Exon trap analysis of a NF1 splice-site mutation in a chronic myelomonocytic leukemia patient

We have previously described a patient with chronic myelomonocytic leukemia who exhibited a mutation (del-10:-8) in the splice-acceptor region in front of the FLR exon of the NF1 tumor suppressor gene. In order to evaluate whether this mutation indeed affects correct splicing of this exon we used an exon trap approach. Our data unequivocally prove the functional relevance of this NF1 mutation. Exon trapping thus represents an attractive strategy to study the consequences of putative splice-site mutations if RNA samples are not available.     

Exon scanning of the entire TSC2 gene for germline mutations in 40 unrelated patients with tuberous sclerosis

Tuberous sclerosis complex (TSC) is a dominantly inherited multisystem disorder resulting in the development of hamartomatous growths in many organs. Genetic heterogeneity has been demonstrated linking the familial cases to either TSC1 at 9q34.3, or TSC2 at 16p13.3. About two-thirds of the TSC cases are sporadic and appear to represent new mutations. While both genes are thought to account for all familial cases, with each representing approximately 50% of the mutations, the proportion of sporadic cases with mutations in TSC1 and TSC2 is yet to be determined. We have examined the entire coding sequence of the TSC2 gene in 20 familial and 20 sporadic cases and identified a total of twenty-one mutations representing 50% and 55% of familial and sporadic cases respectively. Our rate of mutation detection is significantly higher than other published reports. Twenty out of 21 mutations are novel and include 6 missense, 6 nonsense, 5 frameshifts, 2 splice alterations, a 34 bp deletion resulting in abnormal splicing, and an 18 bp deletion which maintains the reading frame. The mutations are distributed throughout the coding sequence with no specific hot spots. There is no apparent correlation between mutation type and clinical severity of the disease. Our results document that at least 50% of sporadic cases arise from mutations in the TSC2 gene. The location of the mutations described here, particularly the missense events, should be valuable for further functional analysis of this tumor suppressor protein.     

Allelic status of 1p, 14q, and 22q and NF2 gene mutations in sporadic schwannomas

Watson is a fully developed suburb of some 30 years in Canberra (the capital city of Australia). A plunge dip using arsenical pesticides for tick control was operated there between 1946 and 1960. Chemical investigations revealed that many soil samples obtained from the study area contained levels of arsenic exceeding the current health-based investigation levels of 100 mg kg-1 set by the National Healthy and Medical Research Council in Australia. For the speciation study, nine composite samples of surface and sub-surface soils and a composite samples of rocks were selected. ICP-MS analysis showed that arsenic levels in these samples ranged from 32 to 1597 mg kg-1. Chemical speciation of arsenic showed that the arsenite (trivalent) components were 0.32-56% in the soil and 44.8% in the rock composite samples. Using a rat model, the absolute bioavailability of these contaminated soils relative to As3+ or As5+ ranged from 1.02 to 9.87% and 0.26 to 2.98%, respectively. An attempt was made to develop a suitable leachate test as an index of bioavailability. However, the results indicated that there was no significant correlation between the bioavailability and leachates using neutral pH water or 1M HC1. Our results indicate that speciation is highly significant for the interpretation of bioavailability and risk assessment data; the bioavailability fractions of arsenic in soils from Watson are small and therefore the healthy impact upon the environment and humans due to this element is limited.     

NF2 gene in neurofibromatosis type 2 patients

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (ezrin-radixin-moesin proteins). Even though penetrance of the disease is >95% and no genetic heterogeneity has been described, point mutations in the NF2 gene have been observed in only 34-66% of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients who express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.     

The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.     

Somatic NF2 gene mutations in familial and non-familial vestibular schwannoma

Vestibular schwannoma occurs both as a sporadic tumour and in the dominantly inherited familial cancer syndrome neurofibromatosis type 2 (NF2). The gene for NF2 has recently been isolated on chromosome 22, and the demonstration of inactivating germline mutations in NF2 patients and NF2 associated tumours suggests that it act as a tumour suppressor. We have investigated 85 sporadic and 2 NF2 associated vestibular schwannomas, and one vagal schwannoma for chromosome 22 allele loss and NF2 gene mutations. A further 7 vestibular schwannomas were investigated for NF2 mutations only. Chromosome 22 allele loss was detected in 34 of 87 vestibular schwannomas and in the vagal nerve schwannoma. Six exons of the NF2 gene were investigated by SSCP analysis in all 95 tumours. Somatic NF2 gene mutations were detected in 13 non-familial vestibular schwannomas and in one of the NF2 vestibular schwannomas. Seven non-familial tumours with an NF2 gene mutation also displayed a chromosome 22 allele loss. Thirteen of the mutations were predicted to produce truncation of the NF2 protein. These results suggest that somatic mutations of the NF2 tumour suppressor gene are a critical step in the pathogenesis of both familial and non-familial vestibular schwannoma and that the mechanism of tumourigenesis complies with a 'two-hit' mutation model.     

