Neuropathology and Molecular Genetics of Neurofibromatosis 2 and Related Tumors

Neurofibromatosis 2 (NF2) is an uncommon, autosomal dominant disorder in which patients are predisposed to neoplastic and dysplastic lesions of Schwann cells (schwannomas and schwannosis), meningeal cells (meningiomas and meningioan-giomatosis) and glial cells (gliomas and glial hamar-tomas). Clinical and genetic criteria that distinguish NF2 from neurofibromatosis 1 have allowed more accurate assignment of specific pathological features to NF2. The NF2 tumor suppressor gene on chromosome 22q12 encodes a widely expressed protein, named merlin, which may link the cytoskeleton and cell membrane. Germline NF2 mutations in NF2 patients and somatic NF2 mutations in sporadic schwannomas and meningiomas have different mutational spectra, but most NF2 alterations result in a truncated, inactivated merlin protein. In NF2 patients, specific mutations do not necessarily correlate with phenotypic severity, although grossly truncating alterations may result in a more severe phenotype. In schwannomas, NF2 mutations are common and may be necessary for tumorigenesis. In meningiomas, NF2 mutations occur more commonly in fibroblastic than meningothelial subtypes, and may cluster in the first half of the gene. In addition, in meningiomas, a second, non- NF2 meningioma locus is probably also involved. Future efforts in NF2 research will be directed toward elucidating the role of merlin in the normal cell and the sequelae of its inactivation in human tumors.     

Germline deletion in a neurofibromatosis type 2 kindred inactivates the NF2 gene and a candidate meningioma locus

Neurofibromatosis type 2 (NF2) is an autosomal dominant diseasewhich predisposes to the development of schwannomas, meningiomas,ependymomas, and juvenile cataracts. The NF2 gene (NF2) hasrecently been isolated and maps to chromosome 22q12 betweenthe loci D22S212 and D22S32. Deletion studies in sporadic andNF2 associated schwannomas and meningiomas, and the presenceof Inactivating mutations in NF2 in patients suggest that itacts as a tumor suppressor gene. A candidate meningioma gene(MEN) has also been isolated from the same Interval. A new highlypolymorphic (CA)n marker, D22S268, which maps very near to NF2,has allowed us to Identify a kindred with three living affectedindividuals, where the disease Is presumably caused by a largegermline deletion. Fluorescence ^{in situ} hybridization and pulsedfield gel electrophoresis confirm the presence of a 700kb deletionwhich includes the neurofllament heavy chain subunit gene locus(NEFH), D22S268, NF2 and the putative MEN gene. The absenceof meningiomas In this pedigree raises doubts as to the existenceof a separate MEN locus In this region. These results supportthe hypothesis that NF2 results from the Inactivation of a tumorsuppressor gene on chromosome 22q.     

Screening for germ-line mutations in the NF2 Gene

Neurofibromatosis type 2 (NF2) is a monogenic dominantly inherited disease that predisposes to the development of tumors of the nervous system, particularly meningiomas and schwannomas. The gene which, when altered, causes NF2, is localized on chromosome 22 and has recently been identified. The NF2 gene is also the site of somatic mutation in tumors, suggesting that it might have a tumor suppressor activity. We here report a screening method for the detection of point mutations in NF2 which takes advantage of denaturing gradient gel electrophoresis (DGGE). This method efficiently screens 95% of the coding sequence and 90% of intron/exon junctions. When applied to 91 unrelated NF2 patients, it enabled the identification of 32 germ-line mutations. Since mutations are found in only one third of the patients, it is expected that mutations or deletions affecting the promoter and/or intronic regions of the NF2 gene occur frequently. The characterized mutations are preferentially located within the 5° half of the gene. Most of them are predicted to lead to the synthesis of a truncated protein. A search for genotype/phenotype correlations showed that, at least in this series of patients, mild manifestations of the disease were associated with mutations which preserve the C-terminal end of the protein.     

