Mosaicism in sporadic neurofibromatosis 2 patients

More than half of neurofibromatosis 2 (NF2) patients represent de novo mutations which could have occurred at either pre-zygotic or post- zygotic stages. A post-zygotic mutation can result in mosaicism. In four sporadic NF2 patients, we found NF2 mutations in only a portion of corresponding leukocytes. In two other sporadic patients, no mutations were found in leukocytes but constitutional NF2 mutations were suggested by identical mutations in different tumors from each patient. We screened leukocyte DNA from a total of 16 inherited and 91 sporadic NF2 patients, and found NF2 mutations in 13 (81%) of the former and in 46 (51%) of the latter cases. The 30% difference in the rate of detection of mutations ( P = 0.051) might be partially explained by mosaicism in a portion of sporadic NF2 patients who carry the mutations in such a fashion that their leukocytes are unaffected. Among sporadic cases, we found mutations more frequently in patients with severe phenotypes (59%) than in patients with mild phenotypes (23%) (difference of 36%, P = 0.007). Mosaicism might be more common in the latter patient group since small populations of mutation-bearing cells can in some cases result in mild phenotypes and can also lead to difficulties in identifying mutations. No mutations were found in eight patients suspected of having NF2. Mosaicism with an extremely small population of affected cells may explain the incomplete phenotypes in some of these patients and the lack of mutations in their leukocytes. These findings suggest that mosaicism is relatively common in NF2 and may have important implications for diagnosis, prognosis and genetic counseling.      

Molecular genetic analysis of chromosome arm 17p and chromosome arm 22q DNA sequences in sporadic pediatric ependymomas

Ependymomas are glial tumors of the brain and spinal cord occurring both sporadically and in a familial syndrome, neurofibromatosis type 2 (NF2). Previous analyses performed on specimens obtained predominantly from adult patients have shown loss of DNA sequences from chromosome arm 22q, which is the location of the NF2 gene. Previously, we documented the consistent loss of chromosome arm 17p DNA in medulloblastoma and astrocytoma, which are the most common brain tumors in children. Although mutation of the TP53 gene located on 17p is the most frequent genetic mutation in all adult tumor types, such mutations are rare in most childhood brain tumors investigated to date. We studied a series of pediatric ependymoma specimens (16 intracranial and 2 spinal) for loss of 17p and 22q DNA sequences and for mutation of the TP53 and NF2 genes. None of the children had the clinical stigmata of NF2. We detected loss of 17p DNA sequences in 9 of the 18 specimens (50%); in 7 of 9 of these specimens (78%), the 144-D6 marker was deleted. In contrast, only 2 of these same 18 specimens (11%) showed loss of 22q DNA. One TP53 gene mutation was detected in a child from a cancer kindred. No mutations were detected in the NF2 gene. Our results suggest that loss of chromosome arm 17p DNA sequences is common in sporadic pediatric ependymomas and that, in contrast to ependymomas in adults, deletion of chromosome arm 22q sequences is rare. Furthermore, TP53 and NF2 gene mutations do not play an important role in the etiology of sporadic pediatric ependymomas. Genes Chromosom Cancer 17:37–44 (1996). © 1996 Wiley-Liss, Inc.     

A variable combination of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene

Signs of neurofibromatosis type 1 (NF1) and Noonan syndrome (NS), two distinct autosomal dominant disorders, occur together in patients reported as Watson syndrome (WS), neurofibromatosis-Noonan syndrome (NFNS), partial LEOPARD syndrome, NS with features of NF1, and NF1 with Noonan-like features. The molecular basis of these combined phenotypes was poorly understood and controversially discussed over several decades. Only recently, there is increasing evidence for WS and NFNS being allelic to NF1 in the majority of patients. In this study we describe seven novel patients from five unrelated families with variable phenotypes of the NF1-NS spectrum which were systematically analyzed for mutations in the disease-causing genes NF1 for NF1 and PTPN11 for NS. Heterozygous mutations or deletions of NF1 were identified in all patients, while no PTPN11 mutation was found. The NF1 mutation segregated with the phenotype in both familial cases. These results support the hypothesis that variable phenotypes of the NF1-NS spectrum represent variants of NF1 in the majority of cases. Constitutive deregulation of the Ras pathway either through activating mutations of PTPN11 or through haploinsufficiency of neurofibromin, which acts as a Ras-inactivating GTP-ase, is probably the common pathogenetic mechanism explaining the phenotypic overlap of NS and NF1.     

Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa

The gene mexR regulates negatively the expression of the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa, and mutations in mexR cause a multiple antibiotic resistance phenotype. Five hundred and forty resistant clones of P. aeruginosa PAO503 were isolated after selection for resistance to chloramphenicol or tetracycline. All isolates showed similar phenotypes and were resistant to tetracycline, chloramphenicol and norfloxacin. Nineteen randomly selected isolates were analyzed. Since mutational analysis by direct sequencing of all regions of interest in several strains is time-consuming and expensive, a screening method, Non-Isotopic RNase Cleavage Assay (NIRCA), was applied to identify mutant genes so that they could be targeted for DNA sequencing. NIRCA is a simple but rapid method for mutational analysis and can be performed in 3-4 h. Results of NIRCA analysis were compared with DNA sequencing. Both NIRCA and DNA sequencing analysis showed mexR gene mutations in 11 of 19 isolates but no alterations in 8 strains. An immunoblot assay showed overexpression of OprN, a component of another multidrug efflux pump, MexE-MexF-OprN, in those eight isolates. Nucleotide sequencing of quinolone resistance-determining regions of DNA gyrase (gyrA) or topoisomerase IV (parC) showed no alterations in any of the 19 mutants. The data indicate that two efflux pump systems, MexA-MexB-OprM and MexE-MexF-OprN, were involved in multidrug resistance including quinolones and that NIRCA is a sensitive method for screening mutations.     

Further genotype – phenotype correlations in neurofibromatosis 2

Selvanathan SK, Shenton A, Ferner R, Wallace AJ, Huson SM, Ramsden RT, Evans DG. Further genotype–phenotype correlations in neurofibromatosis 2. Neurofibromatosis 2 (NF2) is caused by mutations in the NF2 gene predisposing carriers to develop nervous system tumours. Different NF2 mutations result in either loss/reduced protein function or gain of protein function (abnormally behaving mutant allele i.e. truncated protein potentially causing dominant negative effect). We present a comparison between the clinical presentations of patients with mutations that are predicted to produce truncated protein (nonsense/frameshift mutations) to those that results in loss of protein expression (large deletions) to elucidate further genotype–phenotype correlations in NF2. Patients with nonsense/frameshift mutations have a younger age of diagnosis and a higher prevalence/proportion of meningiomas (p = 0.002, p = 0.014), spinal tumours (p = 0.004, p = 0.004) and non‐VIII cranial nerve tumours (p = 0.006, p = 0.003). We also found younger age of diagnosis of vestibular schwannomas (p = 0.007), higher mean numbers of cutaneous lesions (p = 0.003) and spinal tumours (p = 0.006) in these patients. With respect to NF2 symptoms, we found younger age of onset of hearing loss (p = 0.010), tinnitus (p = 0.002), paraesthesiae (p = 0.073), wasting and weakness (p = 0.001) and headaches (p = 0.049) in patients with nonsense/frameshift mutations. Our comparison shows, additional, new correlations between mutations in the NF2 gene and the NF2 disease phenotype, and this further confirms that nonsense/frameshift mutations are associated with more severe NF2 symptoms. Therefore patients with this class of NF2 mutation should be followed up closely.     

Inherited PAX6, NF1 and OTX2 mutations in a child with microphthalmia and aniridia

A girl with aniridia, microphthalmia, microcephaly and café au lait macules was found to have mutations in PAX6, NF1 and OTX2. A novel PAX6 missense mutation (p.R38W) was inherited from her mother whose iris phenotype had not been evident because of ocular neurofibromatosis. Analysis of the NF1 gene in the proband, prompted by the mother's diagnosis and the presence of café au lait spots, revealed a nonsense mutation (p.R192X). Subsequently an OTX2 nonsense mutation (p.Y179X) was identified and shown to be inherited from her father who was initially diagnosed with Leber's congenital amaurosis. Since individual mutations in PAX6, OTX2 or NF1 can cause a variety of severe developmental defects, the proband's phenotype is surprisingly mild. This case shows that patients with complex phenotypes should not be eliminated from subsequent mutation analysis after one or even two mutations are found.     

