Neurofibromatose Typ 2

Neurofibromatosis type 2 (NF2) is a genetic autosomal-dominant disorder characterized by multiple benign tumors of the nervous system. Bilateral vestibular schwannomas, known as acoustic neuromas, are the hallmark of NF2 and can be found in more than 90% of patients, causing progressive hearing loss and not infrequently leading to deafness. Spinal tumors (schwannoma, meningioma and ependymoma) develop with similarly high frequency among NF2 patients, while approximately only 1/3 of these lead to neurological symptoms. Cranial non-vestibular schwannomas and meningiomas are also frequent in NF2 patients. The clinical spectrum of NF2 further includes ophthalmological lesions and polyneuropathy. With a birth incidence of around 1 in 25,000, the genetic cause for NF2 is the heterozygous inactivation of the NF2 tumor suppressor gene on 22q. More than half (50%–80%) of NF2 patients are found to bear de novo mutations which are frequently present in a mosaic fashion (25%–30%). This review presents clinical and genetic aspects of NF2, as well as recent developments in its pharmacological treatment.     

Neurofibromatosis type 2 attributable to gonosomal mosaicism in a clinically normal mother, and identification of seven novel mutations in the NF2 gene

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome that predisposes to the development of bilateral vestibular schwannomas sometimes associated with schwannomas at other locations, meningiomas, ependymomas and juvenile posterior subcapsular lenticular opacities. This disease is caused by inactivating mutations in the NF2 tumour-suppressor gene, located in 22q12. Recently, somatic mosaicism has been demonstrated in some "de novo" NF2 patients. We here report the genetic study of 33 NF2 patients from 33 unrelated Italian families. Twelve mutations were characterised, including seven newly identified mutations and five recurrent ones. Furthermore, we describe one patient with an inactivating mutation that lies in exon 13 but that is present in only a portion of the lymphocytes and, more importantly, a clinically normal individual carrying a somatic/germinal mosaicism for a nonsense mutation in exon 10 of the NF2 gene. Our results confirm the relatively high percentage of mosaicism for mutations in the NF2 gene and establish the importance of evaluating genomic DNA from several tissues, in addition to lymphocytes, so as to identify mosaicism in "de novo" NF2 patients and their relatives. In addition, the demonstration of somatic and/or gonadal mosaicism is an important tool for accurate genetic counselling in families with sporadic cases of NF2.     

Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities.

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.     

Genotype-phenotype correlations for nervous system tumors in neurofibromatosis 2: a population-based study

Neurofibromatosis 2 (NF2) is an autosomal dominant disease that is characterized by tumors on the vestibular branch of the VIII cranial nerve, but other types of nervous system tumors usually occur as well. Genotype-phenotype correlations are well documented for overall NF2 disease severity but have not been definitively evaluated for specific types of non-VIII nerve tumors. We evaluated genotype-phenotype correlations for various types of non-VIII nerve tumors in 406 patients from the population-based United Kingdom NF2 registry, using regression models with the additional covariates of current age and type of treatment center (specialty or nonspecialty). The models also permitted consideration of intrafamilial correlation. We found statistically significant genotype-phenotype correlations for intracranial meningiomas, spinal tumors, and peripheral nerve tumors. People with constitutional NF2 missense mutations, splice-site mutations, large deletions, or somatic mosaicism had significantly fewer tumors than did people with constitutional nonsense or frameshift NF2 mutations. In addition, there were significant intrafamilial correlations for intracranial meningiomas and spinal tumors, after adjustment for the type of constitutional NF2 mutation. The type of constitutional NF2 mutation is an important determinant of the number of NF2-associated intracranial meningiomas, spinal tumors, and peripheral nerve tumors.     