The neurofibromatosis type 2 gene is inactivated in schwannomas

Schwannomas are tumors arising from schwann cells surroundingperipheral nerves. Although most schwannomas are sporadic, theyare seen in approximately 90% of individuals with neuro-fibromatosistype 2 (NF2), an autosomai dominantiy inherited disease withan incidence of 1: 40000 live births. The NF2 gene has recentlybeen isolated on chromosome 22 and encodes a putative membraneorganizing protein named schwannomin. It is believed to actas a tumor suppressor gene based on the high frequency of lossof heterozygosity (LOH) on this autosome in both sporadic andNF2 associated schwannomas and meningiomas and the identificationof inactivating mutation in NF2 patients. In this study we examined61 schwannomas Including 48 sporadic schwannomas (46 of whichare vestlbular schwannomas) and 12 schwannomas obtained fromNF2 patients, for mutations in 10 of the 16 coding exons ofthe NF2 gene. Twelve inactivating mutations were Identified,8 In sporadic tumours and 4 in tumors from people with NF2.These results support the hypothesis that loss of function ofschwannomin is a frequent and fundamental event in the genesisof schwannomas.     

1p and 3p deletions in meningiomas without detectable aberrations of chromosome 22 identified by comparative genomic hybridization

Meningioma is a common tumor of the meninges covering the central nervous system. Although generally a benign tumor, meningioma often recurs and is malignant in 5–10% of all cases. Loss of chromosome 22 loci, and specifically inactivation of the NF2 tumor suppressor gene, is considered one of several critical steps in the tumorigenesis of meningioma. However, cytogenetic and molecular investigations have failed to detect either aberrations of chromosome 22 or mutations in the NF2 gene in approximately 40% of all tumors, thus making it apparent that an alternative mechanism(s) is responsible for the development of a large fraction of meningiomas. This subset of meningiomas is not distinct with regard to clinical and histopathological features from tumors showing deletions on chromosome 22. It is, therefore, important to attempt the elucidation of molecular pathway(s) that may operate in the tumorigenesis of these tumors. We used comparative genomic hybridization (CGH) to identify regions of the genome other than chromosome 22, contributing to the development of meningioma. We analyzed 25 tumors that had undergone detailed LOH analysis on chromosome 22 and were shown to contain no detectable deletions. Two benign, malignancy grade I, meningiomas showed concurrent deletion of 1p and 3p. These results suggest that loss of both 1p and 3p may contribute to meningioma tumorigenesis. This may represent genetic changes that are alternative to deletions on chromosome 22. Genes Chromosomes Cancer 20:419–424, 1997. © 1997 Wiley-Liss, Inc.     

Phenotypic variability associated with 14 splice-site mutations in the NF2 Gene

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder caused by mutations in the NF2 gene. Patients carrying NF2 mutations are predisposed to cerebral and spinal tumors with bilateral vestibular schwannomas as the hallmark. Using single strand conformation polymorphism and temperature gradient gel electrophoresis analysis, we have screened 87 unrelated NF2 patients for mutations in the NF2 gene. In this study, we report phenotypes associated with 14 splice-site mutations carried by 14 propositi and 11 relatives. The mutations were distributed in exons 2, 3, 5, 7, 8, 14, and 15. These splice-site mutations were associated with various phenotypes, from severe to asymptomatic. Phenotypic variation was also observed within families. Mutations downstream from exon 8 resulted more often in mild phenotypes. No meningiomas were found in any of 13 affected or mutation bearing individuals from three families with splice-site mutations of exons 14 and 15. These data suggest that splice-site alteration is a relatively common cause of NF2, and that unlike other mutations the clinical outcomes of splice-site mutations in the NF2 gene are variable. These results add to the growing body of information on genotype–phenotype correlation in NF2. Am. J. Med. Genet. 77:228–233, 1998. © 1998 Wiley-Liss, Inc.     

Molecular genetic analysis of chromosome arm 17p and chromosome arm 22q DNA sequences in sporadic pediatric ependymomas

