Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine

We have created a panel of 29 NF1 variant complementary DNAs (cDNAs) representing missense variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (?/?) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to neurofibromatosis type 1 (NF1) gene or protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to low protein abundance. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTPase activity. We observed that effects on protein abundance and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.     

Construction of cloning‐friendly minigenes for mammalian expression of full‐length human NF1 isoforms

The neurofibromatosis type 1 (NF1) tumor suppressor gene is one of the most frequently mutated genes in human tumors. Research on the NF1 proteins has been partially hindered by the difficulties in cloning and propagating the full‐length coding cDNAs. We have now established a condition for propagating the natural open reading frames (ORFs) and have assembled the ORFs for human NF1 type 1 and 2 isoforms. Furthermore, we were able to eliminate the cDNA cloning toxicity by introducing a mini‐intron. These NF1 minigenes were expressed similarly to the intronless version and could be used to purify full‐length NF1 proteins. The NF1 isoforms expressed from the minigenes showed Ras‐GAP activity in vivo and in vitro, while the type 1 was more potent. Our constructs expand currently available full‐length NF1 constructs and should be valuable tools in expediting the understanding of NF1, particularly the isoform‐specific functions and regulation.     

Neurofibromatosis type 1 (Nf1)‐mutant mice exhibit increased sleep fragmentation

Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder in which affected children and adults are at a higher risk of sleep disorders. In an effort to identify potential sleep disturbances in a small animal model, we used a previously reported Nf1 conditional knockout (Nf1^CKO) mouse strain. In contrast to Nf1 mutant flies, the distribution of vigilance states was intact in Nf1^CKO mice. However, Nf1^CKO mice exhibited increased non‐REM sleep (NREM)‐to‐wake and wake‐to‐NREM transitions. This sleep disruption was accompanied by decreased bout durations during awake and NREM sleep states under both light and dark conditions. Moreover, Nf1^CKO mice have higher percentage delta power during awake and NREM sleep states under all light conditions. Taken together, Nf1^CKO mice phenocopy some of the sleep disturbances observed in NF1 patients and provide a tractable platform to explore the molecular mechanisms governing sleep abnormalities in NF1.     

Neurofibromin regulates G protein-stimulated adenylyl cyclase activity.

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder characterized by multiple benign and malignant nervous system tumors, and by learning defects in 45% of children with NF1 mutations. Studies of neurofibromin, the protein encoded by NF1, have focused on its functions in tumorigenesis and regulation of Ras activity; however, Drosophila NF1 regulates both Ras and cyclic AMP (cAMP) pathways. Expression of a human NF1 transgene rescued cAMP-related phenotypes in NF1 mutant flies (small body size and G protein-stimulated adenylyl cyclase (AC) activity defects), and neuropeptide- and G protein-stimulated AC activity were lower in Nf1-/- as compared to Nf1+/- mouse brains, demonstrating that neurofibromin regulates AC activity in both mammals and flies.     

Assessment of the potential pathogenicity of missense mutations identified in the GTPase‐activating protein (GAP)‐related domain of the neurofibromatosis type‐1 (NF1) gene

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

Neurofibromin regulates somatic growth through the hypothalamic-pituitary axis

To study the role of the neurofibromatosis-1 (NF1) gene in mammalian brain development, we recently generated mice in which Nf1 gene inactivation occurs in neuroglial progenitor cells using the brain lipid binding protein (BLBP) promoter. We found that Nf1(BLBP)CKO mice exhibit significantly reduced body weights and anterior pituitary gland sizes. We further demonstrate that the small anterior pituitary size reflects loss of neurofibromin expression in the hypothalamus, leading to reduced growth hormone releasing hormone, pituitary growth hormone (GH) and liver insulin-like growth factor-1 (IGF1) production. Since neurofibromin both negatively regulates Ras activity and positively modulates cAMP levels, we examined the signaling pathway responsible for these abnormalities. While BLBP-mediated expression of an activated Ras molecule did not recapitulate the body weight and hypothalamic/pituitary defects, treatment of Nf1(BLBP)CKO mice with rolipram to increase cAMP levels resulted in a partial restoration of the body weight phenotype. Furthermore, conditional expression of the Ras regulatory GAP domain of neurofibromin also did not rescue the body weight or Igf1 mRNA defects in Nf1(BLBP)CKO mice. Collectively, these data demonstrate a critical role for neurofibromin in hypothalamic-pituitary axis function and provide further insights into the short stature and GH deficits seen in children with NF1.     

