The genetic spectrum of NF1 variants in 10 unrelated Chinese families with neurofibromatosis type 1.

OBJECTIVES: To investigate the clinical and genetic features in a cohort of Chinese families with neurofibromatosis type 1 (NF1). METHODS: The clinical information of 21 patients with NF1 in 10 families was retrospectively analyzed. To broaden the genetic spectrum of NF1, multiplex ligation-dependent probe amplification analysis was performed first, followed by the whole-exome sequencing, in order to identify pathogenic or potentially pathogenic variants of NF1 gene in 10 unrelated Chinese families. RESULTS: Nine different NF1 variants were identified in all 10 families. Of these, 7 were known pathogenic variants and included the exon 1 deletion, exons 1-58 deletion, c.5401C>T (p.Q1801*), c.2291-2A>C, c.484C>T (p.Q162*), c.4922G>A (p.W1641*) and c.1019_1020del (p.S340Cfs*25). The 2 novel variants were c.5197T>C (p.S1733P) and c.783_797delinsC (p.K261Nfs*25). The p.S1733P variant was classified as a variant of uncertain significance, while p.K261Nfs*25 was classified as pathogenic. Hence, the positive detection rate of NF1 variants was 100% (10/10). While the truncating variants were responsible for 60.0% (6/10) of the cases, the splicing variant was responsible for 10% (1/10) of the cases. CONCLUSION: We identified 2 novel heterozygous variants (c.5197T>C and c.783_797delinsC) in the NF1 gene, which broadens the genetic spectrum of the NF1 gene.

Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-ß RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-ß RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-ß RII-inactivated cancers.

A Comprehensive Overview of NF1 Mutations in Iranian Patients.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutations in the NF1 gene. This disorder shows nearly complete penetrance and high phenotypic variability. We used the whole-exome sequencing technique to identify mutations in 32 NF1 cases from 22 Iranian families. A total of 31 variants, including 30 point mutations and one large deletion, were detected. In eight cases, variants were inherited, while they were sporadic in the remaining. Seven novel variants, including c.5576 T > G, c.6658_6659insC, c.2322dupT, c.92_93insAA, c.4360C > T, c.3814C > T, and c.4565_4566delinsC, were identified. The current study is the largest in terms of the sample size of Iranian NF1 cases with identified mutations. The results can broaden the spectrum of NF1 mutations and facilitate the process of genetic counseling in the affected families.

Revisiting the NPcis mouse model: A new tool to model plexiform neurofibroma.

Neurofibromatosis Type I (NF1) is a rare genetic disorder. NF1 patients frequently develop a benign tumor in peripheral nerve plexuses called plexiform neurofibroma. In the past two decades, tissue-specific Nf1 knockout mouse models were developed using commercially available tissue-specific Cre recombinase and the Nf1 flox mice to mimic neurofibroma development. However, these models develop para-spinal neurofibroma, recapitulating a rare type of neurofibroma found in NF1 patients. The NPcis mouse model developed a malignant version of neurofibroma called malignant peripheral nerve sheath tumor (MPNST) within 3 to 6 months but intriguingly without apparent benign precursor lesion. Here, we revisited the NPcis model and discovered that about 20% display clinical signs similar to Nf1 tissue-specific knockout mice models. However, a systematic histological analysis could not explain the clinical signs we observed although we noticed lesions reminiscent of a neurofibroma in a peripheral nerve, a cutaneous neurofibroma, and para-spinal neurofibroma on rare occasions in NPcis mice. We also observed that 10% of the mice developed a malignant peripheral nerve sheath tumor (MPNST) spontaneously, coinciding with their earring tag identification. Strikingly, half of the sciatic nerves from NPcis mice developed plexiform neurofibroma within 1-6 months when intentionally injured. Thus, we provided a procedure to turn the widely used NPcis sarcoma model into a model recapitulating plexiform neurofibroma.

An Overview of Optic Pathway Glioma With Neurofibromatosis Type 1: Pathogenesis, Risk Factors, and Therapeutic Strategies.

