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We coincidently detected an atypical deletion of at least 1.3-Mb, encompassing the NF1 tumor suppressor gene and several adjacent genes at an apparent heterozygous level in the blood of a 65-year-old female patient. She had multiple subcutaneous tumors that appeared with a certain similarity of subcutaneous neurofibromas, which, however, was revealed as lipomas by histological examination. Comprehensive and exhaustive clinical and radiological examinations did not detect any neurofibromatosis type 1-related clinical symptoms in the patient. Multiplex ligation-dependent probe amplification detected no or only very low level of the 1.3-Mb NF1 deletion in six lipomas and two skin biopsies. Digital polymerase chain reaction estimated the proportion of cells carrying a heterozygous NF1 deletion at 87% in the blood, and 8%, 10%, 13%, 17%, and 20%, respectively, in the five lipomas investigated by this method, confirming our hypothesis of mosaicism. Our findings suggest that de novo cases of genetic disease are potentially mosaic regardless of finding the mutation at an apparently heterozygous level in the blood and that the possibility of mosaicism should be considered in genotype-phenotype studies and genetic counseling., (© 2020 The Authors. Human Mutation Published by Wiley Periodicals, Inc.)

Neurofibromatosis Type 1 (NF1) is a frequent tumor suppressor gene disorder characterized by multiple benign tumors and high risk of malignancy. Internal tumor burden is a major disease-associated manifestation and can be most adequately assessed by magnetic resonance imaging of the whole body. Approximately 5% of NF1 patients have constitutional large NF1-deletions that are generally associated with more severe clinical manifestations. Here, we investigated whether these deletion patients also have more and/or larger internal tumors by assessing internal tumors and their total volume (exclusive of cutaneous and subcutaneous) in 38 NF1 deletion patients (including eight mosaic cases) and 114 age- and gender-matched NF1 patients without deletions. The incidence of internal tumors was significantly lower in mosaic deletion patients (1/8 = 13%) but did not differ between the 30 nonmosaic deletion patients and the 90 age- and gender-matched NF1 patients without large deletions used as controls. Neither the number nor the total volume of tumors per patient differed significantly between the latter two groups. However, extremely high tumor burden (>3,000 ml) was significantly more frequent among nonmosaic NF1 deletion patients than among NF1 patients without large deletions (13% vs. 1%, P = 0.014). Thus, as a group, patients with NF1 deletions do not exhibit a significantly higher internal tumor burden than NF1 patients without such deletions. However, deletion patients can frequently have extremely large internal tumors and thus demand special attention., (Copyright © 2012 Wiley Periodicals, Inc.)

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients., (© 2011 Wiley Periodicals, Inc.)

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties., (© 2011 Wiley Periodicals, Inc.)

Benign peripheral nerve sheath tumors (PNSTs) are a characteristic feature of neurofibromatosis type I (NF1) patients. NF1 individuals have an 8-13% lifetime risk of developing a malignant PNST (MPNST). Atypical neurofibromas are symptomatic, hypercellular PNSTs, composed of cells with hyperchromatic nuclei in the absence of mitoses. Little is known about the origin and nature of atypical neurofibromas in NF1 patients. In this study, we classified the atypical neurofibromas in the spectrum of NF1-associated PNSTs by analyzing 65 tumor samples from 48 NF1 patients. We compared tumor-specific chromosomal copy number alterations between benign neurofibromas, atypical neurofibromas, and MPNSTs (low-, intermediate-, and high-grade) by karyotyping and microarray-based comparative genome hybridization (aCGH). In 15 benign neurofibromas (4 subcutaneous and 11 plexiform), no copy number alterations were found, except a single event in a plexiform neurofibroma. One highly significant recurrent aberration (15/16) was identified in the atypical neurofibromas, namely a deletion with a minimal overlapping region (MOR) in chromosome band 9p21.3, including CDKN2A and CDKN2B. Copy number loss of the CDKN2A/B gene locus was one of the most common events in the group of MPNSTs, with deletions in low-, intermediate-, and high-grade MPNSTs. In one tumor, we observed a clear transition from a benign-atypical neurofibroma toward an intermediate-grade MPNST, confirmed by both histopathology and aCGH analysis. These data support the hypothesis that atypical neurofibromas are premalignant tumors, with the CDKN2A/B deletion as the first step in the progression toward MPNST., (Copyright © 2011 Wiley-Liss, Inc.)

