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PURPOSE: Neurofibromatosis type 2 (NF2) and schwannomatosis (SWN) are genetically distinct tumor predisposition syndromes with overlapping phenotypes. We sought to update the diagnostic criteria for NF2 and SWN by incorporating recent advances in genetics, ophthalmology, neuropathology, and neuroimaging. METHODS: We used a multistep process, beginning with a Delphi method involving global disease experts and subsequently involving non-neurofibromatosis clinical experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing NF2 and SWN. These criteria incorporate mosaic forms of these conditions. In addition, we recommend updated nomenclature for these disorders to emphasize their phenotypic overlap and to minimize misdiagnosis with neurofibromatosis type 1. CONCLUSION: The updated criteria for NF2 and SWN incorporate clinical features and genetic testing, with a focus on using molecular data to differentiate the 2 conditions. It is likely that continued refinement of these new criteria will be necessary as investigators study the diagnostic properties of the revised criteria and identify new genes associated with SWN. In the revised nomenclature, the term "neurofibromatosis 2" has been retired to improve diagnostic specificity. ispartof: GENETICS IN MEDICINE vol:24 issue:9 pages:1967-1977 ispartof: location:United States status: published

Supplementary figure legends

Supplementary Figure 1. ST8814 and S462 cells have markedly different STAT3 signalling profiles after HGF stimulation. Starved ST8814 and S462 cells were stimulated with 20 ng/ml HGF for the indicated time (0 h, 1 h, 2 h, and 3 h). Total and phosphorylated STAT3 (Tyr 705) was determined from protein lysates. β-actin serves as a loading control.

Supplementary Figure 2. Cucurbitacin-I greatly altered the morphology of the S1844.1 and S1507.2 cell lines. Starved S1844.1 and S1507.2 cells were subjected to wound healing assay. Cells were pre-treated with 5 nM Cucurbitacin-I, for 30 min prior to 30 min 20 ng/ml HGF stimulation. Pictures were taking at 0 and 18 h. n=3.

Supplementary Table S2 from High-Resolution DNA Copy Number Profiling of Malignant Peripheral Nerve Sheath Tumors Using Targeted Microarray-Based Comparative Genomic Hybridization

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant condition that predisposes to benign and malignant tumors. The lifetime risk of a malignant peripheral nerve sheath tumor (MPNST) in NF1 is ∼10%. These tumors have a poor survival rate and their molecular basis remains unclear. We report the first comprehensive investigation of DNA copy number across multitude of genes in NF1 tumors using high-resolution array comparative genomic hybridization (CGH), with the aim to identify molecular signatures that delineate malignant from benign NF1 tumors.Experimental Design: We constructed an exon-level resolution microarray encompassing 57 selected genes and profiled DNA from 35 MPNSTs, 16 plexiform, and 8 dermal neurofibromas. Bioinformatic analysis was done on array CGH data to identify concurrent aberrations in malignant tumors.Results: The array CGH profiles of MPNSTs and neurofibromas were markedly different. A number of MPNST-specific alterations were identified, including amplifications of ITGB4, PDGFRA, MET, TP73, and HGF plus deletions in NF1, HMMR/RHAMM, MMP13, L1CAM2, p16INK4A/CDKN2A, and TP53. Copy number changes of HMMR/RHAMM, MMP13, p16INK4A/CDKN2A, and ITGB4 were observed in 46%, 43%, 39%, and 32%, respectively of the malignant tumors, implicating these genes in MPNST pathogenesis. Concomitant amplifications of HGF, MET, and PDGFRA genes were also revealed in MPNSTs, suggesting the putative role of p70S6K pathway in NF1 tumor progression.Conclusions: This study highlights the potential of array CGH in identifying novel diagnostic markers for MPNSTs.

Supplementary Figure 1 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1−/− glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3′-end of CRTAC1 and a deletion of the 5′-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1. [Cancer Res 2009;69(18):7393–401]

Supplementary Figure Legend from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Supplementary Table 1 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Supplementary Tables S1 & S2 from Large-Scale Molecular Comparison of Human Schwann Cells to Malignant Peripheral Nerve Sheath Tumor Cell Lines and Tissues

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)

Supplementary Table 2 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Plexiform Neurofibromas (PN) are a common manifestation of the genetic disorder neurofibromatosis type 1 (NF1). These benign nerve sheath tumors often cause significant morbidity, with treatment options limited historically to surgery. There have been tremendous advances over the past two decades in our understanding of PN, and the recent regulatory approvals of the MEK inhibitor selumetinib are reshaping the landscape for PN management. At present, there is no agreed upon PN definition, diagnostic evaluation, surveillance strategy, or clear indications for when to initiate treatment and selection of treatment modality. In this review, we address these questions via consensus recommendations from a panel of multidisciplinary NF1 experts.