Detection of rearrangements in the NF2 gene using semi-quantitative multiplex fluorescent PCR

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to the development of bilateral vestibular schwannomas (sometimes associated with schwannomas at other locations), meningiomas, and ependymomas. Point mutations that inactivate the NF2 tumor suppressor gene, located in 22q12, have been found in 45-85% of NF2 patients; in addition, large genomic deletions can be found. To evaluate the presence of genomic NF2 rearrangements, we have developed a fluorescent semiquantitative multiplex PCR method. Briefly, short fragments corresponding to the 17 exons, the promoter region, and the 3' end of the NF2 gene were co-amplified by PCR using dye primers. An additional fragment, corresponding to another gene used as an internal control, was systematically amplified in each multiplex PCR. Initially, we validated the method by using monosomic 22q and trisomic 22 samples. The fluorescent multiplex PCR method was then used to analyze 21 NF2 individuals in which single-strand conformational polymorphism (SSCP) analysis and/or direct sequencing had revealed no NF2 point mutations; we were able to detect two deletions and one duplication in NF2 in 3 patients. In conclusion, the method we developed could easily be applied in detecting NF2 deletions and duplications. Discovering genomic duplications is invaluable because they are probably the most difficult molecular alterations to detect with conventional methods and, as a consequence, might be an underestimated cause of NF2.     

Neurofibromatosis type 2 appears to be a genetically homogeneous disease

Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome characterized by the development of vestibular schwannomas and other tumors of the nervous system, including cranial and spinal meningiomas, schwannomas, and ependymomas. The presence of bilateral vestibular schwannomas is sufficient for the diagnosis. Skin manifestations are less common than in neurofibromatosis type 1 (NF1; von Recklinghausen disease). The apparent clinical distinction between NF1 and NF2 has been confirmed at the level of the gene locus by linkage studies; the gene for NF1 maps to chromosome 17, whereas the gene for NF2 has been assigned (in a single family) to chromosome 22. To increase the precision of the genetic mapping of NF2 and to determine whether additional susceptibility loci exist, we have performed linkage analysis on 12 families with NF2 by using four polymorphic markers from chromosome 22 and a marker at the NF1 locus on chromosome 17. Our results confirm the assignment of the gene for NF2 to chromosome 22 and do not support the hypothesis of genetic heterogeneity. We believe that chromosome 22 markers can now be used for presymptomatic diagnosis in selected families. The NF2 gene is tightly linked to the D22S32 locus (maximum lod score 4.12; recombination fraction 0). A CA-repeat polymorphism at the CRYB2 locus was the most informative marker in our families (lod score 5.99), but because the observed recombination fraction between NF2 and CRYB2 was 10 cM, predictions using this marker will need to be interpreted with caution.     

Molecular Analysis of the NF2 Tumor-Suppressor Gene in Schwannomatosis

Patients with multiple schwannomas without vestibular schwannomas have been postulated to compose a distinct subclass of neurofibromatosis (NF), termed "schwannomatosis." To compare the molecular-genetic basis of schwannomatosis with NF2, we examined the NF2 locus in 20 unrelated schwannomatosis patients and their affected relatives. Tumors from these patients frequently harbored typical truncating mutations of the NF2 gene and loss of heterozygosity of the surrounding region of chromosome 22. Surprisingly, unlike patients with NF2, no heterozygous NF2-gene changes were seen in normal tissues. Examination of multiple tumors from the same patient revealed that some schwannomatosis patients are somatic mosaics for NF2-gene changes. By contrast, other individuals, particularly those with a positive family history, appear to have an inherited predisposition to formation of tumors that carry somatic alterations of the NF2 gene. Further work is needed to define the pathogenetics of this unusual disease mechanism.     

Analysis of the neurofibromatosis type 2 gene in different human tumors of neuroectodermal origin

The autosomal dominant syndrome neurofibromatosis type 2 (NF2) is characterized by the development of bilateral vestibular schwannomas, meningiomas, ependymomas and gliomas. The NF2 gene, recently isolated from chromosome 22, is mutated in both sporadic and NF2 tumors such as schwannomas, meningiomas and ependymomas. Mutations of the gene have been described not only in the neoplasms usually associated with NF2, but also in 30% of the melanomas and 41% of the mesotheliomas analyzed. In particular, the finding of mutations in melanomas supports the hypothesis that the NF2 gene is involved in the genesis of several tumor types that arise from the embryonic neural crest. In this study we examined, by single-strand conformation polymorphism (SSCP) analysis, 41 tumors of the central nervous system (11 schwannomas and 30 gliomas), 19 melanomas and 15 Merkel cell carcinoma specimens for mutations in the coding sequence of the NF2 gene. We found three inactivating mutations of the NF2 gene in schwannomas. No alterations of the gene were detected by SSCP analysis of the other tumors. These results confirm the role of NF2 in pathogenesis of schwannomas, but do not define its significance in the genesis of the other neuroectodermal tumors studied. Received: 27 July 1995 / Revised: 11 September 1995     

