Human Molecular Genetics, 2000, Vol. 9, No. 7 1059-1066
© 2000 Oxford University Press
Genetic and cellular defects contributing to benign tumor formation in neurofibromatosis type 1
Departments of Neurology and Pediatrics, University of Pennsylvania, Philadelphia, PA, USA, 1Department of Neurology, Washington University School of Medicine, St Louis, MO, USA, 2Department of Pathology, University of Michigan, Ann Arbor, MI, USA and 3Center for Human Genetics, University Hospital Gasthuisberg, Leuven, Belgium
Received 12 November 1999; Revised and Accepted 1 February 2000.
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ABSTRACT |
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Neurofibromatosis type 1 (NF1) is a common inherited cancer predisposition syndrome. The NF1 gene product, neurofibromin, is hypothesized to function as a tumor suppressor and nearly all NF1 patients develop benign peripheral nerve tumors. These neurofibromas presumably arise from NF1 inactivation in S100+ Schwann cells, but there is no formal proof for this mechanism. We demonstrate that fibroblasts isolated from neurofibromas carried at least one normal NF1 allele and expressed both NF1 mRNA and protein, whereas the S100+ cells typically lacked the NF1 transcript. Our findings further indicate that additional molecular events aside from NF1 inactivation in Schwann cells and/or other neural crest derivatives contribute to neurofibroma formation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Neurofibromatosis type 1 (NF1) is the most common form of neurofibromatosis and one of the most prevalent autosomal dominant diseases in man with a prevalence of ~1/3500 (1,2). Nearly 50% of NF1 patients do not have an affected parent, indicating that the disease frequently results from new mutations. The clinical symptoms are highly variable even within affected families, but the defining features present in most patients by adulthood include café-au-lait (CAL) macules, axillary and inguinal freckling, hamartomas of the iris (Lisch nodules) and benign neurofibromas. The NF1 gene maps to chromosome 17 and is composed of 60 exons spanning 350 kb of genomic DNA (3). Its large (11–13 kb) mRNA encodes a 250 kDa protein called neurofibromin, which contains a functional GTPase activating domain that inhibits the p21-ras proto-oncogene.
The ‘two-hit’ tumor suppressor hypothesis for NF1 predicts that all cells carry a constitutional mutation and a particular cell acquires a second mutation to initiate tumor formation (4). While both alleles are inactivated in NF1-associated malignancies, this has been more difficult to demonstrate for benign neurofibromas (5–10). With the use of multiple intragenic polymorphic markers, somatic loss of an NF1 allele has more recently been found in a number of neurofibromas (11,12). Presumably, these represent the second mutation in the familial cases, but the inactivation of both NF1 alleles has actually been demonstrated in only two neurofibromas to date (10,13).
Schwann cells characteristically express S100 protein and are generally thought to be the progenitors of neurofibromas, which typically contain numerous S100+ cells (14,15). However, these tumors are composed of a mixture of cell types, and both Schwann cells and fibroblasts isolated from neurofibromas (16–18) and from Nf1 knockout mice (19–22) appear biologically abnormal. There have been a few attempts to identify the defective cells genetically in culture, and the loss of an NF1 allele in Schwann cells has been reported in one neurofibroma (23). However, the constitutional mutation was not identified and no mutations were detected in a similar study of Schwann cells cultured from five neurofibromas (24).
Thus, it is still unclear whether both NF1 alleles are inactivated in Schwann cells or whether these cells generally initiate neurofibromas. This study provides evidence that NF1 is specifically inactivated in Schwann cells and/or other neural crest derivatives, but additional molecular events contribute to the cellular pathogenesis of individual neurofibromas in NF1.
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RESULTS |
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Clinical features and NF1 mutations
Five dermal and five spinal tumors were examined (Table 1). Most had the histological features of a neurofibroma, but T8 was classified as a schwannoma. An enlarging neurofibroma (T6) obtained from a pediatric donor was possibly malignant, but the pathology report was unavailable for confirmation. Four of the 10 donors (T3, T4, T5 and T6) fulfilled National Institutes of Health (NIH) diagnostic criteria (25), whereas NF1 was excluded in the medical records for two cases (T8 and T10). Although the clinical symptoms for the remaining donors were compatible with NF1 (T1, T2, T7 and T9), the information was insufficient to determine whether they met strict diagnostic criteria.
