[Analysis of NF1 gene variants in a Chinese pedigree and a sporadic patient with Neurofibromatosis type 1].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree and a sporadic case with Neurofibromatosis type 1 (NF1). METHODS: Clinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from peripheral venous blood samples and subjected to whole exome sequencing. Candidate variants were validated by Sanger sequencing and bioinformatic analysis. RESULTS: All patients from the pedigree were found to harbor a c.3251delC variant in exon 25 of the NF1 gene, whilst a c.4312_4314delGAA variant was found in exon 32 of the NF1 gene in the sporadic case. CONCLUSION: Variants of the NF1 gene may account for the occurrence of NF1 in this pedigree and sporadic case.

Neurofibromin 1 mutations impair the function of human induced pluripotent stem cell-derived microglia.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.

[Analysis of clinical features and variants of NF1 gene in 12 patients with Neurofibromatosis type 1].

OBJECTIVE: To explore the types of NF1 gene variants and clinical characteristics among patients with Neurofibromatosis type I (NF1). METHODS: Clinical data of 12 patients diagnosed at Ningbo Women and Children's Hospital between December 2019 and May 2022 were retrospectively analyzed. The probands and their family members were subjected to high-throughput sequencing, and candidate variants were verified by Sanger sequencing and chromosome microarray analysis. RESULTS: The 12 patients had ranged from 4 months to 27 years old, with a male-to-female ratio of 2 : 1. Cafè-au-lait spots were found in all patients. 83.3% of them also had axillary and/or inguinal freckling, 58.3% had neurofibromas, and 16.7% had congenital pseudarthrosis of the tibia. Five types of NF1 gene variants were identified in the patients, including 5 nonsense variants, 4 frameshift variants, 1 missense variant, 1 splice variant, 1 large deletion involving the whole gene. Six patients were found to harbor de novo variants, 2 had inherited the variants from their parents, and 4 were not verified for their parental origin. The c.3379del (p.Thr1127Glnfs*15) and c.6628_6629del (p.Glu2210Thrfs*10) variants were unreported in literature and databases. CONCLUSION: Most NF1 patients may present with Cafè-au-lait spots initially and are due to pathogenic variant of the NF1 gene. High-throughput sequencing can efficiently identify such variants among the patients and enable the definite diagnosis.

Identification of an NF1 Microdeletion with Optical Genome Mapping.

Neurofibromatosis type 1 (NF1) is a clinically heterogeneous neurocutaneous disorder inherited in autosomal dominant manner. Approximately 5-10% of the cases are caused by NF1 microdeletions involving the NF1 gene and its flanking regions. Microdeletions, which lead to more severe clinical manifestations, can be subclassified into four different types (type 1, 2, 3 and atypical) according to their size, the genomic location of the breakpoints and the number of genes included within the deletion. Besides the prominent hallmarks of NF1, patients with NF1 microdeletions frequently exhibit specific additional clinical manifestations like dysmorphic facial features, macrocephaly, overgrowth, global developmental delay, cognitive disability and an increased risk of malignancies. It is important to identify the genes co-deleted with NF1, because they are likely to have an effect on the clinical manifestation. Multiplex ligation-dependent probe amplification (MLPA) and microarray analysis are the primary techniques for the investigation of NF1 microdeletions. However, based on previous research, optical genome mapping (OGM) could also serve as an alternative method to identify copy number variations (CNVs). Here, we present a case with NF1 microdeletion identified by means of OGM and demonstrate that this novel technology is a suitable tool for the identification and classification of the NF1 microdeletions.

NF1 alterations in cancers: therapeutic implications in precision medicine.

