Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis.

Schwannomatosis is a third major form of neurofibromatosis that has recently been linked to mutations in the SMARCB1 (hSnf5/INI1) tumor suppressor gene. We analyzed the coding region of SMARCB1 by direct sequencing and multiplex ligation-dependent probe amplification (MLPA) in genomic DNA from 19 schwannomatosis kindreds. Microsatellite markers in the SMARCB1 region were developed to determine loss of heterozygosity (LOH) in associated tumors. We detected four alterations in conserved splice acceptor or donor sequences of exons 3, 4 and 6. Two alterations that likely affect splicing were seen in introns 4 and 5. An additional four alterations of unclear pathogenicity were found to segregate on the affected allele in eight families including two non-conservative missense alterations in three families. No constitutional deletions or duplications were detected by MLPA. Nine of 13 tumors examined showed partial LOH of the SMARCB1 region consistent with 'second hits.' Alterations were detected in tumors both with and without somatic NF2 gene changes. These findings support the hypothesis that SMARCB1 is a tumor suppressor for schwannomas in the context of familial disease. Further work is needed to determine its role in other multiple and single tumor syndromes.

Diagnostic criteria for schwannomatosis.

The neurofibromatoses are a diverse group of genetic conditions that share a predisposition to the development of tumors of the nerve sheath. Schwannomatosis is a recently recognized third major form of neurofibromatosis (NF) that causes multiple schwannomas without vestibular tumors diagnostic of NF2. Patients with schwannomatosis represent 2.4 to 5% of all patients requiring schwannoma resection and approximately one third of patients with schwannomatosis have anatomically localized disease with tumors limited to a single limb or segment of spine. Epidemiologic studies suggest that schwannomatosis is as common as NF2, but that familial occurrence is inexplicably rare. Patients with schwannomatosis overwhelmingly present with pain, and pain remains the primary clinical problem and indication for surgery. Diagnostic criteria for schwannomatosis are needed for both clinicians and researchers, but final diagnostic certainly will await the identification of the schwannomatosis locus itself.

Familial schwannomatosis: exclusion of the NF2 locus as the germline event.

BACKGROUND: Schwannomatosis is a recently recognized disorder, defined as multiple pathologically proven schwannomas without vestibular tumors diagnostic of neurofibromatosis 2 (NF2). Some investigators have questioned whether schwannomatosis is merely an attenuated form of NF2. METHODS: The authors identified eight families in which a proband met their diagnostic criteria for schwannomatosis. Archived and prospectively acquired tumor specimens were studied by mutational analysis at the NF2 locus, loss of heterozygosity analysis along chromosome 22, and fluorescent in situ hybridization analysis of NF2 and the more centromeric probe BCR. Linkage analysis could be performed in six of eight families. RESULTS: Clinical characterization of these kindreds showed that no affected family member harbored a vestibular tumor. Molecular analysis of 28 tumor specimens from 17 affected individuals in these kindreds revealed a pattern of somatic NF2 inactivation incompatible with our current understanding of NF2 as an inherited tumor suppressor gene syndrome. Linkage analysis excluded the NF2 locus in two kindreds, and showed a maximum lod score of 6.60 near the more centromeric marker D22S1174. CONCLUSIONS: Schwannomatosis shows clinical and molecular differences from NF2 and should be considered a third major form of neurofibromatosis. Further work is needed to identify the inherited genetic element responsible for familial schwannomatosis.

Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas.

