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

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

Recommendations for the collection and annotation of biosamples for analysis of biomarkers in neurofibromatosis and schwannomatosis clinical trials.

INTRODUCTION: Neurofibromatosis 1 and schwannomatosis are characterized by potential lifelong morbidity and life-threatening complications. To date, however, diagnostic and predictive biomarkers are an unmet need in this patient population. The inclusion of biomarker discovery correlatives in neurofibromatosis 1/schwannomatosis clinical trials enables study of low-incidence disease. The implementation of a common data model would further enhance biomarker discovery by enabling effective concatenation of data from multiple studies. METHODS: The Response Evaluation in Neurofibromatosis and Schwannomatosis biomarker working group reviewed published data on emerging trends in neurofibromatosis 1 and schwannomatosis biomarker research and developed recommendations in a series of consensus meetings. RESULTS: Liquid biopsy has emerged as a promising assay for neurofibromatosis 1/schwannomatosis biomarker discovery and validation. In addition, we review recommendations for a range of biomarkers in clinical trials, neurofibromatosis 1/schwannomatosis-specific data annotations, and common data models for data integration. CONCLUSION: These Response Evaluation in Neurofibromatosis and Schwannomatosis consensus guidelines are intended to provide best practices for the inclusion of biomarker studies in neurofibromatosis 1/schwannomatosis clinical trials, data, and sample annotation and to lay a framework for data harmonization and concatenation between trials.

Management of neurofibromatosis type 1-associated plexiform neurofibromas.

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.

Natural history of NF1 c.2970_2972del p.(Met992del): confirmation of a low risk of complications in a longitudinal study.

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.

High-resolution breakpoint junction mapping of proximally extended D4Z4 deletions in FSHD1 reveals evidence for a founder effect.

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.

First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and advances of new therapeutics.

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.

Breast cancer risk in neurofibromatosis type 1 is a function of the type of NF1 gene mutation: a new genotype-phenotype correlation.

BACKGROUND: Neurofibromatosis type 1 (NF1) predisposes to breast cancer (BC), but no genotype-phenotype correlations have been described. METHODS: Constitutional NF1 mutations in 78 patients with NF1 with BC (NF1-BC) were compared with the NF1 Leiden Open Variation Database (n=3432). RESULTS: No 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 (p<0.05), 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 at <50 years (p=0.041). Eighteen cases had BRCA1/2 testing, revealing one BRCA2 mutation. DISCUSSION: These 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.

Cutaneous neurofibromas: Current clinical and pathologic issues.

OBJECTIVE: To present the current terminology and natural history of neurofibromatosis 1 (NF1) cutaneous neurofibromas (cNF). METHODS: NF1 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. RESULTS: Neurofibromas 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. CONCLUSION: The 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.

Extreme clustering of type-1 NF1 deletion breakpoints co-locating with G-quadruplex forming sequences.

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.

Emerging therapeutic targets for neurofibromatosis type 1.

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 encodes neurofibromin, which functions as a negative regulator of RAS. NF1 is a chronic multisystem 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: We discuss the multisystem nature of NF1 and the signaling pathways affected due to neurofibromin deficiency. 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 for 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 multisystem disorder.

The NF1 somatic mutational landscape in sporadic human cancers.

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 path forward: 2015 International Children's Tumor Foundation conference on neurofibromatosis type 1, type 2, and schwannomatosis.

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.

Telomere erosion in NF1 tumorigenesis.

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.

Confirmation of mutation landscape of NF1-associated malignant peripheral nerve sheath tumors.

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.

Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation. In this study, we postulated that mutations responsible for exerting a modifier effect on FSHD might reside within remotely acting regulatory elements that have the potential to interact at a distance with their cognate gene promoter via chromatin looping. To explore this postulate, genome-wide Hi-C data were used to identify genomic fragments displaying the strongest interaction with the SMCHD1 gene. These fragments were then narrowed down to shorter regions using ENCODE and FANTOM data on transcription factor binding sites and epigenetic marks characteristic of promoters, enhancers and silencers. RESULTS: We identified two regions, located respectively ~14 and ~85 kb upstream of the SMCHD1 gene, which were then sequenced in 229 FSHD/FSHD-like patients (200 with D4Z4 repeat units <11). Three heterozygous sequence variants were found ~14 kb upstream of the SMCHD1 gene. One of these variants was found to be of potential functional significance based on DNA methylation analysis. Further functional ascertainment will be required in order to establish the clinical/functional significance of the variants found. CONCLUSIONS: In this study, we propose an improved approach to predict the possible locations of remotely acting regulatory elements that might influence the transcriptional regulation of their associated gene(s). It represents a new way to screen for disease-relevant mutations beyond the immediate vicinity of the specific disease gene. It promises to be useful for investigating disorders in which mutations could occur in remotely acting regulatory elements.

Evaluation of copy number variation and gene expression in neurofibromatosis type-1-associated malignant peripheral nerve sheath tumours.

BACKGROUND: Neurofibromatosis type-1 (NF1) is a complex neurogenetic disorder characterised by the development of benign and malignant tumours of the peripheral nerve sheath (MPNSTs). Whilst biallelic NF1 gene inactivation contributes to benign tumour formation, additional cellular changes in gene structure and/or expression are required to induce malignant transformation. Although few molecular profiling studies have been performed on the process of progression of pre-existing plexiform neurofibromas to MPNSTs, the integrated analysis of copy number alterations (CNAs) and gene expression is likely to be key to understanding the molecular mechanisms underlying NF1-MPNST tumorigenesis. In a pilot study, we employed this approach to identify genes differentially expressed between benign and malignant NF1 tumours. RESULTS: SPP1 (osteopontin) was the most differentially expressed gene (85-fold increase in expression), compared to benign plexiform neurofibromas. Short hairpin RNA (shRNA) knockdown of SPP1 in NF1-MPNST cells reduced tumour spheroid size, wound healing and invasion in four different MPNST cell lines. Seventy-six genes were found to exhibit concordance between CNA and gene expression level. CONCLUSIONS: Pathway analysis of these genes suggested that glutathione metabolism and Wnt signalling may be specifically involved in NF1-MPNST development. SPP1 is associated with malignant transformation in NF1-associated MPNSTs and could prove to be an important target for therapeutic intervention.

STAT3 and HIF1α Signaling Drives Oncogenic Cellular Phenotypes in Malignant Peripheral Nerve Sheath Tumors.

UNLABELLED: 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.

Recent developments in neurofibromatoses and RASopathies: management, diagnosis and current and future therapeutic avenues.

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/mitogen-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 abnormalities, 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 collaborations directed towards the best practices and therapies for individuals with RASopathies.

Inter-individual differences in CpG methylation at D4Z4 correlate with clinical variability in FSHD1 and FSHD2.

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.

Identification of two novel SMCHD1 sequence variants in families with FSHD-like muscular dystrophy.

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.