Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1.
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Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders in humans and is caused by mutations in the NF1 gene. To date, the majority of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the mRNA level. We have applied a whole NF1 cDNA screening methodology to the study of 80 unrelated NF1 patients and have identified 44 different mutations, 32 being novel, in 52 of these patients. Mutations were detected in 87% of the familial cases, but in 51% of the sporadic ones. At least 15 of the 80 NF1 patients (19%) had recurrent mutations. The study shows that in 50% of the patients in whom the mutations were identified, these resulted in splicing alterations. Most of the splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites. One frameshift, two nonsense and two missense mutations were also responsible for alterations in mRNA splicing. The location and type of mutation within the NF1 gene, and its putative effect at the protein level, do not indicate any relationship to any specific clinical feature of NF1. The high proportion of aberrant spliced transcripts detected in NF1 patients stresses the importance of studying mutations at both the genomic and RNA level. It is possible that part of the clinical variability in NF1 could be due to mutations affecting mRNA splicing, which is the most common molecular defect in NF1. (+info)
NF1 gene and neurofibromatosis 1.
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Neurofibromatosis 1 (NF1), also known as von Recklinghausen disease, is an autosomal dominant condition caused by mutations of the NF1 gene, which is located at chromosome 17q11.2. NF1 is believed to be completely penetrant, but substantial variability in expression of features occurs. Diagnosis of NF1 is based on established clinical criteria. The presentation of many of the clinical features is age dependent. The average life expectancy of patients with NF1 is probably reduced by 10-15 years, and malignancy is the most common cause of death. The prevalence of clinically diagnosed NF1 ranges from 1/2,000 to 1/5,000 in most population-based studies. A wide variety of NF1 mutations has been found in patients with NF1, but no frequently recurring mutation has been identified. Most studies have not found an obvious relation between particular NF1 mutations and the resulting clinical manifestations. The variability of the NF1 phenotype, even in individuals with the same NF1 gene mutation, suggests that other factors are involved in determining the clinical manifestations, but the nature of these factors has not yet been determined. Laboratory testing for NF1 mutations is difficult. A protein truncation test is commercially available, but its sensitivity, specificity, and predictive value have not been established. No general, population-based molecular studies of NF1 mutations have been performed. At this time, it appears that the benefits of population-based screening for clinical features of NF1 would not outweigh the costs of screening. (+info)
NF1 microdeletion syndrome: refined FISH characterization of sporadic and familial deletions with locus-specific probes.
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Two familial and seven sporadic patients with neurofibromatosis 1-who showed dysmorphism, learning disabilities/mental retardation, and additional signs and carried deletions of the NF1 gene-were investigated by use of a two-step FISH approach to characterize the deletions. With FISH of YAC clones belonging to a 7-Mb 17q11.2 contig, we estimated the extension of all of the deletions and identified the genomic regions harboring the breakpoints. Mosaicism accounted for the mild phenotype in two patients. In subsequent FISH experiments, performed with locus-specific probes generated from the same YACs by means of a novel procedure, we identified the smallest region of overlapping (SRO), mapped the deletion breakpoints, and identified the genes that map to each deletion interval. From centromere to telomere, the approximately 0.8-Mb SRO includes sequence-tagged site 64381, the SUPT6H gene (encoding a transcription factor involved in chromatin structure), and NF1. Extending telomerically from the SRO, two additional genes-BLMH, encoding a hydrolase involved in bleomycin resistance, and ACCN1, encoding an amiloride-sensitive cation channel expressed in the CNS-were located in the deleted intervals of seven and three patients, respectively. An apparently common centromeric deletion breakpoint was shared by all of the patients, whereas a different telomeric breakpoint defined a deletion interval of 0.8-3 Mb. There was no apparent correlation between the extent of the deletion and the phenotype. This characterization of gross NF1 deletions provides the premise for addressing correctly any genotype-phenotype correlation in the subset of patients with NF1 deletions. (+info)
A search for evidence of somatic mutations in the NF1 gene.
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Neurofibromatosis type I (NF1) is an autosomal dominant disorder affecting 1 in 3000 people. The NF1 gene is located on chromosome 17q11.2, spans 350 kb of genomic DNA, and contains 60 exons. A major phenotypic feature of the disease is the widespread occurrence of benign dermal and plexiform neurofibromas. Genetic and biochemical data support the hypothesis that NF1 acts as a tumour suppressor gene. Molecular analysis of a number of NF1 specific tumours has shown the inactivation of both NF1 alleles during tumourigenesis, in accordance with Knudson's "two hit" hypothesis. We have studied 82 tumours from 45 NF1 patients. Two separate strategies were used in this study to search for the somatic changes involved in the formation of NF1 tumours. First, evidence of loss of heterozygosity (LOH) of the NF1 gene region was investigated, and, second, a screen for the presence of sequence alterations was conducted on a large panel of DNA derived from matched blood/tumour pairs. In this study, the largest of its kind to date, we found that 12% of the tumours (10/82) exhibited LOH; previous studies have detected LOH in 3-36% of the neurofibromas examined. In addition, an SSCP/HA mutation screen identified five novel NF1 germline and two somatic mutations. In a plexiform neurofibroma from an NF1 patient, mutations in both NF1 alleles have been characterised. (+info)
Evaluation of (18)fluorodeoxyglucose positron emission tomography ((18)FDG PET) in the detection of malignant peripheral nerve sheath tumours arising from within plexiform neurofibromas in neurofibromatosis 1.