Neurofibromatosis 2 (NF2) is a severe autosomal dominant disorder that predisposes to multiple tumours of the nervous system. About half of all patients are founders with clinically unaffected parents. The purpose of the present study was to examine the extent to which mosaicism is present in NF2 founders. A total of 233 NF2 founders with bilateral vestibular schwannomas (BVS) were screened by exon scanning. NF2 mutations were detected in the blood samples of 122 patients (52%). In 10 of the 122 cases, the ratio of mutant to normal alleles was obviously less than 1, suggesting mosaicism. Tumour specimens were available from 35 of the 111 subjects in whom no mutation could be detected in blood specimens. Mutational analysis by exon scanning detected typical NF2 mutations in 21 of the 35 tumours. In nine subjects, the alterations found in tumours could be confirmed to be the constitutional mutation based on finding of identical mutations in pathologically and/or anatomically distinct second tumours. In six other subjects with only a single tumour available, allelic loss of the NF2 gene was found in addition to the mutation in each tumour, suggesting that either the mutation or the deletion of the NF2 gene is probably the constitutional genetic alteration. Our results suggest that failure to find constitutional mutations in blood specimen from these 15 patients was not because of the limitation of the applied screening technique, but the lack of the mutations in their leucocytes, best explained by mosaicism. Extrapolating the rate (15/35 = 43%) of mosaicism in these 35 cases to the 111 NF2 founders with no constitutional NF2 mutations found in their blood, we inferred 48 mosaic subjects (111 x 0.429). Adding the 10 mosaic cases detected directly in blood specimens, we estimate the rate of mosaicism to be 24.8% (58/233) in our cohort of 233 NF2 founders with bilateral vestibular schwannomas.

Gliomas presenting after age 10 in individuals with neurofibromatosis type 1 (NF1).

Children with neurofibromatosis 1 (NF1) often develop low-grade gliomas, but brain tumors are infrequently encountered in adults with NF1. The authors present evidence from two clinical series, one including patients known to have NF1 and another focusing on adults with new onset brain tumors, that suggests an association between NF1 and symptomatic gliomas in older individuals. They also summarize the clinical data on 17 adolescents or adults with NF1 and symptomatic gliomas. The findings suggest that individuals with NF1 are at increased risk of developing gliomas throughout their lives.

Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions.

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.

Establishing priorities in neurofibromatosis research: a workshop summary.

Progress in understanding the biology of the neurofibromatoses (NF1 and NF2) offers hope for the development of new, effective methods of treatment. In May 2000, the National Institute of Neurological Disorders and Stroke (NINDS) hosted a workshop that included leading researchers and clinicians from the NF community. The goal of the meeting was to assess current knowledge and identify priorities for future research. Needs identified included the development of better animal models, further study of the function of the NF1 and NF2 genes, and investigation of the role of modifier genes. The participants agreed that it will also be important to define further the natural history of NF1 and NF2 and to develop an infrastructure to support clinical trials. They also discussed the possible creation of research consortia and NF centers to promote the integration of basic and clinical research.

Monocular elevator paresis in neurofibromatosis type 2.

A retrospective review of 29 consecutive unselected patients referred for neuro-ophthalmic evaluation after the diagnosis of neurofibromatosis type 2 (NF2) showed that four of them had a monocular elevator paresis. In two of the four MRI demonstrated lesions, presumed to be schwannomas, of the third nerve. These findings indicate that monocular elevator paresis is a common neuro-ophthalmic finding in NF2, which the authors suspect is probably a sign of third nerve infiltration or compression by a schwannoma.

Molecular genetic aspects of the phakomatoses: tuberous sclerosis complex and neurofibromatosis 1.

The phakomatoses are a diverse group of diseases characterized by skin lesions in early childhood followed by the development of tumors in many other organs. Tuberous sclerosis complex and neurofibromatosis 1 are of special interest to the neurologist because of their prominent neuro-oncological and neuro-developmental consequences. The cloning of genes responsible for these two diseases has led to the identification of causative mutations, an understanding of basic cellular pathophysiology and the development of animal models. Current laboratory investigations are focused on bringing clinical relevance to these findings, including the prospects of molecular diagnostics and rational therapeutics.

Intramedullary and spinal canal tumors in patients with neurofibromatosis 2: MR imaging findings and correlation with genotype.