Contains fulltext : 248862.pdf (Publisher’s version ) (Closed access) Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis.

PURPOSE: By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS). METHODS: We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended. CONCLUSION: The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS. ispartof: GENETICS IN MEDICINE vol:23 issue:8 pages:1506-1513 ispartof: location:United States status: published

Individuals with the three base pair deletion NM_000267.3(NF1):c.2970_2972del p.(Met992del) have been recognised to present with a milder neurofibromatosis type 1 (NF1) phenotype characterised by café-au-lait macules (CALs) and intertriginous freckling, as well as a lack of cutaneous, subcutaneous and plexiform neurofibromas and other NF1-associated complications. Examining large cohorts of patients over time with this specific genotype is important to confirm the presentation and associated risks of this variant across the lifespan. Forty-one individuals with the in-frame NF1 deletion p.Met992del were identified from 31 families. Clinicians completed a standardised clinical questionnaire for each patient and the resulting data were collated and compared to published cohorts. Thirteen patients have been previously reported, and updated clinical information has been obtained for these individuals. Both CALs and intertriginous freckling were present in the majority of individuals (26/41, 63%) and the only confirmed features in 11 (27%). 34/41 (83%) of the cohort met NIH diagnostic criteria. There was a notable absence of all NF1-associated tumour types (neurofibroma and glioma). Neurofibroma were observed in only one individual—a subcutaneous lesion (confirmed histologically). Nineteen individuals were described as having a learning disability (46%). This study confirms that individuals with p.Met992del display a mild tumoural phenotype compared to those with ‘classical’, clinically diagnosed NF1, and this appears to be the case longitudinally through time as well as at presentation. Learning difficulties, however, appear to affect a significant proportion of NF1 subjects with this phenotype. Knowledge of this genotype–phenotype association is fundamental to accurate prognostication for families and caregivers.

Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disorder. The disease locus for this condition was mapped some 12 years ago and the mutations for the disease are known, but the exact identity of FSHD gene remains elusive. Keywords: facioscapulohumeral muscular dystrophy; mutations; gene; position effect; somatic mosaicism; subtelomeric repeat exchange

ObjectiveTo present the current terminology and natural history of neurofibromatosis 1 (NF1) cutaneous neurofibromas (cNF).MethodsNF1 experts from various research and clinical backgrounds reviewed the terms currently in use for cNF as well as the clinical, histologic, and radiographic features of these tumors using published and unpublished data.ResultsNeurofibromas develop within nerves, soft tissue, and skin. The primary distinction between cNF and other neurofibromas is that cNF are limited to the skin whereas other neurofibromas may involve the skin, but are not limited to the skin. There are important cellular, molecular, histologic, and clinical features of cNF. Each of these factors is discussed in consideration of a clinicopathologic framework for cNF.ConclusionThe development of effective therapies for cNF requires formulation of diagnostic criteria that encompass the clinical and histologic features of these tumors. However, there are several areas of overlap between cNF and other neurofibromas that make distinctions between cutaneous and other neurofibromas more difficult, requiring careful deliberation with input across the multiple disciplines that encounter these tumors and ultimately, prospective validation. The ultimate goal of this work is to facilitate accurate diagnosis and meaningful therapeutics for cNF.

The neurofibromatoses, which include neurofibromatosis type I (NF1), neurofibromatosis type II (NF2), and schwannomatosis, are a group of syndromes characterized by tumor growth in the nervous system. The RASopathies are a group of syndromes caused by germline mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. The RASopathies include NF1, Noonan syndrome, Noonan syndrome with multiple lentigines, Costello syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, capillary malformation arterio-venous malformation syndrome, and SYNGAP1 autism. Due to their common underlying pathogenetic etiology, all these syndromes have significant phenotypic overlap of which one common feature include a predisposition to tumors, which may be benign or malignant. Together as a group, they represent one of the most common multiple congenital anomaly syndromes estimating to affect approximately one in 1000 individuals worldwide. The subcontinent of India represents one of the largest populations in the world, yet remains underserved from an aspect of clinical genetics services. In an effort to bridge this gap, the First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and Advances of New Therapeutics was held in Kochi, Kerala, India. These proceedings chronicle this timely and topical international symposium directed at discussing the best practices and therapies for individuals with neurofibromatoses and RASopathies. Children's Tumor Foundation; Fonds De La Recherche Scientifique ‐ FNRS, Grant/Award Numbers: T002614, T024719F; Manchester NIHR Biomedical Research Centre, Grant/Award Number: IS‐BRC‐1215‐20007; NIH/NIAMS, Grant/Award Number: R01AR062165; Axis Health Biomedicals and the Doctor's Academy; Schiller India; MedGenome; Wales Gene Park; University of Wales Trinity Saint David; Cardiff University; AstraZeneca