Ependymomas are glial tumors of the brain and spinal cord occurring both sporadically and in a familial syndrome, neurofibromatosis type 2 (NF2). Previous analyses performed on specimens obtained predominantly from adult patients have shown loss of DNA sequences from chromosome arm 22q, which is the location of the NF2 gene. Previously, we documented the consistent loss of chromosome arm 17p DNA in medulloblastoma and astrocytoma, which are the most common brain tumors in children. Although mutation of the TP53 gene located on 17p is the most frequent genetic mutation in all adult tumor types, such mutations are rare in most childhood brain tumors investigated to date. We studied a series of pediatric ependymoma specimens (16 intracranial and 2 spinal) for loss of 17p and 22q DNA sequences and for mutation of the TP53 and NF2 genes. None of the children had the clinical stigmata of NF2. We detected loss of 17p DNA sequences in 9 of the 18 specimens (50%); in 7 of 9 of these specimens (78%), the 144-D6 marker was deleted. In contrast, only 2 of these same 18 specimens (11%) showed loss of 22q DNA. One TP53 gene mutation was detected in a child from a cancer kindred. No mutations were detected in the NF2 gene. Our results suggest that loss of chromosome arm 17p DNA sequences is common in sporadic pediatric ependymomas and that, in contrast to ependymomas in adults, deletion of chromosome arm 22q sequences is rare. Furthermore, TP53 and NF2 gene mutations do not play an important role in the etiology of sporadic pediatric ependymomas. Genes Chromosom Cancer 17:37–44 (1996). © 1996 Wiley-Liss, Inc.     

Mapping of a new target region of allelic loss to a 2-cM interval at 22q13.1 in primary breast cancer

Allelic losses on chromosome arm 22q are frequently observed in human meningiomas and in carcinomas of the colon, ovary, and breast. Among 140 primary breast cancers we examined for loss of heterozygosity (LOH) at 16 polymorphic loci on the long arm of chromosome 22, 56 (40%) showed LOH for at least one locus. Eleven of these tumors had retained heterozygosity for markers proximal to the NF2 locus but showed LOH for markers distal to NF2. Deletion mapping indicated a new common region of deletion, 2-cM in extent, at q13.1 between Interleukin 2 receptor β (IL2RB) and D22S279. Our results raise the possibility that one or more tumor suppressor genes associated with breast cancer may exist at 22q13.1. Comparison of these results with clinicohistological data indicated that allelic losses on 22q tend to occur more frequently in tumors of malignant histological types. Genes Chromosomes Cancer 21:108–112, 1998. © 1998 Wiley-Liss, Inc.     

Mapping the breakpoint of a constitutional translocation on chromosome 22 in a patient with NF2

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by development of bilateral acoustic neurinomas and increased incidence of meningiomas. Frequent losses of 1 allele of chromosome 22 in neurinomas and meningiomas has indicated that the gene responsible for NF2 functions as a tumor suppressor. Although the NF2 gene has been mapped within a 13 cM region between D22S1 and D22S28 by linkage analysis, its location with respect to D22S15 is uncertain. We previously reported an NF2 patient with a constitutional balanced translocation t(4;22) (q12;q12.2); the NF2 gene is probably disrupted at the breakpoint. To define the location of this breakpoint on chromosome 22, we performed fluorescence in situ hybridization (FISH) with DNA markers in the NF2 region and determined the physical order of 5 loci: D22S1-NF2-LIF-D22S15-D22S32. © 1993 Wiley-Liss, Inc.     

Regional fine mapping of the β crystallin genes on chromosome 22 excludes these genes as physically linked markers for neurofibromatosis type 2

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disease, characterized by the development of bilateral vestibular schwannomas. The NF2 gene has been assigned to chromosome 22. Cataract and other eye abnormalities are frequently seen in NF2 patients. The specific association of eye abnormalities and NF2 might be caused by a genetic change on chromosome 22 that affects both the NF2 gene and a physically linked crystallin gene. In order to test this hypothesis, we regionally localized the known crystallin genes (i.e. CRYBB2, CRYBB2P1, CRYBB3, and CRYBA4) on chromosome 22. Crystallin gene-specific probes were hybridized to an extended panel of human x rodent somatic cell hybrids containing various portions of chromosome 22. It was found that all crystallin genes map to a very small region on chromosome 22 that is physically separate from the NF2 gene region by at least 160 kb of DNA. In addition, we found that the βB crystallin genes (CRYBB2, CRYBB2P1, and CRYBB3) are clustered on a 300 kb Sacll fragment and that the βA4 crystallin gene (CRYBA4) is not part of this cluster. We conclude that the ocular manifestations in many NF2 patients are probably not the primary consequence of rearrangements on chromosome 22 that involve both the NF2 gene and a nearby β crystallin gene.\\wiley5\wiley$\Wiley-JWPH\final\jwph471     

Universal absence of merlin, but not other ERM family members, in schwannomas.

