Neurofibromatosis Type 1 Gene Research Articles

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.

Not Only RET but NF1 and Chromosomal Instability Are Seen in Young Patients with Sporadic Medullary Thyroid Carcinoma.

Genetic analysis of sporadic medullary thyroid carcinoma (MTC) has revealed somatic variants in RET, RAS, and occasionally other genes. However, around 20% of patients with sporadic MTC lack a known genetic driver. To uncover potential new somatic or germline drivers, we analyze a distinct cohort of patients with sporadic, very early-onset, and aggressive MTC. Germline and somatic DNA exome sequencing was performed in 19 patients, previously tested negative for germline RET variants. Exome sequencing of 19 germline samples confirmed the absence of RET and identified an NF1 pathogenic variant in 1 patient. Somatic sequencing was successful in 15 tumors revealing RET variants in 80%, predominantly p.Met918Thr, which was associated with disease aggressiveness. In RET-negative tumors, pathogenic variants were found in HRAS and NF1. The NF1 germline and somatic variants were observed in a patient without a prior clinical diagnosis of neurofibromatosis type 1, demonstrating that the loss of heterozygosity of NF1 functions as a potential MTC driver. Somatic copy number alterations analysis revealed chromosomal alterations in 53.3% of tumors, predominantly in RET-positive cases, with losses in chromosomes 9 and 22 being the most prevalent. This study reveals that within a cohort of early-onset nonhereditary MTC, RET remains the major driver gene. In RET-negative tumors, NF1 and RAS are drivers of sporadic MTC. In addition, in young patients without a RET germline mutation, a careful clinical evaluation with a consideration of germline NF1 gene analysis is ideal to exclude Neurofibromatosis type 1 (NF1).

Mutation analysis and clinical profile of South African patients with Neurofibromatosis type 1 (NF1) phenotype.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition with complete age-dependent penetrance, variable expressivity and a global prevalence of ∼1/3,000. It is characteriszed by numerous café-au-lait macules, skin freckling in the inguinal or axillary regions, Lisch nodules of the iris, optic gliomas, neurofibromas, and tumour predisposition. The diagnostic testing strategy for NF1 includes testing for DNA single nucleotide variants (SNVs), copy number variants (CNVs) as well as RNA analysis for deep intronic and splice variants, which can cumulatively identify the causative variant in 95% of patients. In the present study, NF1 patients were screened using a next-generation sequencing (NGS) assay targeting NF1 exons and intron/exon boundaries for SNV and NF1 multiple ligation-dependent probe amplification (MLPA) analysis for CNV detection. Twenty-six unrelated Southern African patients clinically suspected of having NF1, based on the clinical diagnostic criteria developed by the National Institute of Health (NIH), were included in the current study. A detection rate of 58% (15/26) was obtained, with SNVs identified in 80% (12/15) using a targeted gene panel and NF1 gene deletion in 20% (3/15) identified using MLPA. Ten patients (38%) had no variants identified, although they met NF1 diagnostic criteria. One VUS was identified in this study in a patient that met NF1 diagnostic criteria, however there was no sufficient information to classify variant as pathogenic. The clinical features of Southern African patients with NF1 are similar to that of the known NF1 phenotype, with the exception of a lower frequency of plexiform neurofibromas and a higher frequency of developmental/intellectual disability compared to other cohorts. This is the first clinical and molecular characterisation of a Southern African ancestry NF1 cohort using both next-generation sequencing and MLPA analysis. A significant number of patients remained without a diagnosis following DNA-level testing. The current study offers a potential molecular testing strategy for our low resource environment that could benefit a significant proportion of patients who previously only received a clinical diagnosis without molecular confirmation.

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.

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

To explore the genetic basis for a Chinese pedigree and a sporadic case with Neurofibromatosis type 1 (NF1). 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. 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. Variants of the NF1 gene may account for the occurrence of NF1 in this pedigree and sporadic case.