INTRODUCTION: NF1 is a tumor suppressor gene encoding neurofibromin, an inhibitor of the RAS/MAPK and PI3K-AKT-mTOR signaling pathways. NF1 germline pathogenic variants cause the tumor predisposition syndrome neurofibromatosis type 1. Targeted therapies (MEK inhibitors) have been approved for benign nerve sheath tumors in neurofibromatosis type 1 patients. NF1 somatic alterations are present in ~5% of all human sporadic cancers. In melanomas, acute myeloid leukemias and lung adenocarcinomas, the NF1 somatic alteration frequency is higher (~15%). However, to date, the therapeutic impact of NF1 somatic alterations is poorly investigated. AREAS COVERED: This review presents a comprehensive overview of targeted therapies and immunotherapies currently developed and evaluated in vitro and in vivo for NF1-altered cancer treatment. A PubMed database literature review was performed to select relevant original articles. Active clinical trials were researched in ClinicalTrials.gov database in August 2022. TCGA and HGMD® databases were consulted. EXPERT OPINION: This review highlights the need to better understand the molecular mechanisms of NF1-altered tumors and the development of innovative strategies to effectively target NF1-loss in human cancers. One of the current major challenges in cancer management is the targeting of tumor suppressor genes such as NF1 gene. Currently, most studies are focusing on inhibitors of the RAS/MAPK and PI3K-AKT-mTOR pathways and immunotherapies.

Generation of two human induced pluripotent stem cell lines from a patient with Neurofibromatosis type 1 (NF1) and pathogenic NF1 gene variant c.1466 A>G BCRTi011-A as well as a first-degree healthy relative (BCRTi010-A).

We describe the generation of two human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) using a non-integrative episomal reprogramming strategy. The first cell line was derived from a NF1 patient with the genetic variant c.1466A>G (BCRTi011-A) which leads to a cryptic splice site and aberrant splicing. The second one was created from a healthy relative of first-degree (BCRTi010-A). The generated iPSC lines were shown to have tri-lineage differentiation potential, a normal karyotype, and expression of pluripotent markers. Both iPSC lines provide a powerful tool for in vitro disease modeling and therapy development.

Superimposed Mosaicism in the Form of Extremely Extended Segmental Plexiform Neurofibroma Caused by a Novel Pathogenic Variant in the NF1 Gene.

Plexiform neurofibromas occurring in approximately 20-50% of all neurofibromatosis type-1 (NF1) cases are histologically benign tumors, but they can be fatal due to compression of vital structures or transformation to malignant sarcomas or malignant peripheral nerve sheath tumors. All sizeable plexiform neurofibromas are thought to result from an early second mutation giving rise to a loss of heterozygosity of the NF1 gene. In this unusual case, a 12-year-old girl presented with a rapidly growing, extremely extensive plexiform neurofibroma with segmental distribution over the entire right arm, extending to the right chest wall and mediastinum, superimposed on classic cutaneous lesions of NF1. After several surgical interventions, the patient was efficiently treated with an oral selective MEK inhibitor, selumetinib, which resulted in a rapid reduction of the tumor volume. Molecular analysis of the NF1 gene revealed a c.2326-2 A>G splice-site mutation in the clinically unaffected skin, peripheral blood sample, and plexiform neurofibroma, which explains the general clinical symptoms. Furthermore, a novel likely pathogenic variant, c.4933dupC (p.Leu1645Profs*7), has been identified exclusively in the girl's plexiform neurofibromas. This second-hit mutation can explain the extremely extensive segmental involvement.

Childhood-Onset Refractory Hypertension Results from Neurofibromatosis Type 1 Caused by a Splicing NF1 Mutation.

INTRODUCTION: Neurofibromatosis type 1 (NF-1) is caused by mutations in the NF1 gene that encodes neurofibromin, a negative regulator of RAS proto-oncogene. Approximately one-third of the reported pathogenic mutations in NF1 are splicing mutations, but most consequences are unclear. The objective of this study was to identify the pathogenicity of splicing mutation in a Chinese family with NF-1 and determine the effects of the pre-mRNA splicing mutation by in vitro functional analysis. METHODS: Next-generation sequencing was used to screen candidate mutations. We performed a minigene splicing assay to determine the effect of the splicing mutation on NF1 expression, and three-dimensional structure models of neurofibromin were generated using SWISS-MODEL and PROCHECK methods, respectively. RESULTS: A pathogenic splicing mutation c.479+1G>C in NF1 was found in the proband characterized by childhood-onset refractory hypertension. In vitro analysis demonstrated that c.479+1G>C mutation caused the skipping of exon 4, leading to a glutamine-to-valine substitution at position 97 in neurofibromin and an open reading frame shift terminating at codon 108. Protein modeling showed that several major domains were missing in the truncated neurofibromin protein. CONCLUSION: The splicing mutation c.479+1G>C identified in a Chinese patient with NF-1 and childhood-onset refractory hypertension caused the skipping of exon 4 and a truncated protein. Our findings offer new evidence for the molecular diagnosis of NF-1.