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The constitutional NF1 mutation was identified in tumor-derived fibroblasts from five donors (Table 1). One entire NF1 allele was deleted in four cases (T1, T2, T3 and T4), which included the only three individuals where large numbers of dermal neurofibromas were specifically noted. In addition to a deletion, T3 fibroblasts carried a single-base-pair substitution in the expressed allele. However, this mutation was probably not pathogenic since isoleucine instead of threonine is found at the same position in Drosophila neurofibromin (GenBank accession no. L26500). These two mutations were also found in skin fibroblasts from the same donor, who died of a malignant sarcoma (26). Two other base-pair substitutions were identified (T6 and T8) that are likely to represent silent coding polymorphisms since there was no evidence for aberrant splicing. The only other pathogenic mutation identified was a stop mutation in exon 30 (T5). No mutations were detected by cDNA sequencing in the five remaining donors, including one who met NIH clinical criteria (T6). Polymorphisms were confirmed by sequencing the relevant exon from genomic DNA, but a random search for mutations by genomic sequencing was not feasible.
NF1 expressed by tumor-derived fibroblasts
NF1 mRNA levels in T1, T2 and T3 fibroblasts represent the amount transcribed from a single allele since these individuals have large constitutional deletions (Fig. 1A). T5 fibroblasts carrying a single stop mutation had similarly low levels, which were markedly lower than those in fibroblasts from normal nerves or from the vestibular schwannoma control (Fig. 1C). NF1 mRNA levels in tumor-derived fibroblasts that expressed two apparently normal copies of the gene varied from either normal (T6 and T8) or reduced (T7, T9 and T10) relative to controls. Except for T6, tumor-derived fibroblasts expressed fibronectin as expected.
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NF1 mRNA from all tumor-derived fibroblasts was translated into protein indistinguishable in molecular weight from normal neurofibromin (Fig. 1B). Despite dramatic differences in mRNA levels, the amount of protein in tumor-derived and normal fibroblasts was fairly similar with two exceptions (Fig. 1C). Even in the fibroblasts with a deletion and a reduction in mRNA levels (T1, T2, T3 and T4), the protein levels were similar to normal controls. However, C1 and T5 fibroblasts had a much lower amount of neurofibromin, which appeared unrelated to mRNA levels.
Characteristics of S100+ cells
S100+ cells predominated (
90%) in 7/10 cultures established under conditions that support the growth of normal human Schwann cells (Fig. 2), but only those from T6 and T8 expanded in culture. While most cells cultured from T3 had the typical bipolar shape of Schwann cells (~80%), many of these were S100–. Furthermore, the S100+ cells from T6, T7 and T8 had unusual morphological features for Schwann cells, and those from T6 were only weakly immunoreactive. The cultures established from T4 (not shown) and T7 contained approximately equal numbers of S100+ cells and contaminating fibroblasts.
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Unexpectedly, the S100+ cells from only four tumors (T1, T5, T9 and T10) were phenotypically similar to normal Schwann cells which express high mRNA levels for myelin P0 (Fig. 1A). Two of these tumors were from individuals that had no evidence of NF1 (Tables 1 and 2). The S100+ cells from five tumors (T2, T3, T6, T7 and T8) were distinctly different from the fibroblast population in NF1 and fibronectin mRNA levels, yet did not express the P0 transcript. Thus, the S100+ cells from only two neurofibromas from NF1 patients (T1 and T5), one dermal and one spinal, were unequivocally identified as Schwann cells.
Absence of NF1 mRNA in S100+ cells
The NF1 transcript was readily detected in Schwann cells from peripheral nerves (Fig. 3A), but the levels were much lower than in normal fibroblasts and were similar to tumor-derived fibroblasts expressing a single allele (Fig. 2B). Because of this disparity, the fibroblast signal obscured that from Schwann cells in a severely contaminated culture (C9). Of the seven tumors that generated highly enriched S100+ cultures, four completely lacked the NF1 transcript (T1, T2, T6 and T8), whereas one had reduced levels (T5) and two had normal or higher levels (T9 and T10). The NF1 mRNA most likely originated from fibroblasts in the cultures from T3 and T7 since the signal was proportional to the degree of contamination (20 and 50%, respectively, from Fig. 2 and additional micrographs). The cell yield from T4 was insufficient for RNA analysis.
