Astrocyte-specific neurofibromatosis 1 gene deletion is insufficient for astrocytoma formation in mice

To gain insight into the role of the NF1 (Neurofibromatosis type 1) gene during neural development and in tumorigenesis, we have utilized the bacteriophage P1, Cre/loxP system to generate a conditional allele at the NF1 locus (NF1 flox) that permits temporal and spatial ablation of function through Cre-mediated recombination. We have been using these mice to assess the scope of NF1 requirement in distinct cell types. At the center of this approach is to identify the cells that give origin to the tumors most frequently found in NF1 patients: neurofibromas, neurofibrosarcomas, and astrocytomas. We have hypothesized that specific stem cells must lose NF1 by LOH to begin this process. I will discuss the consequences of NF1 loss in neurons, Schwann cells, and neural precursors. Distinct tumor phenotypes appear in each case. In malignant tumors, our mouse models indicate that the p53 pathway must also become mutated to cooperate with loss of NF1. Additionally, we have genetic evidence that the haploin-sufficient state is essential for tumor appearance. These data suggest that profilactic therapies preceding tumor appearance should be considered for NF1. Acknowledgements:  Funded by NINDS, NNFF, and DOD.     

COL1A2 gene deletion polymorphism in an Italian and an Ivorian populations

The COL1A2 gene is one of the two genes encoding for the polypeptides of type I collagen, that represent the major constituent of skin, bone, tendons, ligaments, blood vessels, dentin, and many interstitial tissues. The COL1A2 gene deletion polymorphism has been considered as an informative anthropological marker for describing geographically distinct human populations. Aim of the present study was to investigate the genetic variability at COL1A2 locus in two populations, one belonging to Ouangolodougou (n = 133), a village placed in Northern Ivory Coast, and one belonging to Lecco (n = 70), a village placed in a Northern Italy region called Lombardy. For each sampled population no data are available in literature. We reported, for the first time, the presence of the deleted allele among Ivorians (0.06), confirming the low deletion frequency of this polymorphism found in Sub Saharan Africa by other authors. For Italians, frequency analysis of this gene polymorphism (0.28 for the deleted allele) did not show any significant level of differentiation with respect to other Italian and European populations. The article is published in the original.     

A single base deletion in the SLC45A2 gene in a Bullmastiff with oculocutaneous albinism

Oculocutaneous albinism type 4 (OCA4) in humans and similar phenotypes in many animal species are caused by variants in the SLC45A2 gene, encoding a putative sugar transporter. In dog, two independent SLC45A2 variants are known that cause oculocutaneous albinism in Doberman Pinschers and several small dog breeds respectively. For the present study, we investigated a Bullmastiff with oculocutaneous albinism. The affected dog was highly inbred and resulted from the mating of a sire to its own grandmother. We obtained whole genome sequence data from the affected dog and searched specifically for variants in candidate genes known to cause albinism. We detected a single base deletion in exon 6 of the SLC45A2 gene (NM_001037947.1:c.1287delC) that has not been reported thus far. This deletion is predicted to result in an early premature stop codon. It was confirmed by Sanger sequencing and perfectly co‐segregated with the phenotype in the available family members. We genotyped 174 unrelated dogs from diverse breeds, all of which were homozygous wildtype. We therefore suggest that SLC45A2:c.1287delC causes the observed oculocutaneous albinism in the affected Bullmastiff.     

A single base deletion from the C1-inhibitor gene causes type I hereditary angio-oedema.

RFLP analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a C1-inhibitor gene mutation responsible for type I hereditary angio-oedema (HAE). A single base deletion (C-16698) from the eighth exon of the C1-inhibitor gene alters the reading frame of the exon and generates a premature translation termination codon. This represents the first report of this form of C1-inhibitor gene mutation in type I HAE.     

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.     