Icariin Promotes Osteogenic Differentiation in a Cell Model with NF1 Gene Knockout by Activating the cAMP/PKA/CREB Pathway.

Neurofibromatosis type 1 is a rare autosomal dominant genetic disorder, with up to 50% of patients clinically displaying skeletal defects. Currently, the pathogenesis of bone disorders in NF1 patients is unclear, and there are no effective preventive and treatment measures. In this study, we found that knockout of the NF1 gene reduced cAMP levels and osteogenic differentiation in an osteoblast model, and icariin activated the cAMP/PKA/CREB pathway to promote osteoblast differentiation of the NF1 gene knockout cell model by increasing intracellular cAMP levels. The PKA selective inhibitor H89 significantly impaired the stimulatory effect of icariin on osteogenesis in the NF1 cell model. In this study, an osteoblast model of NF1 was successfully constructed, and icariin was applied to the cell model for the first time. The results will help to elucidate the molecular mechanism of NF1 bone disease and provide new ideas for the clinical prevention and treatment of NF1 bone disease and drug development in the future.

Nf1 in heart development: a potential causative gene for congenital heart disease: a narrative review.

Congenital heart disease is the most frequent congenital disorder, affecting a significant number of live births. Gaining insights into its genetic etiology could lead to a deeper understanding of this condition. Although the Nf1 gene has been identified as a potential causative gene, its role in congenital heart disease has not been thoroughly clarified. We searched and summarized evidence from cohort-based and experimental studies on the issue of Nf1 and heart development in congenital heart diseases from various databases. Available evidence demonstrates a correlation between Nf1 and congenital heart diseases, mainly pulmonary valvar stenosis. The mechanism underlying this correlation may involve dysregulation of epithelial-mesenchymal transition (EMT). The Nf1 gene affects the EMT process via multiple pathways, including directly regulating the expression of EMT-related transcription factors and indirectly regulating the EMT process by regulating the MAPK pathway. This narrative review provides a comprehensive account of the Nf1 involvement in heart development and congenital cardiovascular diseases in terms of epidemiology and potential mechanisms. RAS signaling may contribute to congenital heart disease independently or in cooperation with other signaling pathways. Efficient management of both NF1 and cardiovascular disease patients would benefit from further research into these issues.

Germline Neurofibromin 1 mutation enhances the anti-tumour immune response and decreases juvenile myelomonocytic leukaemia tumourigenicity.

Juvenile myelomonocytic leukaemia (JMML) is an aggressive paediatric leukaemia characterized by mutations in five canonical RAS pathway genes, including the NF1 gene. JMML is driven by germline NF1 gene mutations, with additional somatic aberrations resulting in the NF1 biallelic inactivation, leading to disease progression. Germline mutations in the NF1 gene alone primarily cause benign neurofibromatosis type 1 (NF1) tumours rather than malignant JMML, yet the underlying mechanism remains unclear. Here, we demonstrate that with reduced NF1 gene dose, immune cells are promoted in anti-tumour immune response. Comparing the biological properties of JMML and NF1 patients, we found that not only JMML but also NF1 patients driven by NF1 mutations could increase monocytes generation. But monocytes cannot further malignant development in NF1 patients. Utilizing haematopoietic and macrophage differentiation from iPSCs, we revealed that NF1 mutations or knockout (KO) recapitulated the classical haematopoietic pathological features of JMML with reduced NF1 gene dose. NF1 mutations or KO promoted the proliferation and immune function of NK cells and iMacs derived from iPSCs. Moreover, NF1-mutated iNKs had a high capacity to kill NF1-KO iMacs. NF1-mutated or KO iNKs administration delayed leukaemia progression in a xenograft animal model. Our findings demonstrate that germline NF1 mutations alone cannot directly drive JMML development and suggest a potential cell immunotherapy for JMML patients.

New insights into the molecular basis of spinal neurofibromatosis type 1.