This study examined the effects of 22 putative splicing mutations in the NF2 gene by means of transcript analysis and information theory based prediction. Fourteen mutations were within the dinucleotide acceptor and donor regions, often referred to as (AG/GT) sequences. Six were outside these dinucleotide regions but within the more broadly defined splicing regions used in the information theory based model. Two others were in introns and outside the broadly defined regions. Transcript analysis revealed exon skipping or activation of one or more cryptic splicing sites for 17 mutations. No alterations were found for the two intronic mutations and for three mutations in the broadly defined splicing regions. Concordance and partial concordance between the calculated predictions and the results of transcript analysis were found for 14 and 6 mutations, respectively. For two mutations, the predicted alteration was not found in the transcripts. Our results demonstrate that the effects of splicing mutations in NF2 are often complex and that information theory based analysis is helpful in elucidating the consequences of these mutations., (Copyright © 2011 Wiley-Liss, Inc.)

The analysis of monozygotic twins (MZ) concordant for neurofibromatosis type 1 (NF1) has indicated that genetic factors exert a major influence on the clinical variability (e.g. the number of café-au-lait spots and/or neurofibromas) evident in this disease. Here, we report on a pair of monozygotic, dichorionic twins who are phenotypically discordant with respect to NF1. Whereas DNA sequence analysis indicated somatic mosaicism for the NF1 nonsense mutation, c.4108C>T (p.Q1370X), in the affected twin II/1, this lesion was apparently absent in his unaffected brother. The observation of heterozygosity for flanking SNP and microsatellite markers rendered it most unlikely that the observed mosaicism with normal cells was due to mutation reversion brought about either by gene conversion or mitotic recombination. Instead, we conclude that the twinning event, which would have taken place within three days post-fertilization, must have preceded the c.4108C>T mutation which is therefore predicted to have occurred during the blastocyst stage, leading to somatic mosaicism with normal cells lacking the mutation. This is the first reported case of monozygotic twins discordant for NF1 in whom mosaicism for a postzygotic NF1 gene mutation has been observed in the affected but not the unaffected twin., (© 2011 Wiley-Liss, Inc.)

Nonallelic homologous recombination (NAHR) is responsible for the recurrent rearrangements that give rise to genomic disorders. Although meiotic NAHR has been investigated in multiple contexts, much less is known about mitotic NAHR despite its importance for tumorigenesis. Because type-2 NF1 microdeletions frequently result from mitotic NAHR, they represent a good model in which to investigate the features of mitotic NAHR. We have used microsatellite analysis and SNP arrays to distinguish between the various alternative recombinational possibilities, thereby ascertaining that 17 of 18 type-2 NF1 deletions, with breakpoints in the SUZ12 gene and its highly homologous pseudogene, originated via intrachromosomal recombination. This high proportion of intrachromosomal NAHR causing somatic type-2 NF1 deletions contrasts with the interchromosomal origin of germline type-1 NF1 microdeletions, whose breakpoints are located within the NF1-REPs (low-copy repeats located adjacent to the SUZ12 sequences). Further, meiotic NAHR causing type-1 NF1 deletions occurs within recombination hotspots characterized by high GC-content and DNA duplex stability, whereas the type-2 breakpoints associated with the mitotic NAHR events investigated here do not cluster within hotspots and are located within regions of significantly lower GC-content and DNA stability. Our findings therefore point to fundamental mechanistic differences between the determinants of mitotic and meiotic NAHR., (Hum Mutat 31:1163-1173, 2010. © 2010 Wiley-Liss, Inc.)

Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1-REPs B and C. The breakpoints of these approximately 1.0-Mb ("type-3") NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109-bp, respectively, were identified within the LRRC37B-P paralogues of NF1-REPs B and C, and were found to contain sequences capable of non-B DNA formation. Both LCRs contain LRRC37-core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37-containing LCRs at 17q21.31 is known to have mediated the 970-kb polymorphic inversions of the MAPT-locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37-containing LCRs in the human genome.

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type-2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type-2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type-2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs >or=500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type-2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR-mediated somatic type-2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type-2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences., ((c) 2010 Wiley-Liss, Inc.)

In this study, the breakpoints of six large intragenic deletions in the NF2 gene are determined, which had initially been detected by multiplex ligation-dependent probe amplification. While one breakpoint occurred within an exon, the remaining 11 lied in the corresponding flanking introns. Two of the deletions were most likely caused by nonallelic homologous recombination between Alu sequences, while the other four appeared to be the result of nonhomologous endjoining, possibly facilitated by rearrangement-promoting elements at the junctions in some cases. The clinical features of patients with large intragenic deletions and individuals with mutations affecting single or multiple nucleotides of the NF2 gene are relatively similar. However, patients with deletions of the 3' exons 15 and 16 of the NF2 gene did exhibit milder phenotypes, especially with respect to the age of disease onset.

Neurofibromatosis Type I (NF1) is an autosomal dominant disorder characterized by the development of both benign and malignant tumors. The lifetime risk for developing a malignant peripheral nerve sheath tumor (MPNST) in NF1 patients is approximately 10% with poor survival rates. To date, the molecular basis of MPNST development remains unclear. Here, we report the first genome-wide and high-resolution analysis of DNA copy number alterations in MPNST using the 32K bacterial artificial chromosome microarray on a series of 24 MPNSTs and three neurofibroma samples. In the benign neurofibromas, apart from loss of one copy of the NF1 gene and copy number polymorphisms, no other changes were found. The profiles of malignant samples, however, revealed specific loss of chromosomal regions including 1p35-33, 1p21, 9p21.3, 10q25, 11q22-23, 17q11, and 20p12.2 as well as gain of 1q25, 3p26, 3q13, 5p12, 5q11.2-q14, 5q21-23, 5q31-33, 6p23-p21, 6p12, 6q15, 6q23-q24, 7p22, 7p14-p13, 7q21, 7q36, 8q22-q24, 14q22, and 17q21-q25. Copy number gains were more frequent than deletions in the MPNST samples (62% vs. 38%). The genes resident within common regions of gain were NEDL1 (7p14), AP3B1 (5q14.1), and CUL1 (7q36.1) and these were identified in >63% MPNSTs. The most frequently deleted locus encompassed CDKN2A, CDKN2B, and MTAP genes on 9p21.3 (33% cases). These genes have previously been implicated in other cancer conditions and therefore, should be considered for their therapeutic, prognostic, and diagnostic relevance in NF1 tumorigenesis., ((c) 2009 Wiley-Liss, Inc.)

Neurofibromatosis type 1 (NF1), a common autosomal dominant neurogenetic disorder affecting 1 in 4000 individuals worldwide, results from functional inactivation of the 17q11.2-located NF1 gene. Plexiform neurofibroma (PNF) is a congenital benign tumour present in 30-50% of NF1 patients, which in about 10-15% of cases, can develop into a malignant peripheral nerve sheath tumour (MPNST). This study aimed to characterise the NF1 germline and somatic mutations associated with such tumours by DNA analysis in 51 PNFs resected from 44 unrelated NF1 patients. Germline mutations were identified in 35 patients, of which 21 were novel. Somatic NF1 mutations were found in 29 PNF DNAs, which included 9 point mutations, 5 being novel, and 20 tumour DNA samples exhibiting, either loss of heterozygosity (LOH) of the NF1 gene region (16 tumours), or complete or partial NF1 gene deletions analyzed by multiplex ligation-dependent probe amplification (MPLA) analysis. The type of NF1 germline mutations detected in patients with PNF were similar to those detected in most NF1 patients. LOH of the NF1 gene region, as identified by marker analysis and/or MLPA, was detected in only 20/29 (69%) PNFs, compared to the >90% LOH previously found in MPNST. This systematic analysis of the NF1 germline and somatic mutations associated with PNF development suggest that in most such tumours neither the NF1 somatic mutation type, nor its gene location, is influenced by the underlying NF1 germline mutation. Evidence for LOH involving the TP53 gene identified in the PNFs is also reported for the first time.

