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Neurofibromatosis type 1 (NF1) is an autosomal dominant condition with a birth incidence of 1/3,500. Around 50% of cases are due to new mutations. The NF1 gene maps to 17q11.2 and encodes neurofibromin. NF1 is a "classical" tumor suppressor gene. Congenital disseminated NF1 is rare with just two cases previously reported. We present a deceased baby with congenital disseminated NF1 in whom we performed molecular studies. A germline mutation (R461X) in exon 10a of the NF1 gene was found. A 2 bp deletion (3508delCA) in codon 1170 of exon 21 was identified in DNA derived from some tumor tissue. Loss of heterozygosity in NF1 and TP53 was observed in other tumor samples. No microsatellite instability was observed in the tumor samples. This is the first report of molecular analysis of the NF1 locus in a patient with disseminated congenital neurofibromatosis. This case had a de novo germline mutation in NF1 and three documented somatic mutations in the NF1 and TP53 genes in tumor specimens.

Neurofibromatosis type 1 is a common familial cancer syndrome, affecting about 1 in every 4,000 individuals worldwide. We have carried out NF1 gene mutation analysis on DNA isolated from 25 tumours (dermal and plexiform neurofibromas, malignant peripheral nerve sheath tumour, MPNST), obtained at post-mortem from an NF1 patient. Macro and micro sequence alterations of the NF1 gene were studied by dHPLC, microsatellite, RFLP markers and multiplex ligation probe amplification (MLPA). The underlying germline mutation involves a deletion of exons 2 and 3. Of the 25 tumours studied from this patient, characterised somatic mutations were identified in 9 tumours, these were six small deletions (748del T, 2534–2557 del 24bp, 2843delA, 3047–3048 del GT, 4743del G, 7720–7721 delAA), an insertion 649 ins 73 bp, a non-sense mutation R1513X and a single splice site mutation, IVS4C-1 G>A, eight of these represent novel sequence changes in the gene. Evidence for loss of heterozygosity (LOH) was identified in DNA from 7 of the tumours. Each of the tumours analysed contained a different somatic NF1 mutation, indicating that each tumour is the result of an independent somatic event. The somatic mutation detection rate in this study is 64% (16/25), is one of the highest rates in genomic DNA reported so far in a single NF1 patient. Only 68 characterised NF1 somatic mutations have so far been reported and so our data will contribute to NF1 somatic mutational spectrum of the NF1 gene and will be important for understanding the molecular basis of NF1 tumorigenesis.

Neurofibromatosis type 1 (NF1) is characterized by cafe-au-lait spots, skinfold freckling, and cutaneous neurofibromas. No obvious relationships between small mutations (20 bp) of the NF1 gene and a specific phenotype have previously been demonstrated, which suggests that interaction with either unlinked modifying genes and/or the normal NF1 allele may be involved in the development of the particular clinical features associated with NF1. We identified 21 unrelated probands with NF1 (14 familial and 7 sporadic cases) who were all found to have the same c.2970-2972 delAAT (p.990delM) mutation but no cutaneous neurofibromas or clinically obvious plexiform neurofibromas. Molecular analysis identified the same 3-bp inframe deletion (c.2970-2972 delAAT) in exon 17 of the NF1 gene in all affected subjects. The Delta AAT mutation is predicted to result in the loss of one of two adjacent methionines (codon 991 or 992) ( Delta Met991), in conjunction with silent ACA--ACG change of codon 990. These two methionine residues are located in a highly conserved region of neurofibromin and are expected, therefore, to have a functional role in the protein. Our data represent results from the first study to correlate a specific small mutation of the NF1 gene to the expression of a particular clinical phenotype. The biological mechanism that relates this specific mutation to the suppression of cutaneous neurofibroma development is unknown.

Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, represents the third most common human muscular dystrophy. The FSHD disease locus, at chromosome 4q35, is associated with large contractions of the polymorphic repeat sequence array D4Z4. In addition to FSHD disease association with large D4Z4 deletions, a biased interaction with a specific 4qter subtelomeric sequence has been described in patients. Two distinct 4qter subtelomeres, defined as types 4qA and 4qB, have been identified and shown to be equally prevalent in the Caucasian population. In almost all 4q35‐linked patients with FSHD, however, disease expression only occurs when large D4Z4 deletions are located on 4qA‐defined 4qter subtelomeres. Conversely, large D4Z4 repeat contractions situated on 4qB‐defined subtelomeres either are not disease‐causing or exhibit a greatly reduced disease penetrance. This study was initiated to confirm this direct FSHD disease association data by measuring the frequency of type 4qA‐defined and 4qB‐defined subtelomeric sequences in a large cohort of 164 unrelated patients with FSHD from Turkey and the UK, all known to have large D4Z4 deletions. An almost complete association was found between large D4Z4 repeat array deletions located on 4qA‐defined 4qter subtelomeres and disease expression in our large FSHD patient cohort. The observed failure of probes 4qA and 4qB to hybridise to two patient‐derived DNA samples confirms the presence of an additional rare type of 4qter subtelomeric sequence in humans.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominantly inherited conditions. A range of complications has been described, including gastrointestinal manifestations. Gastric carcinoid tumours are associated with multiple endocrine neoplasia, atrophic gastritis and pernicious anaemia but have not been reported in NF1 in the absence of other predisposing factors. We report the occurrence and investigation of a gastric carcinoid tumour in a 23-year-old woman with previously uncomplicated NF1. Analysis of the tumour tissue revealed loss of heterozygosity at the NF1 gene locus but a normal karyotype and an absence of microsatellite instability. A germline NF1 gene nonsense mutation in exon 37 was detected by denaturing high-performance liquid chromatography and DNA sequence analysis. This is the first reported occurrence of a gastric carcinoid tumour in a patient with NF1 in the absence of other predisposing factors such as pernicious anaemia. The analyses indicate that the carcinoid arose through NF1 gene inactivation but in the absence of an inherited NF1 gene microdeletion. This case adds to the range of gastrointestinal tumours that may be encountered in patients with NF1, particularly in those who present with upper gastrointestinal haemorrhage.

