RESUMO
Ependymomas arise from the ependymal cells at different locations throughout the brain and spinal cord. These tumors have a broad age distribution with a range from less than 1 year to more than 80 years. In some intramedullary spinal ependymomas, mutations in the neurofibromatosis 2 (NF2) gene and loss of heterozygosity (LOH) on chromosome arm 22q have been described. Cytogenetic studies have also identified alterations involving chromosome arm 11q, including rearrangements at 11q13, in ependymomas. We analyzed 21 intramedullary spinal, 14 ventricular, 11 filum terminale and 6 intracerebral ependymomas for mutations in the MEN1 gene, which is located at 11q13, and mutations in the NF2 gene, which is located at 22q12, as well as for LOH on 11q and 22q. NF2 mutations were found in 6 tumors, all of which were intramedullary spinal and all of which displayed LOH 22q. Allelic loss on 22q was found in 20 cases and was significantly more frequent in intramedullary spinal ependymomas than in tumors in other locations. LOH 11q was found in 7 patients and exhibited a highly significant inverse association with LOH 22q (p<0.001). A hemizygous MEN1 mutation was identified in 3 tumors, all of which were recurrences from the same patient. Interestingly, the initial tumor corresponded to WHO grade II and displayed LOH 11q but not yet a MEN1 mutation. In 2 subsequent recurrences, the tumor had progressed to anaplastic ependymoma (WHO grade III) and exhibited a nonsense mutation in exon 10 of MEN1 (W471X) in conjunction with LOH 11q. This suggests that loss of wild-type MEN1 may be involved in the malignant progression of a subset of ependymomas. To conclude, our findings provide evidence for different genetic pathways involved in ependymoma formation and progression, which may allow to define genetically and clinically distinct tumor entities.
Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 22/genética , Ependimoma/genética , Genes da Neurofibromatose 2/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Neoplasias da Medula Espinal/genética , Adolescente , Adulto , Idoso , Alelos , Sequência de Bases , Criança , Pré-Escolar , Deleção Cromossômica , Primers do DNA/química , Ependimoma/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
OBJECTIVE: It is hypothesized that transcriptional regulation plays an important role for neurofibromatosis type 2 (NF2) expression in Schwann cells and other cell types. The objective of this study is the isolation and characterization of the transcriptional regulatory elements of the NF2 gene. STUDY DESIGN AND SETTING: A bacterial artificial chromosome library and a partial genomic DNA library were used to isolate the human NF2 gene; NF2 promoter-luciferase constructs were generated, and promoter activities were assayed. This study was carried out in a molecular biology laboratory. RESULTS: A bacterial artificial chromosome clone with an approximately 100-kilobase insert containing nearly the entire human NF2 gene has been isolated. An additional 5' NF2 sequence has also been cloned. Transient transfection experiments demonstrate strong promoter activity from the NF2 5' flanking DNA. CONCLUSIONS: The NF2 gene is approximately 100 kilobases long. Both positive and negative regulatory elements are present in NF2 5' flanking regions. SIGNIFICANCE: Better understanding of the NF2 gene and its regulation will improve molecular diagnostics and ultimately treatment of patients with NF2.
Assuntos
Expressão Gênica , Genes da Neurofibromatose 2/genética , Regiões Promotoras Genéticas , Sequência de Bases , Técnicas de Cultura , DNA Complementar/isolamento & purificação , Genes da Neurofibromatose 2/fisiologia , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.
Assuntos
Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Adulto , Sítios de Ligação , DNA Complementar/química , DNA Complementar/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/química , Genes da Neurofibromatose 2/genética , Humanos , Proteínas de Membrana/genética , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Neurofibromina 2 , Fosfoproteínas/genética , Plasmídeos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Analysis of loss of heterozygosity (LOH) is very important in the study of tumor suppressor genes. However, accurate LOH analysis of tumor suppressor genes is difficult because of dilution by contaminating non-tumor DNA. Thus, enrichment of tumor DNA is required to accurately determine LOH of the tumor. We developed a new application of the fluorescent polymerase chain reaction by coupling it with crypt isolation to accurately assess the incidence of LOH of tumor suppressor genes in 45 colorectal carcinomas. LOH was observed at p53 in 26 of 37 tumors (70.3%), at APC in 13 of 35 (37.1%), at DCC in 16 of 25 (64.0%), at NF-2 in 5 of 23 (21.7%), and at nm23 H-1 in 7 of 30 (23.3%). We could clearly determine LOH of these genes because the crypt isolation technique was used. Although the incidence of LOH at each of these loci, as determined by using this technique, was similar to that obtained in previous studies using conventional methods, this method provides a simpler, more accurate way to assess LOH. In addition, the morphology of the samples can be analyzed before genetic analysis.