BackgroundNeurofibromatosis type 1 (NF1) predisposes to breast cancer (BC), but no genotype-phenotype correlations have been described.MethodsConstitutional NF1 mutations in 78 patients with NF1 with BC (NF1-BC) were compared with the NF1 Leiden Open Variation Database (n=3432).ResultsNo cases were observed with whole or partial gene deletions (HR 0.10; 95% CI 0.006 to 1.63; p=0.014, Fisher’s exact test). There were no gross relationships with mutation position. Forty-five (64.3%; HR 6.4–83) of the 70 different mutations were more frequent than expected (pBRCA1/2 testing, revealing one BRCA2 mutation.DiscussionThese data strongly support the hypothesis that certain constitutional mutation types, and indeed certain specific variants in NF1 confer different risks of BC. The lack of large deletions and excess of nonsenses and missenses is consistent with gain of function mutations conferring risk of BC, and also that neurofibromin may function as a dimer. The observation that somatic NF1 amplification can occur independently of ERBB2 amplification in sporadic BC supports this concept. A prospective clinical-molecular study of NF1-BC needs to be established to confirm and build on these findings, but regardless of NF1 mutation status patients with NF1-BC warrant testing of other BC-predisposing genes.

Neurofibromatosis type 1 (NF1) predisposes to breast cancer (BC), but no genotype-phenotype correlations have been described.ConstitutionalNo cases were observed with whole or partial gene deletions (HR 0.10; 95% CI 0.006 to 1.63; p=0.014, Fisher's exact test). There were no gross relationships with mutation position. Forty-five (64.3%; HR 6.4-83) of the 70 different mutations were more frequent than expected (p0.05), while 52 (74.3%; HR 5.3-83) were significant when adjusted for multiple comparisons (adjusted p≤0.125; Benjamini-Hochberg). Higher proportions of both nonsense and missense mutations were also observed (adjusted p=0.254; Benjamini-Hochberg). Ten of the 11 missense cases with known age of BC occurred at50 years (p=0.041). Eighteen cases hadThese data strongly support the hypothesis that certain constitutional mutation types, and indeed certain specific variants in

The breakpoints of type-1 NF1 deletions encompassing 1.4-Mb are located within NF1-REPa and NF1-REPc, which exhibit a complex structure comprising different segmental duplications in direct and inverted orientation. Here, we systematically assessed the proportion of type-1 NF1 deletions caused by nonallelic homologous recombination (NAHR) and those mediated by other mutational mechanisms. To this end, we analyzed 236 unselected type-1 deletions and observed that 179 of them (75.8%) had breakpoints located within the NAHR hotspot PRS2, whereas 39 deletions (16.5%) had breakpoints located within PRS1. Sixteen deletions exhibited breakpoints located outside of these NAHR hotspots but were also mediated by NAHR. Taken together, the breakpoints of 234 (99.2%) of the 236 type-1 NF1 deletions were mediated by NAHR. Thus, NF1-REPa and NF1-REPc are strongly predisposed to recurrent NAHR, the main mechanism underlying type-1 NF1 deletions. We also observed a non-random overlap between type-1 NF1-deletion breakpoints and G-quadruplex forming sequences (GQs) as well as regions flanking PRDM9A binding-sites. These findings imply that GQs and PRDM9A binding-sites contribute to the clustering of type-1 deletion breakpoints. The co-location of both types of sequence was at its highest within PRS2, indicative of their synergistic contribution to the greatly increased NAHR activity within this hotspot.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited tumor predisposition syndrome with an incidence of one in 3000–4000 individuals with no currently effective therapies. The NF1 gene on chromosome 17 encodes neurofibromin, which functions as a negative regulator of RAS. NF1 is a chronic multi-system disorder affecting many different tissues. Due to cell-specific complexities of RAS signaling, therapeutic approaches for NF1 will likely have to focus on a particular tissue and manifestation of the disease. AREAS COVERED: In this review, we discuss the multi-system nature of NF1 and the signaling pathways affected due to deficiency of neurofibromin. We explore the cell/tissue-specific molecular and cellular consequences of aberrant RAS signaling in NF1 and speculate on their potential as therapeutic targets for the disease. We discuss recent genomic, transcriptomic and proteomic studies combined with molecular, cellular and biochemical analyses which have identified several targets for specific NF1 manifestations. We also consider the possibility of patient-specific gene therapy approaches to tackle NF1. EXPERT OPINION: The emergence of NF1 genotype-phenotype correlations, characterization of cell-specific signaling pathways affected in NF1, identification of novel biomarkers, and the development of sophisticated animal models accurately reflecting human pathology will continue to provide opportunities to develop therapeutic approaches to combat this multi-system disorder.