A small intraexonic deletion within the dystrophin gene suggests a possible mechanism of mutagenesis

A case of Duchenne muscular dystrophy is described with an unusual mutation consisting of a 17-bp deletion within exon 47 of the dystrophin gene. The sequences on either side of the deletion have a high degree of intrastrand base complementarity. It is hypothesised that the mechanism generating the deletion may have been the formation of a hairpin loop structure in a single strand of DNA followed by enzymatic degradation at unpaired regions within the loop. Received: 4 January 1997 / Revised: 21 January 1997     

Brain tumors predominantly express the neurofibromatosis type 1 gene transcripts containing the 63 base insert in the region coding for GTPase activating protein-related domain.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder. A part of the gene for NF1 was cloned and its deduced protein has a domain functionally related to mammalian ras GTP-ase-activating protein (GAP). Human tissues examined express two types of NF1 mRNAs: an originally identified species of NF1 mRNA (type I) and another one containing the 63 base insert in the region coding for GAP-related domain (type II). However relative levels of both mRNAs seem to change under certain conditions. Human brain expresses type I mRNA predominantly, while type II is preferentially expressed in most primary brain tumors (13/16 tumors analyzed). We suggest that higher levels of type II mRNA may be related to the genesis of brain tumors.     

Linkage disequilibrium in the neurofibromatosis 1 (NF1) region: implications for gene mapping.

To test the usefulness of linkage disequilibrium for gene mapping, we compared physical distances and linkage disequilibrium among eight RFLPs in the neurofibromatosis 1 (NF1) region. Seven of the polymorphisms span most of the NF1 gene, while the remaining polymorphism lies approximately 70 kb 3' to a stop codon in exon 49. By using Centre d'Etude du Polymorphisme Humain (CEPH) kindreds, 91-110 unrelated parents were genotyped. A high degree of disequilibrium is maintained among the seven intragenic polymorphisms (r > .82, P < 10(-7)), even though they are separated by as much as 340 kb. The 3' polymorphism is only 68 kb distal to the next polymorphism, but disequilibrium between the 3' polymorphism and all others is comparatively low (magnitude of 4 < .33, P values .27-.001). This result was replicated in three sets of unrelated kindreds: the Utah CEPH families, the non-Utah CEPH families, and an independent set of NF1 families. Trigenic, quadrigenic, three-locus, and four-locus disequilibrium measures were also estimated. There was little evidence of higher-order linkage disequilibrium. As expected for a disease with multiple mutations, no disequilibrium was observed between the disease gene and any of the RFLPs. The observed pattern of high disequilibrium within the gene and a loss of disequilibrium 3' to the stop codon could have implications for gene mapping studies. These are discussed, and guidelines for linkage disequilibrium studies are suggested.     

Lethal osteogenesis imperfecta congenita and a 300 base pair gene deletion for an alpha 1(I)-like collagen.

Broad boned lethal osteogenesis imperfecta is a severely crippling disease of unknown cause. By means of recombinant DNA technology a 300 base pair deletion in an alpha 1(I)-like collagen gene was detected in six patients and four complete parent-child groups including patients with this disease. One from each set of the patients'' clinically unaffected parents also carried the deletion, implying that affected patients were genetic compounds. The study suggests that prenatal diagnosis should be possible with 100% accuracy in subjects without the deletion and with 50% accuracy in those who possess it (who would be either heterozygous--normal, or affected with the disease).     

A single base deletion in the 5'' noncoding region of Theiler''s virus attenuates neurovirulence.

