Homozygous inactivation of NF1 gene in a patient with familial NF1 and disseminated neuroblastoma

Neurofibromatosis type 1 (NF1) patients are susceptible to tumor development. In the present study we describe a child with NF1 and disseminated neuroblastoma whose death resulted from disease progression. The mother had café-au-lait spots suggesting a familial NF1. Neuroblastoma cells showed MYCN amplification and chromosome 1p36 deletion, common features associated with tumor progression in this malignancy. The NF1 gene displayed a germline T --> C transition of intron 14 in both the proband and mother DNA. This mutation, not yet previously described, occurs in a splicing donor site and produces a new mRNA variant observed together with normal NF1 mRNA. Furthermore, the SSCP analysis of the NF1 gene in tumor cells showed a somatic deletion encompassing the intron 26 and 27b of the paternal NF1 allele. Hence, neuroblastoma cells displayed both somatic and germline mutation of the NF1 gene. Our data suggest that, although rare, neuroblastoma in patients with NF1 may display homozygous gene inactivation.     

Recurrent NF1 gene mutation in a patient with oligosymptomatic neurofibromatosis type 1 (NF1).

We report a 21-year-old male with symptomatic optic glioma who does not fulfill the diagnosis of neurofibromatosis 1 (NF1) according to standard NIH criteria. Analysis of the NF1 gene revealed a recurrent mutation in exon 37 (C6792A or Y2264X). This nonsense mutation causes skipping of exon 37 during the splicing process and is predicted to result in a protein shortened by 34 amino acid residues. The mutation was detected in all tissues examined (blood lymphocytes, oral mucosa, and dermal fibroblasts). The same mutation was previously found in 3 patients with clinically confirmed NF1. To our knowledge, this is the first report of an adult patient carrying a putative (non-mosaic) NF1 gene mutation in multiple tissues but not fulfilling the NIH criteria for the clinical diagnosis of NF1.     

Recurrent NF1 gene mutation in a patient with oligosymptomatic neurofibromatosis type 1 (NF1)

We report a 21-year-old male with symptomatic optic glioma who does not fulfill the diagnosis of neurofibromatosis 1 (NF1) according to standard NIH criteria. Analysis of the NF1 gene revealed a recurrent mutation in exon 37 (C6792A or Y2264X). This nonsense mutation causes skipping of exon 37 during the splicing process and is predicted to result in a protein shortened by 34 amino acid residues. The mutation was detected in all tissues examined (blood lymphocytes, oral mucosa, and dermal fibroblasts). The same mutation was previously found in 3 patients with clinically confirmed NF1. To our knowledge, this is the first report of an adult patient carrying a putative (non-mosaic) NF1 gene mutation in multiple tissues but not fulfilling the NIH criteria for the clinical diagnosis of NF1. Am. J. Med. Genet. 86:328–330, 1999.     

Somatic neurofibromatosis type 1 (NF1) inactivation events in cutaneous neurofibromas of a single NF1 patient

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 {"type":"entrez-nucleotide","attrs":{"text":"NM_000267.3","term_id":"270132515","term_text":"NM_000267.3"}}NM_000267.3:c.3113+1G>A in exon 23 of NF1 gene (HGMD ID {"type":"entrez-nucleotide","attrs":{"text":"CS951480","term_id":"159278063","term_text":"CS951480"}}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.     

Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1)

Mosaicism is an important feature of type-1 neurofibromatosis (NF1) on account of its impact upon both clinical manifestations and transmission risk. Using FISH and MLPA to screen 3500 NF1 patients, we identified 146 individuals harboring gross NF1 deletions, 14 of whom (9.6%) displayed somatic mosaicism. The high rate of mosaicism in patients with NF1 deletions supports the postulated idea of a direct relationship between the high new mutation rate in this cancer predisposition syndrome and the frequency of mosaicism. Seven of the 14 mosaic NF1 deletions were type-2, whereas four were putatively type-1, and three were atypical. Two of the four probable type-1 deletions were confirmed as such by breakpoint-spanning PCR or SNP analysis. Both deletions were associated with a generalized manifestation of NF1. Independently, we identified a third patient with a mosaic type-1 NF1 deletion who exhibited segmental NF1. Together, these three cases constitute the first proven mosaic type-1 deletions so far reported. In two of these three mosaic type-1 deletions, the breakpoints were located within PRS1 and PRS2, previously identified as hotspots for nonallelic homologous recombination (NAHR) during meiosis. Hence, NAHR within PRS1 and PRS2 is not confined to meiosis but may also occur during postzygotic mitotic cell cycles.     

Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients

Neurofibromatosis type 1 (NF1), the most common tumor-predisposing disorder in humans, is caused by defects in the NF1 tumor-suppressor gene. Comprehensive mutation analysis applying RNA-based techniques complemented with FISH analysis achieves mutation detection rates of approximately 95% in NF1 patients. The majority of mutations are minor lesions, and approximately 5% are total gene deletions. We found 13 single- and/or multiexon deletions/duplications out of 1,050 detected mutations using our RNA-based approach in a cohort of 1,100 NF1 patients and confirmed these changes using multiplex ligation-dependent probe amplification (MLPA). With MLPA, we found another 12 novel multiexon deletion/duplications in 55 NF1 patients for whom analysis with multiple assays had not revealed a NF1 mutation, including 50 previously analyzed comprehensively. The extent of the 22 deletions and 3 duplications varied greatly, and there was no clustering of breakpoints. We also evaluated the sensitivity of MLPA in identifying deletions in a mosaic state. Furthermore, we tested whether the MLPA P122 NF1 area assay could distinguish between type I deletions, with breakpoints in low-copy repeats (NF1-LCRs), and type II deletions, caused by aberrant recombination between the JJAZ gene and its pseudogene. Our study showed that intragenic deletions and/or duplications represent only approximately 2% of all NF1 mutations. Although MLPA did not substantially increase the mutation detection rate in NF1 patients, it was a useful first step in a comprehensive mutation analysis scheme to quickly pinpoint patients with single- or multiexon deletions/duplications as well as patients with a total gene deletion who will not need full sequencing of the complete coding region.     

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.     

Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P<0.0002), no association was noted between the type of germline and somatic NF1 lesion within the same individual.     

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.     

Biallelic inactivation of NF1 in a sporadic plexiform neurofibroma

Plexiform neurofibromas are a major cause of morbidity in individuals with neurofibromatosis type 1 (NF1). Sporadically, these tumors appear as an isolated feature without other signs of NF1. A role for the NF1 gene in solitary plexiform neurofibromas has never been described. In this study, we report a 13-year-old boy who was diagnosed with a plexiform neurofibroma, without other NF1 diagnostic criteria. The tumor was partially resected and analyzed using different techniques: karyotyping, fluorescence in situ hybridization (FISH), and microarray comparative genomic hybridization (aCGH). Tumor Schwann cell culture and subsequent karyotyping showed a rearrangement involving chromosomes 1 and 17, namely an insertion of chromosomal bands 1p36-35 at 17q11.2. FISH demonstrated that the insertion interrupted the NF1 gene. In addition, a deletion was detected affecting the other NF1 allele. Whole-genome aCGH analysis of the resected tumor confirmed the presence of an 8.28 Mb deletion including the NF1 gene locus in ～15-20% of tumor cells. We conclude that biallelic NF1 inactivation was at the origin of the isolated plexiform neurofibroma in this patient. The insertion is most likely the "first hit" and the large deletion the "second hit."     

Identification of Large NF1 Duplications Reciprocal to NAHR‐Mediated Type‐1 NF1 Deletions

Approximately 5% of all patients with neurofibromatosis type‐1 (NF1) exhibit large deletions of the NF1 gene region. To date, only nine unrelated cases of large NF1 duplications have been reported, with none of the affected patients exhibiting multiple café au lait spots (CALS), Lisch nodules, freckling, or neurofibromas, the hallmark signs of NF1. Here, we have characterized two novel NF1 duplications, one sporadic and one familial. Both index patients with NF1 duplications exhibited learning disabilities and atypical CALS. Additionally, patient R609021 had Lisch nodules, whereas patient R653070 exhibited two inguinal freckles. The mother and sister of patient R609021 also harbored the NF1 duplication and exhibited cognitive dysfunction but no CALS. The breakpoints of the nine NF1 duplications reported previously have not been identified and hence their underlying generative mechanisms have remained unclear. In this study, we performed high‐resolution breakpoint analysis that indicated that the two duplications studied were mediated by nonallelic homologous recombination (NAHR) and that the duplication breakpoints were located within the NAHR hotspot paralogous recombination site 2 (PRS2), which also harbors the type‐1 NF1 deletion breakpoints. Hence, our study indicates for the first time that NF1 duplications are reciprocal to type‐1 NF1 deletions and originate from the same NAHR events.     

Hereditary spinal neurofibromatosis: a rare form of NF1?

We describe a family in which seven members in three generations were affected with a rare spinal neurofibromatosis. The affected adults showed, at the ages of 32, 37, 38, and 61, respectively, multiple spinal neurofibromas symmetrically affecting all spinal roots. Two patients were operated on for histopathologically proven cervical spinal neurofibromas. All patients had café au lait spots, one had several freckles in the axillary area, and two had possible dermal neurofibromas, but iris Lisch-nodules were not present. Other signs of neurofibromatosis types 1 and 2 were absent. A linkage study of the family suggested close linkage to the NF1 locus and excluded it from the NF2 locus. The DNA analysis of histopathologically verified spinal neurofibromas in two patients showed no evidence of LOH at 17q11.2. The findings in the present family, together with those in a family previously described, suggest a clinically distinct form of neurofibromatosis with extensive spinal neurofibromas and café au lait macules, which may be allelic to the NF1 gene.