Neurofibromatosis type 1 (NF1), one of the most common genetic disorders with an estimated prevalence of 1:3000 live births, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and an in-frame 1-amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 patients (129 unrelated probands and 33 affected relatives) carrying a constitutional missense mutation affecting one of five neighboring NF1 codons Leu844, Cys845, Ala846, Leu847 and Gly848, located in the Cysteine-Serine-Rich Domain (CSRD). These recurrent missense mutations affect ~0.8% of unrelated NF1 mutation-positive probands in the UAB cohort. A substantial fraction of these patients presented with a severe phenotype, including plexiform and/or spinal neurofibromas, symptomatic optic pathway gliomas, malignant neoplasms or osseous lesions. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent compared with classic NF1 cohorts (both p

Background Neurofibromatosis type 1 (NF1: Online Mendelian Inheritance in Man (OMIM) #162200) is an autosomal dominantly inherited tumour predisposition syndrome. Heritable constitutional mutations in the NF1 gene result in dysregulation of the RAS/MAPK pathway and are causative of NF1. The major known function of the NF1 gene product neurofibromin is to downregulate RAS. NF1 exhibits variable clinical expression and is characterized by benign cutaneous lesions including neurofibromas and café-au-lait macules, as well as a predisposition to various types of malignancy, such as breast cancer and leukaemia. However, acquired somatic mutations in NF1 are also found in a wide variety of malignant neoplasms that are not associated with NF1. Main body Capitalizing upon the availability of next-generation sequencing data from cancer genomes and exomes, we review current knowledge of somatic NF1 mutations in a wide variety of tumours occurring at a number of different sites: breast, colorectum, urothelium, lung, ovary, skin, brain and neuroendocrine tissues, as well as leukaemias, in an attempt to understand their broader role and significance, and with a view ultimately to exploiting this in a diagnostic and therapeutic context. Conclusion As neurofibromin activity is a key to regulating the RAS/MAPK pathway, NF1 mutations are important in the acquisition of drug resistance, to BRAF, EGFR inhibitors, tamoxifen and retinoic acid in melanoma, lung and breast cancers and neuroblastoma. Other curiosities are observed, such as a high rate of somatic NF1 mutation in cutaneous melanoma, lung cancer, ovarian carcinoma and glioblastoma which are not usually associated with neurofibromatosis type 1. Somatic NF1 mutations may be critical drivers in multiple cancers. The mutational landscape of somatic NF1 mutations should provide novel insights into our understanding of the pathophysiology of cancer. The identification of high frequency of somatic NF1 mutations in sporadic tumours indicates that neurofibromin is likely to play a critical role in development, far beyond that evident in the tumour predisposition syndrome NF1.

The Annual Children's Tumor Foundation International Neurofibromatosis Meeting is the premier venue for connecting discovery, translational and clinical scientists who are focused on neurofibromatosis types 1 and 2 (NF1 and NF2) and schwannomatosis (SWN). The meeting also features rare tumors such as glioma, meningioma, sarcoma, and neuroblastoma that occur both within these syndromes and spontaneously; associated with somatic mutations in NF1, NF2, and SWN. The meeting addresses both state of the field for current clinical care as well as emerging preclinical models fueling discovery of new therapeutic targets and discovery science initiatives investigating mechanisms of tumorigenesis. Importantly, this conference is a forum for presenting work in progress and bringing together all stakeholders in the scientific community. A highlight of the conference was the involvement of scientists from the pharmaceutical industry who presented growing efforts for rare disease therapeutic development in general and specifically, in pediatric patients with rare tumor syndromes. Another highlight was the focus on new investigators who presented new data about biomarker discovery, tumor pathogenesis, and diagnostic tools for NF1, NF2, and SWN. This report summarizes the themes of the meeting and a synthesis of the scientific discoveries presented at the conference in order to make the larger research community aware of progress in the neurofibromatoses.

