative hypertensive crisis, a concept that was underscored in this particular case when the patient was relatively asymptomatic and entirely normotensive preoperatively.

New Model Systems and the Development of Targeted Therapies for the Treatment of Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors.

Neurofibromatosis Type 1 (NF1) is a common genetic disorder and cancer predisposition syndrome (1:3000 births) caused by mutations in the tumor suppressor gene NF1. NF1 encodes neurofibromin, a negative regulator of the Ras signaling pathway. Individuals with NF1 often develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage, some of which progress further to malignant peripheral nerve sheath tumors (MPNSTs). Treatment options for neurofibromas and MPNSTs are extremely limited, relying largely on surgical resection and cytotoxic chemotherapy. Identification of novel therapeutic targets in both benign neurofibromas and MPNSTs is critical for improved patient outcomes and quality of life. Recent clinical trials conducted in patients with NF1 for the treatment of symptomatic plexiform neurofibromas using inhibitors of the mitogen-activated protein kinase (MEK) have shown very promising results. However, MEK inhibitors do not work in all patients and have significant side effects. In addition, preliminary evidence suggests single agent use of MEK inhibitors for MPNST treatment will fail. Here, we describe the preclinical efforts that led to the identification of MEK inhibitors as promising therapeutics for the treatment of NF1-related neoplasia and possible reasons they lack single agent efficacy in the treatment of MPNSTs. In addition, we describe work to find targets other than MEK for treatment of MPNST. These have come from studies of RAS biochemistry, in vitro drug screening, forward genetic screens for Schwann cell tumors, and synthetic lethal screens in cells with oncogenic RAS gene mutations. Lastly, we discuss new approaches to exploit drug screening and synthetic lethality with NF1 loss of function mutations in human Schwann cells using CRISPR/Cas9 technology.

Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations.

SummaryNeurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.