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OBJECTIVES: The ability of (18)fluorodeoxyglucose positron emission tomography ((18)FDG PET) to detect malignant change in plexiform neurofibromas from patients with neurofibromatosis 1 (NF1) was evaluated. METHODS: Eighteen NF1 patients who presented with pain, increase in size, or neurological deficit associated with a plexiform neurofibroma were assessed. Magnetic resonance imaging determined the site and extent of the lesion. Qualitative(18)FDG PET was performed and the standard uptake value (SUV) measured the regional glucose metabolism. Histological confirmation of the diagnosis was obtained in 10 patients. RESULTS: Twenty three plexiform neurofibromas were detected in 18 patients. Seven malignant peripheral nerve sheath tumours, four high grade and three low grade tumours, occurred in five patients. In one patient the clinical and radiological characteristics of the tumour suggested malignancy, but histology was inconclusive. Fifteen benign plexiform neurofibromas were identified in 12 patients and these findings were confirmed histologically in five lesions from four patients. Ten plexiform neurofibromas occurring in eight patients were considered benign on(18)FDG PET and the patients did not undergo surgery. They remained stable or their symptoms improved on clinical follow up (median 9 months). The results of qualitative (18)FDG PET were interpreted as indicating that 13 plexiform neurofibromas were benign and 10 were malignant. No malignant tumours were classified as benign, but two benign tumours were reported as malignant. The SUV was calculated for 20 tumours and was significantly higher in five malignant tumours 5.4 (SD 2.4), than in 15 benign tumours 1.54 (SD 0.7), p=0.002. There was an overlap between benign and malignant tumours in the SUV range 2.7-3.3. CONCLUSIONS: (18)FDG PET is helpful in determining malignant change in plexiform neurofibromas in NF1. Increased separation between benign and malignant lesions could be obtained by calculating the SUV at about 200 minutes after injection of (18)FDG, when the peak activity concentration is obtained in malignant tumours. (+info)
Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1.
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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. (+info)
Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.
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More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16. (+info)
Detailed analysis of the oligodendrocyte myelin glycoprotein gene in four patients with neurofibromatosis 1 and primary progressive multiple sclerosis.
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Neurofibromatosis 1 (NF1) is a common autosomal disorder with a wide range of neurological manifestations. The case histories of five patients, including two siblings, are reported who have both neurofibromatosis 1 and primary progressive multiple sclerosis (PPMS). A further patient with both NF1 and PPMS has since been identified. More recently, a systematic clinical review of 138 unselected adult patients with NF1 identified one patient with a slowly progressive spastic paraparesis and multiple high signal hyperintensities on T2 weighted MRI. Molecular genetic studies suggest a mechanism by which the clinical association of progressive white matter disease and NF1 might arise. The gene for NF1 is located on chromosome 17q, spans 350 kb of genomic DNA, and contains 60 exons. The gene for oligodendrocyte myelin glycoprotein (OMgp) is embedded within intron 27b of the NF1 gene. OMgp is a membrane glycoprotein that appears in the human CNS at the time of myelination. It can be detected immunohistochemically on CNS myelin and on the surface of cultured oligodendrocytes. Structurally, OMgp has the potential to function as an adhesion molecule and could contribute to the interactions between the plasma membranes of oligodendrocytes and axons required for myelination and/or axon survival. This study considers the specific hypothesis that PPMS in patients with NF1 results from concurrent mutation of the OMgp gene. The OMgp genes of four unrelated patients with NF1 and PPMS were examined using a combination of Southern blot, dosage polymerase chain reaction, and chemical cleavage of mismatch. The entire OMgp coding sequence, all intronic sequence, the intron-exon boundaries, and 1 kb of flanking sequence were screened. The DNA from two patients was found to contain an alteration in the OMgp gene resulting in an amino acid change of glycine to aspartic acid at codon 21. It is concluded that PPMS in patients with NF1 can occur without concurrent mutation of the OMgp gene. The glycine to aspartic acid polymorphic alteration at codon 21 is neither sufficient nor necessary for the development of PPMS. (+info)