Identification of NF2 germ-line mutations and comparison with neurofibromatosis 2 phenotypes

Neurofibromatosis 2 (NF2) is an autosomal inherited disorder that predisposes carriers to nervous system tumors. To examine genotype-phenotype correlations in NF2, we performed mutation analyses and gadolinium-enhanced magnetic resonance imaging of the head and full spine in 59 unrelated NF2 patients. In patients with vestibular schwannomas (VSs) or identified NF2 mutations, the mild phenotype was defined as <2 other intracranial tumors and ≤ 4 spinal tumors, and the severe phenotype as either ≥ 2 other intracranial tumors or > 4 spinal tumors. Nineteen mutations were found in 20 (34%) of the patients and were distributed in 12 of the 17 exons of the NF2 gene, including intron-exon boundaries. Seven mutations were frameshift, six were nonsense, four were splice site, two were missense, and one was a 3-bp in frame deletion. The nonsense mutations included one codon 57 and two codon 262 C→T transitions in CpG dinucleotides. The frameshift and nonsense NF2 mutations occurred primarily in patients with severe phenotypes. The two missense mutations occurred in patients with mild phenotypes, and three of the four splice site mutations occurred in families with both mild and severe phenotypes. Truncating NF2 mutations are usually associated with severe phenotypes, but the association of some mutations with mild and severe phenotypes indicates that NF2 expression is influenced by stochastic, epigenetic, or environmental factors. Received: 4 July 1996     

A group of schwannomas with interstitial deletions on 22q located outside the NF2 locus shows no detectable mutations in the NF2 gene

Schwannomas are tumors arising mainly at cranial and spinal nerves. Bilateral vestibular schwannoma is the hallmark of neurofibromatosis type 2 (NF2). The NF2 gene has been cloned and comprehensive analysis of its mutations in schwannomas shows that up to 60% of tumors carry inactivating mutations. Thus, the genetic mechanism behind the development of more than 40% of schwannomas without NF2 mutations is unknown. We have therefore studied tumor tissue from 50 human schwannomas by allelotyping and have found chromosome 22 deletions in over 80% of the cases. We detected 14 cases (27%) that revealed partial deletions of one copy of chromosome 22, i.e., terminal and/or interstitial deletions. We sequenced the NF2 gene in seven of these tumors and detected only one case with mutations. The deletion mapping of chromosome 22 in tumors with partial deletions indicates that several regions, in addition to the NF2 locus, harbor genes involved in schwannoma tumorigenesis. Our findings suggest that heterogeneity in the mechanisms leading to the development of schwannomas probably exists. These findings are in agreement with the recent analysis of schwannomas from familial and sporadic cases of schwannomatosis and point to a possible role of an additional gene, which, in cooperation with the NF2 tumor suppressor, causes schwannomas. Received: 12 November 1998 / Accepted: 1 March 1999     

Klinik und Genetik der Neurofibromatose Typ 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene located on chromosome 17q11.2. NF1 is fully penetrant, meaning that every individual carrying the mutation exhibits symptoms of the disease, although with some considerably variable expressivity. NF1 is characterised by the eponymous neurofibromas, which are benign Schwann cell tumours. Among the other main characteristic features of NF1 are pigmentary anomalies such as café-au-lait spots, axillary or inguinal freckling, and Lisch nodules. NF1 is a member of the class of hereditary cancer syndromes, and patients with NF1 are at increased risk of developing specific NF1-associated tumours. These tumours are caused by the biallelic inactivation of the NF1 tumour suppressor gene, resulting in aberrant Ras regulation. Over the last few years, significant progress has been made in identifying and managing the clinical symptoms of NF1 as well as in developing novel therapeutic approaches.     

NF2 mutation screening by denaturing high-performance liquid chromatography and high-resolution melting analysis

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder caused by mutations in the NF2 gene and predisposing to the development of nervous system. Identification of germline mutations is essential to provide appropriate genetic counseling in NF2 patients, but it represents an extremely challenging task because the vast majority of mutations are unique and spread over the entire coding sequence. Moreover, about 30% of de novo patients are indeed mosaic, and direct sequencing can undetect mutated alleles present in a minority of cells. As most screening techniques do not meet the requirements for efficient NF2 testing, we have developed a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a multiplex ligation-dependent probe amplification approach to screen for gene rearrangements. In addition, we have evaluated high-resolution melting analysis (HRMA) as an exon scanning procedure to identify point mutations in the NF2 gene. The results obtained in 92 NF2 patients expand the NF2 mutational spectrum and indicate DHPLC and HRMA as good systems to screen for point mutations in diseases with a heterogeneous spectrum of alterations.     