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DNA analyses in S100+ cells
Fluorescent in situ hybridization (FISH) demonstrated that one NF1 allele was deleted in the S100+ cells from T1, T2, T3 and T4 as in the corresponding fibroblasts, but none had acquired a second large deletion that could account for the lack of NF1 mRNA (not shown). However, a highly skewed X-inactivation pattern indicated a clonal origin for the neurofibromin-deficient S100+ cells from T1 but not from T2 (not shown). Fibroblasts did not show any skewed X-inactivation. cDNA sequencing to detect more subtle mutations in the S100+ cells was not possible due to the absence of NF1 mRNA or fibroblast contamination, and there was no evidence for loss of heterozygosity (LOH) in the S100+ cells by PCR of CA repeats flanking the NF1 gene or by Southern blotting in the informative cases (not shown).
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DISCUSSION |
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NF1 transcript deficiency
Identifying the defective cell type in the NF1-associated neurofibroma is essential to advancing our understanding of benign tumor formation. Studies of neurofibroma tumor tissue are hampered not only by the size and complexity of the NF1 gene, but also by the presence of functionally normal cells such as perineural cells and mast cells that are recruited into the tumor. By mRNA analyses of enriched cell populations in culture, we found that NF1 was frequently inactivated in the S100+ cells and not the fibroblasts (Table 2). Since all fibroblast populations harbored at least one normal NF1 allele and expressed both NF1 mRNA and protein, we conclude that fibroblasts were not primarily responsible for neurofibroma formation but can not exclude an indirect role for these or other cells. In contrast, the S100+ cells from four neurofibromas completely lacked NF1 mRNA (T1, T2, T6 and T8), and another two appeared deficient after subtracting the contribution of contaminating fibroblasts (T3 and T7). These findings represent the first molecular evidence that the S100+ cells carry an NF1 defect and are primarily responsible for the pathogenesis of neurofibromas.
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Spinal tumors are less common than dermal neurofibromas in NF1 patients and are often asymptomatic (27). These tumors are also associated with variant forms of neurofibromatosis that may or may not be linked to the NF1 locus (28–30). Two of the donors with spinal tumors in this study clearly had NF1 (T5 and T6); however, there was no convincing clinical, genetic or molecular evidence for NF1 involvement in the two other spinal tumors that were classified histologically as neurofibromas (T9 and T10). Moreover, the mRNA analyses indicated that an NF1 defect was responsible for a solitary spinal tumor in a donor who did not meet a single NF1 criterion.
Variations in S100+ cell phenotype
The S100+ cells in neurofibromas are widely assumed to be Schwann cells. While this hypothesis was confirmed in two cases by the co-expression of myelin P0 (Table 2; T1 and T5), the S100+ cells from five of seven cultures with an NF1 transcript deficiency did not express this definitive marker of differentiated Schwann cells (T2, T3, T6, T7 and T8). There are two possible explanations for this finding. First, NF1 may have been inactivated in different neural crest derivatives. Multiple cell types and tumors arising from the neural crest express S100 (14,15), including peripheral neurons, Schwann cells, melanocytes and myofibroblastic cells. The developmental timing of NF1 inactivation could determine which derivative is affected.
It is also possible that the S100+/P0– cells isolated from some neurofibromas were Schwann cells that had de-differentiated. Cell-specific genes, including P0, are transcriptionally repressed by viral and cellular oncogenes (31). However, secondary modifying events appear necessary for P0 repression since NF1 inactivation alone did not disrupt Schwann cell differentiation in Nf1 knockout mice (22) or in two of the neurofibromas (T1 and T5). The likelihood that neurofibromas could be composed of de-differentiated Schwann cells, other neural crest derivatives or their partially transformed counterparts may account for variations in the histological appearance of benign tumors, their growth rate and propensity to become malignant. A more comprehensive evaluation of tumor cell phenotype with an extensive panel of neural crest markers will be necessary to identify the S100+/P0– cells further and determine their degree of differentiation.