Neurofibromatosis type 1 (NF1) was the first RASopathy and is now one of many RASopathies that are caused by germline mutations in genes that encode components of the Ras/mito- gen-activated protein kinase (MAPK) pathway. Their common underlying pathogenetic etiology causes significant overlap in phenotypic features which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, GI and ocular abnormali- ties, and a predisposition to cancer. The proceedings from the symposium “Recent Developments in Neurofibromatoses (NF) and RASopathies: Management, Diagnosis and Current and Future Therapeutic Avenues” chronicle this timely and topical clinical translational research symposium. The overarching goal was to bring together clinicians, basic scientists, physician- scientists, advocate leaders, trainees, students and individuals with Ras pathway syndromes to discuss the most state-of-the-art basic science and clinical issues in an effort to spark collabo- rations directed towards the best practices and therapies for individuals with RASopathies.

Item does not contain fulltext Facioscapulohumeral muscular dystrophy (FSHD: MIM#158900) is a common myopathy with marked but largely unexplained clinical inter- and intra-familial variability. It is caused by contractions of the D4Z4 repeat array on chromosome 4 to 1-10 units (FSHD1), or by mutations in the D4Z4-binding chromatin modifier SMCHD1 (FSHD2). Both situations lead to a partial opening of the D4Z4 chromatin structure and transcription of D4Z4-encoded polyadenylated DUX4 mRNA in muscle. We measured D4Z4 CpG methylation in control, FSHD1 and FSHD2 individuals and found a significant correlation with the D4Z4 repeat array size. After correction for repeat array size, we show that the variability in clinical severity in FSHD1 and FSHD2 individuals is dependent on individual differences in susceptibility to D4Z4 hypomethylation. In FSHD1, for individuals with D4Z4 repeat arrays of 1-6 units, the clinical severity mainly depends on the size of the D4Z4 repeat. However, in individuals with arrays of 7-10 units, the clinical severity also depends on other factors that regulate D4Z4 methylation because affected individuals, but not non-penetrant mutation carriers, have a greater reduction of D4Z4 CpG methylation than can be expected based on the size of the pathogenic D4Z4 repeat array. In FSHD2, this epigenetic susceptibility depends on the nature of the SMCHD1 mutation in combination with D4Z4 repeat array size with dominant negative mutations being more deleterious than haploinsufficiency mutations. Our study thus identifies an epigenetic basis for the striking variability in onset and disease progression that is considered a clinical hallmark of FSHD.

Facioscapulohumeral muscular dystrophy 1 (FSHD1) is caused by a contraction in the number of D4Z4 repeats on chromosome 4, resulting in relaxation of D4Z4 chromatin causing inappropriate expression of DUX4 in skeletal muscle. Clinical severity is inversely related to the number of repeats. In contrast, FSHD2 patients also have inappropriate expression of DUX4 in skeletal muscle, but due to constitutional mutations in SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1), which cause global hypomethylation and hence general relaxation of chromatin. Thirty patients originally referred for FSHD testing were screened for SMCHD1 mutations. Twenty-nine had >11 D4Z4 repeats. SMCHD1 c.1040+1G>A, a pathogenic splice-site variant, was identified in a FSHD1 family with a borderline number of D4Z4 repeats (10) and a variable phenotype (in which a LMNA1 sequence variant was previously described), and SMCHD1 c.2606 G>T, a putative missense variant (p.Gly869Val) with strong in vitro indications of pathogenicity, was identified in a family with an unusual muscular dystrophy with some FSHD-like features. The two families described here emphasise the genetic complexity of muscular dystrophies. As SMCHD1 has a wider role in global genomic methylation, the possibility exists that it could be involved in other complex undiagnosed muscle disorders. Thus far, only 15 constitutional mutations have been identified in SMCHD1, and these two sequence variants add to the molecular and phenotypic spectrum associated with FSHD.