Correlation between large rearrangements and patient phenotypes in NF1 deletion syndrome: an update and review

About 5–10% of neurofibromatosis type 1 (NF1) patients exhibit large genomic germline deletions that remove the NF1 gene and its flanking regions. The most frequent NF1 large deletion is 1.4 Mb, resulting from homologous recombination between two low copy repeats. This “type-1” deletion is associated with a severe clinical phenotype in NF1 patients, with several phenotypic manifestations including learning disability, a much earlier development of cutaneous neurofibromas, an increased tumour risk, and cardiovascular malformations. NF1 adjacent co-deleted genes could act as modifier loci for the specific clinical manifestations observed in deleted NF1 patients. Furthermore, other genetic modifiers (such as CNVs) not located at the NF1 locus could also modulate the phenotype observed in patients with large deletions. In this study, we analysed 22 NF1 deletion patients by genome-wide array-CGH with the aim (1) to correlate deletion length to observed phenotypic features and their severity in NF1 deletion syndrome, and (2) to identify whether the deletion phenotype could also be modulated by copy number variations elsewhere in the genome. We then review the role of co-deleted genes in the 1.4 Mb interval of type-1 deletions, and their possible implication in the main clinical features observed in this high-risk group of NF1 patients.

SOX9 Promotes Collagen VI Secretion by Upregulating PCOLCE in Neurofibroma.

Neurofibromatosis type 1 (NF1) is caused by NF1 gene mutations. Patients with NF1 often have complications with tumors, such as neurofibroma. In order to investigate the pathogenesis of human neurofibroma, a systematic comparison of protein expression levels between Schwann cell-like sNF96.2 cells, which originated from malignant peripheral nerve sheath tumors (MPNST), and normal Schwann cells was performed using 4-D label-free proteomic analysis. In addition, the expression levels and localization of dysregulated proteins were confirmed using a Gene Expression Omnibus (GEO) transcriptomic dataset, Western blot analysis, and immunofluorescence labeling. The effects of SRY-box transcription factor 9 (SOX9) in the neurofibroma and surrounding microenvironment were evaluated in vivo using a tumor transplantation model. The present study observed that SOX9 and procollagen C-endopeptidase enhancer (PCOLCE) were significantly altered. NF1 mutation promoted the nuclear translocation and transcriptional activity of SOX9 in neurofibromas. SOX9 increased collagen VI secretions by enhancing the activation of PCOLCE in neurofibroma cells. These findings might provide new perspectives on the pathophysiological significance of SOX9 in neurofibromas and elucidate a novel molecular mechanism underlying neurofibromas.

Salivary Duct Carcinoma Arising in the Submandibular Gland in a Patient with Neurofibromatosis Type 1.

A 71-year-old man with neurofibromatosis type 1 (NF1) presented to our department with a 1-week history of a painful mass in the left submandibular area. Computed tomography (CT) and magnetic resonance imaging revealed an irregular-shaped tumor with a diameter of 2.0 cm in the left submandibular gland and a metastatic lymph node with a diameter of 1.0 cm adjacent to the tumor. Fluorodeoxyglucose-positron emission tomography/CT revealed increased uptake in the tumor. Fine-needle aspiration cytology revealed atypical cells, suggesting salivary duct carcinoma (SDC). Left neck dissection with resection of the tumor and submandibular gland was performed under general anesthesia. Histologic examination revealed ductal formation with a solid, cystic, cribriform, and papillary structure with intraductal comedonecrosis, diagnosing as SDC originating in the submandibular gland (pT3N1M0 pStage III). Mutational analysis of 160 cancer-related genes by next-generation sequencing (NGS) revealed a germline and frameshift mutation in the NF1 gene (p.R2408Kfs*14) and a somatic and frameshift mutation in the TP53 gene (p.C176Wfs*22). The patient received postoperative radiotherapy to the left neck area at 66 Gy. No evidence of recurrence or metastasis has been observed as of 10 months postoperatively. This is the first reported case of SDC in the submandibular gland in a patient with NF1. The mutational data by NGS may contribute to a better understanding of the oncogenesis of SDC in patients with NF1.