PURPOSE: To determine the appearance of spinal tumors on magnetic resonance (MR) images of patients with neurofibromatosis 2 (NF2), to assess the biologic behavior of these tumors, and to determine the correlation between NF2 germline mutations and these tumors. MATERIALS AND METHODS: Spinal MR images in 49 patients with NF2 were reviewed retrospectively. Intramedullary and intradural extramedullary tumors were counted, and imaging features and growth patterns of intramedullary tumors were determined. Medical records were reviewed for spinal tumor surgery. Data on spinal tumors and NF2 germline mutations in 37 patients from 19 families were analyzed for genotype-phenotype correlation. RESULTS: Thirty-one patients (63%) had spinal tumors: Twenty-six (53%) had intramedullary tumors, 27 (55%) had intradural extramedullary tumors, and 22 (45%) had at least one tumor of each type. Three (12%) patients with intramedullary tumors versus 16 (59%) with extramedullary tumors had undergone surgery for the respective types of tumors. Compared with patients with all other types of mutations, a higher percentage of patients with nonsense and frameshift mutations had intramedullary tumors (P <.025); these patients also had higher mean numbers of all tumors (P <.001), intramedullary tumors (P <.001), and nerve sheath tumors (NSTs) (P <.001). CONCLUSION: In patients with NF2 and spinal tumors, extramedullary tumors (predominantly NSTs) were present in higher numbers and were associated with more surgery than were intramedullary tumors. Our data suggest that the association between nonsense and frameshift mutations and severe NF2 may extend to specific categories of spinal tumors.

Familial occurrence of adiposis dolorosa.

Adiposis dolorosa is a rare condition characterized by multiple painful lipomas. We describe two typically affected siblings who were found to have at least 5 affected first-degree relatives in two generations. Manifestations were remarkably variable, ranging from complete disability to solitary asymptomatic tumors. Review of histopathologic findings did not reveal any features that might distinguish the tumors from common sporadic lipomas. Mutational analysis excluded the 8344 A to G mitochondrial mutation seen in other patients with multiple lipomas. Adiposis dolorosa may be an extreme manifestation of the more common condition of familial multiple lipomas. Further work is needed to define the genetic basis of these conditions.

The parental origin of new mutations in neurofibromatosis 2.

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder characterized by schwannomas and meningiomas that develop after inactivation of both copies of the NF2 gene. Approximately half of all patients with NF2 have unaffected parents and the disease results from new mutations at the NF2 locus. Loss of heterozygosity (LOH) in tumor specimens due to deletions covering the normal NF2 allele can be used to infer the haplotypes surrounding underlying mutations and determine the allelic origin of new mutations. We studied 71 sporadic NF2 patients using both LOH and pedigree analysis and compared the parental origin of the new mutation with the underlying molecular change. In the 45 informative individuals, 31 mutations (69%) were of paternal and 14 (31%) were of maternal origin (P=0.016). Comparison with corresponding constitutional mutations revealed no correlation between parental origin and the type or location of the mutations. However, in 4 of 6 patients with somatic mosaicism the NF2 mutation was of maternal origin. A slight parent of origin effect on severity of disease was found. Further clinical and molecular studies are needed to determine the basis of these unexpected observations.

Schwann cell-onion bulb tumor of the trigeminal nerve: hyperplasia, dysplasia or neoplasia?

Onion bulbs are concentric lamellar structures formed by Schwann or perineurial cells, which may be seen in several generalized or localized diseases of the peripheral nerve. There is debate regarding the pathogenesis of localized tumefactions displaying these microscopic structures. We report the fifth case, to our knowledge, of a Schwann cell-onion bulb tumor, which arose in the trigeminal nerve of a child with an unclassifiable, probably distinct, neurocutaneous syndrome; we also provide evidence for a neoplastic or hamartomatous origin. Molecular studies failed to establish an abnormality in the NF1, NF2, PMP22, or Connexin 32 genes. Similar and previously reported cases are discussed, as well as other onion bulb-forming entities.

Merlin: the neurofibromatosis 2 tumor suppressor.

