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     

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     

Somatic mutations in the neurofibromatosis type 2 gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. Although usually sporadic, they can occur in patients affected by the autosomal dominant syndrome, neurofibromatosis type 2 (NF2). The NF2 gene has recently been isolated from chromosome 22. The presence of germline mutations in NF2 patients and the loss of heterozygosity (LOH) on 22q in NF2 tumors support the hypothesis that the NF2 gene acts as a tumor suppressor. Cytogenetic and LOH studies have suggested that the gene responsible for the development of meningiomas is located in the region of 22q in which the NF2 gene maps. The meningioma gene could therefore be the NF2 gene itself. Recently, somatic mutations of the NF2 gene have been identified in sporadic meningiomas, thus supporting the hypothesis that the NF2 gene is also important in meningioma pathogenesis. In this study, we analyzed sixty-one sporadic meningiomas for LOH of 22q and for mutations in the NF2 gene. LOH was detected in 36 of the 60 informative tumors. Single-strand conformational polymorphism analysis was used to identify nine mutations in five of the eight exons of the NF2 gene studied. The nine tumors with an altered NF2 gene also showed LOH for 22q markers. These results further support the hypothesis that mutations in the NF2 gene are a critical pathogenetic event in at least some meningiomas.     

Analysis of chromosome 22 deletions in neurofibromatosis type 2-related tumors.

The neurofibromatosis type 2 (NF2) gene has been hypothesized to be a recessive tumor suppressor, with mutations at the same locus on chromosome 22 that lead to NF2 also leading to sporadic tumors of the types seen in NF2. Flanking markers for this gene have previously been defined as D22S1 centromeric and D22S28 telomeric. Identification of subregions of this interval that are consistently rearranged in the NF2-related tumors would aid in better defining the disease locus. To this end, we have compared tumor and constitutional DNAs, isolated from 39 unrelated patients with sporadic and NF2-associated acoustic neuromas, meningiomas, schwannomas, and ependymomas, at eight polymorphic loci on chromosome 22. Two of the tumors studied revealed loss-of-heterozygosity patterns, which is consistent with the presence of chromosome 22 terminal deletions. By using additional polymorphic markers, the terminal deletion breakpoint found in one of the tumors, an acoustic neuroma from an NF2 patient, was mapped within the previously defined NF2 region. The breakpoint occurred between the haplotyped markers D22S41/D22S46 and D22S56. This finding redefines the proximal flanking marker and localizes the NF2 gene between markers D22S41/D22S46 and D22S28. In addition, we identified a sporadic acoustic neuroma that reveals a loss-of-heterozygosity pattern consistent with mitotic recombination or deletion and reduplication, which are mechanisms not previously seen in studies of these tumors. This finding, while inconsistent with models of tumorigenesis that invoke single deletions and their gene-dosage effects, lends further support to the recessive tumor-suppressor model.     

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.     

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.     

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.     

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.     

Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas

Background

Mutations in the neurofibromatosis type 2 (NF2) tumor-suppressor gene have been identified in not only NF2-related tumors but also sporadic vestibular schwannomas (VS). This study investigated the genetic and epigenetic alterations in tumors and blood from 30 Korean patients with sporadic VS and correlated these alterations with tumor behavior.

Methodology/Principal Findings

NF2 gene mutations were detected using PCR and direct DNA sequencing and three highly polymorphic microsatellite DNA markers were used to assess the loss of heterozygosity (LOH) from chromosome 22. Aberrant hypermethylation of the CpG island of the NF2 gene was also analyzed. The tumor size, the clinical growth index, and the proliferative activity assessed using the Ki-67 labeling index were evaluated. We found 18 mutations in 16 cases of 30 schwannomas (53%). The mutations included eight frameshift mutations, seven nonsense mutations, one in-frame deletion, one splicing donor site, and one missense mutation. Nine patients (30%) showed allelic loss. No patient had aberrant hypermethylation of the NF2 gene and correlation between NF2 genetic alterations and tumor behavior was not observed in this study.

Conclusions/Significance

The molecular genetic changes in sporadic VS identified here included mutations and allelic loss, but no aberrant hypermethylation of the NF2 gene was detected. In addition, no clear genotype/phenotype correlation was identified. Therefore, it is likely that other factors contribute to tumor formation and growth.     

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.     

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.     

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.     