Breast density in NF1 women: a retrospective study

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by neurofibromin haploinsufficiency due to pathogenic variants in the NF1 gene. Tumor predisposition has long been associated with NF1, and an increased breast cancer (BC) incidence and reduced survival have been reported in recent years for women with NF1. As breast density is another known independent risk factor for BC, this study aims to evaluate the variability of breast density in patients with NF1 compared to the general population. Mammograms from 98 NF1 women affected by NF1, and enrolled onto our monocentric BC screening program, were compared with those from 300 healthy subjects to verify differences in breast density. Mammograms were independently reviewed and scored by a radiologist and using a Computer-Aided Detection (CAD) software. The comparison of breast density between NF1 patients and controls was performed through Chi-squared test and with multivariable ordinal logistic models adjusted for age, body mass index (BMI), number of pregnancies, and menopausal status.breast density was influenced by BMI and menopausal status in both NF1 patients and healthy subjects. No difference in breast density was observed between NF1 patients and the healthy female population, even after considering the potential confounding factors.Although NF1 and a highly fibroglandular breast are known risk factors of BC, in this study, NF1 patients were shown to have comparable breast density to healthy subjects. The presence of pathogenic variants in the NF1 gene does not influence the breast density value.

Gender-Specific Fine Motor Skill Learning Is Impaired by Myelin-Targeted Neurofibromatosis Type 1 Gene Mutation

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene. The clinical presentation of NF1 includes diverse neurological issues in pediatric and adult patients, ranging from learning disabilities, motor skill issues, and attention deficit disorder, to increased risk of depression and dementia. Preclinical research suggests that abnormal neuronal signaling mediates spatial learning and attention issues in NF1; however, drugs that improve phenotypes in models show inconclusive results in clinical trials, highlighting the need for a better understanding of NF1 pathophysiology and broader therapeutic options. Most NF1 patients show abnormalities in their brain white matter (WM) and myelin, and links with NF1 neuropathophysiology have been suggested; however, no current data can clearly support or refute this idea. We reported that myelin-targeted Nf1 mutation impacts oligodendrocyte signaling, myelin ultrastructure, WM connectivity, and sensory–motor behaviors in mice; however, any impact on learning and memory remains unknown. Here, we adapted a voluntary running test—the complex wheel (CW; a wheel with unevenly spaced rungs)—to delineate fine motor skill learning curves following induction of an Nf1 mutation in pre-existing myelinating cells (pNf1 mice). We found that pNf1 mutant females experience delayed or impaired learning in the CW, while proper learning in pNf1 males is predominantly disrupted; these phenotypes add complexity to the gender-dependent learning differences in the mouse strain used. No broad differences in memory of acquired CW skills were detected in any gender, but gene-dose effects were observed at the studied time points. Finally, nitric oxide signaling regulation differentially impacted learning in wild type (WT)/pNf1, male/female mice. Our results provide evidence for fine motor skill learning issues upon induction of an Nf1 mutation in mature myelinating cells. Together with previous connectivity, cellular, and molecular analyses, these results diversify the potential treatments for neurological issues in NF1.

Gene therapy strategies and prospects for neurofibromatosis type 1

To summarize the gene therapy strategies for neurofibromatosis type 1 (NF1) and related research progress. The recent literature on gene therapy for NF1 at home and abroad was reviewed. The structure and function of the NF1 gene and its mutations were analyzed, and the current status as well as future prospects of the transgenic therapy and gene editing strategies were summarized. NF1 is an autosomal dominantly inherited tumor predisposition syndrome caused by mutations in the NF1 tumor suppressor gene, which impair the function of the neurofibromin and lead to the disease. It has complex clinical manifestations and is not yet curable. Gene therapy strategies for NF1 are still in the research and development stage. Existing studies on the transgenic therapy for NF1 have mainly focused on the construction and expression of the GTPase-activating protein-related domain in cells that lack of functional neurofibromin, confirming the feasibility of the transgenic therapy for NF1. Future research may focus on split adeno-associated virus (AAV) gene delivery, oversized AAV gene delivery, and the development of new vectors for targeted delivery of full-length NF1 cDNA. In addition, the gene editing tools of the new generation have great potential to treat monogenic genetic diseases such as NF1, but need to be further validated in terms of efficiency and safety. Gene therapy, including both the transgenic therapy and gene editing, is expected to become an important new therapeutic approach for NF1 patients.