NF‐κB2/p100 deficiency impairs immune responses to T‐cell‐independent type 2 antigens

Formation of the splenic marginal zone (MZ) depends on the alternative NF‐κB signaling pathway. Recently, we reported that unrestricted activation of this pathway in NF‐κB2/p100‐deficient (p100^?/?) knock‐in mice alters the phenotype of MZ stroma and B cells. Here, we show that lack of the p100 inhibitor resulted in an expansion of both MZ B and peritoneal B‐1 cells. However, these cells failed to generate proliferating blasts in response to T‐cell‐independent type 2 (TI‐2) Ags, correlating with dampened IgM and absent IgG3 responses. This phenotype was in part due to increased activity of the NF‐κB subunit RelB. Moreover, p100^?/?→B6 BM chimeras were more susceptible to infection by encapsulated Streptococcus pneumoniae bacteria, pathogens that induce TI‐2 responses. In contrast to the TI‐2 defect, p100 deficiency did not impair immune responses to the TI‐1 Ag LPS and p100^?/? MZ B cells showed normal Ag transportation into B‐cell follicles. Furthermore, p100^?/? MZ B and B‐1 cells failed to respond to TI‐2 Ags in the presence of WT accessory cells. Thus, NF‐κB2/p100 deficiency caused a predominant B‐cell‐intrinsic TI‐2 defect that could largely be attributed to impaired proliferation of plasmablasts. Importantly, p100 was also necessary for efficient defense against clinically relevant TI‐2 pathogens.     

Enzymatic characterization of novel arylsulfatase A variants using human arylsulfatase A‐deficient immortalized mesenchymal stromal cells

Metachromatic leukodystrophy (MLD) is an autosomal‐recessive lysosomal storage disease caused by mutations in the ARSA gene leading to arylsulfatase A (ARSA) deficiency and causing sulfatide accumulation. Main symptoms of the disease are progressive demyelination, neurological dysfunction, and reduced life expectancy. To date, more than 200 different ARSA variants have been reported in MLD patients. Here, we report the biochemical characterization of seven novel pathogenic variants (c.98T > C, c.195delC, c.229G > C, c.545C > G, c.674A > G, c.852T > A, and c.1274A > G), which were found when sequencing a cohort of 31 German MLD families. For that purpose, the ARSA cDNAs carrying the respective mutations inserted by site‐directed mutagenesis were cloned into a MigR1 (MSCV, IRES, GFP, retrovirus‐1) vector. The constructs were overexpressed using retroviral gene transfer in immortalized, human multipotent mesenchymal stromal cells prepared from a patient deficient in ARSA activity (late infantile MLD). In this novel ARSA^?/? cell system, the seven ARSA mutants showed ARSA activity of less than 10% when compared with wild type, which is evidence for the pathogenicity of all seven variants. In conclusion, the system of ARSA^?/?‐immortalized MSC turned out to be a helpful novel tool for the biochemical characterization of ARSA variants.     

Sensory neurons from Nf1 haploinsufficient mice exhibit increased excitability

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by tumor formation. People with NF1 also can experience more intense painful responses to stimuli, such as minor trauma, than normal. NF1 results from a heterozygous mutation of the NF1 gene, leading to decreased levels of neurofibromin, the protein product of the NF1 gene. Neurofibromin is a guanosine triphosphatase activating protein (GAP) for Ras and accelerates the conversion of active Ras-GTP to inactive Ras-GDP; therefore mutation of the NF1 gene frequently results in an increase in activity of the Ras transduction cascade. Using patch-clamp electrophysiological techniques, we examined the excitability of capsaicin-sensitive sensory neurons isolated from the dorsal root ganglia of adult mice with a heterozygous mutation of the Nf1 gene (Nf1+/-), analogous to the human mutation, in comparison to wildtype sensory neurons. Sensory neurons from adult Nf1+/- mice generated a more than twofold higher number of action potentials in response to a ramp of depolarizing current as wild-type neurons. Consistent with the greater number of action potentials, Nf1+/- neurons had lower firing thresholds, lower rheobase currents, and shorter firing latencies than wild-type neurons. Interestingly, nerve growth factor augmented the excitability of wild-type neurons in a concentration-related manner but did not further alter the excitability of the Nf1+/- sensory neurons. These data clearly suggest that GAPs, such as neurofibromin, can play a key role in the excitability of nociceptive sensory neurons. This increased excitability may explain the painful conditions experienced by people with NF1.     