Neurofibromatosis 1 (NF1) is a common neurocutaneous condition with an autosomal dominant pattern of inheritance. The complications are diverse and disease expression varies, even within families. Progress in molecular biology and neuroimaging and the development of mouse models have helped to elucidate the aetiology of NF1 and its clinical manifestations. Furthermore, these advances have raised the prospect of therapeutic intervention for this complex and distressing disease. Members of the United Kingdom Neurofibromatosis Association Clinical Advisory Board collaborated to produce a consensus statement on the current guidelines for diagnosis and management of NF1. The proposals are based on published clinical studies and on the pooled knowledge of experts in neurofibromatosis with experience of providing multidisciplinary clinical and molecular services for NF1 patients. The consensus statement discusses the diagnostic criteria, major differential diagnoses, clinical manifestations and the present strategies for monitoring and management of NF1 complications.

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14Arf, and p16INK4a proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100β protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool. (Cancer Res 2006; 66(5): 2584-91)

Neurofibromatosis type I (NF1) is an autosomal dominant familial tumor syndrome characterized by the presence of multiple benign neurofibromas. In 95% of NF1 individuals, a mutation is found in the NF1 gene, and in 5% of the patients, the germline mutation consists of a microdeletion that includes the NF1 gene and several flanking genes. We studied the frequency of loss of heterozygosity (LOH) in the NF1 region as a mechanism of somatic NF1 inactivation in neurofibromas from NF1 patients with and without a microdeletion. There was a statistically significant difference between these two patient groups in the proportion of neurofibromas with LOH. None of the 40 neurofibromas from six different NF1 microdeletion patients showed LOH, whereas LOH was observed in 6/28 neurofibromas from five patients with an intragenic NF1 mutation (P ¼ 0.0034, Fisher’s exact). LOH of the NF1 microdeletion region in NF1 microdeletion patients would de facto lead to a nullizygous state of the genes located in the deletion region and might be lethal. The mechanisms leading to LOH were further analyzed in six neurofibromas. In two out of six neurofibromas, a chromosomal microdeletion was found; in three, a mitotic recombination was responsible for the observed LOH; and in one, a chromosome loss with reduplication was present. These data show an important difference in the mechanisms of second hit formation in the 2 NF1 patient groups. We conclude that NF1 is a familial tumor syndrome in which the type of germline mutation influences the type of second hit in the tumors. V C 2006 Wiley-Liss, Inc.

Segmental neurofibromatosis type 1 (SNF1), characterized by the regionally limited distribution of neurofibromatosis type 1 (NF1) features, has been attributed to mosaicism for an NF1 gene mutation. The occurrence of classical NF1 in the offspring of a parent with SNF1 suggests that cutaneous mosaicism may be accompanied by gonadal mosaicism. We studied a girl with generalized NF1, and her mother who has SNF1. A recurrent nonsense mutation in exon 31 (R1947X) of the NF1 gene was identified in the lymphocyte DNA of the affected child by denaturing high-performance liquid chromatography and PCR/direct sequencing. DNA sequence analysis failed, however, to identify the R1947X mutation in peripheral lymphocytes, and in keratinocytes and fibroblasts cultured from affected and unaffected skin in the mother. DNA fragments containing exon 31 were then cloned from each cell line and these clones were screened using allele-specific PCR. The R1947X mutation was identified in 29 of 146 clones derived from keratinocytes from the affected region and in 12 of 136 clones derived from fibroblasts from the affected region, but in no clones derived from clinically unaffected tissues. These findings confirm that gonosomal mosaicism can occur in SNF1, with consequent important implications for genetic counselling.

