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PubMed2001

Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 ( NF1) gene.

The identification of mutations in the NF1 gene causing type 1 neurofibromatosis (NF1) has presented a considerable challenge because of the large size of the gene, the lack of significant mutational clustering, the diversity of the underlying pathological lesions and the presence of NF1 pseudogenes. Denaturing high performance liquid chromatography (DHPLC), a high throughput, non-hazardous and largely automated heteroduplex-based technique, is in many ways ideally suited to mutation detection in this condition. DHPLC was therefore optimised for the rapid screening of the 60 exons and splice junctions of the NF1 gene in patients with NF1. The sensitivity of DHPLC was evaluated in a retrospective study of a cohort of 111 unrelated NF1 patients with known germline mutations; 97% of mutations were detected. In a subsequent prospective analysis of 50 unrelated NF1 patients, germline mutations were identified in 34 individuals (68%), 22 of these alterations being novel. This represents the highest rate of mutation detection so far reported for the NF1 gene with a single screening technique and genomic DNA as a target.

Base Sequence↗
PubMed1992

NF1-related locus on chromosome 15.

A neurofibromatosis type I (NF1)-related locus has been identified on chromosome 15. It contains a partial copy of the NF1 GAP-related domain, which is known to interact with the ras protooncogenes. However, the chromosome 15 sequence contains multiple deletions resulting in frameshift mutations and stop codons in several highly conserved sequence blocks. The locus on chromosome 15 therefore represents an NF1 pseudogene. This nonprocessed NF1 pseudogene may produce additional fragments in Southern blotting, pulsed-field gel, and PCR experiments with some NF1 cDNA probes or oligonucleotides. In addition, certain regions of the NF1 gene also cross-hybridize with a locus on chromosome 14. These loci must be considered in mutation analysis of patients with NF1 since aberrant findings may not always reflect changes in the NF1 gene.

Base Sequence↗
PubMed2004

Fluorescence in situ hybridization analysis of allelic losses involving the long arm of chromosome 17 in NF1-associated neurofibromas.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant condition associated with germline mutations of the NF1 gene located at chromosome band 17q11.2. Molecular analysis of a number of NF1-specific tumors has shown the inactivation of both NF1 alleles during tumorigenesis, supporting the tumor suppressor hypothesis for the NF1 gene. Using interphase dual-color fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues, we studied 11 plexiform, 4 cutaneous, and 6 subcutaneous neurofibromas. Cytogenetic analysis was conducted using two probes, one specific for the NF1 region (RP11-229K15) and one for the centromeric region of chromosome 17 as control. No large somatic deletions were found. Only in one of the plexiform neurofibromas loss of a whole chromosome 17 was observed. If we assume that dual-color FISH analysis is sensitive enough to detect the majority of large somatic deletions present, then other mutational mechanisms affecting the NF1 gene are probably involved in neurofibroma formation, and other tumor suppressor genes may play an important role in NF1 tumorigenesis.

Adolescent↗
PubMed1997

Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene.

Neurofibromatosis type 1 (NF1) is among the most common human genetic disorders, having a constellation of cutaneous and skeletal manifestations, intellectual impairment, and an increased risk for a variety of malignancies. The NF1 gene has a high spontaneous mutation rate and is also associated with a variety of sporadic cancers in the general population. While a number of laboratories are involved in a coordinated effort to identify NF1 mutations, an important gap in our knowledge is an understanding of the mechanisms responsible for NF1 mutagenesis. In this present paper we describe our analysis of the sequence environment in the NF1 gene at those sites where small deletions, insertions and nucleotide substitution mutations have been reported. Our objective was to determine whether specific nucleotide sequences commonly occur at these mutation sites within the NF1 gene. We assessed how frequently independent NF1 mutations occur at the site of short direct repeats, single nucleotide repeats (homonucleotides) and at CpG and CpNpG motifs. We have established that homonucleotide and short direct repeats are commonly involved in the majority of small deletions and insertions analysed. Substitution mutations are frequently associated with homonucleotide repeats and methylatable CpG dinucleotides and CpNpG trinucleotides. We suggest that NF1 mutations are acquired and retained by cells through an intricate balancing of repair and replication mechanisms. Such mutations may provide a proliferative advantage for that cell and its progeny.