Assuntos
Adenocarcinoma Mucinoso/genética , Neoplasias Colorretais/genética , Genes Supressores de Tumor/genética , Perda de Heterozigosidade , Núcleosídeo-Difosfato Quinase , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , DNA de Neoplasias/análise , Feminino , Genes APC/genética , Genes DCC/genética , Genes da Neurofibromatose 2/genética , Genes p53/genética , Humanos , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Monoméricas de Ligação ao GTP/genética , Nucleosídeo NM23 Difosfato Quinases , Reação em Cadeia da Polimerase , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Contamination of nontumor tissue makes genetic analysis difficult. For this reason, it is important to obtain pure tumor tissue to ensure accurate genetic analysis. OBJECTIVE: To accurately assess the incidence of mutation of tumor suppressor genes (p53: exon 5-8; APC: mutated cluster region; NF-2 gene: all exons) in 45 colorectal carcinomas. METHODS: We developed an application of the polymerase chain reaction-single-strand conformation polymorphism and DNA sequence by coupling them with crypt isolation. RESULTS: Mutations of p53 and APC genes were found in 24 and 22 of 45 colorectal carcinomas, respectively. No mutation of the NF-2 gene was observed in this cancer. Single-strand conformation polymorphism using a crypt isolation technique showed a clear migrating band and no false-positive data. CONCLUSIONS: The crypt isolation technique is a useful method for accurately analyzing genetic alterations. Furthermore, our proposed method confirmed the morphological findings obtained before the genetic analysis.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , Genes Supressores de Tumor/genética , Adenocarcinoma/patologia , Adulto , Idoso , Colo/patologia , Neoplasias Colorretais/patologia , DNA de Neoplasias/análise , Feminino , Genes APC/genética , Genes da Neurofibromatose 2/genética , Genes p53/genética , Humanos , Mucosa Intestinal/patologia , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Despite intense study of the neurofibromatosis type 2 (NF2) tumor-suppressor protein merlin, the biological properties and tumor-suppressor functions of merlin are still largely unknown. In this study, we examined the molecular activities of NF2-causing mutant merlin proteins in transfected mammalian cells, to elucidate the merlin properties that are critical for tumor-suppressor function. Most important, we found that 80% of the merlin mutants studied significantly altered cell adhesion, causing cells to detach from the substratum. This finding implies a function for merlin in regulating cell-matrix attachment, and changes in cell adhesion caused by mutant protein expression may be an initial step in the pathogenesis of NF2. In addition, five different mutations in merlin caused a significant increase in detergent solubility of merlin compared to wild type, indicating a decreased ability to interact with the cytoskeleton. Although not correlated to the cell-adhesion phenotype, four missense mutations decreased the binding of merlin to the ERM-interacting protein EBP-50, implicating this interaction in merlin inhibition of cell growth. Last, we found that some NF2 point mutations in merlin most closely resembled gain-of-function alleles in their cellular phenotype, which suggests that mutant NF2 alleles may not always act in a loss-of-function manner, as had been assumed, but may include a spectrum of allelic types with different phenotypic effects on the function of the protein. In aggregate, these cellular phenotypes provide a useful assay for identifying the functional domains and molecular partners necessary for merlin tumor-suppressor activity.
Assuntos
Genes da Neurofibromatose 2/genética , Proteínas de Membrana/metabolismo , Neurofibromatose 2/genética , Mutação Puntual/genética , Trocadores de Sódio-Hidrogênio , Alelos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular/genética , Divisão Celular/genética , Linhagem Celular , Tamanho Celular/genética , Citoesqueleto/metabolismo , Drosophila melanogaster/genética , Genes da Neurofibromatose 2/fisiologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neurofibromina 2 , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Solubilidade , TransfecçãoRESUMO
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.