Telomerase activity (TA) and the expression of its enzymatic subunits, which have been demonstrated in many tumors, remain poorly investigated in tumors associated with neurofibromatosis type 1 (NF1). In this study, we analysed the association of TA and the expression of telomerase RNA (TR) and telomerase reverse transcriptase (TERT) in 23 malignant peripheral nerve sheath tumors (MPNST) (17 high grade and 6 low grade tumors), 11 plexiform neurofibromas (PNF) and 6 dermal neurofibromas (DNF). TA was studied using telomerase repeat amplification protocol (TRAP) assay and expression of TR and TERT was investigated using reverse transcription PCR (RT-PCR) and real-time PCR. TA was detected in 14 out of 17 (82%) high grade MPNST, whereas all 6 low grade MPNST and 17 benign tumors were telomerase negative. The TERT transcripts were detected in all high grade MPNST, 50% of the low grade MPNST, and 4 benign tumors. However, the expression level of the TERT strikingly correlated with TA and high grade MPNST. Thus, while TERT expression was similar in both low grade MPNST and PNF (P = 0.115), it was significantly higher in high grade MPNST when compared to either low grade MPNST (P = 0.042), PNF (P = 0.001) or DNF tumors (P = 0.010). These findings indicate that TA and expression level of TERT are potential markers for high grade malignancy in NF1 patients., ((c) 2007 Wiley-Liss, Inc.)

About 10% of neurofibromatosis type 1 (NF1) patients develop malignant peripheral nerve sheath tumors (MPNSTs) and represent considerable patient morbidity and mortality. Elucidation of the genetic mechanisms by which inherited and acquired NF1 disease gene variants lead to MPNST development is important. A study was undertaken to identify the constitutional and somatic NF1 mutations in 34 MPNSTs from 27 NF1 patients. The NF1 germline mutations identified in 22 lymphocytes DNA from these patients included seven novel mutations and a large 1.4-Mb deletion. The NF1 germline mutation spectrum was similar to that previously identified in adult NF1 patients without MPNST. Somatic NF1 mutations were identified in tumor DNA from 31 out of 34 MPNSTs, of which 28 were large genomic deletions. The high prevalence (>90%) of such deletions in MPNST contrast with the =or<20% found in benign neurofibromas and is indicative of the involvement of different mutational mechanisms in these tumors. Coinactivation of the TP53 gene by deletion, or by point mutation along with NF1 gene inactivation, is known to exacerbate disease symptoms in NF1, therefore TP53 gene inactivation was screened. DNA from 20 tumors showed evidence for loss of heterozygosity (LOH) across the TP53 region in 11 samples, with novel TP53 point mutations in four tumors., ((c) 2007 Wiley-Liss, Inc.)

Neurofibromatosis 2 (NF2) is a genetic disorder caused by mutational inactivation of the NF2 gene and is characterized by bilateral vestibular schwannomas, spinal tumors, and other benign tumors of the nervous system. Previously, we found intragenic NF2 mutations in 99 of 188 unrelated NF2 patients by exon-scanning-based methods. Tumor analysis of 22 de novo NF2 patients led to the identification of 12 additional constitutive NF2 mutations. The remaining 77 patients were further examined for large alterations using the newly developed gene dosage assay multiplex ligation-dependent probe amplification (MLPA). One deletion of a single exon, seven deletions of multiple exons, seven deletions involving the 3' or 5' end of the NF2 gene, four deletions involving the whole NF2 gene, and one duplication of three exons were detected. For 47 of the 77 patients, mRNA of adequate quality could be obtained, enabling transcript analysis, which confirmed eight alterations detected by MLPA. In addition, in one family, the mRNA analysis detected an insertion of two exons of another gene. Thus, deletions, duplications, and insertions affecting the NF2 gene were found in 21 cases, which is 11% of the 188 unrelated NF2 patients studied, 16% of the 132 mutations identified, and 27% of the 77 cases in which no intragenic small mutations were detected by exon scanning. The combination of multiple screening techniques facilitated a mutation-detection rate of 100% for the 21 inherited cases in this study., ((c) 2005 Wiley-Liss, Inc.)