Base Sequence↗
PubMed1995

Expression of the neurofibromatosis type 1 (NF1) gene during mouse embryonic development.

The von Recklinghausen neurofibromatosis type 1 (NF1) gene was identified by positional cloning and found to be a tumor suppressor gene expressed most abundantly in brain. One isoform of NF1 (type 2 NF1) contains an additional 21 amino acids inserted into a region of the protein involved in the regulation of p21-ras. To study the role of the NF1 gene in mammalian development, the expression of the NF1 gene and protein product, neurofibromin, during mouse embryonic development was determined. NF1 mRNA and neurofibromin expression was detectable by Northern and Western analysis, respectively, after day 10 of murine embryogenesis and remained elevated throughout development. Type 2 NF1 mRNA expression predominated before day 10, after which time, type 1 (lacking the insertion) NF1 mRNA was the predominant isoform detected. The protein expression of the type 2 isoform was similar to overall neurofibromin expression by Western blot analysis with greatest expression in adult brain. Despite a similar tissue distribution pattern, type 2 neurofibromin was not found to be associated with brain cytoplasmic microtubules in the same fashion as the uninserted type 1 isoform. Collectively, these experiments suggest that the switch from type 2 to type 1 neurofibromin isoform predominance during embryogenesis may have significant functional consequences.

Animals↗
PubMed2000

Nf1 and Gmcsf interact in myeloid leukemogenesis.

The NF1 tumor suppressor gene encodes neurofibromin, a GTPase-activating protein (GAP) for p21ras (Ras). Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML). Some heterozygous Nf1 mutant mice develop a similar myeloproliferative disorder (MPD), and adoptive transfer of Nf1-deficient fetal liver cells consistently induces this MPD. Human JMML and murine Nf1-deficient cells are hypersensitive to granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose cultures. We generated hematopoietic cells deficient in both Nf1 and Gmcsf to test whether GM-CSF is required to drive excessive proliferation of Nf1-/- cells in vivo. Here we show that GM-CSF play a central role in establishing and maintaining the MPD and that recipients engrafted with Nf1-/- Gmcsf-/- hematopoietic cells are hypersensitive to exogenous GM-CSF.

Adoptive Transfer↗
PubMed2002

Nf1 has an essential role in endothelial cells.

Neurofibromatosis type 1 (NF1) or von Recklinghausen neurofibromatosis is a genetic disorder that occurs in 1 of 4000 births and is characterized by benign and malignant tumors. Cardiovascular defects also contribute to NF1, though the pathogenesis is still unclear. Deficiency in neurofibromin (encoded by Nf1) in mice results in mid-embryonic lethality owing to cardiac abnormalities previously thought to be secondary to cardiac neural-crest defects. Using tissue-specific gene inactivation, we show that endothelial-specific inactivation of Nf1 recapitulates key aspects of the complete null phenotype, including multiple cardiovascular abnormalities involving the endocardial cushions and myocardium. This phenotype is associated with an elevated level of ras signaling in Nf1(-/-) endothelial cells and greater nuclear localization of the transcription factor Nfatc1. Inactivation of Nf1 in the neural crest does not cause cardiac defects but results in tumors of neural-crest origin resembling those seen in humans with NF1. These results establish a new and essential role for Nf1 in endothelial cells and confirm the requirement for neurofibromin in the neural crest.