Mechanisms of NF1 inactivation
NF1 mutations were identified in five individuals, but we were unable to find the constitutional mutation in three cases with other evidence of an NF1 defect (Table 2; T6, T7 and T8). Mutations in unsequenced coding or non-coding regions of the gene would have escaped detection or mutations may not exist in fibroblasts in cases of somatic mosaicism. Otherwise, methylation or transcriptional repression could have silenced NF1 expression without a mutation (32,33). Sequencing the coding region was likewise deemed the most practical and efficient strategy for identifying second mutations in the S100+ cells, but was unexpectedly precluded by the absence of NF1 mRNA. Apparently, the second event involves either a mutation that destabilizes the transcript or an epigenetic mechanism of gene silencing. Extensive efforts to identify second NF1 mutations in genomic DNA by conventional techniques, including FISH and LOH, were unsuccessful.
Irrespective of the actual molecular mechanism, the cumulative data from three cases with a large constitutional deletion (Table 2; T1, T2 and T3) were consistent with sequential NF1 inactivation as predicted by the two-hit hypothesis. Fibroblasts isolated from these tumors expressed NF1 mRNA and protein, while the absence of NF1 mRNA in the S100+ cells indicated that the second allele was inactivated and that the protein was not expressed. A non-random pattern of X-inactivation in the S100+ cells from T1, which were also P0+, indicated that this dermal neurofibroma formed from the clonal growth of Schwann cells. Not all neurofibromas appear to be clonal (10), and random X-inactivation in an enriched population of S100+ cells from T2 indicates a polyclonal origin for another dermal neurofibroma with a large constitutional deletion.
One of the most fundamental and perplexing questions raised by this study is why S100+ cells are inherently more susceptible to NF1 inactivation while fibroblasts are resistant. Owing to cell-specific differences in proliferative capacity, proficiency of DNA repair or recombinase activity, for example, certain cell types might have a propensity to acquire a second NF1 mutation. An inherent susceptibility to mutations could easily explain a monoclonal tumor of S100+ cells, but non-random mutations would have to occur with a high frequency to explain a polyclonal tumor. Clearly, the status of both NF1 alleles must be determined to understand the underlying mechanism of neurofibroma formation. The evolution of more reliable screening strategies should improve the detection rate of NF1 mutations (34), but our experience indicates that finding mutations in the second NF1 allele may require the analysis of genomic DNA from the S100+ cells.
Contributing pathogenic factors
This study was designed to test the most commonly held hypothesis that neurofibromas arise from the inactivation of the second NF1 allele in a Schwann cell. Analyses of non-NF1 tumors and normal controls demonstrated that it was technically possible to make this determination, and indeed, the data from T1 precisely matched the predicted results for this mechanism of tumor formation. Conversely, we can infer from the neurofibromas in which the results varied from the predicted outcome that the mechanism of tumor formation was more complex than the original hypothesis. The absence of P0 was particularly significant in this respect because it implies that the timing and mechanism of NF1 inactivation may affect cellular pathology.
Mutational events, such as homologous recombination, contiguous gene deletions or cumulative genetic damage, which disrupted genes necessary for phenotypic gene expression, could account for the absence of P0 observed in the S100+ cells from some tumors. While these are difficult issues to address, the extent of the chromosome 17q loss was determined in three cases with a large constitutional deletion (T1 = D13; T3 = C12; T4 = D14) and was found to span the entire NF1 gene and several neighboring genes (35). Although the role of NF1 flanking genes in the maintenance of cellular phenotype is not yet known, contiguous gene deletions are thought to play a role in the clinical variability of NF1. Several studies have found an association between large deletions and the development of an excessive number of dermal neurofibromas at an early age (36–39), and the three individuals with numerous dermal neurofibromas in our study did have large constitutional deletions.
