Lone B. Andersen

cDNA cloning of the type 1 neurofibromatosis gene : Complete sequence of the NF1 gene product

Von Recklinghausen neurofibromatosis, or type 1 neurofibromatosis (NF1), is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the embryonic neural crest. Portions of the gene have been recently identified by positional cloning, and sequence analysis has shown homology to the GTPase activating protein (GAP) family. In this report we present the results of an extensive cDNA walk resulting in the cloning of the complete coding region of the NF1 transcript. Analysis of the sequences reveals an open reading frame of 2818 amino acids, although alternatively spliced products may code for different protein isoforms. The gene extends for approximately 300 kb on chromosome 17, with its promoter in a CpG-rich island.

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

A Yeast Artificial Chromosome Contig Encompassing the Type 1 Neurofibromatosis Gene

The yeast artificial chromosome (YAC) system (Burke et al., 1987, Science 236: 806-812) allows the direct cloning of large regions of the genome. A YAC contig map of approximately 700 kb encompassing the region surrounding the type 1 neurofibromatosis (NF1) locus on 17q11.2 has been constructed. A single YAC containing the entire NF1 locus has been constructed by homologous recombination in yeast. In the process of contig construction a novel method of YAC end rescue has been developed by YAC circularization in yeast and plasmid rescue in bacteria. YACs containing homology to the NF1 region but mapping to another chromosome have also been discovered. Sequences of portions of the homologous locus indicate that this other locus is a nonprocessed pseudogene.

NF1-related locus on chromosome 15

A neurofibromatosis type I (NF1)-related locus has been identified on chromosome 15. It contains a partial copy of the NF1 GAP-related domain, which is known to interact with the ras protooncogenes. However, the chromosome 15 sequence contains multiple deletions resulting in frameshift mutations and stop codons in several highly conserved sequence blocks. The locus on chromosome 15 therefore represents an NF1 pseudogene. This nonprocessed NF1 pseudogene may produce additional fragments in Southern blotting, pulsed-field gel, and PCR experiments with some NF1 cDNA probes or oligonucleotides. In addition, certain regions of the NF1 gene also cross-hybridize with a locus on chromosome 14. These loci must be considered in mutation analysis of patients with NF1 since aberrant findings may not always reflect changes in the NF1 gene.

A 90 kb DNA deletion associated with neurofibromatosis type 1.

A deletion of 90 kb of DNA has been identified in a patient with neurofibromatosis type 1, using pulsed field gel electrophoresis. The deletion lies between probes 17L1A and AC5 in the critical region of chromosome 17 and represents the only molecular alteration found by PFGE in a series of 90 unrelated patients. The subject showing the deletion is an isolated case, shows typical clinical features, and represents one of the first examples of a molecular deletion to be found in this disorder.

Lineage-specific CaN19 expression in human skin: lack of expression in normal melanocytes

Although expression of CaN19 is reduced in cell lines derived from tumors of diverse origin, its expression in their normal counterparts has not been studied in detail. We find that CaN19 mRNA is expressed at very low to undetectable levels in normal melanocytes as well as malignant melanoma cell lines, confirming that CaN19 expression in normal cells is lineage-dependent.

An EcoRI RFLP in the 5' region of the human NF1 gene

Von Recklinghausen neurofibromatosis or type l neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.