Animals↗
PubMed2000

Microsatellite instability and promoter methylation as possible causes of NF1 gene inactivation in neurofibromas.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. One of the characteristic features of this disease is the development of neurofibromas. Since the NF1 gene is supposed to be a tumour suppressor gene, these neurofibromas should develop upon inactivation of both NF1 alleles. So far, mutation and deletion have been found to be involved in NF1 gene inactivation. However, these inactivating mechanisms explain the development of only a limited fraction of analysed neurofibromas. In this study, we investigated microsatellite instability (MSI) and promoter methylation as potential contributors to NF1 gene inactivation. As site-specific methylation in the NF1 promoter inhibits binding of transcription factors Sp1 and CREB, we studied the methylation status of their binding sites in particular. We analysed 20 neurofibromas and three neurofibrosarcomas, but did not find evidence for microsatellite instability or NF1 promoter methylation in any of the tumours. Thus, our data suggest that both microsatellite instability and promoter methylation are unlikely to be the major causes of NF1 gene inactivation in these tumours.

Base Sequence↗
PubMed1998

NF1 inactivation cooperates with N-ras in in vivo lymphogenesis activating Erk by a mechanism independent of its Ras-GTPase accelerating activity.

We crossed transgenic mice overexpressing the N-ras proto-oncogene (RasTg) with mice carrying one inactivated copy of the NF1 tumor suppressor gene (NF1+/-) to assess their possible cooperation in tumorigenesis. We have found a significant increase in the incidence of lymphomas in animals with both lesions (RasTg NF1+/-), as compared with animals with single lesions. The mechanism of this cooperation appears to be independent of the NF1 GTPase activating activity since the level of Ras-GTP in primary cultures of tumor tissue do not differ among animals with double and with single lesions. Nevertheless, the finding of significantly higher levels of Erk-1 and Erk-2 activation in lymphomas in the RasTg NF1+/- than in the RasTg group suggests that this cooperative effect may be in part explained by increased signaling through the Erk pathways. Consistent with a role for Erk activation in transformation is the additional observation that Erk-1 and Erk-2 activation is significantly increased in lymphomas as compared with normal spleen. This activation is likely to occur by phosphorylation of previously synthesized and inactive Erk proteins since, despite differences in activation, Erk-1 and Erk-2 expression is similar in normal and lymphoid tissue in all groups. The observed cooperation in in vivo lymphomagenesis between N-ras overexpression and NF1 inactivation emphasizes the importance of searching for additional functions for the NF1 protein and of intensifying the screening for NF1 mutations in human lymphomas.

3T3 Cells↗
PubMed1997

Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders.

BACKGROUND: The risk of malignant myeloid disorders in young children with neurofibromatosis type 1 is 200 to 500 times the normal risk. The gene for neurofibromatosis type 1 (NF1) encodes neurofibromin, a protein that negatively regulates signals transduced by Ras proteins. Genetic and biochemical data support the hypothesis that NF1 functions as a tumor-suppressor gene in immature myeloid cells, but inactivation of both NF1 alleles has not been demonstrated in leukemic cells from patients with neurofibromatosis type 1. METHODS: Using an in vitro transcription and translation system, we screened bone marrow samples from 18 children with neurofibromatosis type 1 and myeloid disorders for NF1 mutations that cause a truncated protein. Mutations were confirmed by direct sequencing of genomic DNA from the patients, and from their affected parents, in cases of familial neurofibromatosis type 1. RESULTS: Specimens from 9 of the 18 children contained abnormal peptide fragments, and truncating mutations of the NF1 gene were found in specimens from 8 of these children. The normal NF1 allele was absent in bone marrow samples from five of the eight children. We detected the same mutation in DNA from the affected parent of each child with familial neurofibromatosis type 1. CONCLUSIONS: Both alleles of the NF1 gene are inactivated in leukemic cells in some patients with neurofibromatosis type 1. NF1 appears to function as a tumor-suppressor gene in immature myeloid cells.

Bone Marrow↗
PubMed2007

PU.1, interferon regulatory factor (IRF) 2, and the interferon consensus sequence-binding protein (ICSBP/IRF8) cooperate to activate NF1 transcription in differentiating myeloid cells.

Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2.