Neurofibromatosis type 1 (NF1; MIM# 162200) is a familial cancer syndrome that affects 1 in 3,500 individuals worldwide and is inherited in an autosomal dominant fashion. Malignant Peripheral Nerve Sheath Tumors (MPNSTs) represent a significant cause of morbidity and mortality in NF1 and currently there is no treatment or definite prognostic biomarkers for these tumors. Telomere shortening has been documented in numerous tumor types. Short dysfunctional telomeres are capable of fusion and it is considered that the ensuing genomic instability may facilitate clonal evolution and the progression to malignancy. To evaluate the potential role of telomere dysfunction in NF1-associated tumors, we undertook a comparative analysis of telomere length in samples derived from 10 cutaneous and 10 diffused plexiform neurofibromas, and 19 MPNSTs. Telomere length was determined using high-resolution Single Telomere Length Analysis (STELA). The mean Xp/Yp telomere length detected in MPNSTs, at 3.282 kb, was significantly shorter than that observed in both plexiform neurofibromas (5.793 kb; [p = 0.0006]) and cutaneous neurofibromas (6.141 kb; [p = 0.0007]). The telomere length distributions of MPNSTs were within the length-ranges in which telomere fusion is detected and that confer a poor prognosis in other tumor types. These data indicate that telomere length may play a role in driving genomic instability and clonal progression in NF1-associated MPNSTs.

The commonest tumors associated with neurofibromatosis type 1 (NF1) are benign peripheral nerve sheath tumors, called neurofibromas. Malignant transformation of neurofibromas into aggressive MPNSTs may occur with a poor patient prognosis. A cooperative role of SUZ12 or EED inactivation, along with NF1, TP53, and CDKN2A loss-of-function, has been proposed to drive progression to MPNSTs. An exome sequencing analysis of eight MPNSTs, one plexiform neurofibroma, and seven cutaneous neurofibromas was undertaken. Biallelic inactivation of the NF1 gene was observed in the plexiform neurofibroma and the MPNSTs, underlining that somatic biallelic NF1 inactivation is likely to be the initiating event for plexiform neurofibroma genesis, although it is unlikely to be sufficient for the subsequent MPNST development. The majority (5/8) of MPNSTs in our analyses demonstrated homozygous or heterozygous deletions of CDKN2A, which may represent an early event following NF1 LOH in the malignant transformation of Schwann cells from plexiform neurofibroma to MPNST. Biallelic somatic alterations of SUZ12 was also found in 4/8 MPNSTs. EED biallelic alterations were detected in 2 of the other four MPNSTs, with one tumor having a homozygous EED deletion. A missense mutation in the chromatin regulator KDM2B was also identified in one MPNST. No TP53 point mutations were found in this study, confirming previous data that TP53 mutations may be relatively rare in NF1-associated MPNSTs. Our study confirms the frequent biallelic inactivation of PRC2 subunits SUZ12 and EED in MPNSTs, and suggests the implication of KDM2B.

Objective Neurofibromatosis type 1 (NF1) patients have a 13% risk of developing a malignant peripheral nerve sheath tumor (MPNST). Many MPNSTs are histopathologically complex, with regions exhibiting features of the original benign plexiform neurofibroma (PNF), of an atypical PNF, or of MPNST showing varying degrees of de-differentiation. This study analyzed the genetic alterations associated with this pathological heterogeneity in order to identify the genetic processes involved in transformation from a benign to an aggressive malignant tumor. Methods A histological and molecular analysis of a single MPNST tumor that was subdivided into three histopathologically distinct regions, a benign PNF (region 1), an atypical PNF (region 2), and a high-grade MPNST (region 3), was carried out. Tumor DNA from each region was analyzed in conjunction with the patient’s lymphocyte DNA. Somatic mutation analyses included loss-of heterozygosity (LOH), MLPA analysis, NF1 gene sequencing, and a microarray comparative genomic hybridisation (array CGH) analysis. Results The patient had a germline NF1 splice-site mutation. The NF1-associated LOH analysis found that LOH increased in the three tumor areas, with 9, 42, and 97% LOH evident in regions 1, 2, and 3, respectively. Additional genetic changes, including losses of TP53, RB1, CDKN2A, and of several oncogenes and cell-cycle genes, were found only in the malignant MPNST (region 3). Array CGH also identified genomic gains and losses in DNA from region 3. Discussion This is the first study that correlates the histological and molecular changes associated with MPNST development, confirming the significant cellular and genetic heterogeneity that poses both diagnostic and therapeutic challenges.

Neurofibromatosis type 1 (NF1) and its related disorders (NF1-Noonan syndrome (NFNS) and Watson syndrome (WS)) are caused by heterozygous mutations in the NF1 gene. Pulmonary stenosis (PS) occurs more commonly in NF1 and its related disorders than in the general population. This study investigated whether PS is associated with specific types of NF1 gene mutations in NF1, NFNS and WS. The frequency of different NF1 mutation types in a cohort of published and unpublished cases with NF1/NFNS/WS and PS was examined. Compared with NF1 in general, NFNS patients had higher rates of PS (9/35=26% vs 25/2322=1.1%, P value

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties.