Ras-Mek-Erk Signaling Regulates Nf1 Heterozygous Neointima Formation

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1^+/−) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1^+/− macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1^+/− neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1^+/− mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1^+/− neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes the protein neurofibromin. Neurofibromin negatively regulates Ras activity in multiple cell types by accelerating the hydrolysis of active Ras-GTP to its inactive diphosphate conformation.¹ These loss-of-function mutations accelerate Ras signaling and sensitize vessel wall cells and circulating hematopoietic cells, particularly myeloid progenitors and their differentiated progeny, to growth factors implicated in maintaining vascular wall homeostasis and disease pathogenesis.^1–4 Some patients with NF1 are predisposed to intimal proliferation, termed neointima, leading to debilitating arterial stenosis and tissue ischemia that contribute significantly to the premature mortality observed in this population.⁵Nf1 heterozygous (Nf1^+/−) mice display increased neointima formation, characterized by proliferating vascular smooth muscle cells (VSMCs) and infiltration of bone marrow–derived macrophages after arterial ligation, which is reminiscent of patients with NF1.^5,6 Neurofibromin-deficient endothelial cells, VSMCs, and bone marrow–derived myeloid cells demonstrate preferential activation of the Ras-Erk signaling pathway, without corresponding alterations in Ras–phosphatidylinositol 3-kinase signaling, in response to multiple growth factors in vitro.^2–4,7 This is an interesting observation because lineage-restricted inactivation of a single Nf1 gene in endothelial cells and/or VSMCs does not replicate the striking neointima observed in Nf1 heterozygous mice. However, we recently demonstrated that lineage-specific inactivation of a single Nf1 gene copy in monocytes/macrophages is sufficient to reproduce the enhanced neointima formation observed in Nf1 heterozygous mice compared with wild-type (WT) mice.⁸Based on these observations, we used in vitro and in vivo systems of macrophage function to test the hypothesis that Nf1 heterozygous macrophage function and mobilization to sites of inflammation are directly controlled by Ras-Erk signaling and that use of a specific and long-acting inhibitor of Ras-Erk signaling, under evaluation in multiple phase 1 and 2 clinical trials for cancer and preclinical models of NF1 malignancy,^1,9–12 will reduce neointima formation after mechanical injury.     

Neurofibroma-associated growth factors activate a distinct signaling network to alter the function of neurofibromin-deficient endothelial cells

Genetic inactivation of tumor suppressor genes initiates human cancers. However, interaction of accessory cells with the tumor-initiating cell within the microenvironment is often required for tumor progression. This paradigm is relevant to understanding neurofibroma development in neurofibromatosis type I patients. Somatic inactivation of the Nf1 tumor suppressor gene, which encodes neurofibromin, is necessary but not sufficient to initiate neurofibroma development. In contrast, neurofibromas occur with high penetrance in mice in which Nf1 is ablated in Schwann cells in the context of a heterozygous mutant (Nf1+/-) microenvironment. Neurofibromas are highly vascularized, and recent studies suggest that Nf1+/- mice have increased angiogenesis in vivo. However, the function of neurofibromin in human endothelial cells (ECs) and the biochemical mechanism by which neurofibromin regulates neoangiogenesis are not known. Utilizing Nf1+/- mice, primary human ECs and endothelial progenitor cells harvested from NF1 patients, we identified a discrete Ras effector pathway, which alters the proliferation and migration of neurofibromin-deficient ECs in response to neurofibroma-derived growth factors both in vitro and in vivo. Thus, these studies identify a unique biochemical pathway in Nf1+/- ECs as a potential therapeutic target in the neurofibroma microenvironment.     

Characterization of Pheochromocytomas in a Mouse Strain with a Targeted Disruptive Mutation of the Neurofibromatosis Gene Nf1

Patients with neurofibromatosis type 1 (NF1) show an increased frequency of pheochromocytomas. TheNF1 gene encodes a GTPase-activating protein that controls the activity ofras proteins in intracellular signalling. A mouse strain with a knockout mutation of Nf1, the murine counterpart ofNF1, has recently been constructed. This mutation, designated Nf1ⁿ³¹, has been shown to be associated with the frequent development of pheochromocytomas in heterozygous animals. Pheochromocytomas are extremely rare in wild-type mice. We have characterized the tumors to assess their relevance as a model for human pheochromocytomas. The frequency of pheochromocytomas was determined in inbred compared to outbred mice carrying the Nf1ⁿ³¹ mutation. Paraffin sections of pheochromocytomas from seven mice were stained immunohistochemically for the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) to infer their profiles of catecholamine synthesis, and for chromogranin A (CGA) to infer their content of secretory granules. Cultured cells from a representative tumor were studied in vitro to assess proliferation and neuronal differentiation. Pheochromocytomas arose in approx 15% of Nf1ⁿ³¹ mice with a mixed genetic background, but were absent in inbred mice. Approximately one-fourth of the tumors were bilateral. The tumors exhibited variable morphology. All included cells that appeared well differentiated and resembled normal chromaffin cells in that they expressed TH, PNMT, and CGA. Focal neuronal differentiation was also observed. In cell culture, the tumor cells ceased to proliferate and the majority underwent terminal differentiation into TH-positive cells with neuronal morphology. The phenotype of pheochromocytomas in mice with the Nf1ⁿ³¹ mutation resembles that of human pheochromocytomas, particularly with respect to their ability to produce epinephrine, as inferred from positive staining for PNMT. The tumors also resemble both normal and neoplastic human adrenal medulla with respect to their extensive differentiation into neuron-like cells in vitro. This change in phenotype may be related toras activation. These neoplasms may be valuable both as models for the pathobiology of adrenal medullary neoplasia, and as a source of epinephrine-producing pheochromocytoma cells lines, for which adequate models currently do not exist.     