Identification of hub genes and drug candidates for NF2-related vestibular schwannoma by bioinformatics tools.

Neurofibromatosis type 2 (NF2)-related vestibular schwannoma (NF2-VS) is a rare genetic disorder that results in bilateral acoustic neuromas. However, the exact pathogenesis of the disease is still unclear. This study aims to use bioinformatics analyses to identify potential hub genes and therapeutic. We retrieved the mRNA expression profiles (GSE108524 and GSE141801) of NF2-VS from the database, and selected the leading 25% genes with the most variance across samples for weighted correlation network analysis. Subsequently, we conducted gene ontology term and Kyoto Encyclopedia of Genes and Genomes signaling network enrichment analyses. The STRING database was employed for protein-protein interaction (PPI) axis construction. The mRNA-miRNA modulatory network was generated via the miRTarBase database. Differentially expressed genes (DEGs) were identified via the R package "limma" in both datasets, and hub genes were screened via intersection of common DEGs, candidate hub genes from the PPI axis, and candidate hub genes from the key module. Finally, common DEGs were uploaded onto the connectivity map database to determine drug candidates. Based on our observations, the blue module exhibited the most significant relation to NF2-VS, and it included the NF2 gene. Using enrichment analysis, we demonstrated that the blue modules were intricately linked to modulations of cell proliferation, migration, adhesion, junction, and actin skeleton. Overall, 356 common DEGs were screened in both datasets, and 33 genes carrying a degree > 15 were chosen as candidate hub genes in the PPI axis. Subsequently, 4 genes, namely, GLUL, CAV1, MYH11, and CCND1 were recognized as real hub genes. In addition, 10 drugs with enrichment scores < -0.7 were identified as drug candidates. Our conclusions offered a novel insight into the potential underlying mechanisms behind NF2-VS. These findings may facilitate the identification of novel therapeutic targets in the future.

Drug Responses in Plexiform Neurofibroma Type I (PNF1) Cell Lines Using High-Throughput Data and Combined Effectiveness and Potency.

Background: Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by heterozygous germline NF1 gene mutations that predispose patients to developing plexiform neurofibromas, which are benign but often disfiguring tumors of the peripheral nerve sheath induced by loss of heterozygosity at the NF1 locus. These can progress to malignant peripheral nerve sheath tumors (MPNSTs). There are no approved drug treatments for adults with NF1-related inoperable plexiform neurofibromas, and only one drug (selumetinib), which is an FDA-approved targeted therapy for the treatment of symptomatic pediatric plexiform neurofibromas, highlighting the need for additional drug screening and development. In high-throughput screening, the effectiveness of drugs against cell lines is often assessed by measuring in vitro potency (AC50) or the area under the curve (AUC). However, the variability of dose-response curves across drugs and cell lines and the frequency of partial effectiveness suggest that these measures alone fail to provide a full picture of overall efficacy. Methods: Using concentration-response data, we combined response effectiveness (EFF) and potency (AC50) into (a) a score characterizing the effect of a compound on a single cell line, S = log[EFF/AC50], and (b) a relative score, ΔS, characterizing the relative difference between a reference (e.g., non-tumor) and test (tumor) cell line. ΔS was applied to data from high-throughput screening (HTS) of a drug panel tested on NF1-/- tumor cells, using immortalized non-tumor NF1+/- cells as a reference. Results: We identified drugs with sensitivity, targeting expected pathways, such as MAPK-ERK and PI3K-AKT, as well as serotonin-related targets, among others. The ΔS technique used here, in tandem with a supplemental ΔS web tool, simplifies HTS analysis and may provide a springboard for further investigations into drug response in NF1-related cancers. The tool may also prove useful for drug development in a variety of other cancers.

