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Title: Mutational analysis of the NF1 GAP-related domain in neuroectodermal tumors

Abstract

To try to contribute to the more precise characterization of the function of the NF1 gene in tumorigenesis we have analyzed the most conserved region of its coding sequence, the GAP-related domain (NF1 GRD), which is attributed with tumor suppressor function. The rationale for the study was based on the likelihood of finding structural alterations resulting in loss of function of this region, in situations such as tumors of neuroepithelial tissues. In these situations, the activity of the NF1 gene product, neurofibromis, a GTPase activating protein, seems to be crucial in regulating the mechanisms of signal transduction mediated by p21 ras. We have studied the NF1 GRD region by PCR amplification of each exon (exons 21-27a) followed by subsequent PAGE and SSCP analysis of the amplification products in 60 primary sporadic neuroectodermal tumors. Our sample included: 14 neuroblastoma, 11 glioblastoma, 8 medulloblastoma, 7 ependimoma, 6 peripheral PNET, 1 ganglioneuroma, 1 glioma, 1 Ewing sarcoma, 1 meningioma and 1 schwannoma. We have not identified structural alterations of the NF1 GRD region in the tumors analysed, with one possible exception now in the process of being characterized. We can conclude that the loss of the NF1 gene tumor suppressor function that mightmore » lead or contribute to the development of malignancies in tissues of neuroectodermal origin is not due to structural abnormalities of the region of the gene interacting with p21 ras, either as a negative regulator or as a downstream effector of it. These data, together with the observation that the oncogene ras 21 is not typically mutated in neuroectodermal tumors, and that GTP-ras has been found normally regulated in neurofibromis-deficient melanoma and neuroblastoma cell lines, seem to support the hypothesis that the antioncogene activity of the NF1 gene could be totally independent from its interaction with ras.« less

Authors:
; ;  [1]
  1. Universita di Padova (Italy) [and others
Publication Date:
OSTI Identifier:
133593
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-0322
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; STRUCTURE-ACTIVITY RELATIONSHIPS; GENE MUTATIONS; PATIENTS; NERVOUS SYSTEM DISEASES; NEOPLASMS; SKIN; POLYMERASE CHAIN REACTION; PROTEINS