Motor deficits and neurofibromatosis type 1 (NF1)‐associated MRI impairments in a mouse model of NF1

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized inter alia by cognitive and motor dysfunction and appearance of high‐signal foci on T2‐weighted images in the brain. Nf1^+/? mice are useful models for studying aspects of NF1, including cognitive deficits. Here we assessed their motor performance and used quantitative transverse T2 relaxation MRI to identify structural abnormalities in their brains. Nf1^+/? mice exhibited both enhanced and reduced T2 signals in distinct brain regions compared to wild‐type mice, and their motor performance was impaired. As in NF1 patients, enhanced T2 signals in Nf1^+/? mice were observed in the thalamus and basal ganglia. Reduced T2 signals were seen in motor‐associated regions along the motor pathway, predominantly in the white matter of the cerebral peduncle and the optic tract. Correlation analysis between T2 signals and motor performance suggested that the motor deficits are associated with impairments in the cerebral peduncle and the amygdala. Copyright © 2010 John Wiley & Sons, Ltd.     

Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908‐321C>G). AMOs were designed to target the newly created 5′ splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence‐specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild‐type levels, we measured the amount of Ras‐GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO‐dependent decrease in Ras‐GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been usedsuccessfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders. Hum Mutat 30, 454–462, 2009. © 2009 Wiley‐Liss, Inc.     

Protective dendritic cell responses against listeriosis induced by the short form of the deubiquitinating enzyme CYLD are inhibited by full‐length CYLD

The deubiquitinating enzyme CYLD is an important tumor suppressor and inhibitor of immune responses. In contrast to full‐length CYLD, the immunological function of the naturally occurring short splice variant of CYLD (sCYLD) is insufficiently described. Previously, we showed that DCs, which lack full‐length CYLD but express sCYLD, exhibit augmented NF‐κB and DC activation. To explore the function of sCYLD in infection, we investigated whether DC‐specific sCYLD regulates the pathogenesis of listeriosis. Upon Listeria monocytogenes infection of CD11c‐Cre Cyld^{ex7/8 fl/fl} mice, infection of CD8α⁺ DCs, which are crucial for the establishment of listeriosis in the spleen, was not affected. However, NF‐κB activity of CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs was increased, while activation of ERK and p38 was normal. In addition, CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs produced more TNF, IL‐10, and IL‐12 upon infection, which led to enhanced stimulation of IFN‐γ‐producing NK cells. In addition CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs presented Listeria Ag more efficiently to CD8⁺ T cells resulting in a stronger pathogen‐specific CD8⁺ T‐cell proliferation and more IFN‐γ production. Collectively, the improved innate and adaptive immunity and survival during listeriosis identify the DC‐specific FL‐CYLD/sCYLD balance as a potential target to modulate NK‐cell and Ag‐specific CD8⁺ T‐cell responses.     

Association of Genes in the NF‐κB Pathway with Antibody‐Positive Primary Sjögren's Syndrome

