Evidence for the complete inactivation of the NF2 gene in the majority of sporadic meningiomas.

Meningiomas are common central nervous system tumours which present usually in the 4th and 5th decades of life. Loss of constitutional heterozygosity on chromosome 22 in 60% of sporadic meningiomas has implied the involvement of a tumour suppressor gene. The neurofibromatosis type 2 gene (NF2), a prime candidate for involvement in meningioma, was screened for point mutations. After examining eight of the 16 known NF2 exons in 151 meningiomas, 24 inactivating mutations were characterized. Significantly, these aberrations were exclusively detected in tumours which lost the other chromosome 22 allele. These results provide strong evidence that the suppressor gene on chromosome 22, frequently inactivated in meningioma, is the NF2 gene, and suggest that another gene is involved in the development of 40% of meningiomas.

Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin-ezrin-radixin family of cytoskeleton-associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Neurofibromatosis 2 tumour suppressor schwannomin interacts with betaII-spectrin.

NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein. Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations. The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types.

Neurofibromatosis type 2: a new mechanism of tumor suppression.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease which predisposes primarily to CNS tumors such as schwannomas (vestibular and spinal), meningiomas, ependymomas and juvenile posterior lenticular opacities. Allelic losses on chromosome 22q first suggested the existence of a tumor suppressor on this autosome in accordance with Knudson's 'two hit' model. The gene was identified by positional cloning and found to encode a novel protein schwannomin (also known as merlin), with high sequence similarity to the band 4.1 family of proteins. This similarity suggested a new mechanism of tumor suppression since it was the first time a structural protein had been associated with a human tumor. Mutation analysis confirmed that inactivation of the NF2 gene occurred in NF2 tumors and a majority of sporadic schwannomas and meningiomas. Expression and functional studies have provided additional information on the possible involvement of this novel tumor suppressor in cell differentiation, embryogenesis and growth suppression.

Molecular cloning and characterization of alternatively spliced transcripts of the mouse neurofibromatosis 2 gene.

The human neurofibromatosis 2 (NF2) gene has recently been isolated and predicted to encode a novel protein named merlin. Based on its high homology to the moesin-ezrin-radixin family of proteins, it may be involved in mediating interactions between the plasma membrane and the cytoskeleton. Here we report the isolation and characterization of multiple transcript isoforms of the mouse NF2 gene. The full length coding complementary DNA sequence of transcript isoform I is 1788 base pairs in length, shares 90% sequence identity with the human NF2 complementary DNA, and encodes a putative protein of 596 amino acids sharing 98% homology with the human merlin protein. Transcript isoforms II and III carry a 45- and 16-base pair insertion, respectively, at nucleotide 1740 at the 3' end, generated by two different modes of alternative splicing; both insertions introduce premature termination codons. Thus, transcript isoforms II and III predict proteins of 591 and 584 amino acids with altered COOH-termini of more hydrophilic character as compared to isoform I. Northern blot analysis and reverse transcription-polymerase chain reaction analysis indicate that the mouse NF2 gene is widely expressed in different tissue types and that the alternative transcripts are variantly expressed. The results presented here indicate high conservation of the NF2 gene during evolution and suggest a possible role for the COOH-terminus in mouse merlin function.

CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene.

Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.

Clonal origin of recurrent meningiomas.

Meningiomas are common intracranial and intraspinal tumors. They are treated primarily by surgical resection. Meningioma recurrence following surgery is frequent despite advances in microneurosurgery. However, it is not clear whether recurrent meningiomas, close or distant to the primary resection site, arise from incomplete resection, dissemination of tumor fragments or from independent tumor growth. In order to address the question of clonality in recurring meningiomas, we examined a series of five patients with a total of 14 tumors for X-chromosome inactivation in the tumor tissues. Four patients with a total of 11 meningiomas were informative for polymorphisms either in the PGK or the AR genes. All recurrent meningiomas were found to be clonal with respect to the primary lesions. This finding suggests a common molecular pathogenesis of primary meningioma and subsequent recurrences (p<0.01). In a sixth patient, we analyzed the NF2 gene for mutations in the primary and 5 recurrent meningiomas. All six lesions carried the identical NF2 mutation, strongly indicating a common origin for these tumors. We conclude that recurrent meningiomas usually arise from dissemination of tumor fragments, most likely at the time of the first surgical resection. Our data should alert to the potential of meningioma cells for seeding during surgical procedures.

