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.     

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.     

Flanking markers bracket the neurofibromatosis type 2 (NF2) gene on chromosome 22.

Neurofibromatosis 2 or bilateral acoustic neurofibromatosis (NF2) is a severe autosomal dominant disorder characterized by the development of multiple tumors of the nervous system, including meningiomas, gliomas, neurofibromas, ependymomas, and particularly acoustic neuromas. Polymorphic DNA markers have revealed frequent loss of one copy of chromosome 22 in the tumor types associated with NF2. Family studies have demonstrated that the primary defect in NF2 is linked to DNA markers on chromosome 22, suggesting that it involves inactivation of a tumor suppressor gene. We have employed a combination of multipoint linkage analysis and examination of deletions in primary tumor specimens to precisely map the NF2 locus between flanking polymorphic DNA markers on chromosome 22. The 13-cM region bracketed by these markers corresponds to 13% of the genetic length of the long arm of chromosome 22 and is expected to contain less than 5 x 10(6) bp of DNA. The delineation of flanking markers for NF2 should permit accurate presymptomatic and prenatal diagnosis for the disorder and greatly facilitate efforts to isolate the defective gene on the basis of its location.     

Loss of heterozygosity and the origin of meningioma

Summary In some human tumors, loss of particular genes manifested indirectly by loss of heterozygosity for specific RFLPs seems to uncover either heterozygous deletions leading to a gene dosis effect or homozygous deletions due to a silent allele at the corresponding locus, both causing the loss of regulatory functions (antioncogenes suppressor genes). Meningioma, a benign human tumor derived from the coverings of brain and spinal cord, is associated with complete loss, rarely deletion, of one chromosome 22. About 60% of meningiomas exhibit monosomy 22 in all or part of cells; however, about 40% display a normal karyotype. Comparison of constitutional and tumor genomes from 12 patients showed loss of heterozygosity on 22 in three cases, suggesting the involvement of events at the DNA level.     

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.     

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     

Equal parental origin of chromosome 22 losses in human sporadic meningioma: no evidence for genomic imprinting.

Inactivation of tumor suppressor genes can occur either by mutation at the gene locus or by loss of part or all of the chromosome region containing the gene. The latter is most frequently detected by DNA markers as loss of heterozygosity in the tumor tissue. In several reports, the paternal homologue was preferentially retained in embryonal tumors associated with loss of particular chromosomal regions, suggesting genomic imprinting of the corresponding tumor suppressor loci. To explore the generality of these findings and the possible role of genomic imprinting in adult tumors of the nervous system, we have determined the parental origin of chromosome 22 loss in sporadic meningioma. Of nine cases studied, five tumors retained the maternally derived chromosome 22 homologue while four retained the paternally derived chromosome 22. Thus, in contrast to the embryonal tumors, the meningioma locus on chromosome 22 is inactivated by random mutation in sporadic adult meningiomas.     

Identification of New Members of the Gas2 and Ras Families in the 22q12 Chromosome Region

Monitoring of loss of heterozygosity in human tumors has suggested that the 22q12 region may contain another tumor suppressor gene(s) in addition to the NF2 gene. The genomic sequencing of a 40-kb region bounded by the EWS and BAM22 genes and containing a CpG-rich region has identified two new genes in the q12 region of chromosome 22. One of them, GAR22, is closely related to mouse Gas2, a gene isolated as being negatively regulated by serum and growth factors and exhibiting a high expression in growth arrested mouse fibroblasts. The other, RRP22, belongs to the Ras superfamily, in which it defines a new subgroup. Its expression appears to be strictly limited to the central nervous system. Growth-arrest-specific and Ras-related genes are likely candidates to be involved in tumorigenic processes. Although no mutation was observed in a small set of meningiomas and schwannomas, alteration of these new genes should now be searched in other tumors with frequent allelic losses on chromosome 22 not associated with NF2 mutation.     