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P

The 2011 annual meeting of the Children’s Tumor Foundation, the annual gathering of the neurofibromatosis (NF) research and clinical communities, was attended by 330 participants who discussed integration of new signaling pathways into NF research, the appreciation for NF mutations in sporadic cancers, and an expanding pre-clinical and clinical agenda. NF1, NF2, and schwannomatosis collectively affect approximately 100,000 persons in US, and result from mutations in different genes. Benign tumors of NF1 (neurofibroma and optic pathway glioma) and NF2 (schwannoma, ependymoma, and meningioma) and schwannomatosis (schwannoma) can cause significant morbidity, and there are no proven drug treatments for any form of NF. Each disorder is associated with additional manifestations causing morbidity. The research presentations described in this review covered basic science, preclinical testing, and results from clinical trials, and demonstrate the remarkable strides being taken toward understanding of and progress toward treatments for these disorders based on the close interaction among scientists and clinicians.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy after the dystrophinopathies and myotonic dystrophy and is associated with a typical pattern of muscle weakness. Most patients with FSHD carry a large deletion in the polymorphic D4Z4 macrosatellite repeat array at 4q35 and present with 1-10 repeats whereas non-affected individuals possess 11-150 repeats. An almost identical repeat array is present at 10q26 and the high sequence identity between these two arrays can cause difficulties in molecular diagnosis. Each 3.3-kb D4Z4 unit contains a DUX4 (double homeobox 4) gene that, among others, is activated upon contraction of the 4q35 repeat array due to the induction of chromatin remodelling of the 4qter region. A number of 4q subtelomeric sequence variants are now recognised, although FSHD only occurs in association with three 'permissive' haplotypes, each of which is associated with a polyadenylation signal located immediately distal of the last D4Z4 unit. The resulting poly-A tail appears to stabilise DUX4 mRNAs transcribed from this most distal D4Z4 unit in FSHD muscle cells. Synthesis of both the DUX4 transcripts and protein in FSHD muscle cells induces significant cell toxicity. DUX4 is a transcription factor that may target several genes which results in a deregulation cascade which inhibits myogenesis, sensitises cells to oxidative stress and induces muscle atrophy, thus recapitulating many of the key molecular features of FSHD.

The neurofibromatoses (NF) encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect 100,000 Americans; over 2 million persons worldwide; and are caused by mutation of tumor suppressor genes. Individuals with NF1 in particular may develop tumors anywhere in the nervous system; additional manifestations can include learning disabilities, bone dysplasia, cardiovascular defects, unmanageable pain, and physical disfigurement. Ultimately, the NFs can cause blindness, deafness, severe morbidity, and increased mortality and NF1 includes a risk of malignant cancer. Today there is no treatment for the NFs (other than symptomatic); however, research efforts to understand these genetic conditions have made tremendous strides in the past few years. Progress is being made on all fronts, from discovery studies-understanding the molecular signaling deficits that cause the manifestations of NF-to the growth of preclinical drug screening initiatives and the emergence of a number of clinical trials. An important element in fuelling this progress is the sharing of knowledge, and to this end, for over 20 years the Children's Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share ideas and build collaborations. The 2010 NF Conference held in Baltimore, MD June 5-8, 2010 hosted over 300 NF researchers and clinicians. This paper provides a synthesis of the highlights presented at the Conference and as such, is a "state-of-the-field" for NF research in 2010.

Neurofibromatosis type 1, (NF1) is a complex, autosomal dominant disorder characterized by benign and malignant tumors which result from NF1 gene mutations. The molecular mechanisms that underlie NF1 tumorigenesis are still poorly understood although inactivation of other modifying loci in conjunction with NF1 mutations is postulated to be involved. These modifying loci may include deficiencies in mismatch repair genes and elements involved in cell cycle regulation (TP53, RB1, and CDKN2A). We have analyzed the somatic mutations in 89 cutaneous neurofibromas derived from three unrelated NF1 patients with high tumor burden, by loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, and CDKN2A genes, by assessing microsatellite instability (MSI), by direct sequencing of the NF1, TP53, and several mismatch repair (MMR) genes and by multiplex ligation-dependent probe amplification of the NF1 and TP53 genes. The aim was both to assess the possible clonality of these tumors and also to assess the involvement of other potential genetic loci in the development of these neurofibromas. Somatic NF1 mutations were identified in 57 (64%) of neurofibroma samples. Each mutation was distinct demonstrating the independent origin of each tumor. While somatic LOH of the TP53 gene was identified in four tumors, no specific deletions or sequence variations were identified. LOH of markers flanking the RB1 gene was also found in one tumor but no CDKN2A mutations were detected. Although evidence of MSI was seen in 21 tumors, no MMR gene alterations were identified. The identification of LOH involving TP53 and RB1 loci is a novel finding in benign cutaneous neurofibromas possibly demonstrating an alternative underlying molecular mechanism associated with the development of these benign tumors from this cohort of patients.