Spinal neurofibromatosis (SNF) is a form of neurofibromatosis type 1 (NF1) characterized by bilateral neurofibromas involving all spinal roots. The pathogenic mechanisms determining the SNF form are currently unknown. To verify the presence of genetic variants possibly related to SNF or classic NF1, we studied 106 sporadic NF1 and 75 SNF patients using an NGS panel of 286 genes encoding RAS pathway effectors and neurofibromin interactors and evaluated the expression of syndecans (SDC1, SDC2, SDC3, SDC4), the NF1 3' tertile interactors, by quantitative real-time PCR. We previously identified 75 and 106 NF1 variants in SNF and NF1 cohorts, respectively. The analysis of the distribution of pathogenic NF1 variants in the three NF1 tertiles showed a significantly higher prevalence of NF1 3' tertile mutations in SNF than in the NF1 cohort. We hypothesized a potential pathogenic significance of the 3' tertile NF1 variants in SNF. The analysis of syndecan expression on PBMCs RNAs from 16 SNF, 16 classic NF1 patients and 16 healthy controls showed that the expression levels of SDC2 and SDC3 were higher in SNF and NF1 patients than in controls; moreover, SDC2, SDC3 and SDC4 were significantly over expressed in patients mutated in the 3' tertile compared to controls. Two different mutational NF1 spectra seem to characterize SNF and classic NF1, suggesting a pathogenic role of NF1 3' tertile and its interactors, syndecans, in SNF. Our study, providing new insights on a possible role of neurofibromin C-terminal in SNF, could address effective personalized patient management and treatments.

Novel, heterozygous, de novo pathogenic variant (c.4963delA: p.Thr1656Glnfs*42) of the NF1 gene in a Chinese family with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) presents an autosomal dominant, haploinsufficient, and multisystemic disorder with patches of skin café-au-lait spots, lisch nodules in the iris, even tumors in the peripheral nervous system or fibromatous skin. In this study, a Chinese young woman who suffered from NF1 disease with first-trimester spontaneous abortion was recruited. Analysis for whole exome sequencing (WES), Sanger sequencing, short tandem repeat (STR), and co-segregation was carried out. As results, a novel, heterozygous, de novo pathogenic variant (c.4963delA:p.Thr1656Glnfs*42) of the NF1 gene in the proband was identified. This pathogenic variant of the NF1 gene produced a truncated protein that lost more than one-third of the NF1 protein at the C-terminus including half of the CRAL-TRIO lipid-binding domain and nuclear localization signal (NLS), thus leading to pathogenicity (ACMG criteria: PVS1 + PM2 + PM2). Analysis for NF1 conservation in species revealed high conservation in different species. Analysis of NF1 mRNA levels in different human tissues showed low tissue specificity, which may affect multiple organs presenting other symptoms or phenotypes. Moreover, prenatal NF1 gene diagnosis showed both alleles as wild types. Thus, this NF1 novel variant probably underlays the NF1 pathogenesis in this pedigree, which would help for the diagnosis, genetic counseling, and clinical management of this disorder.

Malignant peripheral nerve sheath tumor on a patient with a maternally inherited novel NF1 gene pathogenic germline variant: Case report.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by pathogenic variants in NF1, which negatively regulates the RAS pathway. Knowledge of the genotype-phenotype correlation in this disease is an important tool for prognostic evaluation and early detection of malignant peripheral nerve sheath tumors (MPNST), present in approximately 10% of these patients. We present the case of a teenager with a left jaw MPNST and a previously unreported germline pathogenic variant on NF1. CASE PRESENTATION: An 11-year-old female with a NF1 clinical diagnosis was referred to our hospital with a MPNST in an advanced state. A previously unreported NF1 pathogenic variant was obtained (GRCh37: NM_182493.2 c.3299C>G, p.Ser1100*). Despite great efforts from the surgical and medical teams, the tumor progression couldn't be halted, resulting in the patient's death. DISCUSSION: As MPNSTs are refractory to current treatment regimens, early diagnosis, and development of new therapies, such as MEK inhibitors, is necessary for reducing morbidity and mortality within NF1 patients. This increases the importance of a more widespread genetic testing strategy. CONCLUSION: The report of a novel NF1 pathogenic variant in a patient with maternally inherited neurofibromatosis type 1 and a MPNST increases the knowledge of the genotype-phenotype correlation in the disease.