Optic pathway gliomas (OPGs) are most predominant pilocytic astrocytomas, which are typically diagnosed within the first decade of life. The majority of affected children with OPGs also present with neurofibromatosis type 1 (NF1), the most common tumor predisposition syndrome. OPGs in individuals with NF1 primarily affect the optic pathway and lead to visual disturbance. However, it is challenging to assess risk in asymptomatic patients without valid biomarkers. On the other hand, for symptomatic patients, there is still no effective treatment to prevent or recover vision loss. Therefore, this review summarizes current knowledge regarding the pathogenesis of NF1-associated OPGs (NF1-OPGs) from preclinical studies to seek potential prognostic markers and therapeutic targets. First, the loss of the NF1 gene activates 3 distinct Ras effector pathways, including the PI3K/AKT/mTOR pathway, the MEK/ERK pathway, and the cAMP pathway, which mediate glioma tumorigenesis. Meanwhile, non-neoplastic cells from the tumor microenvironment (microglia, T cells, neurons, etc.) also contribute to gliomagenesis via various soluble factors. Subsequently, we investigated potential genetic risk factors, molecularly targeted therapies, and neuroprotective strategies for tumor prevention and vision recovery. Last, potential directions and promising preclinical models of NF1-OPGs are presented for further research. On the whole, NF1-OPGs develop as a result of the interaction between glioma cells and the tumor microenvironment. Developing effective treatments require a better understanding of tumor molecular characteristics, as well as multistage interventions targeting both neoplastic cells and non-neoplastic cells.

Inflammatory rhabdomyoblastic tumor, pheochromocytoma, and pulmonary hamartoma in a patient with neurofibromatosis type 1: a case report.

BACKGROUND: Inflammatory rhabdomyoblastic tumors are relatively recently recognized soft tissue tumors with a low malignant potential. Here, we present a case of concurrent inflammatory rhabdomyoblastic tumor (IRMT), adrenal pheochromocytoma, and pulmonary hamartoma in a patient with neurofibromatosis type 1 (NF1). To our knowledge, this is the first time that this constellation of tumors has been described in the literature. CASE PRESENTATION: A female patient in her late 20s with known NF1 was diagnosed with an inflammatory rhabdomyoblastic tumor, pheochromocytoma, and pulmonary hamartoma in a short succession. IRMT was found to harbor a near-haploid genome and displayed a typical immunohistochemical profile as well as a focal aberrant p53 expression pattern. CONCLUSIONS: This case report strengthens the theory that defects in the tumor suppressor NF1 play a central role in the pathogenesis of inflammatory rhabdomyoblastic tumors and that IRMT may be part of the spectrum of neurofibromatosis type 1 related tumors.

NF1+/ex42del miniswine model the cellular disruptions and behavioral presentations of NF1-associated cognitive and motor impairment.

Cognitive or motor impairment is common among individuals with neurofibromatosis type 1 (NF1), an autosomal dominant tumor-predisposition disorder. As many as 70% of children with NF1 report difficulties with spatial/working memory, attention, executive function, and fine motor movements. In contrast to the utilization of various Nf1 mouse models, here we employ an NF1+/ex42del miniswine model to evaluate the mechanisms and characteristics of these presentations, taking advantage of a large animal species more like human anatomy and physiology. The prefrontal lobe, anterior cingulate, and hippocampus from NF1+/ex42del and wild-type miniswine were examined longitudinally, revealing abnormalities in mature oligodendrocytes and astrocytes, and microglial activation over time. Imbalances in GABA: Glutamate ratios and GAD67 expression were observed in the hippocampus and motor cortex, supporting the role of disruption in inhibitory neurotransmission in NF1 cognitive impairment and motor dysfunction. Moreover, NF1+/ex42del miniswine demonstrated slower and shorter steps, indicative of a balance-preserving response commonly observed in NF1 patients, and progressive memory and learning impairments. Collectively, our findings affirm the effectiveness of NF1+/ex42del miniswine as a valuable resource for assessing cognitive and motor impairments associated with NF1, investigating the involvement of specific neural circuits and glia in these processes, and evaluating potential therapeutic interventions.

Dietary intervention rescues a bone porosity phenotype in a murine model of Neurofibromatosis Type 1 (NF1).

Neurofibromatosis type 1 (NF1) is a complex genetic disorder that affects a range of tissues including muscle and bone. Recent preclinical and clinical studies have shown that Nf1 deficiency in muscle causes metabolic changes resulting in intramyocellular lipid accumulation and muscle weakness. These can be subsequently rescued by dietary interventions aimed at modulating lipid availability and metabolism. It was speculated that the modified diet may rescue defects in cortical bone as NF1 deficiency has been reported to affect genes involved with lipid metabolism. Bone specimens were analyzed from wild type control mice as well as Nf1Prx1-/- (limb-targeted Nf1 knockout mice) fed standard chow versus a range of modified chows hypothesized to influence lipid metabolism. Mice were fed from 4 weeks to 12 weeks of age. MicroCT analysis was performed on the cortical bone to examine standard parameters (bone volume, tissue mineral density, cortical thickness) and specific porosity measures (closed pores corresponding to osteocyte lacunae, and larger open pores). Nf1Prx1-/- bones were found to have inferior bone properties to wild type bones, with a 4-fold increase in the porosity attributed to open pores. These measures were rescued by dietary interventions including a L-carnitine + medium-chain fatty acid supplemented chow previously shown to improve muscle histology function. Histological staining visualized these changes in bone porosity. These data support the concept that lipid metabolism may have a mechanistic impact on bone porosity and quality in NF1.

