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Background Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with various skeletal abnormalities occurring as part of a complex phenotype. Tibial dysplasia, which typically presents as anterolateral bowing of the leg with subsequent fracture and nonunion (pseudarthrosis), is a serious but infrequent osseous manifestation of NF1. Over the past several years, results from clinical and experimental studies have advanced our knowledge of the role of NF1 in bone. On the basis of current knowledge, we propose a number of concepts to consider as a theoretical approach to the optimal management of tibial pseudarthrosis. Methods A literature review for both clinical treatment and preclinical models for tibial dysplasia in NF1 was performed. Concepts were discussed and developed by experts who participated in the Children's Tumor Foundation sponsored International Bone Abnormalities Consortium meeting in 2011. Results Concepts for a theoretical approach to treating tibial pseudarthrosis include: bone fixation appropriate to achieve stability in any given case; debridement of the "fibrous pseudarthrosis tissue" between the bone segments associated with the pseudarthrosis; creating a healthy vascular bed for bone repair; promoting osteogenesis; controlling overactive bone resorption (catabolism); prevention of recurrence of the "fibrous pseudarthrosis tissue"; and achievement of long-term bone health to prevent recurrence. Conclusions Clinical trials are needed to assess effectiveness of the wide variation of surgical and pharmacologic approaches currently in practice for the treatment of tibial pseudarthrosis in NF1. Level of evidence Level V, expert opinion.

Neurofibromatosis type 1 (NF1) is a multi-system disease caused by mutations in the NF1 gene encoding a Ras-GAP protein, neurofibromin, which negatively regulates Ras signaling. Besides neuroectodermal malformations and tumors, the skeletal system is often affected (e.g. scoliosis and long bone dysplasia) demonstrating the importance of neurofibromin for development and maintenance of the musculoskeletal system. Here, we focus on the role of neurofibromin in skeletal muscle development. Nf1 gene inactivation in the early limb bud mesenchyme using Prx1-cre (Nf1(Prx1)) resulted in muscle dystrophy characterized by fibrosis, reduced number of muscle fibers and reduced muscle force. This was caused by an early defect in myogenesis affecting the terminal differentiation of myoblasts between E12.5 and E14.5. In parallel, the muscle connective tissue cells exhibited increased proliferation at E14.5 and an increase in the amount of connective tissue as early as E16.5. These changes were accompanied by excessive mitogen-activated protein kinase pathway activation. Satellite cells isolated from Nf1(Prx1) mice showed normal self-renewal, but their differentiation was impaired as indicated by diminished myotube formation. Our results demonstrate a requirement of neurofibromin for muscle formation and maintenance. This previously unrecognized function of neurofibromin may contribute to the musculoskeletal problems in NF1 patients.

The skeleton is frequently affected in individuals with neurofibromatosis type 1, and some of these bone manifestations can result in significant morbidity. The natural history and pathogenesis of the skeletal abnormalities of this disorder are poorly understood and consequently therapeutic options for these manifestations are currently limited. The Children's Tumor Foundation convened an International Neurofibromatosis Type 1 Bone Abnormalities Consortium to address future directions for clinical trials in skeletal abnormalities associated with this disorder. This report reviews the clinical skeletal manifestations and available preclinical mouse models and summarizes key issues that present barriers to optimal clinical management of skeletal abnormalities in neurofibromatosis type 1. These concepts should help advance optimal clinical management of the skeletal abnormalities in this disease and address major difficulties encountered for the design of clinical trials.

The oncogene E2a-Pbx1 is formed by the t(1;19) translocation, which joins the N-terminal transactivation domain of E2a with the C-terminal homeodomain of PBX1. The goal of this work was to elucidate the mechanisms by which E2a-Pbx1 can lead to deregulated target gene expression. For reporter constructs it was shown that E2a-Pbx1 can activate transcription through homodimer elements (TGATTGAT) or through heterodimer elements with Hox proteins (e.g. TGATTAAT). We show a novel mechanism by which E2a-Pbx1 activates transcription of EF-9 using a promoter in intron 1 of the EF-9 gene, resulting in an aminoterminal truncated transcript. Our results indicate that the LDFS motif of E2a is essential for the transactivation of EF-9, but dispensable for transactivation of fibroblast growth factor 15. The E2a LDFS motif was also essential for proliferation of NIH3T3 fibroblasts but was dispensable for the E2a-Pbx1-induced differentiation arrest of myeloid progenitors.