NF1-Related MicroRNA Gene Polymorphisms and the Susceptibility to Soft Tissue Sarcomas: A Case-Control Study.

Soft tissue sarcomas (STS) are rare malignant tumors of mesenchymal origin, which are easy to metastasize and relapse and are a great threat to human health. In our previous study, the abnormal expression of neurofibromin 1 (NF1) is observed in tumor tissue of STS, and the NF1 gene is regulated by miRNAs. The study aimed to assess the association between NF1-related miRNA gene polymorphisms and the risk of STS. In this case-control study, the information and peripheral blood were collected from 169 patients with STS and 170 healthy controls. Six single-nucleotide polymorphisms of the NF1-related miRNAs were investigated and genotyped using a Sequenom MassARRAY® matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform. The association between the polymorphisms and the risk of STS was estimated using unconditional logistic regression analysis. There was a significant statistical difference on genotype distribution of miR-199a2 rs12139213 between the case group and the control group (p = 0.026). Comparing with individuals with wild-type AA, individuals with the AT/TT genotype had a 1.753-fold (odds ratio [OR] = 1.753, 95% confidence interval [CI] = 1.090-2.819, p = 0.021) increased risk of STS and 1.907-fold (OR = 1.907, 95% CI = 1.173-3.102, p = 0.009) increased risk of STS adjusted for age and smoking status. Individuals with the AG/GG genotype for miR24-3p rs4743988 displayed a significantly reduced risk of STS compared with individuals with homozygous mutations AA (OR = 0.605, 95% CI = 0.376-0.973, p = 0.038). Individuals carrying the AT/TT genotype for miR-199a2 rs12139213 or the AA genotype for miR24-3p rs4743988 may be susceptible to STS, which could be potential biomarkers for the diagnosis of STS.

Neurofibromatosis-Noonan syndrome and growth deficiency in an Iranian girl due to a pathogenic variant in NF1 gene.

BACKGROUND: Mutations in NF1 gene could cause allelic disorders with clinical spectrum of Neurofibromatosis type 1 to Noonan syndrome. Here, a 7-year-old Iranian girl is described with Neurofibromatosis-Noonan syndrome due to a pathogenic variant in NF1 gene. METHODS: Clinical evaluations were performed along with genetic testing using whole exome sequencing (WES). The variant analysis including pathogenicity prediction was also done using bioinformatics tools. RESULTS: The chief compliant of the patient was short stature and lack of proper weight gain. Other symptoms were developmental delay, learning disability, inadequate speech skill, broad forehead, hypertelorism, and epicanthal folds, low set ears and webbed neck. A small deletion, c.4375-4377delGAA, was found in NF1 gene using WES. This variant was classified as pathogenic according to ACMG. CONCLUSIONS: NF1 variants may show variable phenotypes among the patients; identifying such variants is helpful in therapeutic management of the disease. WES is considered as an appropriate test to diagnose Neurofibromatosis-Noonan syndrome.

Neurofibromatosis type 1 system-based manifestations and treatments: a review.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is a genetic disorder caused by a mutation in the NF1 gene. This disease presents with various system-based manifestations, including cardiac, musculoskeletal, and neuronal issues, which have been well-studied in previous research and have prompted the development of current and emerging treatments. These treatments, mainly medications targeting specific manifestations of NF1, aim to mitigate the negative impacts of the disease on patients' lives. NF1 is associated with an increased risk of malignancy and a significant decrease in life expectancy. In this paper, we review the current and emerging treatments for NF1 in relation to its system-based manifestations. METHODS: We conducted an extensive literature search using specific keywords through databases such as PubMed, Scopus, and Cochrane. The articles we found were compiled and subjected to strict inclusion and exclusion criteria. RESULTS: Pharmacological advances have led to the development of products that hold promise as future treatments for NF1. Given the diverse manifestations that can affect multiple organ systems in patients with NF1, it is important to consider a variety of treatment options to achieve optimal results. However, one of the major challenges in diagnosing and treating NF1 is that patients present asymptomatically, making it necessary to rely on clinical features for diagnosis. CONCLUSION: In conclusion, NF1 is a complex disease with varying manifestations and a growing field of pharmacologic treatments. The information presented in this article synthesizes current knowledge and available therapies for NF1.