Assuntos
Proteínas de Membrana/análise , Neurofibromatose 2/genética , Alelos , Éxons , Genes da Neurofibromatose 2/genética , Humanos , Immunoblotting , Perda de Heterozigosidade , Mutação/genética , Neurofibromina 2 , Linhagem , FenótipoRESUMO
The molecular mechanisms of carcinogenesis in pancreatic cancer are still poorly understood, although the inactivation of tumor suppressor genes at multiple loci is suspected. We investigated the loss of heterozygosity (LOH) on chromosome 22 in pancreatic cancer by means of a PCR-based microsatellite analysis of archival paraffin-embedded histological sections in order to better define deleted region(s) and to test whether the NF-2 gene is involved. Using a panel of thirteen markers that spanned the long arm of chromosome 22, loss of heterozygosity was identified for at least one locus in 37% of investigated pancreatic adenocarcinomas. These deletions are clustered into two separate areas of the chromosome 22--one proximal to the NF-2 gene and one distal. The NF-2 gene itself is not involved. These regions are likely locations of tumor suppressor genes that may contribute to the development of pancreatic cancer.
Assuntos
Adenocarcinoma/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 22/genética , Deleção de Genes , Perda de Heterozigosidade/genética , Neoplasias Pancreáticas/genética , Genes da Neurofibromatose 2/genética , Genes Supressores de Tumor/genética , Marcadores Genéticos , Humanos , Repetições de Microssatélites , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Mecanismo Genético de Compensação de Dose , Neoplasias Meníngeas/genética , Meningioma/genética , Análise Mutacional de DNA , Feminino , Genes da Neurofibromatose 2/genética , Humanos , Imageamento por Ressonância Magnética , Mutação/genética , Recidiva Local de Neoplasia/genética , Fosfoglicerato Quinase/genética , Polimorfismo Conformacional de Fita Simples , Receptores Androgênicos/genéticaRESUMO
Neurofibromatosis type 2 (NF2) is an autosomally inherited disorder, caused by a mutation in NF2 tumor suppressor gene on chromosome 22q12, being characterized by multiple intracranial tumors including schwannomas, meningiomas and ependymomas. The protein encoded by the NF2 gene has a similarity to ezrin, radixin and moesin (ERM) proteins that link membrane proteins to the cytoskeleton. It has been reported that the majority of NF2 gene mutations are nonsense mutations that result in a premature termination of translation. We have developed and established a yeast-based stop codon assay for detection of NF2 gene premature terminating mutations. This assay utilizes an autonomously replicating yeast vector that expresses an NF2::ADE2 chimera protein, which gives a normal white colony when a sample NF2 cDNA is homologously recombinated, while it gives a red colony when the sample cDNA contains a nonsense mutation. The assay gave 8.0 +/- 3.5 (mean +/- SD) background red colonies when tested on clinical samples which did not contain an NF2 gene mutation. A total of 16 schwannomas (including three NF2 cases) were tested by the assay. NF2 gene mutations were detected as red colonies more than 10% in 13 of the 16 cases. Sequence analyses of plasmids recovered from the red colonies showed single base substitutions giving stop codons in five cases, and base deletions leading to frameshift and premature termination in four. Additionally, in-frame exon skippings were found in three cases. One case that gave 14% red colonies did not show a clonal mutation. This study demonstrates that the newly established assay is capable of an efficient detection of nonsense mutations of NF2 gene in clinical samples.
Assuntos
Códon de Terminação/análise , Genes da Neurofibromatose 2/genética , Mutação , Saccharomyces cerevisiae/genética , Regiões Terminadoras Genéticas , Códon de Terminação/genética , Análise Mutacional de DNA/métodos , Humanos , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Análise de Sequência de DNARESUMO
We previously reported NF2 mutations in malignant mesothelioma (MM) cell lines and corresponding primary tumors. We have now generated polyclonal antibodies that specifically recognize the C-terminus of the NF2 protein. Western blot analysis was performed on 25 MM cell lines, 14 of which showed no NF2 expression. Single-strand conformation polymorphism and DNA sequence analyses revealed NF2 mutations in each of these 14 cell lines. To explore the mechanism of inactivation of NF2, loss of heterozygosity analysis was performed with two microsatellite markers located in the vicinity of the NF2 locus in chromosome band 22q12. Eighteen of the 25 cell lines (72%) showed losses at one or both loci tested. All cases exhibiting mutation and/or aberrant expression of NF2 showed allelic losses, suggesting that inactivation of NF2 in MM occurs via a two-hit mechanism.