NF2 (neurofibromatosis 2, encoding the merlin protein) gene mutations and chromosome 22q loss have been demonstrated in the majority of sporadic and NF2-associated schwannomas, but many schwannomas fail to demonstrate genetic evidence of biallelic NF2 gene inactivation. In addition, the role of the merlin-related ERM family members (ezrin, radixin, and moesin) remains unclear in these tumors. We therefore studied expression of NF2-encoded merlin as well as ezrin, radixin, and moesin in 22 vestibular and peripheral schwannomas that had been evaluated for NF2 mutations and chromosome 22q loss. Western blotting and immunohistochemistry with antibodies directed against the amino and carboxy termini of merlin demonstrated loss of merlin expression in all studied schwannomas, including 12 tumors lacking genetic evidence of biallelic NF2 gene inactivation. Western blotting with antibodies directed against ezrin, radixin, and moesin, however, showed expression of these proteins in all schwannomas. In addition, immunohistochemistry with an antibody to moesin revealed widespread expression in tumor and endothelial cells. These data indicate that the specific loss of merlin is universal to schwannomas and is not linked to loss of ezrin, radixin, or moesin expression.     

Analysis of the neurofibromatosis 2 gene reveals molecular variants of meningioma.

There is evidence from cytogenetic and loss of heterozygosity studies for the involvement of a tumor suppressor gene on chromosome 22 in the formation of meningiomas. Recently, the NF2 gene, which causes neurofibromatosis type 2 and which is located in the affected region on chromosome 22, has been identified. A previous study on 8 of the 17 exons of the NF2 gene described mutations in 16% of meningiomas. We have analyzed the entire coding region of the NF2 gene in 70 sporadic meningiomas and identified 43 mutations in 41 patients. These resulted predominantly in immediate truncation, splicing abnormalities, or an altered reading frame of the predicted protein product. Although there was no evidence for distinct hotspots, all mutations occurred in the first 13 exons, the region of homology with the filopodial proteins moesin, ezrin, and radixin. The association of loss of heterozygosity on chromosome 22 with mutations in the NF2 gene was significant. These data suggest that NF2 represents the meningioma locus on chromosome 22. NF2 mutations occurred significantly more frequently in fibroblastic meningioma (70%) and transitional meningioma (83%) than in meningiothelial meningioma (25%), thus indicating a differential molecular pathogenesis of these meningioma variants.     

Evaluation of NF2 gene deletion in sporadic schwannomas, meningiomas, and ependymomas by chromogenic in situ hybridization

Fluorescence in situ hybridization, loss of heterozygosity testing, and comparative genomic hybridization have been used to detect NF2 gene alterations in both sporadic and neurofibromatosis type 2 (NF2)-associated central nervous system tumors. In this study, we performed chromogenic in situ hybridization (CISH) and immunohistochemistry to evaluate for NF2 gene deletion in a group of sporadic meningiomas, schwannomas, and ependymomas. Twenty-two sporadic tumors, including 9 ependymomas, 10 meningiomas, and 3 schwannomas, were studied. CISH and immunohistochemistry were performed using the NF2 gene deletion probe and NF2 polyclonal antibody. Deletion of the NF2 gene was identified in 11 (50%) tumors, including 60% (6/10) of meningiomas, 33% (3/9) of ependymomas, and 67% (2/3) of schwannomas. The remaining 11 (50%) cases were diploid. Overall, immunoexpression of NF2 protein was observed in 50% (11/22) tumors, and concordance between CISH and immunohistochemistry was observed in 73% of cases. Our results support previous observations that schwannomas and meningiomas, and to a lesser degree, ependymomas, express a high incidence of NF2 gene deletion, which supports the hypothesis that NF2 gene plays an important role in their tumorigenesis. In addition, we have validated CISH as an efficient, economic, and reliable method for routinely assessing NF2 gene deletion in these tumors.     

NF2 gene analysis distinguishes hemangiopericytoma from meningioma.