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     

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 Biological Determinations of Meningioma Progression and Recurrence

Meningiomas are tumors that arise from the coverings of the brain or spinal cord. 5% of the cases turn into malignant forms with aggressive clinical behavior and increased risk of tumor recurrence. One hundred and five patients with meningiomas were operated by open surgery. To investigate predictors of meningioma recurrence in total 124 samples of 105 patients were investigated by iFISH. Dual-probe hybridization was performed to access chromosomal alterations of chromosomes 1p-, 9p- and 22q. Additionally, methylation of TIMP3 and p16 was analyzed with MS-PCR. Of the 105 investigated tumors 59.1% (62/105) were WHO grade I, 33.3% (35/105) were WHO grade II and 7.7% (8/105) were anaplastic meningiomas (grade III), respectively. The histopathological data correlates with the recurrence rate of the investigated meningiomas. Hypermethylation of TIMP3 was detected in 13.3% of all meningiomas: 10.9% in WHO grade I meningiomas, 25.0% in grade II and 14.3% in grade III meningiomas, respectively. No correlation of TIMP3 hypermethylation with tumor recurrence or WHO grade (p = 0.2) was observed. Interestingly, deletion of 1p36 emerged as a significant predictor of shorter overall survival (log rank test, p<0.001), whereas TIMP3 promoter methylation had no significant effect on overall survival (log rank test, p = 0.799). The results of the current study support the finding that the deletion of chromosome 1p is an independent marker of meningioma recurrence and progression (p = 0.0097). Therefore the measurement of genetic aberrations in meningiomas allows in a combined histological approach a more precise assessment of the prognosis of meningiomas than histopathology alone.     

Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11

A panel of glial tumors consisting of 11 low grade gliomas, 9 anaplastic gliomas, and 29 glioblastomas were analyzed for loss of heterozygosity by examining at least one locus for each chromosome. The frequency of allele loss was highest among the glioblastomas, suggesting that genetic alterations accumulate during glial tumor development. The most common genetic alteration detected involved allele losses of chromosome 10 loci; these losses were observed in all glioblastomas and in three of the anaplastic gliomas. In order to delineate which chromosome 10 region or regions were deleted in association with glial tumor development, a deletion mapping analysis was performed, and this revealed the partial loss of chromosome 10 in eight glioblastomas and two of the anaplastic gliomas. Among these cases, three distinct regions of chromosome 10 were indicated as being targeted for deletion: one telomeric region on 10p and both telomeric and centromeric locations on 10q. These data suggest the existence of multiple chromosome 10 tumor suppressor gene loci whose inactivation is involved in the malignant progression of glioma.     

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     

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.     

Association between Inflammatory Infiltrates and Isolated Monosomy 22/del(22q) in Meningiomas

Meningiomas contain highly variable levels of infiltrating tissue macrophages (TiMa) and other immune cells. In this study we investigated the potential association between the number and immunophenotype of inflammatory and other immune cells infiltrating the tumor as evaluated by multiparameter flow cytometry, and the clinico-biological, cytogenetic and gene expression profile (GEP) of 75 meningioma patients. Overall, our results showed a close association between the amount and cellular composition of the inflammatory and other immune cell infiltrates and the cytogenetic profile of the tumors. Notably, tumors with isolated monosomy 22/del(22q) showed greater numbers of TiMa, NK cells and (recently)-activated CD69⁺ lymphocytes versus meningiomas with diploid and complex karyotypes. In addition, in the former cytogenetic subgroup of meningiomas, tumor-infiltrating TiMa also showed a more activated and functionally mature phenotype, as reflected by a greater fraction of CD69⁺, CD63⁺, CD16⁺ and CD33⁺ cells. GEP at the mRNA level showed a unique GEP among meningiomas with an isolated monosomy 22/del(22q) versus all other cases, which consisted of increased expression of genes involved in inflammatory/immune response, associated with an M1 TiMa phenotype. Altogether, these results suggest that loss of expression of specific genes coded in chromosome 22 (e.g. MIF) is closely associated with an increased homing and potentially also anti-tumoral effect of TiMa, which could contribute to explain the better outcome of this specific good-prognosis cytogenetic subgroup of meningiomas.     

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.     

Search for putative suppressor genes in meningioma: significance of chromosome 22

Summary Cytogenetic and molecular studies of various solid tumors have indicated that a series of different chromosomal regions may be deleted in the tumor genome. Usually, losses of heterozygosity are observed and, from this finding, the presence of specific genes acting as tumor suppressors has been deduced. In particular tumors, however, only a single chromosome site appears to be affected. Therefore, we have carried out a study of human meningioma, investigating 7 such putative suppressor regions by applying twelve site-specific DNA markers. In 6 out of 19 tumors, we exclusively found loss of heterozygosity for markers of the long arm of chromosome 22; none of the tumors showed statistically significant additional allelic losses for the regions 1p, 3p, 5p, 5q, 11p, 13q, 17p. Our data support the long-standing observation that only losses of or within chromosome 22 are associated with the development of meningiomas. Other suppressor regions are apparently not involved.     

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.