Genetic/epigenetic effects in NF1 microdeletion syndrome: beyond the haploinsufficiency, looking at the contribution of not deleted genes.

NF1 microdeletion syndrome, accounting for 5-11% of NF1 patients, is caused by a deletion in the NF1 region and it is generally characterized by a severe phenotype. Although 70% of NF1 microdeletion patients presents the same 1.4 Mb type-I deletion, some patients may show additional clinical features. Therefore, the contribution of several pathogenic mechanisms, besides haploinsufficiency of some genes within the deletion interval, is expected and needs to be defined. We investigated an altered expression of deletion flanking genes by qPCR in patients with type-1 NF1 deletion, compared to healthy donors, possibly contributing to the clinical traits of NF1 microdeletion syndrome. In addition, the 1.4-Mb deletion leads to changes in the 3D chromatin structure in the 17q11.2 region. Specifically, this deletion alters DNA-DNA interactions in the regions flanking the breakpoints, as demonstrated by our 4C-seq analysis. This alteration likely causes position effect on the expression of deletion flanking genes.Interestingly, 4C-seq analysis revealed that in microdeletion patients, an interaction was established between the RHOT1 promoter and the SLC6A4 gene, which showed increased expression. We performed NGS on putative modifier genes, and identified two "likely pathogenic" rare variants in RAS pathway, possibly contributing to incidental phenotypic features.This study provides new insights into understanding the pathogenesis of NF1 microdeletion syndrome and suggests a novel pathomechanism that contributes to the expression phenotype in addition to haploinsufficiency of genes located within the deletion.This is a pivotal approach that can be applied to unravel microdeletion syndromes, improving precision medicine, prognosis and patients' follow-up.

Comprehensive genomic profiling from C-CAT database unveiled over 80% presence of oncogenic drivers in anaplastic thyroid carcinoma including BRAF, RAS family, NF1, and FGFR1.

OBJECTIVE: Anaplastic thyroid carcinoma (ATC) is considered a very aggressive carcinoma and has been difficult to treat with therapeutic strategies. This study examines the landscape of genomic alteration in ATC, including the BRAF V600E mutation, and its clinical implications. DESIGN, PATIENTS AND MESUREMENT: A retrospective observational study was conducted using collected at the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) in Japan, utilizing comprehensive genomic profiling data from 102 ATC cases. Additionally, AACR-GENIE data from 267 cases were analysed for validation. Statistical methods, including the conditional Kendall tau statistic and χ2 tests, were employed for survival analysis and gene mutation comparisons. RESULTS: Among 102 ATCs, BRAF, RAS, and other driver mutations were found in 83 cases (81.2%). The prevalence of BRAF V600E mutations was as high as 60%. Co-mutation analysis identified different genomic profiles in the BRAF, RAS, and wild-type groups. Despite the diverse molecular backgrounds, no significant differences in clinical variables and overall survival were observed. The analysis considering left-side amputation suggested that RAS mutations had a poorer prognosis. In the BRAF/RAS wild-type group, FGFR1 and NF1 were identified as driver mutations, with an accumulation of copy number variations and less TERT promoter mutations. This molecular subgrouping was also supported by the AACR-GENIE data. CONCLUSIONS: Comprehensive genomic analysis of ATC in Japan revealed distinct molecular subgroups, highlighting the importance of BRAF V600E mutations, particularly V600E, as potential therapeutic targets and suggest the relevance of tailor-made therapeutic strategies based on genomic profiling.

MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells.

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-ß 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.

Past, Present, and Future Therapeutic Strategies for NF-1-Associated Tumors.

PURPOSE OF REVIEW: Neurofibromatosis type 1 (NF-1) is a cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene that encodes the neurofibromin protein, which functions as a negative regulator of Ras signaling. We review the past, current, and future state of therapeutic strategies for tumors associated with NF-1. RECENT FINDINGS: Therapeutic efforts for NF-1-associated tumors have centered around inhibiting Ras output, leading to the clinical success of downstream MEK inhibition for plexiform neurofibromas and low-grade gliomas. However, MEK inhibition and similar molecular monotherapy approaches that block Ras signaling do not work for all patients and show limited efficacy for more aggressive cancers such as malignant peripheral nerve sheath tumors and high-grade gliomas, motivating novel treatment approaches. We highlight the current therapeutic landscape for NF-1-associated tumors, broadly categorizing treatment into past strategies for serial Ras pathway blockade, current approaches targeting parallel oncogenic and tumor suppressor pathways, and future avenues of investigation leveraging biologic and technical innovations in immunotherapy, pharmacology, and gene delivery.