The histogenesis of dural-based or "central" hemangiopericytomas (cHPCs) remains controversial. Some authors consider these tumors variants of meningiomas while others consider them akin to peripheral hemangiopericytomas (pHPCs). Meningiomas frequently have mutations in the neurofibromatosis 2 (NF2) gene, providing a molecular marker for meningiomas and other NF2-related tumors. We therefore analyzed the NF2 gene in cHPCs, pHPCs, and meningiomas to determine whether cHPCs are more similar at the molecular genetic level to meningiomas or pHPCs. Using paraffin-embedded archival material from 28 cHPCs (including three primary and recurrent tumors), 10 pHPCs, and 26 meningiomas, we scanned all 17 exons of the NF2 gene and flanking intronic sequences for mutations with single strand conformation polymorphism analysis and DNA sequencing. No NF2 mutations were found in either cHPCs or pHPCs, whereas 35% of meningiomas had NF2 gene alterations (P < 0.001). The NF2 gene mutations in meningiomas were all truncating mutations, consistent with previous studies. Our findings suggest that cHPCs are distinct from meningiomas at the molecular genetic level and support prior clinico-pathological data that distinguish these tumor-entities.     

Somatic NF2 gene mutations in familial and non-familial vestibular schwannoma

Vestibular schwannoma occurs both as a sporadic tumour and Inthe dominantly inherited familial cancer syndrome neuroflbromatosistype 2 (NF2). The gene for NF2 has recently been Isolated onchromosome 22, and the demonstration of inactivating germllnemutations In NF2 patients and NF2 associated tumours suggeststhat it act as a tumour suppressor. We have investigated 85sporadic and 2 NF2 associated vestibular schwannomas, and onevagal schwannoma for chromosome 22 allele loss and NF2 genemutations. A further 7 vestibular schwannomas were Investigatedfor NF2 mutations only. Chromosome 22 allele loss was detectedin 34 of 87 vestibular schwannomas and In the vagal nerve schwannoma.Six exons of the NF2 gene were Investigated by SSCP analysisin all 95 tumours. Somatic NF2 gene mutations were detectedIn 13 non-familial vestibular schwannomas. and in one of theNF2 vestibular schwannomas. Seven non-famlllal tumours withan NF2 gene mutation also displayed a chromosome 22 allele loss.Thirteen of the mutations were predicted to produce truncationof the NF2 protein. These results suggest that somatic mutationsof the NF2 tumour suppressor gene are a critical step In thepathogenesis of both famlllal and non-famlllal vestibular schwannomaand that the mechanism of tumourigenesis complles with a ‘two-hit’mutation model.     

Molecular characterization of chromosome 22 deletions in schwannomas.

Schwannomas are tumors of the cranial, spinal, and peripheral nerve sheaths that originate from Schwann cells. Acoustic neurinomas are the most frequent cranial schwannomas. They might develop sporadically or in the context of neurofibromatosis type 2 (NF2). Loss of part or all of chromosome 22 is frequently found in acoustic schwannomas, suggesting that the NF2 gene is a tumor suppressor gene involved in the genesis of these tumors. Only a few spinal schwannomas have been molecularly characterized so far, showing that chromosome 22 loss might also occur in these tumors. Here we present the molecular analysis of chromosome 22 in 23 acoustic schwannomas and nine schwannomas of other locations (including other cranial nerves and spinal and peripheral nerves). Most of these tumors were from sporadic cases. Multiple schwannomas of various locations were analyzed in two patients with NF2. We found partial or complete monosomy for chromosome 22 in 22% of the acoustic schwannomas and 55% of the non-acoustic schwannomas. The tumors with partial monosomy included four with terminal deletions and one with a deletion of the centromeric part of the long arm of chromosome 22. The region between the beta B2-1 crystallin locus (CRYB2A) and the myoglobin locus (MB) was commonly deleted in these tumors. Our studies suggest that a schwannoma-related tumor suppressor gene within this region, which might be the NF2 gene, is involved in the development of schwannomas of various locations in the nervous system. Our studies indicate that the second hit in the genesis of different schwannomas within one (predisposed) NF2 patient occurs independently and via different mechanisms.     