Ritscher-Schinzel syndrome (RSS)/3C (cranio-cerebro-cardiac) syndrome (OMIM#220210) is a rare and clinically heterogeneous developmental disorder characterized by intellectual disability, cerebellar brain malformations, congenital heart defects, and craniofacial abnormalities. A recent study of a Canadian cohort identified homozygous sequence variants in the KIAA0196 gene, which encodes the WASH complex subunit strumpellin, as a cause for a form of RSS/3C syndrome. We have searched for genetic causes of a phenotype similar to RSS/3C syndrome in an Austrian family with two affected sons. To search for disease-causing variants, whole-exome sequencing (WES) was performed on samples from two affected male children and their parents. Before WES, CGH array comparative genomic hybridization was applied. Validation of WES and segregation studies was done using routine Sanger sequencing. Exome sequencing detected a missense variant (c.1670A>G; p.(Tyr557Cys)) in exon 15 of the CCDC22 gene, which maps to chromosome Xp11.23. Western blots of immortalized lymphoblastoid cell lines (LCLs) from the affected individual showed decreased expression of CCDC22 and an increased expression of WASH1 but a normal expression of strumpellin and FAM21 in the patients cells. We identified a variant in CCDC22 gene as the cause of an X-linked phenotype similar to RSS/3C syndrome in the family described here. A hypomorphic variant in CCDC22 was previously reported in association with a familial case of syndromic X-linked intellectual disability, which shows phenotypic overlap with RSS/3C syndrome. Thus, different inactivating variants affecting CCDC22 are associated with a phenotype similar to RSS/3C syndrome.European Journal of Human Genetics advance online publication, 11 June 2014; doi:10.1038/ejhg.2014.109.

Recently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development.

Neurofibromatosis type 1 (NF1) (MIM#162200) is a relatively frequent genetic condition that predisposes to tumor formation. The main types of tumors occurring in NF1 patients are cutaneous and subcutaneous neurofibromas, plexiform neurofibromas, optic pathway gliomas, and malignant peripheral nerve sheath tumors. To search for somatic mutations in cutaneous (dermal) neurofibromas, whole-exome sequencing (WES) was performed on seven spatially separated tumors and two reference tissues (blood and unaffected skin) from a single NF1 patient. Validation of WES findings was done using routine Sanger sequencing or Sequenom IPlex SNP genotyping. Exome sequencing confirmed the existence of a known familial splice-site mutation NM_000267.3:c.3113+1G>A in exon 23 of NF1 gene (HGMD ID CS951480) in blood, unaffected skin, and all tumor samples. In five out of seven analyzed tumors, we additionally detected second-hit mutations in the NF1 gene. Four of them were novel and one was previously observed. Each mutation was distinct, demonstrating the independent origin of each tumor. Only in two of seven tumors we detected an additional somatic mutation that was not associated with NF1. Our study demonstrated that somatic mutations of NF1 are likely the main drivers of cutaneous tumor formation. The study provides evidence for the rareness of single base pair level alterations in the exomes of benign NF1 cutaneous tumors.European Journal of Human Genetics advance online publication, 8 October 2014; doi:10.1038/ejhg.2014.210.

Background Metacarpal 4–5 fusion (MF4; MIM %309630) is a rare congenital malformation of the hand characterised by the partial or complete fusion of the fourth and fifth metacarpals. The anomaly occurs as an isolated trait or part of a genetic syndrome. Methods To search for disease-causing mutation, whole exome sequencing (WES) was performed on samples from a single trio. Before WES, molecular screening including gene sequencing and array comparative genomic hybridisation was applied. Validation of WES and segregation studies were done using routine Sanger sequencing. Results Exome sequencing detected a nonsense mutation (c.C535T; p.R179X) in exon 3 of the FGF16 gene, which maps to chromosome Xq21.1. Mutational screening of the FGF16 gene performed in an unrelated proband of different ethnicity showed another nonsense mutation in exon 3 (c.C470A; p.S157X). Conclusions This study shows that truncating mutations of FGF16 are associated with X-linked recessive metacarpal 4–5 fusion. The study provides evidence for the involvement of FGF16 in the fine tuning of the human skeleton of the hand.

Background Neurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients. Methods In situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients. Results Increased expression of Mia was found in Nf1-deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden. Conclusions MIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies.

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1-/- cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1-/- cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis. © 2011 Kolanczyk et al., published_or_final_version

The human lysozyme (LZM) gene is highly methylated in LZM-nonexpressor immature myeloid and in nonmyeloid cells and unmethylated only in LZM-expressing cells. Extended methylation analyses of the CpG-poor 5′ flanking region and of the exon 4 CpG island (both containing Alu elements) of the LZM gene were now performed. Marked demethylation was noted after treatment of AML1/ETO-positive Kasumi-1 cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-azaCdR), not associated with cellular differentiation. LZM mRNA in Kasumi-1, but not in several AML1/ETO-negative myeloid cell lines, was specifically and independently up-regulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Increased chromatin accessibility within the 5′ LZM gene was observed concomitantly with 5-azaCdR-induced demethylation. In contrast, TSA treatment had no effect on chromatin accessibility, but, as shown by chromatin immunoprecipitation, resulted in increased acetylation of histones H3 and H4. Repression of LZM transcription is mediated by conditional AML1/ETO expression in an inducible cell line model (U-937), and is reversed by siRNA “knock-down” of AML1/ETO in Kasumi-1 cells (Dunne et al., Oncogene 25: 2006). Antagonization of LZM repression following conditional expression of AML1/ETO was achieved by TSA. In conclusion, we demonstrate complex interactions between DNA methylation and histone modifications in mediating LZM repression, which implicate AML1/ETO as one component involved in local chromatin remodeling. Interestingly, inhibitors of DNMTs and HDACs independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.