Mutant products of the NF2 tumor suppressor gene are degraded by the ubiquitin-proteasome pathway

Neurofibromatosis type 2 (NF2), a syndrome associated with multiple tumors of the nervous system, mostly schwannomas, is caused by mutations in the NF2 tumor suppressor gene that encodes schwannomin (Sch). Here we examined NF2 pathogenetic mutations that result in misfolding of the FERM domain. We found that these mutant forms of Sch were efficiently degraded by the ubiquitin-proteasome pathway. In transfected cells, Sch Delta F118 was 3-fold more efficiently degraded than the related molecule ezrin bearing the equivalent mutation. In heterozygous Nf2 knock-out mouse fibroblasts, endogenous mutant Sch Delta 81-121, but not wild type Sch, was also degraded by proteasomes. We further show that this degradation pathway is functional in primary Schwann cells. We analyzed Sch Delta 39-121 expressed in a transgenic mouse model of NF2 and found that Sch Delta 39-121, but not the endogenous wild type Sch, was unstable due to proteasome-mediated degradation. Altogether these results suggest that degradation of mutant Sch mediated by the ubiquitin-proteasome pathway is a physiopathological pathway contributing to the loss of Sch function in NF2 patients.     

Bevacizumab Treatment for Meningiomas in NF2: A Retrospective Analysis of 15 Patients

Bevacizumab treatment can result in tumor shrinkage of progressive vestibular schwannomas in some neurofibromatosis 2 (NF2) patients but its effect on meningiomas has not been defined.To determine the clinical activity of bevacizumab against NF2-related meningiomas, we measured changes in volume of meningiomas in NF2 patients who received bevacizumab for treatment of progressive vestibular schwannomas. A radiographic response was defined as a 20% decrease in tumor size by volumetric MRI analysis. In addition, we determined the expression pattern of growth factors associated with tumor angiogenesis in paraffin-embedded tissues from 26 unrelated meningiomas. A total of 48 meningiomas in 15 NF2 patients were included in this study with a median follow up time of 18 months. A volumetric radiographic response was seen in 29% of the meningiomas (14/48). Tumor shrinkage was not durable: the median duration of response was 3.7 months and the median time to progression was 15 months. There was no significant correlation between pre-treatment growth rate and meningioma response in regression models. Tissue analysis showed no correlation between tumor microvascular density and expression of VEGF pathway components. This data suggests that, in contrast to schwannomas, activation of VEGF pathway is not the primary driver of angiogenesis in meningiomas. Our results suggest that a minority of NF2-associated meningiomas shrink during bevacizumab therapy and that these responses were of short duration. These results are comparable to previous studies of bevacizumab in sporadic meningiomas.     

Predictors of the risk of mortality in neurofibromatosis 2

To evaluate clinical and molecular predictors of the risk of mortality in people with neurofibromatosis 2 (NF2), we analyzed the mortality experience of 368 patients from 261 families in the United Kingdom NF2 registry, using the Cox proportional-hazards model and the jackknife method. Age at diagnosis, intracranial meningiomas, and type of treatment center were informative predictors of the risk of mortality. In Cox models, the relative risk of mortality increased 1.13-fold per year decrease in age at diagnosis (95% confidence interval [CI] 1.08-1.18) and was 2.51-fold greater in people with meningiomas compared with those without meningiomas (95% CI 1.38-4.57). The relative risk of mortality in patients treated at specialty centers was 0.34 compared with those treated at nonspecialty centers (95% CI 0.12-0.98). In a separate model, the relative risk of mortality in people with constitutional NF2 missense mutations was very low compared with those with other types of mutations (nonsense or frameshift mutations, splice-site mutations, and large deletions), but the CI could not be well quantified because there was only one death among people with missense mutations. We conclude that age at diagnosis, the strongest single predictor of the risk of mortality, is a useful index for patient counseling and clinical management (as are intracranial meningiomas). To ensure optimal care, we recommend that people with NF2 be referred to specialty treatment centers.     