Phenotypic variability associated with 14 splice-site mutations in the NF2 Gene

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. Am. J. Med. Genet. 77:228–233, 1998. © 1998 Wiley-Liss, Inc.     

Screening for germ-line mutations in the NF2 Gene

Neurofibromatosis type 2 (NF2) is a monogenic dominantly inherited disease that predisposes to the development of tumors of the nervous system, particularly meningiomas and schwannomas. The gene which, when altered, causes NF2, is localized on chromosome 22 and has recently been identified. The NF2 gene is also the site of somatic mutation in tumors, suggesting that it might have a tumor suppressor activity. We here report a screening method for the detection of point mutations in NF2 which takes advantage of denaturing gradient gel electrophoresis (DGGE). This method efficiently screens 95% of the coding sequence and 90% of intron/exon junctions. When applied to 91 unrelated NF2 patients, it enabled the identification of 32 germ-line mutations. Since mutations are found in only one third of the patients, it is expected that mutations or deletions affecting the promoter and/or intronic regions of the NF2 gene occur frequently. The characterized mutations are preferentially located within the 5° half of the gene. Most of them are predicted to lead to the synthesis of a truncated protein. A search for genotype/phenotype correlations showed that, at least in this series of patients, mild manifestations of the disease were associated with mutations which preserve the C-terminal end of the protein.     

Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas

Schwannomatosis is characterized by the onset of multiple intracranial, spinal, or peripheral schwannomas, without involvement of the vestibular nerve, which is instead pathognomonic of neurofibromatosis type 2 (NF2). Recently, a schwannomatosis family with a germline mutation of the SMARCB1 gene on chromosome 22 has been described. We report on the molecular analysis of the SMARCB1 and NF2 genes in a series of 21 patients with schwannomatosis and in eight schwannomatosis-associated tumors from four different patients. A novel germline SMARCB1 mutation was found in one patient; inactivating somatic mutations of NF2, associated with loss of heterozygosity (LOH) of 22q, were found in two schwannomas of this patient. This is the second report of a germline SMARCB1 mutation in patients affected by schwannomatosis and the first report of SMARCB1 mutations associated with somatic NF2 mutations in schwannomatosis-associated tumors. The latter observation suggests that a four-hit mechanism involving the SMARCB1 and NF2 genes may be implicated in schwannomatosis-related tumorigenesis.     

NF1 Molecular Characterization and Neurofibromatosis Type I Genotype–Phenotype Correlation: The French Experience

Neurofibromatosis type 1 (NF1) affects about one in 3,500 people in all ethnic groups. Most NF1 patients have private loss‐of‐function mutations scattered along the NF1 gene. Here, we present an original NF1 investigation strategy and report a comprehensive mutation analysis of 565 unrelated patients from the NF‐France Network. A NF1 mutation was identified in 546 of the 565 patients, giving a mutation detection rate of 97%. The combined cDNA/DNA approach showed that a significant proportion of NF1 missense mutations (30%) were deleterious by affecting pre‐mRNA splicing. Multiplex ligation‐dependent probe amplification allowed the identification of restricted rearrangements that would have been missed if only sequencing or microsatellite analysis had been performed. In four unrelated families, we identified two distinct NF1 mutations within the same family. This fortuitous association points out the need to perform an exhaustive NF1 screening in the case of molecular discordant‐related patients. A genotype–phenotype study was performed in patients harboring a truncating (N = 368), in‐frame splicing (N = 36), or missense (N = 35) mutation. The association analysis of these mutation types with 12 common NF1 clinical features confirmed a weak contribution of the allelic heterogeneity of the NF1 mutation to the NF1 variable expressivity.     