BACKGROUND: Segmental duplications flanking the neurofibromatosis type 1 (NF1) gene locus on 17q11 mediate most gene deletions in NF1 patients. However, the large size of the gene and the complexity of the locus architecture pose difficulties in deletion analysis. We report the construction and application of the first NF1 locus specific microarray, covering 2.24 Mb of 17q11, using a non-redundant approach for array design. The average resolution of analysis for the array is approximately 12 kb per measurement point with an increased average resolution of 6.4 kb for the NF1 gene. METHODS: We performed a comprehensive array-CGH analysis of 161 NF1 derived samples and identified heterozygous deletions of various sizes in 39 cases. The typical deletion was identified in 26 cases, whereas 13 samples showed atypical deletion profiles. RESULTS: The size of the atypical deletions, contained within the segment covered by the array, ranged from 6 kb to 1.6 Mb and their breakpoints could be accurately determined. Moreover, 10 atypical deletions were observed to share a common breakpoint either on the proximal or distal end of the deletion. The deletions identified by array-CGH were independently confirmed using multiplex ligation-dependent probe amplification. Bioinformatic analysis of the entire locus identified 33 segmental duplications. CONCLUSIONS: We show that at least one of these segmental duplications, which borders the proximal breakpoint located within the NF1 intron 1 in five atypical deletions, might represent a novel hot spot for deletions. Our array constitutes a novel and reliable tool offering significantly improved diagnostics for this common disorder.

We report a case that draws attention to a hitherto undescribed association of neurofibromatosis type 1 (NF1) with juvenile polyp, congenital intrahepatic portosystemic venous shunt, multiple subcutaneous lipomas, and horseshoe kidney. Our patient has fulfilled the National Institutes of Health consensus conference criteria for NF1 by having neurofibromas, axillary freckling, Lisch nodules, and café-au-lait spots. There is no family history of NF1 and his 7-year-old son has no stigmata of NF1. On the other hand, the patient's family had a presumably dominant inheritance of horseshoe kidney: the father, proband, sister, and son of the other sister had a horseshoe kidney. The patient was investigated for mutations in the NF1 gene and PTEN, but no germline mutations were detected. The differential diagnosis for such a collection of hamartomatous, cutaneous, and vascular disorders includes the Proteus, Bannayan-Riley-Ruvalcaba, and Cowden syndromes. None of these diagnoses was convincingly confirmed in this patient.

The regulatory regions of the neurofibromatosis type 1 (NF1) gene have scarcely been screened either for mutations of potential pathological importance or for functional polymorphisms. To address this question, a 987 bp sequence spanning the promoter and 5' flanking sequence of the human NF1 gene was screened for sequence variants in 570 unrelated NF1 patients and 105 controls. Five novel sequence variants were identified, comprising a 14 bp deletion at -142 within the promoter region, three single nucleotide substitutions in the 5'UTR (C + 247T, C + 261G, G + 462C), and a substitution (C + 514T) at the 5' end of the coding region that served to generate a Stop codon. The latter is likely to be of pathological significance since it is predicted to lead to the synthesis of a truncated protein. The functional significance of three of the other variants (14 bp del, C + 261G, G + 462C) was explored by luciferase reporter gene expression and electrophoretic mobility shift assays. The del14 variant demonstrated allele-specific protein binding without altered reporter gene expression and the G + 462C allele showed slightly decreased reporter gene expression.

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.

Therapeutic options are limited for neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNST) and clinical trials using drug agents have so far been unsuccessful. This lack of clinical success is likely attributed to high levels of intratumoral molecular heterogeneity and variations in signal transduction within MPNSTs. To better explore the variance of malignant signaling properties within heterogeneous MPNSTs, four MPNST cell lines (ST8814, S462, S1844.1, and S1507.2) were used. The data demonstrate that small-molecule inhibition of the MET proto-oncogene and mTOR had variable outcome when preventing wound healing, cell migration, and invasion, with the S462 cells being highly resistant to both. Of interest, targeted inhibition of the STAT3 transcription factor suppressed wound healing, cell migration, invasion, and tumor formation in all four MPNST lines, which demonstrates that unlike MET and mTOR, STAT3 functions as a common driver of tumorigenesis in NF1-MPNSTs. Of clinical importance, STAT3 knockdown was sufficient to block the expression of hypoxia-inducible factor (HIF)1α, HIF2α, and VEGF-A in all four MPNST lines. Finally, the data demonstrate that wound healing, cell migration, invasion, and tumor formation through STAT3 are highly dependent on HIF signaling, where knockdown of HIF1α ablated these oncogenic facets of STAT3. Implications: This research reveals that aberrant STAT3 and HIF1a activity drives tumor progression in MPNSTs, indicating that inhibition of the STAT3/HIF1α/VEGF-A signaling axis is a viable treatment strategy. Mol Cancer Res; 13(7); 1149–60. ©2015 AACR.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P, We thank the patients, their family and their referral physicians for the information. We thank the genetics counselor students who helped to re-contact all referral physicians for confirming information. This work was supported though the Children's Tumor Foundation by the Isaac and Sadie Fuchs Genotype-Phenotype Study (to L.M.) and by internal funds from the Medical Genomics Laboratory at UAB.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited neuromuscular disorder after Duchenne muscular dystrophy and myotonic dystrophy. The gene underlying FSHD was mapped to chromosome 4q35 in 1990 and was shown to be closely linked to locus D4F104S1. Although D4F104S1-associated deletions are closely associated with FSHD, the identity and location of the FSHD gene (or genes) still remain elusive, as does the mechanistic basis of the disease. In addition, although approximately 5% of FSHD families fail to exhibit linkage to 4q35, a putative second locus remains unidentified. The search for the FSHD gene has been hampered both by sequence homologies between the 4q35 candidate region and other chromosomal regions and by the presence of many highly repetitive sequences. Molecular diagnosis for FSHD is usually offered with 98% accuracy but because of its complexity, a much more simple test would be preferable. Indeed, the identification of the FSHD gene itself should potentiate major improvements in diagnostic testing.

