Mutational and functional analysis of the neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20–27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD. Received: 13 July 1996 / Revised: 6 August 1996     

Recurrence of a nonsense mutation in the NF1 gene causing classical neurofibromatosis type 1

Summary The gene responsible for von Recklinghausen neurofibromatosis (NF1) has recently been identified, and several point mutations and deletions have been described. The availability of intron-exon boundaries of several exons of the NF1 gene facilitates the search for mutations in affected patients. We have analysed 38 patients for mutations in exon 4 of the NF1 gene, and found one patient with a CT transition at base position 1087 of the cDNA, changing an arginine codon to a stop codon, at amino acid position 365. Sequencing of other members of the family, including both parents, did not show the mutation, confirming that this mutation is responsible for this sporadic NF1 case. As the mutation described here was previously identified in an independent case by others, this case represents a recurrence of this mutation and suggests that codon 365 might be a hot spot for mutations in the NF1 gene. Thus, a specific search for this mutation should be performed when studying NF1 sporadic or familiar cases for genetic analysis.     

Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations of the NF1 gene, is characterized by multiple neurofibromas, pigmentation anomalies, and a variety of other possible complications, including an increased risk of malignant neoplasias. Tumorigenesis in NF1 is believed to follow the two-hit hypothesis postulated for tumor-suppressor genes. Loss of heterozygosity (LOH) has been shown to occur in NF1-associated malignancies and in benign neurofibromas, but only few of the latter yielded a positive result. Here we describe a systematic approach of searching for somatic inactivation of the NF1 gene in neurofibromas. In the course of these studies, two new intragenic polymorphisms of the NF1 gene, a tetranucleotide repeat and a 21-bp duplication, could be identified. Three tumor-specific point mutations and two LOH events were detected among seven neurofibromas from four different NF1 patients. Our results suggest that small subtle mutations occur with similar frequency to that of LOH in benign neurofibromas and that somatic inactivation of the NF1 gene is a general event in these tumors. The spectrum of somatic mutations occurring in various tumors from individual NF1 patients may contribute to the understanding of variable expressivity of the NF1 phenotype.     

Progress towards identifying the neurofibromatosis (NF1) gene

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder of humans. Linkage analysis has recently mapped the NF1 gene to the proximal long arm of chromosome 17. The identification of two NF1 patients with balanced translocations has now allowed the location of the gene to be narrowed to a few hundred kilobases of chromosome band 17q11.2, using a combination of somatic cell hybrid technology, linking clones and pulsed field gel electrophoresis.     

A novel RsaI polymorphism within intron 39 of the neurofibromatosis type 1 (NF1) gene

Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Analysis of single-strand conformation polymorphisms of exons 10 and 11 of the LDL receptor gene from familial hypercholesterolemia heterozygotes indicated the presence of two mutations, which were characterized by DNA sequencing. One mutation (N466) was a 3-bp deletion in exon 10 that deletes Asn in codon 466. The other (intron 11_+1,GT) was a splice donor mutation at position +1 of intron 11.     

Phenotypic and genetic characterization of novel variant in the NF1 gene underlying neurofibromatosis type 1 in five Chinese families

Science China Life Sciences -     

Characterization and significance of nine novel mutations in exon 16 of the neurofibromatosis type 1 (NF1) gene

Nine novel mutations have been characterized as the result of screening exon 16 of the human NF1 gene in 465 unrelated neurofibromatosis type 1 patients. These lesions include three nonsense and two missense mutations, two deletions, one duplication, and one mutation in the 5′ splice site of intron 16. Although exon 16 is the largest NF1 exon, no mutations have so far been reported in this region. This apparent paucity of lesions may be due either to a reduced functional importance of exon 16 or a screening bias or both. However, consideration of the mutability of exon 16 in comparison with other exons suggests that, at least for single base pair substitutions, no such factors need be invoked. Any previous lack of exon 16 mutations in this category would be explicable in terms of a lower propensity to mutate for codons in this gene region. Received: 1 November 1996 / Revised: 5 December 1996     

A de novo nonsense mutation in exon 28 of the neurofibromatosis type l (NF1) gene

We have screened a total of 105 unrelated patients with neurofibromatosis type l (NF1) for mutations in exon 28 of the NF1 gene using heteroduplex analysis and single strand conformation polymorphism analysis. One novel mutation has been identified and characterised. This mutation involves a 13-bp deletion (AAACTGGCTGAGC or AACTGGCTGAGCA) from base position 5077 (or 5078) to 5089 (or 5090) of the cDNA coding sequence. This alteration leads to a reading frame shift with a premature amber termination signal (TAG) at codon 1694. In addition, there is a change from lysine to threonine at codon 1693. The truncated gene product is estimated to be 1125 amino acid residues shorter than the predicted normal protein (2818 amino acids).     