A missense mutation in the neurofibromatosis 2 gene occurs in patients with mild and severe phenotypes.

We identified a missense mutation (T185-->C, Phe62-->Ser) in the neurofibromatosis 2 (NF2) gene in a family with mild and severe NF2 phenotypes. This mutation was previously reported in an unrelated family in which all affected individuals had mild phenotypes. These data demonstrate a lack of correlation between NF2 genotype and NF2 phenotype for this mutation.

Mild familial neurofibromatosis 2 associates with expression of merlin with altered COOH-terminus.

OBJECTIVE: To understand molecular details of the pathogenesis of a very mild and homogenous form of neurofibromatosis 2 (NF2). BACKGROUND: Inactivation of the NF2 tumor suppressor gene leads to the development of multiple nervous system tumors, accompanied by loss of the NF2 gene product, merlin, or schwannomin. The severity of disease varies between patients, and the biologic basis of this variation is poorly understood. METHODS: We studied the genotype-phenotype correlation in a large pedigree with extremely mild and uniform disease manifesting as slowly growing bilateral vestibular nerve schwannomas of late onset. RESULTS: The tumors demonstrated a low proliferation rate and loss of the wild-type NF2 allele. The disease is caused by a novel mutation in the NF2 gene at intron 15 splice donor site (1737 + 3 a --> t), which was identified in all carriers by the minisequencing method. The mutation resulted in splicing out of exon 15 and production of two transcripts: a novel mutant transcript with exon 16 and overexpression of isoform III, normally detected at a low level. Both transcripts encode for the COOH-terminus of isoform III. Immunoblotting of patient fibroblasts and tumor tissue demonstrated variant merlin with altered COOH-terminus. CONCLUSIONS: The mutational skip of exon 15 and the expression of a protein with the COOH-terminus of isoform III correlates with the exceptionally mild NF2, and suggests tumor suppressor activity for isoform III. The detection of expressed mutant proteins may provide useful information for prediction of the clinical outcome of individual mutations.

Presymptomatic DNA and MRI diagnosis of neurofibromatosis 2 with mild clinical course in an extended pedigree.

Neurofibromatosis 2 (NF2), a dominantly inherited disorder, typically manifests as bilateral vestibular schwannomas and predisposes to other nervous system tumors. In this study, we present a large pedigree with a benign course of NF2 (mild Gardner type) characterized by slowly growing vestibular schwannomas but few other manifestations. The family was thoroughly investigated with neurologic, ophthalmologic, and neuro-otologic methods including gadolinium-enhanced MRI of the head and spine and DNA linkage analysis. In the clinical analysis of 22 family members, MRI was superior to neuro-otologic methods in the detection of asymptomatic tumors. Based on the DNA linkage analyses we identified the NF2 mutation carriers with a high degree of certainty. These DNA markers (CRYB2, NEFH, D22S268, and D22S280) can also be used for presymptomatic diagnosis in other NF2 families. Early detection of NF2 gene mutation carriers has become possible using linkage analysis in familial NF2. MRI screening of carriers will reveal presymptomatic vestibular schwannomas (and other CNS tumors), making early intervention possible, but an efficient treatment strategy to prevent deafness has not yet been established.

Phenotypic variability in monozygotic twins with neurofibromatosis 2.

Mutations in the neurofibromatosis 2 (NF2) tumor suppressor gene on chromosome 22q12 cause a clinically variable autosomal dominant syndrome characterized by bilateral vestibular schwannomas (VSs), other nervous system tumors, and early onset lenticular cataracts. We studied three pairs of monozygotic (MZ) twins with NF2, all with bilateral VSs, to separate genetic from nongenetic causes of clinical variability. The evaluation included gadolinium-enhanced high-resolution magnetic resonance imaging of the head and spine, neuro-ophthalmic examination with slit lamp, physical examination, and zygosity testing with microsatellite markers. Each MZ pair was concordant for general phenotypic subtype (mild or severe) and often for the affected organ systems. However, the MZ pairs were discordant for some features of disease presentation or progression. For example, all three pairs were discordant for presence or type of associated cranial tumors. We hypothesize that phenotypic differences between NF2 MZ twins are at least partly due to stochastic processes, such as the loss of the second NF2 allele or alleles of other genes.