Neurofibromatosis type 1 (NFI) is a common autosomal dominant disorder characterised by café-au-lait spots, neurofibromas and iris hamartomas. Since the NF1 gene product neurofibromin contains a GAP-related domain involved in the down-regulation of p21(ras) oncogene activity, the NF1 gene has come to be regarded as a tumour-suppressor gene. One common mechanism of tumour-suppressor gene inactivation during tumorigenesis is promoter hypermethylation, this "epi-mutation" being functionally equivalent to a second-hit somatic mutation. To assess the importance of promoter hypermethylation in NF1 gene inactivation in NF1-related tumours, the methylation status of the NF1 promoter region was determined by bisulphite-modified genomic sequencing in NF1-specific tumours and peripheral blood lymphocytes (PBL) from both NF1 patients and normal controls. Tumour-specific CpG methylation of six distinct CpG sites was identified at positions -609, -429, 406, -383, -331 and -315 relative to the transcriptional start site. However, since all other CpG sites were unmethylated in all tissues examined, it is unlikely that CpG hypermethylation within the NF1 promoter represents a common mutational mechanism leading to neurofibroma formation.

A number of frequently unrecognised vascular manifestations have been described in patients with neurofibromatosis type 1 (NF1), including involvement of the great vessels, cerebral, visceral and renal arteries. Rarely, changes in the coronary arteries have been reported in adults with NF1. We report on a 16-year-old boy affected by NF1 with dysmorphic features and three aneurysms in the mid-portion of the left descending coronary artery disclosed by chance during investigation for a malignant peripheral nerve sheath tumour. Molecular analysis detected a gross de novo deletion in the NF1 gene. The boy had had no previous cardiac symptoms but died suddenly after developing signs and symptoms suggestive of myocardial infarction. Conclusion To the best of our knowledge, this represents the first report of multiple lesions in the coronary arteries in a child affected by neurofibromatosis type 1 with a known deletion of the neurofibromatosis type 1 gene.

Dideoxy fingerprinting was used to analyse the 5' flanking region of the neurofibromin (NF1) gene in a panel of 380 neurofibromatosis type 1 (NF1) patients. Five polymorphisms/rare variants were identified at positions -412, - 402, + 16, + 25 and + 132, but control data indicated that these were unlikely to be of pathological significance. Promoter mutations in the NF1 gene are not, therefore, a common cause of NF1. This notwithstanding, a reporter gene assay was performed to determine if these variants could affect the expression of the NF1 gene, and all three changes in the 5'-untranslated region (UTR) (+ 16, + 25, + 132) were found to be associated with a 60-70% increase in reporter gene expression.

This study outlines the molecular DNA findings derived from 12 separate prenatal diagnoses offered to families with a history of facioscapulohumeral muscular dystrophy. A high risk of the fetus being affected was identified in five pregnancies. Several practical problems are discussed, particularly those arising from the quality and quantity of DNA made available for molecular diagnosis. Evidence of the 4q35 and 10q26 telomeric exchanges is present in 20 per cent of the general population and the specificity of the test is 95 per cent. The eventual isolation and functional characterization of the FSHD gene should allow us to unravel many of the complexities currently associated with the molecular diagnosis of this disorder.

We present the clinical and ophthalmological findings, genetic analysis, and therapy of two siblings with NF1 and bilateral OPG. In genetic analysis, a heteroduplex profile was detected in exon 4b of the NF1 gene for the affected patients and mother. Sequencing of the DNA samples identified a C > T nucleotide change in exon 4b (c484CAG > TAG). This nonsense mutation resulted in a change of glutamine to a stop codon (Q162X) and is a novel NF1 gene alteration.

Denaturing high-performance liquid chromatography (DHPLC) is a novel high-capacity technique for detecting new mutations. We have evaluated the sensitivity and specificity of this method in a blind analysis of exon H of the factor IX gene and exon 16 of the neurofibromatosis type 1 gene. Under a single set of conditions for each exon, 55/55 individuals carrying 48 unique mutations were correctly identified as were 55/55 individuals with wildtype alleles. We conclude that DHPLC is a highly sensitive and specific method for mutation detection.