Neurofibromatosis Type I (NF1) is an autosomal dominant disorder characterized by the development of both benign and malignant tumors. The lifetime risk for developing a malignant peripheral nerve sheath tumor (MPNST) in NF1 patients is approximately 10% with poor survival rates. To date, the molecular basis of MPNST development remains unclear. Here, we report the first genome-wide and high-resolution analysis of DNA copy number alterations in MPNST using the 32K bacterial artificial chromosome microarray on a series of 24 MPNSTs and three neurofibroma samples. In the benign neurofibromas, apart from loss of one copy of the NF1 gene and copy number polymorphisms, no other changes were found. The profiles of malignant samples, however, revealed specific loss of chromosomal regions including 1p35-33, 1p21, 9p21.3, 10q25, 11q22-23, 17q11, and 20p12.2 as well as gain of 1q25, 3p26, 3q13, 5p12, 5q11.2-q14, 5q21-23, 5q31-33, 6p23-p21, 6p12, 6q15, 6q23-q24, 7p22, 7p14-p13, 7q21, 7q36, 8q22-q24, 14q22, and 17q21-q25. Copy number gains were more frequent than deletions in the MPNST samples (62% vs. 38%). The genes resident within common regions of gain were NEDL1 (7p14), AP3B1 (5q14.1), and CUL1 (7q36.1) and these were identified in >63% MPNSTs. The most frequently deleted locus encompassed CDKN2A, CDKN2B, and MTAP genes on 9p21.3 (33% cases). These genes have previously been implicated in other cancer conditions and therefore, should be considered for their therapeutic, prognostic, and diagnostic relevance in NF1 tumorigenesis.

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1−/− glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3′-end of CRTAC1 and a deletion of the 5′-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1. [Cancer Res 2009;69(18):7393–401]

Many disease-causing splicing mutations described in the literature produce changes in splice sites (SS) or in exon-regulatory sequences. The delineation of these splice aberrations can provide important insights into novel regulation mechanisms. In this study, we evaluated the effect of patient variations in neurofibromatosis type 1 (NF1) exon 29 and its 5'SS surrounding area on its splicing process. Only two of all nonsense, missense, synonymous and intronic variations analyzed in this study clearly altered exon 29 inclusion/exclusion levels. In particular, the intronic mutation +5g>a had the strongest effect, resulting in total exon exclusion. This finding prompted us to evaluate the exon 29 5'SS in relation to its ability to bind U1 snRNP. This was performed by direct analysis of the ability of U1 to bind to wild-type and mutant donor sites, by engineering an in vitro splicing system to directly evaluate the functional importance of U1 snRNA base pairing with the exon 29 donor site, and by coexpression of mutant U1 snRNP molecules to try to rescue exon 29 inclusion in vivo. The results revealed a low dependency on the presence of U1 snRNP, and suggest that exon 29 donor site definition may depend on alternative mechanisms of 5'SS recognition.

DNA from 164 unrelated Duchenne muscular dystrophy patients was screened with cDNA probes from the dystrophin gene. Molecular deletions were demonstrated in 82 (50%) subjects. Sixty-two deletions (76%) were detected using cDNA probes Cf56a (cDNA 8) and Cf56b (cDNA 6-7) which map to the centre of the gene, while 22 deletions (27%) mapped to the 5' end of the gene. In three subjects, the deletion extended from the 5' end to the centre of the gene. One deletion was identified by probe 47-4 (cDNA 5b-7) alone. In six of the deletions, junction fragments of altered size were observed. Using the three cDNA probes, RW2kb, Cf56a (cDNA 8) and Cf56b (cDNA 6-7), 99% of the deletions were detected. This will have implications for prenatal diagnosis in deletion families. Unlike Becker muscular dystrophy, where the deletions are more homogeneous, the deletions in the present study were heterogeneous both in size and position. No correlation between intelligence and either site or extent of deletion was found.

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.