Botulinum toxin A, adjunctive therapy for refractory headaches associated with pericranial muscle tension

A set of neurofibromatosis type 1 (NF1) patients was screened for large NF1 gene deletions by comparing patient and parent genotypes at 10 intragenic polymorphic loci. Of 67 patient/parent sets (47 new mutation patients and 20 familial cases), five (7.5%) showed loss of heterozygosity (LOH), indicative of NF1 gene deletion. These five patients did not have severe NF1 manifestations, mental retardation, or dysmorphic features, in contrast to previous reports of large NF1 deletions. All five deletions were de novo and occurred on the maternal chromosome. However, two patients showed partial LOH, consistent with somatic mosaicism for the deletion, suggesting that mosaicism may be more frequent in NF1 than previously recognised (and may have bearing on clinical severity). We suggest that large NF1 deletions (1) are not always associated with unusual clinical features, (2) tend to occur more frequently on maternal alleles, and (3) are an important mechanism for constitutional and somatic mutations in NF1 patients.     

Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion

A partial physical map has been constructed of the human class Mu glutathione S-transferase genes on chromosome 1p13.3. The glutathione S-transferase genes in this cluster are spaced about 20 kilobase pairs (kb) apart, and arranged as 5'-GSTM4-GSTM2-GSTM1-GSTM5-3'. This map has been used to localize the end points of the polymorphic GSTM1 deletion. The left repeated region is 5 kb downstream from the 3'-end of the GSTM2 gene and 5 kb upstream from the beginning of the GSTM1 gene; the right repeated region is 5 kb downstream from the 3'-end of the GSTM1 and 10 kb upstream from the 5'-end of the GSTM5 gene. The GSTM1-0 deletion produces a novel 7.4-kb HindIII fragment with the loss of 10.3- and 11.4-kb HindIII fragments. The same novel fragment was seen in 13 unrelated individuals (20 null alleles), suggesting that most GSTM1-0 deletions involve recombinations between the same two regions. We have cloned and sequenced the deletion junction that is produced at the GSTM1-null locus; the 5'- and 3'-flanking regions are more than 99% identical to each other and to the deletion junction sequence over 2.3 kb. Because of the high sequence identity between the left repeat, right repeat, and deletion junction regions, the crossing over cannot be localized within the 2.3-kb region. The 2.3-kb repeated region contains a reverse class IV Alu repetitive element near one end of the repeat.     

Mosaicism in sporadic neurofibromatosis 2 patients

More than half of neurofibromatosis 2 (NF2) patients represent de novo mutations which could have occurred at either pre-zygotic or post-zygotic stages. A post-zygotic mutation can result in mosaicism. In four sporadic NF2 patients, we found NF2 mutations in only a portion of corresponding leukocytes. In two other sporadic patients, no mutations were found in leukocytes but constitutional NF2 mutations were suggested by identical mutations in different tumors from each patient. We screened leukocyte DNA from a total of 16 inherited and 91 sporadic NF2 patients, and found NF2 mutations in 13 (81%) of the former and in 46 (51%) of the latter cases. The 30% difference in the rate of detection of mutations ( P = 0.051) might be partially explained by mosaicism in a portion of sporadic NF2 patients who carry the mutations in such a fashion that their leukocytes are unaffected. Among sporadic cases, we found mutations more frequently in patients with severe phenotypes (59%) than in patients with mild phenotypes (23%) (difference of 36%, P = 0.007). Mosaicism might be more common in the latter patient group since small populations of mutation-bearing cells can in some cases result in mild phenotypes and can also lead to difficulties in identifying mutations. No mutations were found in eight patients suspected of having NF2. Mosaicism with an extremely small population of affected cells may explain the incomplete phenotypes in some of these patients and the lack of mutations in their leukocytes. These findings suggest that mosaicism is relatively common in NF2 and may have important implications for diagnosis, prognosis and genetic counseling.     