Mutation screening in neurofibromatosis type 1 (NF1) families has long been hampered by the complexity of the NF1 gene. By using a novel multi-track screening strategy, 67 NF1 families (54 two-generation, 13 three-generation) with a de novo mutation in the germline of the first generation were studied with two extragenic and 11 intragenic markers. The pathological lesion was identified in 31 cases. Loss of heterozygosity (LOH) in the affected individual revealed a gross gene deletion in 15 of the two-generation families; in 12 (80%) of them, the deletion was maternally derived. Eleven patients with a gross deletion exhibited developmental delay, ten had dysmorphic features and six manifested a learning disability. No gross deletion was apparent in any of the 13 three-generation families, suggesting that such lesions are subject to more intense selection. In these families, the new mutation was of paternal origin in 11 kindreds and the underlying mutational event could be characterised in three of them.

A major advance in the molecular diagnosis of facioscapulohumeral muscular dystrophy is the recently reported elimination of confounding DNA fragments arising from homologous sequences located at 10q26. In order to evaluate the specificity and sensitivity of this important diagnostic test, we have compared a group of 130 patients fulfilling the diagnostic criteria for FSHD with 200 control subjects not known to have an increased risk of having an FSHD mutation. Among the FSHD cases the smallest BlnI/EcoRI fragment sizes ranged from 10 to > 48 kb with 94.6% (95% CI 89.2-97.8%) of cases having fragment sizes of 34 kb or less. Among the 400 chromosomes from controls the smallest BlnI/EcoRI fragment observed with the EcoRI/BlnI double restriction enzyme digest was 38 kb +/- 2 kb, suggesting a test specificity at a fragment size < 34 kb of or very near to 100% (lower 95% CI 98.2%). Test sensitivity at < 34 kb is estimated at 94.6% (95% CI 89.2-97.8%), all outliers having fragments > 38 kb. The Southern blot analysis with DNA probe p13E-11 has created a valuable molecular diagnostic test for FSHD.

The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types1. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer2, 3. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras4. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.

Neurofibromatosis type 1 (NF1) is a familial tumour predisposition syndrome with no current treatment regime. Neurofibromin, the NF1 gene product, is a Ras-GAP protein that downregulates Ras and when inactivated leads to increased cell growth, proliferation and eventually tumorigenesis. The Ras family proteins have a central role in cell biology and are of prime importance in cancer development.This article reviews recent advances from the past 5 years, covering aspects of molecular and clinical diagnosis of NF1, genotype/phenotype studies, the RASopathies, NF1 tumorigenesis, mouse NF1 models and potential therapies. What reader will gain: The reader is introduced to recent advances in NF1 that focus on clinical and molecular diagnosis. The reader should gain a better understanding of the molecular basis of NF1 and of other NF1-like syndromes, and the challenges presented for clinical management.A molecular diagnosis can usually be made in most NF1 patients using a battery of gene screening techniques. The sensitivity and specificity of mutation detection will increase with the introduction of new technologies and new bioinformatic tools. Pathogenic somatic mutations of the NF1 gene are increasingly being identified in tumours not usually associated with NF1, a clear indication that neurofibromin has a much wider biological role and has an importance far beyond NF1.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20-27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD.

We report the first visible cytogenetic deletion involving the NF1 gene in a patient with sporadic neurofibromatosis, dysmorphic features, and marked developmental delay. The combined evidence of molecular and cytogenetic techniques based on dosage reduction, hemizygosity for microsatellite markers, high resolution G banding, and FISH analysis, predicts this deletion to be approximately 7 Mb in size. Our findings highlight the importance of conducting a detailed cytogenetic and FISH analysis in patients with NF1 who have additional dysmorphic features or particularly severe learning difficulties.

Neurofibromatosis type 1 (NF1), also called von Recklinghausen disease or peripheral neurofibromatosis, is a common autosomal dominant disorder characterised by multiple neurofibromas, café au lait spots, and Lisch nodules of the iris, with a variable clinical expression. The gene responsible for this condition, NF1, has been isolated by positional cloning. It spans over 350 kb of genomic DNA in chromosomal region 17q11.2 and encodes an mRNA of 11-13 kb containing at least 59 exons. NF1 is widely expressed in a variety of human and rat tissues. Four alternatively spliced NF1 transcripts have been identified. Three of these transcript isoforms (each with an extra exon: 9br, 23a, and 48a, respectively) show differential expression to some extent in various tissues, while the fourth isoform (2.9 kb in length) remains to be examined. The protein encoded by NF1, neurofibromin, has a domain homologous to the GTPase activating protein (GAP) family, and downregulates ras activity. The identification of somatic mutations in NF1 from tumour tissues strongly supports the speculation that NF1 is a member of the tumour suppressor gene family. Although the search for mutations in the gene has proved difficult, germline mutation analysis has shown that around 82% of all the fully characterised NF1 specific mutations so far predict severe truncation of neurofibromin. Further extensive studies are required to elucidate the gene function and the mutation spectrum. This should then facilitate the molecular diagnosis and the development of new therapy for the disease.