中国人遗传性非腺瘤病性结直肠癌家系hMSH2和hMLH1基因突变分析

目的 分析符合不同临床标准的中国遗传性非腺瘤病性结直肠癌(HNPCC)家系hMSH2和hMLH1基因种系突变状况,评价不同临床标准预示突变检测的敏感性。方法应用DNA直接测序对24个符合Amsterdam标准、15个符合日本标准家系先证者和19个符合Bethesda指导纲要患者(字系中仅1例患者)进行hMSH2和hMLH1基因种系突变检测。对检出突变的家系进行家庭成员的突变筛选。并对检出突变患者进行肿瘤组织突变的检测。结果在16例家系先证者中检测到6个hMSH2突变和11个hMLHl种系突变,其中12个突变是国际上尚未报道过的新突变。突变位于不同外显子中,其中6个突变位于hMLHl第14-16外显子。Amsterdam标准家系突变阳性率为50％(12／24),以日本标准所筛家系突变阳性率为3／15,以上两组家系以外的Bethesda指导纲要患者突变阳性率为1／19。突变类型包括移码突变、无义突变、剪接异常、框架内插入或缺失以及错义突变。基因突变与疾病共分离,检出突变家系先证者的肿瘤组织错配修复基因表现出3种不同基因型：(1)野生型等位基因丢失;(2)肿瘤组织基因型与生殖细胞一致;(3)突变型等位基因丢失。结论中国人HNPCC家系hMSH2和hMLHl突变谱广泛,突变类型多样,hMLHl突变较hMSH2突变多见,突变较为集中于hMLHl外显子14-16。不同临床标准预示突变的敏感性不同。突变基因型与疾病表现型共分离。家系成员中尚未发病的突变携带者应予密切监测。     

Frequent mutations of NF2 and allelic loss from chromosome band 22q12 in malignant mesothelioma: evidence for a two-hit mechanism of NF2 inactivation.

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.     

Mutations of the neurofibromatosis type 2 gene and lack of the gene product in vestibular schwannomas

Schwannomas are common tumors of the nervous system and arefrequently found in patients with neurofibromatosis (NF) 2.Although loss of heterozygosity in NF2 tumors suggests thatthe NF2 gene functions as a tumor suppressor gene, the NF2 geneshows amino acid sequence homology to structural proteins inone of which dominantly acting mutations have been described.We performed a mutational analysis in 30 vestibular schwannomasand examined the effect of mutations on the NF2 protein. Wedetected 18 mutations in 30 vestibular schwannomas of whichseven contained loss or mutation of both NF2 alleles. Most mutationswere predicted to result in a truncated protein. Mutationalhot spots were not identified. Immunocytochemical studies usingantibodies to the NF2 protein showed complete absence of stainingin tumor Schwann cells, whereas staining was observed in normalvestibular nerve. These data indicate that loss of NF2 proteinfunction is a necessary step in schwannoma pathogenesis andthat the NF2 gene functions as a recessive tumor suppressorgene.     

Somatic NF2 gene mutations in familial and non-familial vestibular schwannoma

Vestibular schwannoma occurs both as a sporadic tumour and Inthe dominantly inherited familial cancer syndrome neuroflbromatosistype 2 (NF2). The gene for NF2 has recently been Isolated onchromosome 22, and the demonstration of inactivating germllnemutations In NF2 patients and NF2 associated tumours suggeststhat it act as a tumour suppressor. We have investigated 85sporadic and 2 NF2 associated vestibular schwannomas, and onevagal schwannoma for chromosome 22 allele loss and NF2 genemutations. A further 7 vestibular schwannomas were Investigatedfor NF2 mutations only. Chromosome 22 allele loss was detectedin 34 of 87 vestibular schwannomas and In the vagal nerve schwannoma.Six exons of the NF2 gene were Investigated by SSCP analysisin all 95 tumours. Somatic NF2 gene mutations were detectedIn 13 non-familial vestibular schwannomas. and in one of theNF2 vestibular schwannomas. Seven non-famlllal tumours withan NF2 gene mutation also displayed a chromosome 22 allele loss.Thirteen of the mutations were predicted to produce truncationof the NF2 protein. These results suggest that somatic mutationsof the NF2 tumour suppressor gene are a critical step In thepathogenesis of both famlllal and non-famlllal vestibular schwannomaand that the mechanism of tumourigenesis complles with a ‘two-hit’mutation model.     

Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas

Neurofibromatosis 2 (NF2) is a severe autosomal dominant disorder that predisposes to multiple tumours of the nervous system. About half of all patients are founders with clinically unaffected parents. The purpose of the present study was to examine the extent to which mosaicism is present in NF2 founders. A total of 233 NF2 founders with bilateral vestibular schwannomas (BVS) were screened by exon scanning. NF2 mutations were detected in the blood samples of 122 patients (52%). In 10 of the 122 cases, the ratio of mutant to normal alleles was obviously less than 1, suggesting mosaicism. Tumour specimens were available from 35 of the 111 subjects in whom no mutation could be detected in blood specimens. Mutational analysis by exon scanning detected typical NF2 mutations in 21 of the 35 tumours. In nine subjects, the alterations found in tumours could be confirmed to be the constitutional mutation based on finding of identical mutations in pathologically and/or anatomically distinct second tumours. In six other subjects with only a single tumour available, allelic loss of the NF2 gene was found in addition to the mutation in each tumour, suggesting that either the mutation or the deletion of the NF2 gene is probably the constitutional genetic alteration. Our results suggest that failure to find constitutional mutations in blood specimen from these 15 patients was not because of the limitation of the applied screening technique, but the lack of the mutations in their leucocytes, best explained by mosaicism. Extrapolating the rate (15/35 = 43%) of mosaicism in these 35 cases to the 111 NF2 founders with no constitutional NF2 mutations found in their blood, we inferred 48 mosaic subjects (111 x 0.429). Adding the 10 mosaic cases detected directly in blood specimens, we estimate the rate of mosaicism to be 24.8% (58/233) in our cohort of 233 NF2 founders with bilateral vestibular schwannomas.     

Germline and somatic NF1 gene mutations in plexiform neurofibromas

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.     

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DNA.     

The heterogeneous nature of germline mutations in NF1 patients with malignant peripheral serve sheath tumours (MPNSTs)

Malignant peripheral nerve sheath tumours (MPNSTs) are a major cause of mortality in patients with neurofibromatosis 1 (NF1). We have analysed lymphocyte DNA samples from 30 NF1 patients with MPNSTs to determine their underlying constitutional NF1 gene mutations. Mutations were detected in 27/30 (90%) of these patients. NF1 mutations identified included nonsense, missense, frameshift, splice site mutation and single or multi-exonic deletions and with no obvious clustering of the mutations across the gene. Fourteen of the mutations represent novel gene changes. There did not appear to be any relationship between the mutation type and the level of clinical severity observed. Of the 20 patients with high grade MPNSTs, seven patients had small (<20 bp) and multi-exonic deletions and three had small insertions (<20 bp). Several studies have suggested that NF1 patients with a constitutional 1.5 Mb deletion of the NF1 gene have an increased risk of developing malignant peripheral nerve sheath tumours (MPNSTs). None of our patients had a 1.5 Mb deletion. Larger prospective studies are needed to ascertain whether there is a different spectrum of NF1 mutations in NF1 patients with high grade compared to low grade MPNSTs and of patients with the 1.5Mb deletion, in order to determine the true frequency of MPNST in this sub-group of NF1 patients.     

Comprehensive NF1 screening on cultured Schwann cells from neurofibromas

Neurofibromatosis type 1 (NF1) is mainly characterized by the occurrence of benign peripheral nerve sheath tumors or neurofibromas. Thorough investigation of the somatic mutation spectrum has thus far been hampered by the large size of the NF1 gene and the considerable proportion of NF1 heterozygous cells within the tumors. We developed an improved somatic mutation detection strategy on cultured Schwann cells derived from neurofibromas and investigated 38 tumors from nine NF1 patients. Twenty-nine somatic NF1 lesions were detected which represents the highest NF1 somatic mutation detection rate described so far (76%). Furthermore, our data strongly suggest that the acquired second hit underlies reduced NF1 expression in Schwann cell cultures. Together, these data clearly illustrate that two inactivating NF1 mutations, in a subpopulation of the Schwann cells, are required for neurofibroma formation in NF1 tumorigenesis. The observed somatic mutation spectrum shows that intragenic NF1 mutations (26/29) are most prevalent, particularly frameshift mutations (12/29, 41%). We hypothesize that this mutation signature might reflect slightly reduced DNA repair efficiency as a trigger for NF1 somatic inactivation preceding tumorigenesis. Joint analysis of the current and previously published NF1 mutation data revealed a significant difference in the somatic mutation spectrum in patients with a NF1 microdeletion vs. non-microdeletion patients with respect to the prevalence of loss of heterozygosity events (0/15 vs. 41/81). Differences in somatic inactivation mechanism might therefore exist between NF1 microdeletion patients and the general NF1 population.     

Germline and somatic NF1 gene mutation spectrum in NF1-associated malignant peripheral nerve sheath tumors (MPNSTs)

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.