Molecular targets for NF1-associated malignant peripheral nerve sheath tumor

Malignant Peripheral Nerve Sheath Tumor (MPNST) is a soft-tissue neurosarcoma. It can occur sporadically, after radiotherapy or in patients with Neurofibromatosis 1 (NF1). The hereditary disorder, NF1, is a common cancer predisposition syndrome. The main genetic feature is the mutation of the NF1 tumor suppressor gene that is inherited in an autosomal dominant, progressive manner. Mutations of the NF1 gene increase the activity of Ras signaling and cause the development of different types of tumors, including subcutaneous and plexiform neurofibromas. These can have further mutations that mediate the transformation into MPNST. Somatic mutations that have been observed are the loss of cell cycle regulators of the CDKN2A gene, and the inactivation of Polycomb Repressive Complex 2 (PRC2), mainly embryonic ectoderm development (EED) or suppressor of zeste 12 homologue (SUZ12). Other molecular pathways that have been targeted for treatment are dual MAPK-mTOR targeting, p53 protein, and MEK-ERK pathway. To advance the therapies focused on delaying or inhibiting malignant tumor formation in NF1, we need to understand the implications of the molecular and genetic pathway that are involved in the transformation into MPNST.     

Identification of three neurofibromatosis type 2 (NF2) gene mutations in vestibular schwannomas

Vestibular schwannomas (VSs) are common benign tumors of Schwann cell origin and are frequently found in patients with neurofibromatosis type 2 (NF2). We analyzed 15 sporadic VSs for mutations in the NF2 gene. We detected mutations in three of the tumors, two of which contained loss of heterozygosity (LOH). One of the tumors contained a novel mutation, a 19-bp deletion in exon 4. The two other tumors contained an identical mutation, a complete exon 4 deletion. The exon 4 deletion represents the second most frequently reported mutation of the NF2 gene in VSs.     

Identification of mutations in theNF2 gene in Polish patients with neurofibromatosis type 2

Point mutation and loss of heterozygosity (LOH) analyses were performed in 12 Polish patients with a classic symptom of NF2 - bilateral vestibular schwannomas (BVS). In 5 patients (41.7%), germline mutations were found in the NF2 gene: 2 previously reported substitutions (c.592C>T and c.52C>T) and 3 novel mutations (c.1001_1002insG, c.1029_1030insCC, c.774_778dupGAATG). In addition, LOH analysis of 30 tumour samples from 10 patients revealed a molecular basis of NF2 in 3 patients (25%) that did not have any germline mutation. The molecular defects in sporadic cases of NF2 are still being discussed.     

Regulation of mTOR complex 2 signaling in neurofibromatosis 2-deficient target cell types

Inactivating mutations in the neurofibromatosis 2 (NF2) tumor suppressor gene results in the development of schwannomas and meningiomas. Using NF2-deficient meningioma cells and tumors, together with the normal cellular counterparts that meningiomas derive, arachnoid cells, we identified merlin as a novel negative regulator of mTOR complex 1 (mTORC1). We now show that merlin positively regulates the kinase activity of mTORC2, a second functionally distinct mTOR complex, and that downstream phosphorylation of mTORC2 substrates, including Akt, is reduced upon acute merlin deficiency in cells. In response to general growth factor stimulation, Akt signaling is attenuated in merlin RNA interference-suppressed human arachnoid and Schwann cells by mechanisms mediated by hyperactive mTORC1 and impaired mTORC2. Moreover, Akt signaling is impaired differentially in a cell type-dependent manner in response to distinct growth factor stimuli. However, contrary to activation of mTORC1, the attenuated mTORC2 signaling profiles exhibited by normal arachnoid and Schwann cells in response to acute merlin loss were not consistently reflected in NF2-deficient meningiomas and schwannomas, suggesting additional genetic events may have been acquired in tumors after initial merlin loss. This finding contrasts with another benign tumor disorder, tuberous sclerosis complex, which exhibits attenuated mTORC2 signaling profiles in both cells and tumors. Finally, we examined rapamycin, as well as the mTOR kinase inhibitor, Torin1, targeting both mTOR complexes to identify the most efficacious class of compounds for blocking mTOR-mediated signaling and proliferation in merlin-deficient meningioma cells. These studies may ultimately aid in the development of suitable therapeutics for NF2-associated tumors.