Cell Differentiation↗
PubMedDate not supplied

No aberrant methylation of neurofibromatosis 1 gene (NF1) promoter in pilocytic astrocytoma in childhood.

Tumors of the central nervous system are the most frequent solid tumors in childhood. With 30-40% of this heterogenous group, low-grade astrocytomas represent the most common subtype. Neurofibromatosis type 1 (NF1) is strongly associated with the development of pilocytic astrocytoma (PA), frequently appearing as optic glioma. Neurofibromatosis 1 gene (NF1 ) fulfills the criteria of a tumor suppressor gene and is deleted or mutated heterozygously in patients with NF1. This suggests an involvement in the development of PA. To clarify whether silencing of NF1 by promoter methylation plays a role in PA and especially in optic glioma, the authors investigated the methylation status in 30 PA, 6 of which had optic glioma. However, no methylation was found at the NF1 promoter region in PA. To rule out that silencing of NF1 by promoter methylation is restricted to higher-grade astrocytomas, 15 pediatric WHO II degree and IV degree astrocytomas were analyzed: 12 astrocytomas II and 3 glioblastomas displayed no NF1 promoter methylation. The authors conclude that NF1 silencing by methylation plays no role in low-grade astrocytoma.

Adolescent↗
PubMed2003

Elevated risk for MPNST in NF1 microdeletion patients.

An NF1 microdeletion is the single most commonly reported mutation in individuals with neurofibromatosis type 1 (NF1). Individuals with an NF1 microdeletion have, as a group, more neurofibromas at a younger age than the group of all individuals with NF1. We report that NF1 microdeletion individuals additionally have a substantially higher lifetime risk for the development of malignant peripheral nerve sheath tumors than individuals with NF1 who do not have an NF1 microdeletion. This should be taken into account in the medical follow-up of individuals with an NF1 microdeletion.

Adolescent↗
PubMed2002

Mutational and expression analysis of the NF1 gene argues against a role as tumor suppressor in sporadic pilocytic astrocytomas.

Children with neurofibromatosis type I (NF1) have a highly increased risk for developing optic nerve gliomas. Several lines of evidence support the notion that the NF1 gene functions as tumor suppressor in these pilocytic astrocytomas and therefore it is tempting to hypothesize that the NF1 gene plays a similar role in sporadic pilocytic astrocytomas. We searched for possible mechanisms of inactivation of the NF1 gene in pilocytic astrocytomas of different locations. Protein truncation testing (PTT) did not render indication for inactivating mutations in 10 analyzed tumors. Further, loss of heterozygosity analysis revealed maintenance of heterozygosity for 3 intragenic markers in 11 informative cases. Using a real-time PCR-based assay we showed that total NF1 transcript levels are high in pilocytic astrocytomas and that the NF1 type I and type II expression ratios in pilocytic astrocytomas are comparable to ratios in normal brain tissue and high-grade gliomas. Consequently, the data presented here argue against altered NF1 gene expression and the involvement of the NF1 gene in the tumorigenesis of sporadic pilocytic astrocytomas.

Adolescent↗
PubMed1996

Chromosome 17 allelic loss and NF1-GRD mutations do not play a significant role as molecular mechanisms leading to melanoma tumorigenesis.