Assuntos
Cromossomos Humanos Par 22/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes da Neurofibromatose 2/genética , Perda de Heterozigosidade/genética , Mesotelioma/genética , Mutação/genética , Western Blotting , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neurofibromina 2 , Polimorfismo Conformacional de Fita Simples , Células Tumorais CultivadasAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 22 , Neoplasias Meníngeas/genética , Meningioma/genética , Distinções e Prêmios , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Córtex Cerebral/patologia , Progressão da Doença , Genes da Neurofibromatose 2/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Meníngeas/patologia , Meningioma/patologia , PrognósticoRESUMO
Ependymal tumors are heterogeneous with regard to morphology, localization, age at first clinical manifestation, and prognosis. Several molecular alterations have been reported in these tumors, including allelic losses on chromosomes 10, 17, and 22 and mutations in the NF2 gene. However, in contrast to astrocytic gliomas, no consistent molecular alterations have been associated with distinct types of ependymal tumors. To evaluate whether morphological subsets of ependymomas are characterized by specific genetic lesions, we analyzed a series of 62 ependymal tumors, including myxopapillary ependymomas, subependymomas, ependymomas, and anaplastic ependymomas, for allelic losses on chromosome arms 10q and 22q and mutations in the PTEN and NF2 genes. Allelic losses on 10q and 22q were detected in 5 of 56 and 12 of 54 tumors, respectively. Six ependymomas carried somatic NF2 mutations, whereas no mutations were detected in the PTEN gene. All six of the NF2 mutations occurred in ependymomas of WHO grade II and were exclusively observed in tumors with a spinal localization (P = 0.0063). These findings suggest that a considerable fraction of spinal ependymomas are associated with molecular events involving chromosome 22 and that mutations in the NF2 gene may be of primary importance for their genesis. Furthermore, our data suggest that the more favorable clinical course of spinal ependymomas may relate to a distinct pattern of genetic alterations different from that of intracerebral ependymomas.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22 , Ependimoma/genética , Proteínas de Membrana/genética , Neoplasias da Medula Espinal/genética , Neoplasias da Coluna Vertebral/genética , Proteínas Supressoras de Tumor , Adolescente , Adulto , Idoso , Alelos , Pré-Escolar , Cromossomos Humanos Par 10 , Feminino , Genes da Neurofibromatose 2/genética , Humanos , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Mutação , Neurofibromina 2 , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Polimorfismo Conformacional de Fita SimplesRESUMO
We cloned novel splice variants Mer150, Mer151 and Mer162 of the neurofibromatosis 2 (NF2) tumor suppressor, which demonstrate a tissue-specific and development-specific expression pattern. Isoform Mer150 is created by cryptic splicing from exon 8 to 14 and represents an N-terminal truncation of 259 residues. Mer151 is characterized by in-frame splicing out of several exons and a modified C-terminus due to a frameshift in exons 13+14 and premature termination. Mer162 represents a head-to-tail isoform resulting from in-frame skipping of exons 5-16. As a common feature, the alpha-helical domain and a variable proportion of the ERM homology domain are spliced out in these isoforms. To investigate differences in subcellular localization, we expressed epitope-tagged cDNA constructs of the wild-type NF2 as well as of the three alternatively spliced transcripts in NIH 3T3 cells by nuclear microinjection or lipid-mediated transfection. Subcellular localization of Mer151 in filopodia and ruffling membranes was similar to the wild-type NF2. Mer151, however, was targeted to the nucleus, which was not observed for wild-type NF2, Mer150 or Mer162. A putative nuclear localization signal created by alternative splicing was identified in Mer151. In contrast to Mer151, Mer150 and Mer162 were not found in regions of the plasma membrane, but localized to a granular intracellular compartment. The results suggest that the recently described actin-binding domain in exon 10, but not the presence or absence of exons 2+3, is relevant for subcellular targeting. Although the NF2 protein is known as a cytoskeletal linker, additional functions in a cytoplasmic compartment and in the nucleus may exist.