Loss of heterozygosity on chromosome 22 in human gliomas does not inactivate the neurofibromatosis type 2 gene.

The molecular genetic alterations that underlie development of gliomas, the most common neoplasm of the human central nervous system, include activation of cellular proto-oncogenes as well as inactivation of tumor suppressor genes. Although research has identified some affected loci, others clearly remain to be identified. We have investigated loss of heterozygosity on chromosome 22 in a panel of sporadic gliomas, and have assessed the possibility that inactivation of the neurofibromatosis type 2 (NF2) tumor suppressor gene on 22q plays a role in development of sporadic gliomas in humans. Loss of heterozygosity for loci on chromosome 22 loci was observed in 15 of 47 informative blood-tumor pairs, although no common area of loss of heterozygosity shared by all of these tumors could be identified. The most frequently affected segment, distal to the NF2 locus and bounded proximally by D22S15 and distally by a gene for myoglobin, was shared by as many as 11 tumors. Loss of heterozygosity at the NF2 locus was observed in 10 tumors. No rearrangements of the NF2 gene could be detected by Southern analysis of restriction endonuclease-digested genomic DNA, and no abnormally migrating bands were detected on single strand conformation analysis of individual exons of the NF2 gene. Thus, although frequent loss of heterozygosity on chromosome 22 suggests that inactivation of a tumor suppressor gene on this chromosome plays a role in development of gliomas, there is no evidence that inactivation of the NF2 gene is implicated in this process, confirming the results of other studies of the NF2 gene in human gliomas. The identity of the putative tumor suppressor gene on 22q involved in development of gliomas remains unknown.

Detection of gains and losses in 18 meningiomas by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to examine gains and losses in 18 meningioma tumors that had been previously analyzed for loss of heterozygosity (LOH) at 22q12. Partial or complete losses were seen by CGH in only 9 of 18 cases on chromosome 22. This compares with 11 of 18 losses of single or more loci by LOH. The discrepancy in these results in probably explained by the increased sensitivity of LOH by using microsatellite markers that are able to detect small deletions, whereas losses on the order of 10-15 megabases are required for confident identification by CGH. There was no consistent pattern of gains or losses by CGH, including those tumors that lacked LOH at 22q12. In one tumor of interest in which CGH and LOH studies failed to demonstrate loss on chromosome 22, CGH identified an area of amplification at 17q22-23.

Frequent type 2 neurofibromatosis gene transcript mutations in sporadic intramedullary spinal cord ependymomas.

OBJECTIVE: To further investigate the role of Type 2 neurofibromatosis (NF2) gene transcript mutations in the sporadically occurring counterparts of NF2-associated tumors. METHODS: Reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis, single strand conformation polymorphism analysis, and automated deoxyribonucleic acid sequence analysis were used to screen for mutations in the NF2 gene transcript in seven unrelated patients with sporadic intramedullary spinal cord ependymomas. RESULTS: Five of seven intramedullary spinal cord ependymomas harbored detectable mutations. All of these mutations occurred in the region of the transcript that is homologous to known cytoskeletal proteins and resulted in significant truncation of the predicted protein product. CONCLUSION: Mutations of the NF2 transcript occur in the majority of sporadic intramedullary spinal cord ependymomas. These mutations are frequent in a region of the transcript that is homologous to a family of cytoskeletal proteins, and they probably render the protein product inactive. These results add to the body of knowledge concerning the role of the NF2 gene transcript in tumorigenesis.

Combined molecular genetic studies of chromosome 22q and the neurofibromatosis type 2 gene in central nervous system tumors.