Allelic loss in human cutaneous melanoma has been detected on chromosomes 1p, 6q, 9p, 10q, and 11q. Chromosome 17 contains important tumor suppressor genes such as p53, NM23, and neurofibromatosis type 1 (NF1), which have been implicated in melanoma tumorigenesis. The role of p53 has already been studied by a number of laboratories, showing contrasting results. In the present study, two restriction fragment length polymorphism (RFLP) probes for the NM23 and NF1 genes, together with five other RFLP and four variable number of tandem repeat chromosome 17 probes, were investigated at the loss of heterozygosity (LOH) level in a Southern blot-based assay. The NF1 gene was also tested for LOH by a polymerase chain reaction (PCR)-based approach in two different experiments, using a dinucleotide repeat polymorphic probe at locus D17S250 (17q11.2-q12), and an Alu probe intragenic to the NF1 gene (17q11.2). A PCR single-strand conformation polymorphism assay was included in the study for mutation detection at the NF1-GTPase-activating protein-related domain (GRD). A total of 68 melanocytic tumors were analyzed. LOH was detected in 9 of 87 informative cases (10%). LEW301 (17p11.2-pcen) presented the highest LOH frequency (22%). NM23 showed LOH in 17% of the informative cases, while NF1 did not show either LOH in the Southern blot- and PCR-based experiments or mutations at the NF1-GRD. These results are in concordance with those of previous smaller studies, but when compared with higher LOH frequencies obtained from other chromosomes, these findings indicate that the LOH values found in our study can most likely be attributed to background effect. Thus, chromosome 17 LOH is likely to play and unimportant role as a genetic event in melanoma tumorigenesis. Nevertheless, NF1 merits further study, since homozygous deletions have been detected at this locus in melanoma cell lines.

Alleles↗
PubMed2006

Connective tissue dysplasia in five new patients with NF1 microdeletions: further expansion of phenotype and review of the literature.

Approximately 5% of patients with neurofibromatosis type 1 (NF1) have deletions of the entire NF1 gene. The phenotype usually includes early onset, large number of neurofibromas, presence of congenital anomalies, cognitive deficiency, and variable dysmorphic features and growth abnormalities. Connective tissue abnormalities are not generally recognised as a part of NF1 microdeletion syndrome, but mitral valve prolapse, joint laxity, and/or soft skin on the palms have been reported in a few patients. We describe clinical findings in six newly diagnosed patients with NF1 microdeletions, five of whom presented with connective tissue abnormalities. A literature review of the clinical findings associated with NF1 microdeletion was also performed. Our report confirms that connective tissue dysplasia is common in patients with NF1 microdeletions. Given the potential for associated cardiac manifestation, screening by echocardiogram may be warranted. Despite the large number (>150) of patients with known NF1 microdeletions, the clinical phenotype remains incompletely defined. Additional reports of patients with NF1 microdeletions, including comprehensive clinical and molecular information, are needed to elucidate possible genotype-phenotype correlation.

Adolescent↗
PubMed1993

Neurofibromatosis type 1 (NF1): knowledge, experience, and reproductive decisions of affected patients and families.

Eighty-one subjects (56 affected patients and 25 parents of isolated affected cases) from 63 families with neurofibromatosis type 1 (NF1) on the North Western Regional Genetic Family Register (NWRGFR) were interviewed. Patients were interviewed either before (n = 26) or after (n = 55) genetic counselling. In the group as a whole, knowledge of the clinical features and the genetic aspects of the condition was poor (mean score 7 within the range of 0 to 18). The following factors were significantly associated with higher knowledge: (1) genetic counselling, (2) higher social class, (3) child with NF1, (4) when NF1 had influenced reproductive decisions, (5) young age at diagnosis, and (6) member of a patient support group. The majority of the affected subjects perceived themselves to be more severely affected than by medical classification, with persons who had been diagnosed later in life, had a child with NF1, or who were concerned about the cosmetic aspects of the disease perceiving themselves to be more severely affected. Assessment of the psychosocial effects of NF1 at different stages of life showed that 63% of affected subjects experienced difficulties at school and 48% said that the condition, particularly cosmetic aspects, caused anxiety during adolescence (n = 54). These difficulties may have contributed to later problems with career attainment and confidence in relationships. Seventy-seven percent of parents stated that their child was experiencing difficulties at school relating to NF1 (n = 51). Of the subjects at risk of having a child with NF1 and who knew about NF1 before having their family (n = 32), 45% said that it had influenced their reproductive decisions. Of 29 subjects who were still considering children, 41% wished to have prenatal diagnosis in a future pregnancy, but only three subjects stated that they would terminate an affected pregnancy.

Adolescent↗
PubMed1996

Molecular genetics of neurofibromatosis type 1 (NF1).

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

Animals↗