Neurofibromatosis type 1 is one of the most common inherited disorders with an incidence of 1 in 3000. The search for NF1 mutations has been hampered by the overall size of the gene, the large number of exons, and the high mutation rate. To date, fewer than 90 mutations have been reported to the NF1 mutation analysis consortium and the details on 76 mutations have been published. We have identified five new mutations using single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) and three intragenic deletions with the microsatellite markers. Of the five new mutations, two were in exon 27a, two in exon 45, and one in exon 49 and these include 4630delA, 4572delC, R7846X, T7828A, and one in the 3' untranslated region (3' UTR). The two nucleotide alterations in exon 27a and the one in exon 45 are predicted to produce a truncated protein.

The wide range of severity in facioscapulohumeral muscular dystrophy (FSHD) complicates genetic advice, although onset age is youngest and severity is greatest in isolated cases. From 14 of 16 large FSHD families which are 4q35 linked, and from 25 of 34 isolated cases exhibiting a de novo D4F104S1 DNA fragment, we find a correlation between proband age at onset and FSHD-associated D4F104S1 fragment size (r = 0.56; P 85% of FSHD maps to 4q35, will facilitate genetic counseling. We propose that quantitative variation in a uniform mutation mechanism influences age at onset, but by deletion rather than expansion of DNA.

Facioscapulohumeral dystrophy (FSHD) is an autosomal-dominant neuromuscular disorder with a prevalence of 1 in 20,000. The DNA marker p13E-11 (D4F104S1) detects a de novo DNA rearrangement in the majority of sporadic and FSHD cases. These rearrangements consist of deletions of multiple copies of tandem repeat (D4Z4). We have studied 34 new mutation FSHD families of which 26 showed a de novo fragment with p13E-11. In three of the remaining eight families without a de novo fragment, germinal mosaicism was noted. In each case, the proband had inherited a small EcoR1 fragment from the clinically unaffected mother; however, the hybridization signal intensity of this fragment in the mother's DNA was significantly reduced in all three families. This is the first study to describe such mosaicism in FSHD families using DNA analysis and therefore has a considerable significance for genetic counseling and prenatal diagnosis.

A 20 year old male patient with sporadic neurofibromatosis type 1 (NF1) is described with a large deletion (1.5 Mb) involving theNF1 gene, dysmorphism, mental retardation, and unusual ocular and skeletal features. Several NF1 patients with a large NF1 deletion and associated dysmorphism, and a large

MicroRNAs (miRNAs) are a class of non-coding RNA, which have recently been shown to have a wide variety of regulatory functions in relation to gene expression. Since their identification nearly 20 years ago, miRNAs have been found to play an important role in cancer, including in neurofibromatosis type 1 (NF1)-associated tumours. NF1 is the most commonly inherited tumour predisposition syndrome and can lead to malignancy via the development of malignant peripheral nerve sheath tumours (MPNSTs). Although the mechanisms by which benign neurofibromas develop into MPNSTs still remain to be elucidated, it is becoming increasingly clear that miRNAs play a key role in this process and have the potential to be used as both diagnostic and prognostic markers of tumorigenesis.

Neurofibromatosis type-1 (NF1), resulting from NF1 gene loss of function, is characterized by an increased risk of developing benign and malignant peripheral nerve sheath tumors (MPNSTs). Whereas the cellular heterogeneity of NF1-associated tumors has been well studied, the molecular heterogeneity of MPNSTs is still poorly understood. Mutational heterogeneity within these malignant tumors greatly complicates the study of the underlying mechanisms of tumorigenesis. We have explored this molecular heterogeneity by performing loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, PTEN, and CDKN2A genes on sections of 10 MPNSTs derived from 10 unrelated NF1 patients. LOH data for the TP53 gene was found to correlate with the results of p53 immunohistochemical analysis in the same tumor sections. Further, approximately 70% of MPNSTs were found to display intra-tumoral molecular heterogeneity as evidenced by differences in the level of LOH between different sections of the same tumor samples. This study constitutes the first systematic analysis of molecular heterogeneity within MPNSTs derived from NF1 patients. Appreciation of the existence of molecular heterogeneity in NF1-associated tumors is important not only for optimizing somatic mutation detection, but also for understanding the mechanisms of NF1 tumorigenesis, a prerequisite for the development of specifically targeted cancer therapeutics.

Neurofibromatosis type-1 (NF1) is caused by constitutional mutations of the NF1 tumor-suppressor gene. Although ∼85% of inherited NF1 microlesions constitute truncating mutations, the remaining ∼15% are missense mutations whose pathological relevance is often unclear. The GTPase-activating protein-related domain (GRD) of the NF1-encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1- GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1-GRD expression vector and activated Ras was assayed by an enzyme-linked immunosorbent assay (ELISA). Ten NF1-GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP-bound Ras in comparison to wild-type NF1 protein. The remaining five NF1-GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild-type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1-GRD structure and function. HumMutat 33:1687–1696, 2012. C � 2012 Wiley Periodicals, Inc.