Monosomy of chromosome 22 or deletions of 22q have been described in meningiomas and astrocytic tumors, the incidence of which is increased in Type 2 neurofibromatosis. Recently, the gene for neurofibromatosis Type 2 (NF2) has been identified at Chromosome 22q12, and a tumor suppression role has been suggested. Because there have been only a few studies of the NF2 gene on central nervous system tumors other than vestibular schwannomas, we investigated the potential role of NF2 as a tumor suppressor gene in a group of sporadic meningiomas and astrocytomas. Forty-four tumors (26 meningiomas and 18 astrocytic tumors of different grades) were screened for NF2 mutations for the entire 17 exons by the polymerase chain reaction-single-strand conformation polymorphism method. In addition, 37 tumors and their respective constitutional deoxyribonucleic acid were analyzed for loss of heterozygosity of 22q alleles by four polymorphic microsatellite markers. Seven inactivating mutations were found in Exons 4, 5, 6, and 10 in 7 of 26 (27%) meningiomas, but none were found in astrocytic tumors. Altogether, 69% of meningiomas and 20% of astrocytic tumors revealed a loss of heterozygosity of 22q markers. All tumors with NF2 mutations showed concurrent loss of alleles on 22q, thus fulfilling Knudson's criteria for tumor suppressor genes in meningiomas. We conclude that inactivation of the NF2 gene is involved in the pathogenesis of a proportion of meningiomas but not in astrocytic tumors. Because many meningiomas and some astrocytic tumors had allelic loss of 22q but intact NF2, there is a possibility that other tumor suppressor genes exist on 22q and may be involved in the pathogenesis of central nervous system tumors.

Reduced expression of schwannomin/merlin in human sporadic meningiomas.

OBJECTIVE: The neurofibromatosis type 2 gene is frequently mutated in sporadic meningiomas. The protein product of the neurofibromatosis type 2 gene is called schwannomin or merlin. Its expression in leptomeningeal cells from which meningiomas are derived and the characteristics of mutated forms in meningiomas, to our knowledge, have not been previously studied. METHODS: Immunoblotting and immunoprecipitation experiments with two specific antibodies were used to determine the size and subcellular distribution of schwannomin/merlin in rabbit and human brain tissue and established human leptomeningeal LTAg2B cells. Immunoblotting was used to determine the expression level of schwannomin/merlin in 14 human sporadic meningiomas. RESULTS: Both antibodies detect a protein of approximately 66 kDa, which is predominantly expressed in the Triton X-100-insoluble fraction of the brain and LTAg2B cells. The levels of schwannomin/merlin were severely reduced in eight tumors (57%) when compared with the expression levels in the human brain, LTAg2B cells, and the remaining six meningiomas. All six tumors with the normal schwannomin/merlin expression were of meningotheliomatous type. In contrast, all other histological types and one meningotheliomatous tumor with psammoma bodies were deficient in the 66-kDa schwannomin/merlin. Although nonsense mutations leading to premature stop codons are common in the neurofibromatosis type 2 gene in meningiomas, we found no evidence of truncated schwannomin/merlin forms in the tumors analyzed. CONCLUSION: The absence of complete schwannomin/merlin in almost 60% of primary sporadic meningiomas seems to be an important factor in meningioma tumorigenesis. The development of meningotheliomatous meningiomas is probably linked to alterations in other oncogenes or tumor suppressor genes.

Molecular genetic investigation of the neurofibromatosis type 2 tumor suppressor gene in sporadic meningioma.

The authors investigated the role of somatic mutations of the neurofibromatosis type 2 (NF2) gene in sporadic meningioma. Neurofibromatosis 2 is a dominantly inherited familial tumor syndrome predisposing affected patients to a variety of central nervous system tumors including vestibular schwannoma and meningioma. Neurofibromatosis type 2 is caused by germline mutations in the NF2 tumor suppressor gene. In addition, the authors and others have reported that somatic NF2 gene mutations occur frequently in nonfamilial vestibular schwannoma. In this study, molecular genetic analysis was performed on 23 nonfamilial meningiomas. Paired DNA samples extracted from the blood and tumors of the patients were analyzed for loss of heterozygosity (LOH) in the region of the NF2 gene on chromosome 22 using closely linked DNA markers. The NF2 gene mutations were sought by single-stranded conformation polymorphism analysis and DNA sequencing. Fourteen (61%) of 23 meningiomas showed LOH in the region of the NF2 gene on chromosome 22. Somatic NF2 gene mutations were detected in eight meningiomas (35%) after screening all 17 exons. All tumors with NF2 gene mutations showed simultaneous chromosome 22 LOH. Review of the histopathological findings of the cases studied did not demonstrate any predominance of genetic abnormalities in a particular histological type of meningioma. These results are compatible with the hypothesis that the NF2 gene acts as a tumor suppressor and that its inactivation is important in the pathogenesis of sporadic meningioma.

