M. Culver

A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations.

Abstract Overlapping cDNA clones from the translocation break-point region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5′ end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T→C transition changing a leucine to a proline; another NF1 allele harbors a C→T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.

Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus

Three new neurofibromatosis type 1 (NF1) mutations have been detected and characterized. Pulsed-field gel and Southern blot analyses reveal the mutations to be deletions of 190, 40, and 11 kb of DNA. The 11 kb deletion does not contain any of the previously characterized genes that lie between two NF1 translocation breakpoints, but it does include a portion of a rodent/human conserved DNA sequence previously shown to span one of the translocation breakpoints. By screening cDNA libraries with the conserved sequence, we identified a number of cDNA clones from the translocation breakpoint region (TBR), one of which hybridizes to an approximately 11 kb mRNA. The TBR gene crosses at least one of the chromosome 17 translocation breakpoints found in NF1 patients. Furthermore, the newly characterized NF1 deletions remove internal exons of the TBR gene. Although these mutations might act by compromising regulatory elements affecting some other gene, these findings strongly suggest that the TBR gene is the NF1 gene.

The neurofibromatosis type 1 gene encodes a protein related to GAP

cDNA walking and sequencing have extended the open reading frame for the neurofibromatosis type 1 gene (NF1). The new sequence now predicts 2485 amino acids of the NF1 peptide. A 360 residue region of the new peptide shows significant similarity to the known catalytic domains of both human and bovine GAP (GTPase activating protein). A much broader region, centered around this same 360 amino acid sequence, is strikingly similar to the yeast IRA1 product, which has a similar amino acid sequence and functional homology to mammalian GAP. This evidence suggests that NF1 encodes a cytoplasmic GAP-like protein that may be involved in the control of cell growth by interacting with proteins such as the RAS gene product. Mapping of the cDNA clones has confirmed that NF1 spans a t(1;17) translocation mutation and that three active genes lie within an intron of NF1, but in opposite orientation.

The gene encoding the oligodendrocyte-myelin glycoprotein is embedded within the neurofibromatosis type 1 gene.

In the course of efforts to identify the neurofibromatosis type 1 gene (NF1), three genes were found embedded within an intron of NF1. The cDNA sequence of one of these genes (OMGP) encodes oligodendrocyte-myelin glycoprotein. OMGP spans at least 2.7 kb of genomic DNA, and it maps within 4 kb of the breakpoint of a balanced chromosomal translocation carried by an individual with NF1. OMGP is similar in genomic structure to two other expressed genes, EVI2A and EVI2B, which lie approximately 20 and 5 kb telomeric of the OMGP locus, respectively. All three genes have the same transcriptional orientation and are contained within one intron of NF1, which is transcribed off the opposite strand. Whether altered expression of OMGP might play a role in the clinical heterogeneity of NF1 is as yet unclear.

Identification and characterization of transcripts from the neurofibromatosis 1 region: the sequence and genomic structure of EVI2 and mapping of other transcripts.

Mapping of the EVI2 gene between the translocation breakpoints of two patients with neurofibromatosis type 1 (NF1), combined with the likely role of its murine homolog in neoplastic disease, implicates EVI2 as a possible candidate for the NF1 gene. We report here the expression of a 1.6-kb EVI2 transcript in normal human brain and peripheral blood mononuclear cells. Sequencing studies predict an EVI2 protein of 232 amino acids that contains an N-terminal signal peptide, an extracellular domain with five potential glycosylation sites, a single hydrophobic transmembrane domain with a leucine zipper, and a hydrophilic cytoplasmic domain. These features are all well-conserved with respect to the mouse Evi-2 protein and are consistent with the hypothesis that EVI2 is a membrane protein that may complex with itself and/or other proteins within the membrane, perhaps to function as part of a cell-surface receptor. In the course of these studies we have also identified three other transcripts (classes of cDNAs) from the NF1 region. Two of these transcripts map between the NF1 translocation breakpoints; the remaining transcript maps just outside this region.

The Human Homolog of Murine Evi-2 Lies Between Two von Recklinghausen Neurofibromatosis Translocations

Von Recklinghausen neurofibromatosis (NF1) is one of the most common inherited human disorders. The genetic locus that harbors the mutation(s) responsible for NF1 is near the centromere of chromosome 17, within band q11.2. Translocation breakpoints that have been found in this region in two patients with NF1 provide physical landmarks and suggest an approach to identifying the NF1 gene. As part of our exploration of this region, we have mapped the human homolog of a murine gene (Evi-2) implicated in myeloid tumors to a location between the two translocation breakpoints on chromosome 17. Cosmid-walk clones define a 60-kb region between the two NF1 translocation breakpoints. The probable role of Evi-2 in murine neoplastic disease and the map location of the human homolog suggest a potential role for EVI2 in NF1, but no physical rearrangements of this gene locus are apparent in 87 NF1 patients.

Evolutionary and functional mitogenomics associated with the genetic restoration of the Florida panther

Florida panthers are endangered pumas that currently persist in reduced patches of habitat in South Florida, USA. We performed mitogenome reference-based assemblies for most parental lines of the admixed Florida panthers that resulted from the introduction of female Texas pumas into South Florida in 1995. With the addition of 2 puma mitogenomes, we characterized 174 single nucleotide polymorphisms (SNPs) across 12 individuals. We defined 5 haplotypes (Pco1-Pco5), one of which (Pco1) had a geographic origin exclusive to Costa Rica and Panama and was possibly introduced into the Everglades National Park, Florida, prior to 1995. Haplotype Pco2 was native to Florida. Haplotypes Pco3 and Pco4 were exclusive to Texas, whereas haplotype Pco5 had an undetermined geographic origin. Phylogenetic inference suggests that haplotypes Pco1-Pco4 diverged ~202000 (95% HPDI = 83000-345000) years ago and that haplotypes Pco2-Pco4 diverged ~61000 (95% HPDI = 9000-127000) years ago. These results are congruent with a south-to-north continental expansion and with a recent North American colonization by pumas. Furthermore, pumas may have migrated from Texas to Florida no earlier than ~44000 (95% HPDI = 2000-98000) years ago. Synonymous mutations presented a greater mean substitution rate than other mitochondrial functional regions: nonsynonymous mutations, tRNAs, rRNAs, and control region. Similarly, all protein-coding genes were under predominant negative selection constraints. We directly and indirectly assessed the presence of potential deleterious SNPs in the ND2 and ND5 genes in Florida panthers prior to and as a consequence of the introduction of Texas pumas. Screenings for such variants are recommended in extant Florida panthers.

Erratum: A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations (Cell 62, 193-201, 1990; July 13 issue)

[This corrects the article on p. 492 in vol. 41.].