Loss of chromosome 22 and absence of NF2 gene mutation in a case of multiple meningiomas

Multiple meningiomas are rare, and only 13 cases have been subjected to molecular genetic analysis to detect mutations of the tumor-suppressor gene neurofibromatosis type 2 (NF2) located on chromosome 22. Most of these cases display NF2 gene mutations parallel to loss of the chromosome 22 homolog, indicating that inactivation of this gene may represent an early event in the development of multiple meningiomas. We report a case of a 61-year-old woman who developed multiple (dorsal and intracranial) meningiomas. Cytogenetic and molecular genetic studies demonstrated the loss of a copy of chromosome 22 in the 5 meningiomas studied and the absence of NF2 gene mutations in 4 of those available for this molecular analysis. These findings, together with similar data from 2 previously reported cases, suggest the participation of a tumor-suppressor gene other than NF2 on chromosome 22 in the pathogenesis of a subgroup of multiple meningiomas.     

Update from the 2011 International Schwannomatosis Workshop: From genetics to diagnostic criteria

Schwannomatosis is the third major form of neurofibromatosis and is characterized by the development of multiple schwannomas in the absence of bilateral vestibular schwannomas. The 2011 Schwannomatosis Update was organized by the Children's Tumor Foundation ( www.ctf.org ) and held in Los Angeles, CA, from June 5–8, 2011. This article summarizes the highlights presented at the Conference and represents the “state‐of‐the‐field” in 2011. Genetic studies indicate that constitutional mutations in the SMARCB1 tumor suppressor gene occur in 40–50% of familial cases and in 8–10% of sporadic cases of schwannomatosis. Tumorigenesis is thought to occur through a four‐hit, three‐step model, beginning with a germline mutation in SMARCB1 (hit 1), followed by loss of a portion of chromosome 22 that contains the second SMARCB1 allele and one NF2 allele (hits 2 and 3), followed by mutation of the remaining wild‐type NF2 allele (hit 4). Insights from research on HIV and pediatric rhabdoid tumors have shed light on potential molecular pathways that are dysregulated in schwannomatosis‐related schwannomas. Mouse models of schwannomatosis have been developed and promise to further expand our understanding of tumorigenesis and the tumor microenvironment. Clinical reports have described the occurrence of intracranial meningiomas in schwannomatosis patients and in families with germline SMARCB1 mutations. The authors propose updated diagnostic criteria to incorporate new clinical and genetic findings since 2005. In the next 5 years, the authors expect that advances in basic research in the pathogenesis of schwannomatosis will lead toward clinical investigations of potential drug therapies. © 2013 Wiley Periodicals, Inc.     

Evaluation of NF2 and NF1 tumor suppressor genes in distinctive gastrointestinal nerve sheath tumors traditionally diagnosed as benign schwannomas: s study of 20 cases

A significant percentage of conventional schwannomas, whether sporadic or associated with neurofibromatosis 2 (NF2), show loss of heterozygosity (LOH) at NF2 and/or NF2 inactivating mutations. Similarly, a significant percentage of neurofibromas show LOH at NF1 and/or NF1 inactivating mutations. There are no molecular genetic data on gastrointestinal (GI) nerve sheath tumors traditionally diagnosed as benign schwannomas, rare neoplasms possibly derived from the schwannian elements dispersed between the smooth muscle fibers. In this study, we analyzed 1 esophageal, 16 gastric, 1 small intestinal, and 2 colonic tumors of such type. Histologically, all were spindle cell neoplasms positive for S-100 protein, vimentin, and glial fibrillary acidic protein, and negative for smooth muscle markers, KIT, CD34, neurofilament proteins, and HMB45. Focal or extensive lymphoid cuffs, often containing germinal centers, were present in most cases. None of the patients had NF2 or NF1. Chromosomes 22 and 17, particularly NF2 and NF1 loci, were analyzed for LOH in all GI tumors and for comparative purposes in 10 conventional schwannomas. LOH on 22q was seen in 40% of conventional schwannomas but in only 5% (1 of 20) of GI schwannomas. PCR amplification followed by direct sequencing of PCR products failed to identify mutations in NF2 coding sequences (exons 1-15) in 13 cases, including a case with LOH on 22q. Losses on 17q involving NF1 were seen in both GI and conventional schwannomas in 50% and 33% of analyzed tumors, respectively. LOH at NF1 might be one of the genetic features seen in peripheral nerve sheath tumors from different locations and should be interpreted with caution. However, lack of NF2 alterations strongly supports the hypothesis that GI schwannomas represent a morphologically and genetically distinct group of peripheral nerve sheath tumors that are different from conventional schwannomas.