A total of 500 unselected unrelated neurofibromatosis 1 (NF1) patients were screened for deletions of the NF1 gene. After excluding 67 patients with known intragenic NF1 mutations, the remaining 433 were genotyped using six intragenic and one distal microsatellite marker for the NF1 gene. A total of 28 patients were hemi- or homozygous for all seven markers and were thus considered as candidates for NF1 deletion with a calculated probability of 99.99%. Metaphase or interphase cells were available from 23 of these 28 individuals for molecular cytogenetics. Fluorescence in situ hybridization (FISH) confirmed an NF1 deletion in 22 (96%) of the 23 patients. Thus, a constitutional deletion of the NF1 gene is responsible for the disease phenotype in at least 4.4% of the 500 unselected NF1 patients. Genotyping using multiple microsatellite markers may provide a simple, inexpensive, and efficient strategy for screening deletions of the NF1 gene, and can as well be applied for other large genes., (Copyright 2004 Wiley-Liss, Inc.)

Neurofibromatosis type 1 (NF1) patients have 10% of lifetime risk for developing malignant peripheral nerve sheath tumors (MPNST), one of the most aggressive cancers. We examined the spectrum of constitutional NF1 mutations among 24 NF1 patients with MPNST. We found mutations in 18 patients: four megabase deletions involving the NF1 gene, 13 truncating mutations, and only one missense mutation. One deletion included both exonic and intronic sequences. No typical splicing mutation was found. Five of these mutations were novel: c.3686delA, c.197&#95;204+9del17, c.3044T>C (p.Leu1015Pro), c.2497delT, and c.6020&#95;6027dup. The proportion of megabase deletions of the NF1 gene found in patients with MPNST (17%=4/24) was higher than that in a group of unselected NF1 patients (5.4%=27/500)., (Copyright 2003 Wiley-Liss, Inc.)

Measuring correlation in clinical traits among relatives is important to our understanding of the causes of variable expressivity in Mendelian diseases. Random effects models are widely used to estimate intrafamilial correlations, but such models have limitations. We incorporated survival techniques into a random effects model so that it can be used to estimate intrafamilial correlations in continuous variables with right censoring, such as age at onset. We also describe a negative-binomial gamma mixture model to determine intrafamilial correlations of discrete (e.g., count) data. We demonstrate the utility of these methods by analyzing intrafamilial correlations among patients with neurofibromatosis 2 (NF2), an autosomal-dominant disease caused by mutations of the NF2 tumor-suppressor gene. We estimated intrafamilial correlations in age at first symptom of NF2, age at onset of hearing loss, and number of intracranial meningiomas in 390 NF2 nonprobands from 153 unrelated families. A significant intrafamilial correlation was observed for each of the three features: age at onset (0.35; 95% confidence interval (CI) 0.23-0.47), age at onset of hearing loss (0.51; 95% CI, 0.35-0.64), and number of meninginomas (0.29; 95% CI, 0.15-0.43). Significant correlations were also observed for age at first symptom within NF2 families with truncating mutations (0.41; 95% CI, 0.06-0.68) or splice-site mutations (0.29; 95% CI, 0.03-0.51), for age at onset of hearing loss within families with missense mutations (0.67; 95% CI, 0.18-0.89), and for number of meningiomas within families with splice-site mutations (0.39; 95% CI, 0.13-0.66). Our findings are consistent with effects of both allelic and nonallelic familial factors on the clinical variability of NF2., (Copyright 2002 Wiley-Liss, Inc.)