Finally, there were some data from this study to suggest that cell-specific differences in NF1 expression may be relevant to neurofibroma formation. Entire gene deletions markedly reduced the amount of NF1 mRNA in fibroblasts, yet these cells maintained a level of neurofibromin similar to controls. In contrast, the levels of NF1 mRNA in normal Schwann cells were lower than in normal fibroblasts and similar to the levels found in fibroblasts carrying an NF1 mutation (Fig. 3B). Owing to their inherently lower level of NF1 mRNA, Schwann cells may be more severely affected by a stop mutation (T5) or variations in genetic background (C1), which appeared to disrupt protein expression in fibroblasts (Fig. 1C). Furthermore, the translation of NF1 mRNA into protein was a slow and inefficient process in a rat Schwann cell line (40), which would further compromise the ability of certain cells to compensate for a mutation. These findings raise the possibility that factors affecting NF1 mRNA or protein expression could have an impact on the pathogenesis of a mutation. While there is no direct evidence for this type of mechanism, it would be consistent with a polyclonal tumor origin since all cells of a certain type would be affected.
In summary, our cellular analyses support the concept that NF1 gene inactivation is essential for benign tumor formation in NF1, but variations in the phenotype of the defective cells suggest that additional factors are likely to be important in determining the cellular pathology in a given tumor. Future studies aimed at defining these additional factors will have a great impact on our understanding of the molecular pathogenesis of benign tumors in NF1.
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MATERIALS AND METHODS |
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Tissue acquisition
Neurofibromas were obtained from the Department of Neurosurgery or the Cancer Center tissue bank at the University of Michigan, the NIH Tissue Bank at the University of Miami or Washington University Neurofibromatosis Clinic. A vestibular neuroma was obtained to provide a non-NF1 tumor control. Normal peripheral nerves were obtained from non-NF1 patients undergoing dorsal rhizotomy or were removed at autopsy. Tissue received from distant locations was shipped on ice in medium containing 10% fetal bovine serum (FBS) buffered with 15 mM HEPES pH 7.4. All tissue was obtained and used in accordance with NIH and University Institutional Review Board guidelines. Clinical records and pathology reports were reviewed to ascertain whether donors met diagnostic criteria for NF1 (25).
Cell culture
Tissue was digested with 200 U/ml collagenase and 0.8 U/ml dispase (Boehringer Mannheim, Indianapolis, IN), and a portion of the cell suspension was seeded onto uncoated plastic dishes. Highly enriched fibroblast cultures were generated in 3–5 passages in DM (Dulbecco’s modified Eagle’s medium, 4.5 g/l glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2.5 µg/ml amphotericin) with 20% FBS. Fibroblasts were positively identified by immunofluorescent staining with monoclonal antibodies to human fibroblast surface protein (1B10; Sigma, St Louis, MO). The remaining suspension was seeded in Opti-MEM (Gibco BRL, Gaithersburg, MD) onto 10 cm plastic dishes or glass coverslips pre-adsorbed with 10 µg/ml poly-L-lysine (41). After attachment, the cells were maintained in DM containing 10% FBS, 50 ng/ml rhGGF2 (Cambridge NeuroScience, Cambridge, MA), 0.5 µM forskolin and 0.5 mM isobutylmethylxanthine (42). Cells on coverslips were fixed and stained for S100 by indirect immunofluorescence (41) and the percentage of contaminating fibroblasts was determined by counting S100-negative cells under phase and/or fluorescent optics.
Northern hybridization
Total cellular RNA was isolated by acidic phenol extraction with RNAzol B (Tel-Test, Inc., Friendswood, TX). RNA samples (2–10 µg) were size fractionated by electrophoresis in 1.25% agarose gels containing 2 M formaldehyde, transferred to Hybond N nitrocellulose filters by capillary flow and cross-linked to the filter with UV light. cDNA inserts for NF1p5, myelin P0, fibronectin (clone FN771; ATTC, Rockville, MD) and ß-actin were labeled by random priming (DNA labeling kit; Boehringer Mannheim). Two sets of filters were hybridized at 42°C under high-stringency conditions with the same batch of probes and exposed to Kodak XAR film with an intensifying screen at –70°C. The hybridization signal for NF1 and ß-actin was estimated by scanning densitometry (Molecular Dynamics SI, Sunnyvale, CA), and arbitrary optical density units for NF1/ß-actin represent the normalized level of NF1 mRNA.