Neurofibromatosis type 1 (NF1) affects 1/3,000–4,000 individuals worldwide and is caused by the inactivation of the NF1 tumour suppressor gene which encodes the protein neurofibromin. Consistent with Knudson’s two-hit hypothesis, NF1 patients harbouring a heterozygous germline NF1 mutation develop several NF1-associated tumours upon somatic inactivation of the second (wild-type) NF1 allele. Although the identification of somatic mutations in NF1 patients has always been problematic on account of the extensive cellular heterogeneity manifested by neurofibromas, their classification is a prerequisite for understanding the complex molecular mechanisms underlying NF1 tumorigenesis. Here, we have performed a meta-analysis of known somatic NF1 mutations identified in a range of NF1-associated neoplasms including peripheral nerve sheath tumours (neurofibromas), malignant peripheral nerve sheath tumours (MPNSTs), astrocytomas, gastrointestinal stromal tumours, gastric carcinoid, juvenile myelomonocytic leukaemia, glomus tumours, neuroblastomas, breast cancer and phaeochromocytomas. Comparison of the frequencies of the different types of somatic NF1 mutation so far observed with those of their previously reported germline counterparts reveals significant differences (p = 0.00014), with the somatic mutational spectrum being characterised by a greater proportion of nonsense mutations and a smaller proportion of splice-site mutations. Finally, C>T and G>A changes in CpG and CpHpG oligonucleotides resulting in missense/nonsense mutations in the NF1 gene were significantly overrepresented in the soma as compared to the germline (p = 0.007).

Inherited mutations of the NF1 tumour suppressor gene give rise to the tumour predisposition syndrome neurofibromatosis type 1 (NF1). The NF1 gene, located at chromosome 17q11.2, spans 283 kb and contains 61 exons that give rise to a 12-kb mRNA transcript encoding neurofibromin, a key negative regulator of the cellular Ras signalling pathway. The NF1 locus is characterised by one of the highest mutation rates known for any human disorder. Nearly 1,300 different inherited mutations of the NF1 gene have been reported to date as a cause of NF1. These vary in size from deletions spanning more than a megabase to subtle single base-pair substitutions that alter an encoded amino acid or the function of a splice junction. Here, we review the known germline NF1 mutational spectrum and show how the nature, location and frequency of different types of inherited NF1 mutation are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment.

A variety of different somatic copy number alterations (SCNAs) have been reported in NF1-associated malignant peripheral nerve sheath tumours (MPNSTs). Indeed, SCNA analysis, based upon different platforms, is beginning to identify specific regulatory pathways associated with MPNSTs, thereby providing us with new potential targets for therapeutic intervention. Extensive studies have also been undertaken on gene expression in NF1-associated tumours but not yet in parallel with SCNA analysis. Although in some cancers, SCNAs are associated with the altered regulation of genes, an integrative study of SCNAs and gene expression is currently lacking in the context of NF1-associated tumours. It is to be expected that such an integrative study will aid in the identification not only of driver genes associated with NF1 tumourigenesis but also cancer-specific biological pathways and hence potential therapeutic targets. It is envisaged that a combination of next generation sequencing and powerful new bioinformatic tools will provide new insights into how high SCNA affects gene expression and the biological pathways.

A dominantly inherited muscular dystrophy with onset in the shoulder girdle and later progression to the lower limbs is described. The disorder was clinically distinguishable from known facioscapulohumeral, scapulohumeral and limb girdle syndromes. A 38 kb allele detected by probe p13E-11 (D4F104S1) segregated with the disease. Linkage analysis gave a maximum lod score of z = 1.61 at theta = 0.01 with the 4q35 markers D4S184 (affected only analysis z = 1.20 at theta = 0.01) suggesting probable allelism with facioscapulohumeral muscular dystrophy.

Neurobromatosis type 1 (NF1) is one of the commonest genetic disorders in humans. The gene for NF1 was cloned in 1990. The protein encoded by the gene (neurofibromin) has extensive sequence homology with GTPase-activating protein (GAP). Despite screening the whole coding region of the gene for large and medium size rearrangements and approximately 40% of the coding region of the gene for small alterations, only 45 germ-line mutations have been reported in more than 500 unrelated patients. Of these, 25 mutations involve small changes in the gene, of which 17 (68%) result in the formation of an inappropriate stop codon. A “hot spot” for mutations has not been identified. The high mutation rate at this locus and the general difficulty in identifying mutations are discussed. A complete understanding of the structure and function of the NF1 gene awaits further detailed studies of both naturally occurring and in vitro-generated mutations. © 1994 Wiley-Liss, Inc.

Somatic gene mutations constitute key events in the malignant transformation of human cells. Somatic mutation can either actively speed up the growth of tumour cells or relax the growth constraints normally imposed upon them, thereby conferring a selective (proliferative) advantage at the cellular level. Neurofibromatosis type-1 (NF1) affects 1/3,000-4,000 individuals worldwide and is caused by the inactivation of the NF1 tumour suppressor gene, which encodes the protein neurofibromin. Consistent with Knudson's two-hit hypothesis, NF1 patients harbouring a heterozygous germline NF1 mutation develop neurofibromas upon somatic mutation of the second, wild-type, NF1 allele. While the identification of somatic mutations in NF1 patients has always been problematic on account of the extensive cellular heterogeneity manifested by neurofibromas, the classification of NF1 somatic mutations is a prerequisite for understanding the complex molecular mechanisms underlying NF1 tumorigenesis. Here, the known somatic mutational spectrum for the NF1 gene in a range of NF1-associated neoplasms --including peripheral nerve sheath tumours (neurofibromas), malignant peripheral nerve sheath tumours, gastrointestinal stromal tumours, gastric carcinoid, juvenile myelomonocytic leukaemia, glomus tumours, astrocytomas and phaeochromocytomas -- have been collated and analysed.