Assuntos
Processamento Alternativo , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Membrana/genética , Isoformas de Proteínas/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Arginina/genética , Sequência de Bases , Transporte Biológico , Linhagem Celular Transformada , Códon de Terminação/genética , DNA Complementar/química , DNA Complementar/genética , Éxons/genética , Feminino , Fibroblastos , Mutação da Fase de Leitura , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes da Neurofibromatose 2/genética , Variação Genética , Humanos , Membranas Intracelulares/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Neurofibromina 2 , Isoformas de Proteínas/metabolismo , Pseudópodes/metabolismo , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Deleção de Sequência , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Genes da Neurofibromatose 2/genética , Terapia Genética/métodos , Proteínas de Membrana/biossíntese , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/terapia , Meningioma/genética , Meningioma/terapia , Proteínas de Neoplasias/biossíntese , Apoptose , Western Blotting , Divisão Celular , Citometria de Fluxo , Vetores Genéticos , Humanos , Neoplasias Meníngeas/patologia , Meningioma/patologia , Neurofibromina 2 , Simplexvirus , Fatores de Tempo , Resultado do Tratamento , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder predisposing to multiple neoplastic lesions with the hallmark of schwannoma arising at the eighth cranial nerve. NF2 shows a distinct clinical variability, with a mild and a severe form of the disease. The NF2 gene is mutated in constitutional DNA of affected patients from NF2 families and in sporadic cases. Comprehensive mutation analyses in patients with severe and mild phenotypes revealed mutations in only 34%-66%. In the remaining fraction, the genetic mechanism behind the development of NF2 is unknown. Analyses of germline mutations do not provide a conclusive explanation for the observed clinical heterogeneity of NF2. It can therefore be hypothesized that other factors, e.g., modifier gene(s), contribute to the development of a more severe NF2 phenotype. We report a mentally retarded patient with the severe form of NF2 who displays a 7.4 million base pair deletion on chromosome 22. We performed a full genetic characterization of this case using heterozygozity analysis of 41 markers from chromosome 22, detailed FISH mapping of deletion breakpoints, allelotyping of all other chromosomes, and sequencing of the NF2 gene in tumor DNA. Two genomically large deletions similar in size (700-800 kb), which encompass the entire NF2 gene, have been reported previously in mildly affected NF2 patients. The centromeric breakpoints of these deletions were similar to the centromeric breakpoint in the present case. However, the deletion in our patient extends over a much larger distance toward the telomere of 22q. Our results support the existence of NF2 modifier gene(s) and suggest that such a putative locus maps to a 6.5-MB interval on 22q, between D22S32 and the MB gene.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Genes da Neurofibromatose 2/genética , Neurofibromatose 2/genética , Adulto , Criança , Genes da Neurofibromatose 2/fisiologia , Marcadores Genéticos , Humanos , Masculino , Fenótipo , Polimorfismo de Fragmento de RestriçãoRESUMO
Atualmente neurofibromatose faz parte de um grupo de doenças heterogêneas. A neurofibromatose NF-1 é a mais comum das facomatoses ocorrendo em aproximadamente 90 por cento dos casos. A anomalia anastomótica mais comum entre as áreas supridas pela artéria carótida e a artéria vertebral é uma artéria de grosso calibre que se localiza ao nível do seio cavernoso. Esta comunicaçäo vascular na opiniäo de muitos pesquisadores é a primitiva artéria trigeminal. É apresentada uma paciente com neurofibromatose tipo NF-1 periférico que apresentou uma cefaléia explosiva seguida de agitaçäo psicomotora. A punçäo lombar revelou-se hemorrágica e o estudo angiográfico revelou um aneurisma de artéria carótida interna direita e uma larga comunicaçäo entre os sistemas carótido-basilar ao nível do seio cavernoso