Western blotting
Cell pellets were lysed in RIPA buffer (50 mM Tris–HCl pH 8, 137 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS) with protease inhibitors (aprotinin, leupeptin and benzamide), and the protein concentration of the clarified supernatant was measured (BioRad protein assay, Hercules, CA). Protein samples (20 µg) were separated by 8% SDS–PAGE and transferred onto nitrocellulose filters. Filters were cut horizontally, guided by Rainbow molecular weight markers (Amersham, Arlington Heights, IL), and the top piece was incubated with the WA15a anti-neurofibromin antibody (43), while the bottom piece was incubated with a rabbit polyclonal anti-
-tubulin antibody (Sigma; diluted 1:1000). Filters were washed and processed for chemiluminescent detection (Amersham).
NF1 sequencing
cDNA was generated from mRNA with M-MLV reverse transcriptase (BRL, Gaithersburg, MD) and used to amplify the NF1 coding region from position 48 to 8464 in eight overlapping PCRs (primers and PCR conditions are available from the authors upon request). Each PCR fragment was sequenced using a solid-phase sequencing protocol. Exons 23a and 48a were sequenced starting from genomic DNA (Purgene DNA isolation kit; Gentra Systems, Minneapolis, MN). Coding polymorphisms in exon 5 and 13 or newly discovered silent mutations were used to check for transcription of both NF1 alleles in the cDNA. Base pair changes detected by RT–PCR were confirmed by sequencing the relevant exon from genomic DNA. Nucleotides and amino acids were numbered according to the sequence provided by the NNFF International NF1 Mutation Analysis Consortium and mutations were deposited in the database (44).
FISH
Fixed interphase cells were prepared on glass slides using standard cytogenetic techniques and two-color FISH was performed to detect large NF1 deletions. Cosmids or P1 clones from the NF1 gene region (cFF13, cP5 and P1-60) were labeled with biotin-11-dUTP by nick translation. Control probes (Oncor, Gaithersburg, MD) bracketing the NF1 locus (D17S379 at 17p13.3 and RARA at 17q21.1) were labeled with digoxigenin-16-ATP. FISH was performed on some specimens after immunofluorescent labeling of S100.
X-chromosome inactivation
The X-inactivation pattern was compared between tumor fibroblasts and S100+ cells isolated from the two female donors carrying a constitutional deletion (Table 1; Id# 0608 and 0720). HpaII-digested and undigested DNA was used as a template for amplification of the (CAG)n repeat in exon 1 of the androgen-receptor gene. Resulting PCR products were separated and quantified on an ALF automated sequencer (Pharmacia, Piscataway, NJ) as described (45).
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ACKNOWLEDGEMENTS |
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We thank the patients who donated tissue, Lisa Baumbach, the University of Miami Tissue Bank (NICHD #NOI-HD-8-3284) and the University of Michigan Cancer Center for assistance with obtaining tissue samples, Miriam Lerner and Andrew Coyle for technical support, David Viskochil, Francis Collins, Don Cleveland and Greg Lemke for generously providing cosmids and plasmids, and M. Marchionni for kindly supplying rhGGF2. This work was supported in part by grants from the NIH (NS33130 and NS08075 to J.L.R. and NS33494 to D.H.G.) and from the FWO-Vlaanderen and the University of Leuven (G.0238.98 and A3255 to E.L.).
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NOTE ADDED IN PROOF |
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Genomic DNA from Schwann cell and fibroblast cultures from the two tumors with no evidence of NF1 involvement (T9 and T10) was sent to Mia MacCollin (MGH Neuroscience Center, Boston, MA) for single-strand conformational polymorphism analysis of the NF2 gene. No deviations from normal patterns were detected in the 17 coding exons of NF2, suggesting that mutations in genes other than NF1 or NF2 give rise to variant forms of neurofibromatosis with benign Schwann cell tumors.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Neurology Research, 502C Abramson Center, 3400 Civic Center Blvd, Philadelphia, PA 19104-4318, USA. Tel: +1 215 590 2406; Fax: +1 215 590 3779; Email: jlr@mail.med.upenn.edu
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