Familial meningioma: analysis of expression of neurofibromatosis 2 protein Merlin. Report of two cases.

Meningiomas are primarily benign brain tumors thought to arise through multistep tumorigenesis, involving both the activation of oncogenes and the loss of tumor suppressor genes. The recently isolated neurofibromatosis 2 (NF2) tumor suppressor gene has been found to be mutated in a large proportion of meningiomas. Almost all cases of familial meningioma occur in association with NF2. Familial meningioma in isolation from NF2 (sporadic) is exceedingly rare, with only 14 reports since 1959. The authors report the existence of a family lacking any stigmata of NF2, in which two members had spinal meningiomas. Tumor specimens were subjected to immunocytochemical analysis for the NF2 protein product Merlin, which has been implicated in the tumorigenesis of meningioma. Merlin immunoreactivity was present in both tumor specimens, implying that the NF2 tumor suppressor gene was not deleted in these tumors. This supports the hypothesis that a second tumor suppressor gene locus, other than NF2, acts in the formation of familial sporadic meningioma. The results are discussed in the context of putative oncogenic mechanisms of familial meningiomas.

Inhibition of NF2-negative and NF2-positive primary human meningioma cell proliferation by overexpression of merlin due to vector-mediated gene transfer.

OBJECT: The absence of in vitro models of neurofibromatosis Type 2 (NF2)-defective meningiomas has limited investigative efforts to study the biological effects of this gene in the pathogenesis of these tumors. The goals of this report are to show that gene transfer vectors can efficiently express the wild-type NF2 transgene into primary meningioma cells and to determine effects on cellular proliferation. METHODS: In this study, the authors have compared the transducing capacities of a retrovirus, an adenovirus, and a herpes simplex virus amplicon vector for use in primary human meningioma cells harvested from human tumors excised from patients with and without NF2. Transduction efficiencies with the latter vector approached 100% and it was selected to transfer the wild-type NF2 transgene into these cells. Western blot analysis confirmed that vector-mediated gene transfer mediated the expression of the NF2-encoded polypeptide merlin. Overexpression of merlin significantly inhibited the proliferation of both NF2-negative and NF2-positive human meningioma cells when compared to the proliferation of cells transduced with a control vector. CONCLUSIONS: This study demonstrates the feasibility of using vector-mediated gene transfer to study wild-type NF2 gene function in short-term cultures of primary human meningioma cells.

Multiple schwannomas: schwannomatosis or neurofibromatosis type 2?

OBJECT: The aim of this study was to clarify the clinical outcome of schwannomatosis, a rare condition characterized by multiple nonvestibular schwannomas in the absence of meningiomas, intraspinal ependymomas, and other clinical signs of neurofibromatosis type 2 (NF2). METHODS: Nine patients with schwannomatosis treated at one institution are presented and their clinical course during a median follow-up time of 9.9 years is discussed. The patients were typically middle-aged at the time of their first operation (median 43.5 years), none had a positive family history of schwannomatosis or NF2, and none showed cutaneous or ocular signs of NF2. On histopathological examination the tumors from the patients with schwannomatosis showed a lobular appearance and frequent Verocay bodies, signs indicating NF2, more often than 20 sporadic schwannomas that were investigated as controls. Two patients died of unrelated causes at 3.2 and 9.9 years, respectively, of follow up. Magnetic resonance images of the head and spine were obtained in seven patients at the end of the follow-up period. New spinal schwannomas were detected in one patient and a residual schwannoma in three. No germline mutations of the NF2 gene were found in these seven patients. Two additional patients originally included in the schwannomatosis group who were 8.6 and 11.7 years old at initial surgery had NF2. One was diagnosed at follow-up review and the other developed a fulminant disease that led to death in 4 years. CONCLUSIONS: The clinical course, long-term outcome, and genetic mechanism of schwannomatosis differ from that of NF2.