Viral chimeras have been constructed through in vitro manipulations of the infectious cDNA clones of two prototypes of Theiler's murine encephalomyelitis virus: (i) the virulent GDVII strain and (ii) the less virulent BeAn and VL strains. Previous studies have suggested that the phenotypic differences in virulence between the BeAn and GDVII strains map to both the 5' noncoding and the coat protein regions of these viral genomes. It is shown here that attenuation mapped to the 5' noncoding region is due, at least in part, to an inadvertent deletion resulting from a cloning artifact of one C nucleotide out of four between positions 876 and 879 in the BeAn sequences. The in vitro growth characteristics in BHK-21 cells, however, do not reflect the large differences in neurovirulence between chimeras that are identical except for the deleted C. Another chimera with a mutation at position 877 and a deletion at 976 is also attenuated. The wild-type sequences from the less virulent strains BeAn and VL between nucleotides 1 and 933, in an otherwise GDVII chimera, do not attenuate virulence. Sequences of the 500 nucleotides of the 5' noncoding region proximal to the translation initiation codon were obtained for nine additional Theiler's virus strains. The attenuating deletions are discussed in the context of these sequences and the proposed secondary structures for the 5' noncoding region.     

A refined genetic map of the region of chromosome 17 surrounding the von recklinghausen neurofibromatosis (NF1) gene

The von Recklinghausen neurofibromatosis (NF1) gene has been mapped to the pericentromeric region of chromosome 17. We conducted linkage analyses of NF1 by using 10 polymorphic DNA markers from this chromosomal region. We ascertained 20 American Caucasian NF1 families (163 individuals, 98 NF1 affected) in Michigan and Ohio and also studied a large family ascertained primarily in North Carolina. The following markers were used in this study: HHH202, TH17.19, D17Z1, ERBA1, EW203, EW206, EW207, EW301, CRI-L581, and CRI-L946. NF1 did not recombine with either TH17.19 or HHH202 in any of the informative meioses surveyed (maximum lod scores of 17.04 and 7.21, respectively, at a recombination fraction of .00), indicating that these markers map very close to the NF1 gene. We also report evidence of three instances of recombination between NF1 and the centromeric marker D17Z1 (maximum lod score of 13.43 at a recombination fraction of .04), as well as two crossovers between pairs of marker loci. We find no evidence of locus heterogeneity, and our results support the localization of the NF1 gene to proximal chromosome 17q.     

Novel 12-bp deletion in the coding region of the bovineNPM1 gene affects growth traits

The nucleophosmin 1 gene (NPM1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in the control of various aspects of cell growth and homeostasis. In this study, the coding region of theNPM1 gene was screened in 1035 individuals of 4 Chinese cattle breeds by DNA sequencing and polyacrylamide gel electrophoresis. A novel 12-bp deletion mutation was identified in the coding region of theNPM1 gene. The PCR products of primerNPM1-P2 exhibited 3 genotypes and 2 alleles: 178 bp (denoted asW) and 166 bp (denoted asD). GenotypeDD and alleleD were predominant in the studied populations. Association analysis with growth traits in the Nanyang breed (N = 265) showed that the animals with genotypeDD had significantly greater birth weight, body weight, body length, and heart girth than those with genotypeWD (P < 0.01 orP < 0.05) at birth and after 6 months and 12 months, but not at 18 and 24 months of age. Results of this study suggest that theNPM1 gene is a candidate gene for growth traits in cattle.     

Single base pair germ-line deletion in the p53 gene in a cancer predisposed family

A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Because this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppressor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated denaturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitutional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the formation of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.     

Molecular characterization of an atypical beta-thalassemia caused by a large deletion in the 5'' beta-globin gene region.

We describe a Canadian family of Czechoslovakian descent that came to our attention because of an HbA2 percentage approximately twice that of an average case of heterozygous beta-thalassemia. This unique phenotype suggested to us the possibility of a novel genetic mechanism being responsible for their beta-thalassemia. To investigate this possibility, we mapped, cloned, and sequenced the mutant beta-globin allele. This molecular analysis demonstrated the presence of a unique 4,237 base pair (bp) deletion extending from 3.3 kilobases (kb) 5' of the beta-globin mRNA cap site to approximately the middle of beta IVS-2. This truncated beta-globin gene further extends the heterogeneity of mutations known to cause beta-thalassemia and delineates new sequences involved in nonhomologous recombination events in the beta-globin gene region.