Is vestibular schwannoma really a benign tumor? Case report and review

Introduction: Vestibular schwannoma (VS) is a benign tumor that develops from Schwann cells of the eighth cranial pair, mainly in the cerebellopontine angle. Case Presentation: We report the case of a 30-year-old female patient who developed left otalgia associated with neglected tinnitus, the evolution of which was marked by the development of a static cerebellar syndrome and a behavioral disorder, whose brain MRI revealed a locally advanced process in the cerebellopontine angle at the expense of the vestibulocochlear nerve, in favor of a VS, complicated by involvement of the tonsils, which unfortunately led to the patient’s death. Discussion: VS, formerly known as acoustic neuroma, is an extra-axial intracranial tumor that accounts for over 80% of pontocerebellar angle tumors, and is secondary in the majority of cases to inactivation of the neurofibromatosis type 2 (NF2) tumor suppressor gene, either by mutation of the NF2 gene or loss of chromosome 22q. In the majority of cases, it is unilateral and solitary, but in almost 8% of cases, it is associated with NF2. Cerebral MRI is the examination of choice for the detection, characterization, and diagnosis of VS without the need for biopsy, mainly with T1-weighted sequences before and after gadolinium injection. Treatment is based essentially on surgery or radiosurgery, depending on the size, impact, and expertise of the treatment team. Conclusion: VS remains an important intracranial tumor entity, which can be life-threatening in cases of advanced local invasion.

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.

Antiseizure effect of MEK inhibitor in a child with neurofibromatosis type 1-Developmental and epileptic encephalopathy and optic pathway glioma.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder due to a mutation in NF1 gene, resulting in phenotypically heterogeneous systemic manifestations. Patients with NF1 are prone to develop neoplasms of the central nervous system (CNS) and are particularly at risk for optic pathway gliomas (OPG). Epilepsy is another recognized neurologic complication in patients with NF1, with a prevalence estimated between 4% and 14%. Several case reports and early phase clinical trials have demonstrated that the mitogen-activated protein kinase inhibitors (MEKi) are effective in NF1-low-grade gliomas (LGGs), but their influence on seizure activity in humans has not been established. Here, we report a patient with NF1 and developmental and epileptic encephalopathy (DEE) harboring pharmacoresistant tonic seizures, and progressive optic pathway glioma (OPG). By using a MEKi therapy for her OPG, we observed an end to epileptic seizures as well as a significant improvement of interictal EEG abnormalities, despite a lack of tumor reduction. MEK inhibitor therapy should be considered for patients with NF1 and refractory epilepsy.

A common single nucleotide variant in the cytokine receptor-like factor-3 (CRLF3) gene causes neuronal deficits in human and mouse cells.

Single nucleotide variants in the general population are common genomic alterations, where the majority are presumed to be silent polymorphisms without known clinical significance. Using human induced pluripotent stem cell (hiPSC) cerebral organoid modeling of the 1.4 megabase Neurofibromatosis type 1 (NF1) deletion syndrome, we previously discovered that the cytokine receptor-like factor-3 (CRLF3) gene, which is co-deleted with the NF1 gene, functions as a major regulator of neuronal maturation. Moreover, children with NF1 and the CRLF3L389P variant have greater autism burden, suggesting that this gene might be important for neurologic function. To explore the functional consequences of this variant, we generated CRLF3L389P-mutant hiPSC lines and Crlf3L389P-mutant genetically engineered mice. While this variant does not impair protein expression, brain structure, or mouse behavior, CRLF3L389P-mutant human cerebral organoids and mouse brains exhibit impaired neuronal maturation and dendrite formation. In addition, Crlf3L389P-mutant mouse neurons have reduced dendrite lengths and branching, without any axonal deficits. Moreover, Crlf3L389P-mutant mouse hippocampal neurons have decreased firing rates and synaptic current amplitudes relative to wild type controls. Taken together, these findings establish the CRLF3L389P variant as functionally deleterious and suggest that it may be a neurodevelopmental disease modifier.