Two CA/GT repeat polymorphisms in intron 27 of the human neurofibromatosis type 1 (NF1) gene

We describe two polymorphic microsatellites in intron 27 of the neurofibromatosis type l (NF1) gene. The microsatellites consist of TG/AC and AC/TG dinucleotide repeats detecting five and seven alleles and with heterozygosities of 0.46 and 0.72, respectively. These microsatellites are useful tools both for direct and indirect genetic analysis of NF1.     

Close flanking markers for neurofibromatosis type I (NF1).

A genetic linkage study with 16 polymorphic DNA markers spanning the region 17p11-17q24 in 22 NF1 families is presented. Close linkage between NF1 and eight pericentromeric markers (HHH202, EW206, CRI-L946, EW203, EW301, FG2, p17H8, and CRI-L581) has been found, probe HHH202 being the closest marker to NF1. Genetic heterogeneity has been excluded. The study of multiply informative meioses suggests that the probes HHH202 and RW206 are flanking markers for NF1. The most likely order on the basis of multiply informative meioses and multipoint mapping is pter-pA10.41-EW301-cen-HHH202-NF1-EW206-++ +EW207-qter.     

High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene

Detailed analyses of 20 patients with sporadic neurofibromatosis type 1 (NF1) microdeletions revealed an unexpected high frequency of somatic mosaicism (8/20 [40%]). This proportion of mosaic deletions is much higher than previously anticipated. Of these deletions, 16 were identified by a screen of unselected patients with NF1. None of the eight patients with mosaic deletions exhibited the mental retardation and facial dysmorphism usually associated with NF1 microdeletions. Our study demonstrates the importance of a general screening for NF1 deletions, regardless of a special phenotype, because of a high estimated number of otherwise undetected mosaic NF1 microdeletions. In patients with mosaicism, the proportion of cells with the deletion was 91%-100% in peripheral leukocytes but was much lower (51%-80%) in buccal smears or peripheral skin fibroblasts. Therefore, the analysis of other tissues than blood is recommended, to exclude mosaicism with normal cells in patients with NF1 microdeletions. Furthermore, our study reveals breakpoint heterogeneity. The classic 1.4-Mb deletion was found in 13 patients. These type I deletions encompass 14 genes and have breakpoints in the NF1 low-copy repeats. However, we identified a second major type of NF1 microdeletion, which spans 1.2 Mb and affects 13 genes. This type II deletion was found in 8 (38%) of 21 patients and is mediated by recombination between the JJAZ1 gene and its pseudogene. The JJAZ1 gene, which is completely deleted in patients with type I NF1 microdeletions and is disrupted in deletions of type II, is highly expressed in brain structures associated with learning and memory. Thus, its haploinsufficiency might contribute to mental impairment in patients with constitutional NF1 microdeletions. Conspicuously, seven of the eight mosaic deletions are of type II, whereas only one was a classic type I deletion. Therefore, the JJAZ1 gene is a preferred target of strand exchange during mitotic nonallelic homologous recombination. Although type I NF1 microdeletions occur by interchromosomal recombination during meiosis, our findings imply that type II deletions are mediated by intrachromosomal recombination during mitosis. Thus, NF1 microdeletions acquired during mitotic cell divisions differ from those occurring in meiosis and are caused by different mechanisms.     

A novel and very peculiar HincII polymorphism in the 5' region of the human neurofibromatosis type 1 (NF1) gene

We report a HincII polymorphism in the 5' end of the neurofibromatosis type 1 gene (NF1) as detected with a probe made of exons 1 to 4a (nucleotides 2 to 401 of the cDNA). This HincII site is most probably in an intron. Evidence presented suggests the probe reveals not one but two similar polymorphisms.     

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.