Recurrent spontaneous pneumothorax in an NF1 patient with a novel causative variant: broadening genotype-phenotype correlations.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance, most commonly known to affect the skin and eyes. Although lung involvement in the form of cysts and bullae occurs in up to 20% of adults, the seemingly intuitive association of NF1 and spontaneous pneumothorax is not widely recognised among clinicians. Here, we report the second case of recurring spontaneous pneumothorax in the context of NF1 with a confirmed molecular diagnosis. In both cases, the NF1 variants featured a premature stop codon in the C-terminal protein domain. Interestingly, our patient had mild skin symptoms, suggesting that spontaneous pneumothorax may not be correlated with cutaneous disease severity. More genotype-phenotype correlation studies are needed for NF1 in general and for its link to spontaneous pneumothorax in particular.

Generation of a human iPSC line CIPi004-A from a patient with neurofibromatosis type 1 and epilepsy harboring a heterozygous mutation in NF1 gene.

The NF1 gene is related to neurofibromatosis type 1 (NF1), which is an autosomal dominant disorder associated with multisystem involvement and epilepsy susceptibility. A human induced pluripotent stem cell (iPSC) line was derived from a pediatric patient with NF1 and epilepsy, harboring a heterozygous NF1 gene mutation. The iPSC line exhibits high levels of pluripotency markers, maintains the NF1 gene mutation, and demonstrates the capacity to undergo differentiation potential in vitro into three germ layers. The iPSC line will serve as a valuable resource for investigating the underlying mechanisms and conducting drug screening related to NF1 and NF1-associated epilepsy.

Whole-exome sequencing revealed a likely pathogenic variant in NF1 causing neurofibromatosis type I and Arrhythmogenic Cardiomyopathy.

BACKGROUND: Neurofibromatosis type I (NF1) is a genetic disorder characterized by the tumor's development in nerve tissue. Complications of NF1 can include pigmented lesions, skin neurofibromas, and heart problems such as cardiomyopathy. In this study, we performed whole-exome sequencing (WES) on an Iranian patient with NF1 to identify the genetic cause of the disease. METHODS: Following clinical assessment, WES was used to identify genetic variants in a family with a son suffering from NF1. No symptomatic manifestations were observed in other family members. In the studied family, in silico and segregation analysis were applied to survey candidate variants. RESULTS: Clinical manifestations were consistent with arrhythmogenic cardiomyopathy (ACM). WES detected a likely pathogenic heterozygous missense variant, c.3277G > A:p.Val1093Met, in the NF1 gene, confirmed by PCR and Sanger sequencing. The patient's parents and brother had a normal sequence at this locus. CONCLUSIONS: Although there is no cure for NF1, genetic tests, such as WES, can detect at-risk asymptomatic family members. Furthermore, cardiac evaluation could also help these patients before heart disease development.

Drosophila Contributions towards Understanding Neurofibromatosis 1.

Neurofibromatosis 1 (NF1) is a multisymptomatic disorder with highly variable presentations, which include short stature, susceptibility to formation of the characteristic benign tumors known as neurofibromas, intense freckling and skin discoloration, and cognitive deficits, which characterize most children with the condition. Attention deficits and Autism Spectrum manifestations augment the compromised learning presented by most patients, leading to behavioral problems and school failure, while fragmented sleep contributes to chronic fatigue and poor quality of life. Neurofibromin (Nf1) is present ubiquitously during human development and postnatally in most neuronal, oligodendrocyte, and Schwann cells. Evidence largely from animal models including Drosophila suggests that the symptomatic variability may reflect distinct cell-type-specific functions of the protein, which emerge upon its loss, or mutations affecting the different functional domains of the protein. This review summarizes the contributions of Drosophila in modeling multiple NF1 manifestations, addressing hypotheses regarding the cell-type-specific functions of the protein and exploring the molecular pathways affected upon loss of the highly conserved fly homolog dNf1. Collectively, work in this model not only has efficiently and expediently modelled multiple aspects of the condition and increased understanding of its behavioral manifestations, but also has led to pharmaceutical strategies towards their amelioration.

p53 modulates kinase inhibitor resistance and lineage plasticity in NF1-related MPNSTs.

Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.

STING activation reprograms the microenvironment to sensitize NF1-related malignant peripheral nerve sheath tumors for immunotherapy.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-activating protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in the development of multiple neoplasms, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in patients with NF1. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune checkpoint blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell-rich tumor microenvironment. While MPNSTs are noninflamed "cold" tumors, here, we converted MPNSTs into T cell-inflamed "hot" tumors by activating stimulator of IFN genes (STING) signaling. Mouse genetic and human xenograft MPNST models treated with a STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNSTs.

Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors.

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.