Neurofibromatosis type-1 (NF1) is associated with the growth of benign and malignant tumors. Approximately 15% of NF1 patients develop malignant peripheral nerve sheath tumors (MPNSTs), underlining the need to identify specific diagnostic/prognostic biomarkers associated with MPNST development. The Affymetrix Genome-Wide Human single-nucleotide polymorphism (SNP) Array 6.0 was used to perform SNP genotyping and copy number alteration (CNA), loss-of-heterozygosity (LOH), and copy number neutral–LOH (CNN–LOH) analyses of DNA isolated from 15 MPNSTs, five benign plexiform neurofibromas (PNFs), and patient-matched lymphocyte DNAs. MPNSTs exhibited high-level CNN-LOH, with recurrent changes occurring in MPNSTs but not PNFs. CNN–LOH was evident in MPNSTs but occurred less frequently than genomic deletions. CNAs involving the ITGB8, PDGFA, Ras-related C3 botulinum toxin substrate 1 (RAC1) (7p21-p22), PDGFRL (8p22-p21.3), and matrix metallopeptidase 12 (MMP12) (11q22.3) genes were specific to MPNSTs. Pathway analysis revealed the MPNST-specific amplification of seven Rho–GTPase pathway genes and several cytoskeletal remodeling/cell adhesion genes. In knockdown experiments employing short-hairpin RAC1, ROCK2, PTK2, and LIMK1 RNAs to transfect both control and MPNST-derived cell lines, cell adhesion was significantly increased in the MPNST cell lines, whereas wound healing, cell migration, and invasiveness were reduced, consistent with a role for these Rho–GTPase pathway genes in MPNST development and metastasis. These results suggest new targets for therapeutic intervention in relation to MPNSTs. Hum Mutat 33:763–776, 2012. © 2012 Wiley Periodicals, Inc.

Cancer stem cells (CSCs) are believed to be the regenerative pool of cells responsible for repopulating tumors. Gaining knowledge about the signaling characteristics of CSCs is important for understanding the biology of tumors and developing novel anti-cancer therapies. We have identified a subpopulation of cells positive for CD133 (a CSC marker) from human primary malignant peripheral nerve sheath tumor (MPNST) cells which were absent in non-malignant Schwann cells. CD133 was also found to be expressed in human tissue samples and mouse MPNST cells. CD133+ cells were capable of forming spheres in non-adherent/serum-free conditions. The activation levels of Ras and its downstream effectors such as ERK, JNK, PI3K, p38K, and RalA were significantly increased in this population. Moreover, the CD133+ cells showed enhanced invasiveness which was linked to the increased expression of β-Catenin and Snail, two important proteins involved in the epithelial to mesenchymal transition, and Paxilin, a focal adhesion protein. Among other important characteristics of the CD133+ population, endoplasmic reticulum stress marker IRE1α was decreased, implying the potential sensitivity of CD133+ to the accumulation of unfolded proteins. Apoptotic indicators seemed to be unchanged in CD133+ cells when compared to the wild (unsorted) cells. Finally, in order to test the possibility of targeting CD133+ MPNST cells with Ras pathway pharmacological inhibitors, we exposed these cells to an ERK inhibitor. The wild population was more sensitive to inhibition of proliferation by this inhibitor as compared with the CD133+ cells supporting previous studies observing enhanced chemoresistance of these cells.

Background Neurofibromatosis type 1 (NF1) affects 1 in 2500 people, and 15% of these may develop an optic pathway glioma (OPG). OPGs behave differently in NF1, and, given their frequency, surveillance is important. However, this is difficult because of the additional complications these patients may have, such as learning difficulties. Management is also different given that NF1 results from loss of function of tumour suppressor gene. A genotype–phenotype correlation may help to determine who is at risk of developing these tumours, aid focused screening, and shed light on response to treatments. Methods As part of a long-term follow-up study of patients with NF1 OPGs, the authors assessed genotype–phenotype correlation. Fluorescein in situ hybridisation was performed to identify large deletions, and then a full gene screen for mutations, by denaturing high-performance liquid chromatography. Results 80 patients with NF1 OPGs were identified, and molecular analyses were performed in a subset of 29. A clustering of pathogenic changes in the 5′ tertile of the gene was found. The authors combined these results with those for another two NF1 OPG cohorts and collectively found the same trend. When compared with a control population of NF1 patients without an OPG, the OR of a mutation being present in the 5′ tertile was 6.05 (p=0.003) in the NF1 OPG combined cohorts. Conclusion It is possible that genotype is a significant determinant of the risk of development of OPGs in NF1.