Incidence and classification of pediatric diffuse parenchymal lung diseases in Germany

Neurofibromatosis type 2 (NF2) is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000) is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular schwannomas may also cause dizziness or imbalance as a first symptom. Nausea, vomiting or true vertigo are rare symptoms, except in late-stage disease. The other main tumours are schwannomas of the other cranial, spinal and peripheral nerves; meningiomas both intracranial (including optic nerve meningiomas) and intraspinal, and some low-grade central nervous system malignancies (ependymomas). Ophthalmic features are also prominent and include reduced visual acuity and cataract. About 70% of NF2 patients have skin tumours (intracutaneous plaque-like lesions or more deep-seated subcutaneous nodular tumours). Neurofibromatosis type 2 is a dominantly inherited tumour predisposition syndrome caused by mutations in the NF2 gene on chromosome 22. More than 50% of patients represent new mutations and as many as one-third are mosaic for the underlying disease-causing mutation. Although truncating mutations (nonsense and frameshifts) are the most frequent germline event and cause the most severe disease, single and multiple exon deletions are common. A strategy for detection of the latter is vital for a sensitive analysis. Diagnosis is based on clinical and neuroimaging studies. Presymptomatic genetic testing is an integral part of the management of NF2 families. Prenatal diagnosis and pre-implantation genetic diagnosis is possible. The main differential diagnosis of NF2 is schwannomatosis. NF2 represents a difficult management problem with most patients facing substantial morbidity and reduced life expectancy. Surgery remains the focus of current management although watchful waiting with careful surveillance and occasionally radiation treatment have a role. Prognosis is adversely affected by early age at onset, a higher number of meningiomas and having a truncating mutation. In the future, the development of tailored drug therapies aimed at the genetic level are likely to provide huge improvements for this devastating condition.     

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.     

Mutational analysis and expression studies of the neurofibromatosis type 2 (NF2) gene in a patient with a ring chromosome 22 and NF2

The case of a seriously disabled and retarded female patient with neurofibromatosis type 2 (NF2) is reported. She suffered from bilateral vestibular schwannomas, multiple intracranial meningiomas and neurinomas. The constitutional karyotype of the patient was 46,XX, r(22)/45,XX,–22. A constitutional G to A transition in the proximal 3′ untranslated region of isoforms 1 and 2 was identified in the patient’s NF2 gene and shown not to affect differential splicing or mRNA stability. The instability of the ring chromosome 22 with the associated loss of tumor suppressor genes on chromosome 22, in particular the loss of the NF2 gene, are assumed to have caused multiple tumorigenesis in this patient Received: 7 February 1997 / Accepted: 26 February 1997     

Mutational spectrum in the neurofibromatosis type 2 gene in sporadic and familial schwannomas

Using a heteroduplex approach and direct sequencing, we have completed the screening of approximately 88% of the neurofibromatosis type 2 (NF2)-coding sequence of DNA extracted from 33 schwannomas from NF2 patients and from 29 patients with sporadic schwannomas. The extensive screening has resulted in the identification of 33 unique mutations. Similarly to other human genes, we have shown that the CpG sites are more highly mutable in the NF2 gene. The frequency, distribution, and types of mutations were shown to differ between the sporadic and familial tumors. The majority of the mutations resulted in protein truncation and were consistent with more severe phenotype, however three missense mutations were identified during this study and were all associated with milder manifestations of the disease. Received: 25 September 1995 / Revised: 19 December 1995     

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.     

The mouse ortholog of the human SMARCB1 gene encodes two splice forms

The human SMARCB1 gene (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1, previously named the INI1/hSNF5 gene) is a tumor suppressor gene located on chromosome 22q11.2 and is inactivated in malignant rhabdoid tumors. By using an EST-based approach, we cloned two splice forms of the Smarcb1 gene in mouse and a longer splice form of the human ortholog. Proteins corresponding to the longer (385 aa) and the shorter (376 aa) forms are 100% conserved between human and mouse. Meningiomas and schwannomas are tumors frequently deleting various regions on chromosome 22, including the SMARCB1 locus. We therefore directly sequenced seven SMARCB1 exons (90% of the open reading frame) in search for mutations in 41 meningiomas and 23 schwannomas. No inactivating mutations were observed, which suggests that the SMARCB1 gene is not involved in the pathogenesis of these tumors.     

A missense mutation in the NF2 gene results in moderate and mild clinical phenotypes of neurofibromatosis type 2

Since the identification of the NF2 tumor suppressor gene in 1993, various mutations have been found in NF2-related tumors and in lymphocytes from NF2 patients. Most of the reported mutations result in truncated gene products. Missense mutations affecting the tumor suppressor are rare. These missense mutations would provide valuable information for the understanding of the function of the tumor suppressor, since they should affect critical parts of the protein. In this study we describe a novel point mutation in exon 15 of the NF2 gene, which is found in lymphocyte DNA of two NF2 patients from one family. This mutation is expected to result in a substitution of Pro for Gln at codon 538. Though both of the two patients developed bilateral vestibular schwannomas, the first patient showed onset of the disease at the age of 31 years and presented with various central, peripheral and abdominal tumors, while the second patient showed later onset of clinical symptoms (at age 52 years) and presented with only two additional small spinal tumors.