Exon 5 mutations in the p53 gene in relapsed childhood acute lymphoblastic leukemia

Thirty seven children with relapsed acute lymphoblastic leukemia (ALL), 25 B-lineage and 12 T-lineage, were analyzed for p53 alterations at different stages of the disease. Loss of heterozygosity (LOH) was detected in the relapse phase in three patients. p53 mutations were identified by single strand conformation polymorphism (SSCP) and sequencing analyzes in seven of the 37 ALL patients (19%); three B-lineage (12%) and four T-lineage (33%). Most of the mutations were identified in the relapse phase. In two exceptional cases, one of the mutations was indicated as a germ line and the other was already present at diagnosis. No p53 mutation was identified in any of the other 20 available bone marrow samples obtained at diagnosis. No correlation between the p53 status and clinical outcome could be determined. The majority of the mutations (four out of seven, 57%) were clustered at exon 5. Our data implicate that p53 exon 5 is a frequent site of mutations in relapsed childhood ALL.     

The product of the NF2 tumour suppressor gene localizes near the plasma membrane and is highly expressed in muscle cells

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.     

Multiple schwannomas in the radial nerve

We present a case of multiple schwannomas in the radial nerve. The occurrence of multiple schwannomas in a single major nerve is very rare. Magnetic resonance imaging was useful in detecting the tumours. As schwannomas may be multiple without clinical symptoms, we recommend MR imaging of the entire limb when schwannomas occur in a major nerve in the upper extremity.     

Exon organisation of the mouse gene encoding the Adrenoleukodystrophy related protein (ALDRP)

ALDR is one of the four genes encoding an ATP Binding Cassette (ABC) hemi-transporter of the peroxisomal membrane so far identified in mammalian cells. The best known of these is X-ALD, whose dysfunction has been causally associated with X-linked adrenoleukodystrophy. ALDR and X-ALD protein product are closely related and we show here that this striking conservation is maintained at the genomic level. Although extending to a larger genomic region, the organisation of the mouse ALDR gene mirrors exactly that of X-ALD. This supports further the hypothesis that among the four known peroxisomal ABC hemi-transporters ALDRP is the most likely candidate as a modifier contributing to the phenotypic variability of X-linked adrenoleukodystrophy.     

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.     

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading

To demonstrate regional activation in the rat cerebral cortex related to stress-evoked neuroendocrine response, Fos expression in both the cerebral cortex and hypothalamic paraventricular nucleus (PVN) was immunohistochemically examined in two experimental groups; a lipopolysaccharide (LPS) intraperitoneally injected group for inflammatory stress and a restraint group for emotional stress. The LPS injection (100 microg/100 g b.w.) and restraint (for 30 min) had similar effect on Fos-like immunoreactivity (Fos-LI) in PVN with regard to the number of immunoreactive nuclei and their distribution pattern, while the times to maximize Fos-LI were different. Numerical analysis of cortical Fos-LI in untreated rats showed a distinct region-specific pattern. Statistical analysis revealed no significant increase in Fos-LI density in any cortical regions in the LPS group, but restraint resulted in a dramatic and region-specific increase. A significant increase was detected in the prefrontal cortex (the cingulate, orbital and agranular insular cortex), the frontal area 2, the agranular retrosplenial cortex, the parietal cortex, and the medial and lateral occipital area 2. These results indicate that cortical activation relevant to specific functions may be involved in stress-specific neural circuitry.     

Quantification analysis of splice signal sequences. Exon skipping in beta-globin gene of thalassemia

Nucleotide sequences of human beta-globin and its mutant genes were analyzed by quantification method. In the mutant, a GT-->AT change occurs at the 5'-splice site of the second intron, causing skipping of the whole second exon during RNA splicing. A mechanism of exon skipping was proposed on the basis of the intensity of 3'-splice signal sequence.     

Exon scrambling of MLL transcripts occur commonly and mimic partial genomic duplication of the gene

The aim of this study was to test the hypothesis that prolactin may up- and down-regulate prolactin receptor gene expression in the anterior pituitary gland and hypothalamus respectively. Experiments were carried out in bantams (Gallus domesticus). Comparisons were made of concentrations of PRLR mRNA in the anterior pituitary gland and basal and preoptic hypothalamus in adult males and females held on long days (low vs high plasma prolactin); in 3-week-old juvenile male and females on short days (high vs low plasma prolactin); in 8-week-old juvenile male and females on short days (both low plasma prolactin); in adult laying, incubating, and out-of-lay (high, very high, and low plasma prolactin, respectively); in adult cockerels exposed to long or short days (high vs low prolactin); and in adult hens exposed to long or short days (high vs low prolactin). There was a sex difference in anterior pituitary and basal hypothalamic PRLR mRNA, with lower values in both tissues in females than in males. Compared with laying and out-of-lay hens, anterior pituitary and basal hypothalamic PRLR mRNA concentrations in incubating hens were increased and decreased, respectively. In adult birds of either sex held on long or short days, there was no difference in pituitary PRLR mRNA, while basal hypothalamic PRLR mRNA was lower on short days. PRLR mRNA in the preoptic hypothalamus was not affected by sex, reproductive state, or photoperiod. It is concluded that there is no consistent relationship between plasma prolactin, in the physiological range, and the concentration of PRLR mRNA in the anterior pituitary gland, basal hypothalamus, and preoptic hypothalamus.     

Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.