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.

Cotargeting Phosphoinositide 3-Kinase and Focal Adhesion Kinase Pathways Inhibits Proliferation of NF2 Schwannoma Cells.

Neurofibromatosis Type 2 (NF2) is a tumor predisposition syndrome caused by germline inactivating mutations in the NF2 gene encoding the merlin tumor suppressor. Patients develop multiple benign tumor types in the nervous system including bilateral vestibular schwannomas (VS). Standard treatments include surgery and radiation therapy, which may lead to loss of hearing, impaired facial nerve function, and other complications. Kinase inhibitor monotherapies have been evaluated clinically for NF2 patients with limited success, and more effective nonsurgical therapies are urgently needed. Schwannoma model cells treated with PI3K inhibitors upregulate activity of the focal adhesion kinase (FAK) family as a compensatory survival pathway. We screened combinations of 13 clinically relevant PI3K and FAK inhibitors using human isogenic normal and merlin-deficient Schwann cell lines. The most efficacious combination was PI3K/mTOR inhibitor omipalisib with SRC/FAK inhibitor dasatinib. Sub-GI50 doses of the single drugs blocked phosphorylation of their major target proteins. The combination was superior to either single agent in promoting a G1 cell-cycle arrest and produced a 44% decrease in tumor growth over a 2-week period in a pilot orthotopic allograft model. Evaluation of single and combination drugs in six human primary VS cell models revealed the combination was superior to the monotherapies in 3 of 6 VS samples, highlighting inter-tumor variability between patients consistent with observations from clinical trials with other molecular targeted agents. Dasatinib alone performed as well as the combination in the remaining three samples. Preclinically validated combination therapies hold promise for NF2 patients and warrants further study in clinical trials.

Mutational Spectrum and Genotype-phenotype Correlations in Neurofibromatosis Type 1 Patients from North Macedonia: Identification of Ten Novel NF1 Pathogenic Variants.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, characterized by multiple café-au-lait macules, axillary and inguinal freckling, tumors of the nervous system, and iris hamartomas. More than 3,100 different pathogenic variants have been reported in the NF1 gene, including missense, nonsense, frameshift, in-frame, splicing, and large deletions. To determine the NF1 mutational spectrum in patients with NF1 from the Republic of North Macedonia. A cohort study. Molecular analyses included reverse transcription and cDNA sequencing of the NF1 gene and next-generation sequencing using the TruSight Cancer panel, along with the multiple ligation probe amplification method to detect single nucleotide variants and copy number variations. Direct DNA sequencing was also used for the family member analysis. Our 9-year study of patients suspected of having NF1 in the Republic of North Macedonia encompassed molecular characterization of 30 cases of the disease. We identified 28 unique pathogenic NF1 variants (NM_001042492.3), of which ten were novel: c.208delA; c.341_364del; c.1480_1481delTT; c.2325+1G>C; c.2495_2496dupAC; c.2533_2541del; c.4517delC; c.5844C>G; c.6971delA; c.7605_7606delGAinsAT. In addition to the variant spectrum analysis, our research revealed two positive genotype-phenotype correlations. One between the clinical manifestation of cognitive impairment and gross deletions in the NF1 gene, and the other between cognitive impairment and truncating variants located in the RAS-GAP functional domain. This is the first study of NF1 patients in the Republic of North Macedonia, and it contributes ten novel variants to the global spectrum of pathogenic NF1 variants. It also corroborates the crucial importance of NF1 genetic testing for a prompt and precise diagnosis, particularly in younger patients.