Primary Sjögren's syndrome (SS) is a systemic autoimmune inflammatory disease characterized by focal lymphocytic infiltrates in the lachrymal and salivary glands and autoantibodies against the SSA/Ro and SSB/La antigens. Experimental studies have shown an activation of NF‐κB in primary SS. NF‐κB activation results in inflammation and autoimmunity and is regulated by inhibitory and activating proteins. Genetic studies have shown an association between multiple autoimmune diseases and TNFAIP3 (A20) and TNIP1 (ABIN1), both repressors of NF‐κB and of IKBKE (IKKε), which is an NF‐κB activator. The aim of this study was to analyse single nucleotide polymorphisms (SNPs) in the IKBKE, NFKB1, TNIP1 and TNFAIP3 genes for association with primary SS. A total of 12 SNPs were genotyped in 1105 patients from Scandinavia (Sweden and Norway, n = 684) and the UK (n = 421) and 4460 controls (Scandinavia, n = 1662, UK, n = 2798). When patients were stratified for the presence of anti‐SSA and/or anti‐SSB antibodies (n = 868), case–control meta‐analysis found an association between antibody‐positive primary SS and two SNPs in TNIP1 (P = 3.4 × 10^?5, OR = 1.33, 95%CI: 1.16–1.52 for rs3792783 and P = 1.3 × 10^?3, OR = 1.21, 95%CI: 1.08–1.36 for rs7708392). A TNIP1 risk haplotype was associated with antibody‐positive primary SS (P = 5.7 × 10^?3, OR = 1.47, 95%CI: 1.12–1.92). There were no significant associations with IKBKE, NFKB1 or TNFAIP3 in the meta‐analysis of the Scandinavian and UK cohorts. We conclude that polymorphisms in TNIP1 are associated with antibody‐positive primary SS.     

Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma

Low-grade brain tumors (pilocytic astrocytomas) arising in the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome are hypothesized to result from a combination of germline and acquired somatic NF1 tumor suppressor gene mutations. However, genetically engineered mice (GEM) in which mono-allelic germline Nf1 gene loss is coupled with bi-allelic somatic (glial progenitor cell) Nf1 gene inactivation develop brain tumors that do not fully recapitulate the neuropathological features of the human condition. These observations raise the intriguing possibility that, while loss of neurofibromin function is necessary for NF1-associated low-grade astrocytoma development, additional genetic changes may be required for full penetrance of the human brain tumor phenotype. To identify these potential cooperating genetic mutations, we performed whole-genome sequencing (WGS) analysis of three NF1-associated pilocytic astrocytoma (PA) tumors. We found that the mechanism of somatic NF1 loss was different in each tumor (frameshift mutation, loss of heterozygosity, and methylation). In addition, tumor purity analysis revealed that these tumors had a high proportion of stromal cells, such that only 50%–60% of cells in the tumor mass exhibited somatic NF1 loss. Importantly, we identified no additional recurrent pathogenic somatic mutations, supporting a model in which neuroglial progenitor cell NF1 loss is likely sufficient for PA formation in cooperation with a proper stromal environment.NF1 is one of the most common autosomal dominant tumor predisposition syndromes in which affected individuals develop brain tumors. In this regard, 15%–20% of children with NF1 develop World Health Organization (WHO) grade I pilocytic astrocytomas (PAs) (Listernick et al. 1994). These low-grade glial neoplasms typically arise in children <7 yr of age and most commonly occur in the optic pathway (optic nerves, chiasm, and tracts) or in the brainstem (Listernick et al. 1997). Similar to other tumor predisposition syndromes, children with NF1 are born with one mutated, nonfunctional copy of the NF1 gene, such that loss of the remaining allele in appropriate progenitor cells enables tumorigenesis. Consistent with this “two hit” hypothesis, previous studies of NF1-associated PA (NF1-PA) have demonstrated loss of heterozygosity at the DNA level (Kluwe et al. 2001; Gutmann et al. 2003) and loss of NF1 protein (neurofibromin) expression (Gutmann et al. 2000).While NF1 loss in neuroglial progenitors is necessary for PA formation in individuals with NF1, genetically engineered mice (GEM) with conditional loss of Nf1 gene expression in neuroglial progenitor cells fail to develop brain tumors (Bajenaru et al. 2002; Zhu et al. 2005). This failure to develop gliomas reflects the need for cooperating Nf1^+/− non-neoplastic cells in the tumor microenvironment. In this regard, similar to children with NF1 harboring one nonfunctional and one functional NF1 allele in every cell of their bodies, Nf1^+/− mice (one functional and one inactivated Nf1 allele) with absent neuroglial progenitor Nf1 expression develop optic gliomas with nearly 100% penetrance (Bajenaru et al. 2003). Similar to their human counterparts, these murine brain tumors exhibit low levels of proliferation, increased numbers of endothelial cells and microglia, and nuclear pleomorphism (Bajenaru et al. 2005; Kim et al. 2010).Interestingly, these mouse optic gliomas do not fully recapitulate the classic histopathological features of the human tumors, in that they lack Rosenthal fibers and eosinophilic granular bodies (Louis et al. 2007), raising the intriguing possibility that other genetic alterations exist in human NF1-PA. The purpose of the current study was to employ advanced whole-genome sequencing technologies to establish the genomic landscape of human NF1-PA to facilitate the development of improved approaches to the diagnosis and management of these brain tumors.