Lee B. Jacoby

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 × 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.

Frequency and distribution of NF2 mutations in schwannomas

Sporadic and inherited schwannomas were scanned for the nature, frequency, and distribution of mutations in the NF2 locus encoding the merlin tumor suppressor protein on 22q. Of 58 tumors, 47% displayed loss of heterozygosity for NF2, leaving a total of 89 NF2 alleles to be examined. Pathogenic alterations were identified in 62 of these alleles, including 36 frameshifts with premature termination, 14 nonsense mutations, and 12 changes presumed to affect splicing. Effects of ten of the latter were confirmed in the NF2 transcript and indicated that activation of cryptic splice sites in coding sequence is another frequent mechanism leading to truncation of merlin. The mutations were relatively evenly distributed across both the protein 4.1 superfamily (exons 1–9) and the α‐helical (exons 10–15) domains of merlin, but they did not occur at all in exons 16 and 17, which encode the proteins alternative COOH‐termini. The data support the “two‐hit” tumor suppressor model for formation of schwannomas and indicate that loss of merlin function can be achieved by truncation at various locations in the protein. However, the absence of mutations in exons 16 and 17 suggests that an inactivating mutation affecting only one of the merlins alternative termini may not be sufficient to eliminate tumor suppressor function. Genes Chromosom Cancer 17:45–55 (1996).

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.

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 merlins 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 merlins 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 merlins 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.

Neurofibromatosis 2: loss of merlin's protective spell.

Schwannomas and meningiomas occur as multiple tumors in sufferers of neurofibromatosis 2 (NF2) and as solitary tumors in the general population due to the inactivation of a gene at chromosome 22q12. In 1993, a location cloning approach revealed this tumor suppressor, dubbed merlin, as a novel member of a family of proteins that link elements of the cytoskeleton and the cell membrane. Subsequent investigations have confirmed merlins role in tumor formation, but have yet to reveal its mechanism of action.

The molecular biology of human glial tumors

Abstract Recent advances in molecular genetics have provided new insights into the mechanisms responsible for the development of the most prevalent tumors of the human nervous system. The results of these studies suggest that a common molecular mechanism — loss of regions of chromosome 22 possibly containing a tumor suppressor gene — is associated with several tumor types including Schwannomas, neurofibromas, meningiomas, and astrocytomas. This mechanism appears to be similar for both the solitary tumors that sporadically occur in the general population as well as for tumors in patients with one form of the genetic disorder neurofibromatosis. These results may ultimately be linked with parallel investigations of tumor-associated growth factors known to be mitogenic for the cells forming these neoplasms, and with reports of amplification of several oncogenes in malignant astrocytomas. Investigations into the relationship between recessive tumor suppressor genes and dominant oncogenes are providing new models of multi-step tumorigenesis in the nervous system. These research efforts may provide new techniques for diagnosis and treatment of patients with these tumors and ultimately lead to new insights into mechanisms controlling normal development and differentiation of the nervous system.

Clonal analysis of a case of multiple meningiomas using multiple molecular genetic approaches: Pathology case report

OBJECTIVE Multiple meningiomas are uncommon brain tumors occurring concurrently in several intracranial locations in the same patient. In the present study, we determined the clonality, methylation status of deoxyribonucleic acid, and relationship of genetic alterations in eight meningiomas from one female patient. METHODS Six molecular genetic techniques, including two methylation-based clonality assays and one transcription-based clonality assay, methylation analysis of CpG islands by methylation-specific polymerase chain reaction, loss of heterozygosity, microsatellite instability, and mutational analysis of the NF2 gene on chromosome 22, were used in comparative investigations on clonality and genetic alterations. RESULTS The presence of clonal tumor cells was demonstrated by 1) loss of the same copy of chromosome 22 in all eight tumors; 2) transcription of the human AR gene from the same allele in six of eight tumors; 3) a common unmethylated allele at the AR locus in all eight tumors; and 4) the identical single-basepair insertion mutation in exon 9 of the NF2 gene in six of eight tumors. In addition, loss of a copy of the X chromosome in one tumor nodule and microsatellite instability in another nodule were observed. CONCLUSION Taken together, this case of multiple meningiomas was most likely monoclonal in origin. Loss of chromosome 22 was an early event during the development of multiple meningiomas and was followed by mutations at the NF2 locus. Later events, including loss of the X chromosome, variation of AR gene expression, or microsatellite instability, may also have played a role in the development of multiple meningiomas in this patient.

The parental origin of new mutations in neurofibromatosis 2

ABSTRACTNeurofibromatosis 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.

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.

The NF2 gene and merlin protein in human osteosarcomas

0S1 Low Parosteal No Yes 0S2 Low Parosteal No Yes 0S3 High Giant cell rich, Paget’s No NA 0S4 High Osteoblastic No Yes 0S5 High Osteoblastic No Yes 0S6 High Mixed osteoblastic/chondroblastic No Yes 0S7 High Giant cell rich No Yes 0S8 High Mixed osteoblastic/chondroblastic No Yes 0S9 High Osteoblastic No NA 0S10 High Mixed osteoblastic/chondroblastic No No 0S11 High Osteoblastic No NA 0S12 High Osteoblastic No Yes 0S13 High Osteoblastic No Yes 0S15 High Osteoblastic No NA 0S16 High Chondroblastic No NA 0S17 High Telangiectatic No NA 0S18 High Osteoblastic No Yes 0S19 High Osteoblastic No Yes 0S20 High Osteoblastic No Yes 0S21 High Osteoblastic No NA 0S22 High Osteoblastic No Yes 0S23 High Osteoblastic No Yes