In recent years, it has become clear that the ERMs occupy a crucial position as protein linkers that both respond to and participate in reorganization of membrane-cytoskeletal interactions. With the identification of new binding partners, the ERMs are also implicated in linked regulation of the activities of particular membrane proteins. Thus, they reside at a junction in a complex web of interactions that must respond to stimuli from both outside and inside the cell. As expected from its structural motifs, merlin behaves in a manner similar to the ERM proteins, but with some notable differences. Chief among these is the absence of intramolecular interaction to mask intermolecular interaction domains in isoform 2. The full range of merlin's intermolecular interactions remains to be delineated, but it can be expected from the comparison to ERMs that merlin also sits within a web of interactions that may involve multiple partners and signaling pathways, some of which it shares with the ERMs. Defining merlin's tumor suppressor function will likely require identifying those differences that are peculiarly important in the target cell types of NF2. However, the fact that inactivation of merlin in the mouse by targeted mutagenesis produces a variety of malignant tumors with a high rate of metastasis [33] suggests that merlin's suppression of tumor formation may involve different partners and pathways in different cell types and genetic backgrounds. Consequently, the disruptions due to merlin inactivation in the progression of malignant mesothelioma may represent a tumor suppressor role operating by a different pathway than that in schwannoma or meningioma.

Allelic expression of the NF2 gene in neurofibromatosis 2 and schwannomatosis.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by formation of multiple schwannomas and meningiomas due to inactivating mutations in the NF2 tumor suppressor gene on chromosome 22. We describe a polymorphism in the 3' untranslated region of the NF2 gene that is informative in about one-third of individuals. This polymorphism permitted an assessment of the relative expression of NF2 transcripts in lymphoblastoid cell RNA from 22 unrelated NF2 patients heterozygous for a germline NF2 mutation, along with 6 schwannomatosis patients, and 14 unaffected controls. Unequal allelic expression (1.8- to 20-fold) was detected in 15 of the NF2 cases, but in none of the schwannomatosis or control individuals. Underexpression of the NF2 mutant allele was documented for all 6 nonsense or frameshift mutations, 3 of 6 splice mutations, and 1 of 4 missense mutations, which, unexpectedly, was shown to alter the NF2 transcript and create a premature stop codon. In contrast, equal expression or slight overexpression of NF2 mutant alleles was observed for 2 in-frame deletions, 2 splice alterations, and 3 missense mutations. In the remaining 5 cases, the allele representing the mutant transcript was not known. Thus, truncating NF2 mutations, which are the most frequent alterations in NF2 patients and NF2-associated tumors, were associated with underexpression of the mutant allele, whereas the less common in-frame alterations usually showed normal or slight overexpression of the mutant transcript.

Loss of the NF2 gene and merlin occur by the tumorlet stage of schwannoma development in neurofibromatosis 2.

Loss of the neurofibromatosis 2 (NF2) gene-encoded protein merlin is a universal finding in sporadic and NF2-associated schwannomas. Certain NF2 patients may develop numerous minute Schwann cell tumorlets of the spinal nerve roots in addition to larger, frank schwannomas and thereby provide an opportunity to investigate the timing of NF2 gene/merlin loss in Schwann cell tumorigenesis. We studied an NF2 patient with a germline NF2 gene frameshift mutation who had many Schwann cell tumorlets and schwannomas. Loss of heterozygosity studies of DNA from microdissected specimens showed allelic loss of the NF2 region of chromosome 22q in tumorlets as well as schwannomas. Immunohistochemistry further demonstrated loss of merlin expression in tumorlets as well as schwannomas, with intact expression in adjacent nerve. Thus, loss of both NF2 alleles and merlin occur early in Schwann cell tumorigenesis, before the tumorlet stage. The study of tumorlets and schwannomas in such patients may also provide an opportunity to elucidate mechanisms responsible for the subsequent growth of Schwann cell lesions into symptomatic tumors.