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder caused by genetic alterations of the NF1 gene on 17q11.2. About 30% of NF1 patients develop plexiform neurofibromas (PNFs), which often cause severe clinical deficits. To determine whether there is a certain genotype underlying PNFs or subtypes of PNFs, we screened 42 NF1 patients from 41 families with PNFs for mutations in the NF1 gene. In 33 out of the 41 (80%) unrelated patients NF1 mutations were found, 24 are novel while the other 9 have been described in previous studies. The 33 mutations included 23 nonsense and frameshift, six splice and four missense mutations. The tumors in these patients had various sizes and features/growth characteristics. No correlation was found between the type or location of the NF1 mutations and size, location or feature of the PNFs, suggesting that many types of NF1 mutations can lead to development of PNFs., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2001 Wiley-Liss, Inc.)

Neurofibromas, the hallmark of neurofibromatosis 1, are composed mainly of Schwann cells and fibroblasts. Inactivation of both NF1 alleles is the cause of these benign tumors, but it is unknown which cell type is the progenitor. In this study, we selectively cultured Schwann cells from an NF1-associated neurofibroma. Fibroblasts were also obtained by culturing the tumor cells under standard conditions. Using four intragenic markers, we genotyped the NF1 locus in the original tumor and in the derived Schwann cells and fibroblasts. Loss of heterozygosity for two informative markers, which indicates loss of one NF1 allele, was found in Schwann cells but not in fibroblasts. This result suggests that genetic alterations of the NF1 gene in Schwann cells are responsible for the development of neurofibromas.

Previous experimental observations indicate that inhibition of voltage-dependent K+ currents suppresses proliferation of normal Schwann cells. In the present study we tested the opposite relationship, i.e., whether Schwann cells from tumors with abnormally high rates of proliferation would have an increase in membrane K+ currents. Whole-cell membrane currents were studied in cultured cells from schwannomas of two neurofibromatosis type 2 (NF2) patients (n = 53), one patient with a sporadic schwannoma (n = 22), and two control subjects (n = 41). Five different types of voltage-dependent membrane currents were found in all of the Schwann cells tested. Membrane depolarization activated outward K+ and Cl- currents; quinidine was found to block the K+ current (IC50 approximately 1 microM), and NPPB reduced the Cl- current. Ba2+-sensitive inward rectifier K+ currents, fast Na+ currents, and a transient, inactivating K+ current were less frequently observed. On average, NF2 cells were found to have statistically significant higher membrane potential and larger non-inactivating K+ outward current as compared to controls. Electrophysiological parameters of Schwann cells from a sporadic schwannoma showed a tendency for larger outward currents; however, the difference did not reach statistical significance. Together the data support the suggestion of a possible link between K+ outward current and proliferation of Schwann cells.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder caused by mutations in the NF2 gene. Patients carrying NF2 mutations are predisposed to cerebral and spinal tumors with bilateral vestibular schwannomas as the hallmark. Using single strand conformation polymorphism and temperature gradient gel electrophoresis analysis, we have screened 87 unrelated NF2 patients for mutations in the NF2 gene. In this study, we report phenotypes associated with 14 splice-site mutations carried by 14 propositi and 11 relatives. The mutations were distributed in exons 2, 3, 5, 7, 8, 14, and 15. These splice-site mutations were associated with various phenotypes, from severe to asymptomatic. Phenotypic variation was also observed within families. Mutations downstream from exon 8 resulted more often in mild phenotypes. No meningiomas were found in any of 13 affected or mutation bearing individuals from three families with splice-site mutations of exons 14 and 15. These data suggest that splice-site alteration is a relatively common cause of NF2, and that unlike other mutations the clinical outcomes of splice-site mutations in the NF2 gene are variable. These results add to the growing body of information on genotype-phenotype correlation in NF2.