Citation Formats

Vinanzi, C., Basso, G., and Perilongo, G. Mutational analysis of the NF1 GAP-related domain in neuroectodermal tumors. United States: N. p., 1994. Web.
Vinanzi, C., Basso, G., & Perilongo, G. Mutational analysis of the NF1 GAP-related domain in neuroectodermal tumors. United States.
Vinanzi, C., Basso, G., and Perilongo, G. Thu . "Mutational analysis of the NF1 GAP-related domain in neuroectodermal tumors". United States. doi:.
@article{osti_133593,
title = {Mutational analysis of the NF1 GAP-related domain in neuroectodermal tumors},
author = {Vinanzi, C. and Basso, G. and Perilongo, G.},
abstractNote = {To try to contribute to the more precise characterization of the function of the NF1 gene in tumorigenesis we have analyzed the most conserved region of its coding sequence, the GAP-related domain (NF1 GRD), which is attributed with tumor suppressor function. The rationale for the study was based on the likelihood of finding structural alterations resulting in loss of function of this region, in situations such as tumors of neuroepithelial tissues. In these situations, the activity of the NF1 gene product, neurofibromis, a GTPase activating protein, seems to be crucial in regulating the mechanisms of signal transduction mediated by p21 ras. We have studied the NF1 GRD region by PCR amplification of each exon (exons 21-27a) followed by subsequent PAGE and SSCP analysis of the amplification products in 60 primary sporadic neuroectodermal tumors. Our sample included: 14 neuroblastoma, 11 glioblastoma, 8 medulloblastoma, 7 ependimoma, 6 peripheral PNET, 1 ganglioneuroma, 1 glioma, 1 Ewing sarcoma, 1 meningioma and 1 schwannoma. We have not identified structural alterations of the NF1 GRD region in the tumors analysed, with one possible exception now in the process of being characterized. We can conclude that the loss of the NF1 gene tumor suppressor function that might lead or contribute to the development of malignancies in tissues of neuroectodermal origin is not due to structural abnormalities of the region of the gene interacting with p21 ras, either as a negative regulator or as a downstream effector of it. These data, together with the observation that the oncogene ras 21 is not typically mutated in neuroectodermal tumors, and that GTP-ras has been found normally regulated in neurofibromis-deficient melanoma and neuroblastoma cell lines, seem to support the hypothesis that the antioncogene activity of the NF1 gene could be totally independent from its interaction with ras.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 1994},
month = {Thu Sep 01 00:00:00 EDT 1994}
}
  • Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes,more » a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.« less
  • A mutation search in the NF1 gene, using heteroduplex analysis of exons amplified from 52 patients with NF1, identified one patient with an unusual heteroduplex pattern in exon 24. The heteroduplex pattern was also present in the patient`s affected father, but not in her unaffected mother or in 50 individuals with and 50 without NF1. We characterized the familial mutation by cloning and sequencing the 159 bp exon 24, which encodes part of a GAP-related domain, and about 40 bp of each flanking intron. The mutation responsible for the heteroduplex formation is a single base deletion in codon 1416 thatmore » causes a frameshift and the immediate formation of a stop codon. Exon 24 was amplified using PCR conditions and primers previously reported to amplify only from the NF1 locus on chromosome 17. In our family however, multiple unrelated clones each with sequence differing from wild-type by one 3 bp deletion and 10 single base changes suggested that the clones contained sequence from one of the NF1-related loci on chromosomes 2, 12, 14, 15, 21, or 22. We sequenced the PCR product from monochromosomal somatic cell hybrids and found the chromosome 15 product to be identical to the {open_quotes}mutant{close_quotes} clones. The sequence has {approximately}95% homology with the NF1 exon 24 but only {approximately}10% homology with published sequence for a chromosome 15 NF1-related locus amplified from a neuroblastoma. A possible second NF1-related locus on chromosome 15 could therefore also confound NF1 mutation analysis.« less
  • Neurofibromatosis type I (NF1) is a common disorder that predisposes to neoplasia in tissues derived from the embryonic neural crest. The NF1 gene encodes a tumor suppressor that most likely acts through the interaction of its GTPase-activating protein (GAP)-related domain (GRD) with the product of the ras protooncogene. We have previously identified a site in the NF1 mRNA, within the first half of the NF1 GRD, which undergoes base-modification editing. Editing at that site changes a C to a U, thereby introducing an in-frame stop codon. NF1 RNA editing has been detected in all cell types studied, to date. Inmore » order to investigate the role played by editing in NF1 tumorigenesis, we analyzed RNA from 19 NF1 and 4 non-NF1 tumors. We observed varying levels of NF1 mRNA editing in different tumors, with a higher range of editing levels in more malignant tumors (e.g., neurofibrosarcomas) compared to benign tumors (cutaneous neurofibromas). Plexiform neurofibromas have an intermediate range of levels of NF1 mRNA editing. We also compared tumor and nontumor tissues from several NF1 individuals, to determine the extent of variability present in the constitutional levels of NF1 mRNA editing and to determine whether higher levels are present in tumors. The constitutional levels of NF1 mRNA editing varied slightly but were consistent with the levels observed in non-NF1 individuals. In every case, there was a greater level of NF1 mRNA editing in the tumor than in the nontumor tissue from the same patient. These results suggest that inappropriately high levels of NF1 mRNA editing does play a role in NF1 tumorigenesis and that editing may result in the functional equivalent of biallelic inactivation of the NF1 tumor suppressor. 24 refs., 4 figs., 2 tabs.« less
  • A putative tumor suppressor gene on the short arm of human chromosome 9 has been identified recently and named as multiple tumor suppressor 1 (MTS1). MTS1 is identical to the previously identified cyclin-dependent kinase-4 inhibitor gene p16, a cell cycle regulatory protein. Frequent homozygous deletions of MTS1 gene has been documented recently in cell lines derived from different types of tumors including breast tumors, suggesting that MTS1 is a tumor suppressor gene that is probably involved in a variety of human tumors. To determine the frequency of MTS1 mutations in primary breast tumors, we screened 39 primary breast tumors (16more » lobular carcinoma and 23 ductal carcinoma) and 5 established breast tumor cell lines by utilizing single stranded conformational polymorphism (SSCP) analysis. SSCP analysis was carried out for all 3 exons of the MTS1 gene utilizing primers in the flanking intronic sequences. Two of the five breast cancer tumor cell lines analyzed exhibited deletion of the entire MTS1 gene. However, only one of the thirty-nine primary breast tumors revealed a potential SSCP variation in exon 2 of the MTS1 gene which is currently characterized by sequencing. SSCP analysis also revealed two intragenic polymorphisms, one in exon 2 and one in the 3{prime} untranslated region, that could be used to assay allelic loss directly at the MTS1 locus. These results suggest that the mutation of the MTS1 gene may not be a critical genetic change in the formation of primary breast cancer, and the deletions observed in breast tumor cell lines may be due to product of cell growth in vitro.« less
  • Despite recent progress, such as the identification of PRAD1/cyclin D1 as a parathyroid oncogene, it is likely that many genes involved in the molecular pathogenesis of parathyroid tumors remain unknown. Individuals heterozygous for inherited mutations in the extracellular Ca{sup 2+}-sensing receptor gene that reduce its biological activity exhibit a disorder termed familial hypocalciuric hypercalcemia or familial benign hypercalcemia, which is characterized by reduced responsiveness of parathyroid and kidney to calcium and by PTH-dependent hypercalcemia. Those who are homozygous for such mutations present with neonatal severe hyperparathyroidism and have marked parathroid hypercellularity. Thus, the Ca{sup 2+}-sensing receptor gene is a candidatemore » parathyroid tumor suppressor gene, with inactivating mutations plausibly explaining set-point abnormalities in the regulation of both parathyroid cellular proliferation and PTH secretion by extracellular Ca{sup 2+} similar to those seen in hyperparathyroidism. Using a ribonuclease A protection assay that has detected multiple mutations in the Ca{sup 2+}-sensing receptor gene in familial hypocalciuric hypercalcemia and covers more than 90% of its coding region, we sought somatic mutations in this gene in a total of 44 human parathyroid tumors (23 adenomas, 4 carcinomas, 5 primary hyperplasias, and 12 secondary hyperplasias). No such mutations were detected in these 44 tumors. Thus, our studies suggest that somatic mutation of the Ca{sup 2+}-sensing receptor gene does not commonly contribute to the pathogenesis of sporadic parathyroid tumors. As such, PTH set-point dysfunction in parathroid tumors may well be secondary to other clonal proliferative defects and/or mutations in other components of the extracellular Ca{sup 2+}-sensing pathway. 29 refs., 2 figs.« less