A study of radiation necrosis and edema in the canine brain using positron emission tomography and magnetic resonance imaging

Evidence derived from sequence comparisons and the genomic organization of the murine Antennapedia-class homeobox gene clusters suggest that they arose from a primordial cluster through a process of gene duplication and divergence followed by cluster duplication. A large chromosomal domain surrounding the ancestral homeobox cluster also appears to have been duplicated and has remained relatively stable since the divergence of humans and rodents. To test the extent of the duplicated chromosomal domain, we have initiated physical mapping studies of the regions surrounding the four murine homeobox clusters using pulsed-field gel electrophoresis and yeast artificial chromosome cloning. In this study, we present a long-range restriction map of mouse chromosome 11 spanning 1500 kb in the region surrounding the Hox-b cluster. We have determined that the gene for the nerve growth factor receptor is tightly linked to the Hox-b complex and is located within 50 kb of the Hox-b 1 gene at the 3' end of the cluster. Four yeast artificial chromosomes have been isolated and characterized by the polymerase chain reaction, long-range restriction mapping, and Southern blotting. Two clones of 150 and 300 kb contain the entire Hox-b cluster and the nerve growth factor receptor gene. A 440-kb clone contains the 3' end of the Hox-b cluster, the nerve growth factor receptor gene, and extends downstream. A 210-kb clone contains the 5' end of the Hox-b cluster and extends upstream. These clones confirm the pulsed-field restriction map of uncloned mouse DNA and represent a contig of approximately 600 kb of cloned material from mouse chromosomes 11.     

Mutations in von Recklinghausen neurofibromatosis: an hypothesis

Von Recklinghausen neurofibromatosis or neurofibromatosis type I (NF1) is a relatively frequent (1/3,000 livebirths) autosomal dominant condition. Some unusual aspects are noted in this disorder: new mutations are frequent and almost all are of paternal origin without parental age effect. The recurrence of NF1 among children of healthy parents is rare as opposed to other dominant disorders. I propose that in NF1 (1) new mutations occur often in somatic cells or in late germinal cells, however, they occur very rarely in early germinal cells leading to germinal mosaicism and (2) the individual with somatic mosaicism presents symptoms of the disease. Therefore, an NF1 patient with an apparent new mutation is often a somatic mosaic for the mutation and if the mosaic is also present in germinal cells some of his children will be affected. This hypothesis may explain the unusual aspects of mutation in NF1.     

A 20.7 kb deletion within the factor VIII gene associated with LINE-1 element insertion

Large deletions within the factor VIII gene account for approximately 5% of the mutations causing haemophilia A. The characterization of such mutations can provide insights into the molecular mechanisms of these and other deletions in man. We have analyzed a 20.7 kb deletion spanning exons 15 to 20 within the factor VIII gene in a patient with severe haemophilia A. Long range PCR was used to investigate the extent of the deletion and to provide a template for sequencing across the deletion breakpoint. A 38-base insertion homologous to the 3' region of a LINE-1 (L1) element was detected at the breakpoint of the deletion. Normal sequence at the 5' breakpoint in intron 14 was homologous to an L1 flanking region and normal sequence at the 3' breakpoint in intron 20 was homologous to an adjacent sequence within the same L1 flanking region. A molecular mechanism for the deletion involving retrotransposition of a readthrough product of an L1 element plus its 3' flanking region is suggested.     

Sj?gren''s syndrome associated with autoimmune hepatitis. A case report

We report on a 4-year-old girl with obesity and hyperphagia whose peripheral blood cytogenetic analysis showed mosaicism for a deletion of band 1p36.33. Terminal 1p deletions are rarely reported and this patient represents the first identified case of mosaicism. Given the subtlety of the cytogenetic abnormality and the possibility of mosaicism, the incidence of such deletions has probably been underestimated. While a characteristic phenotype associated with this karyotypic abnormality was described recently, the present report highlights the additional clinical findings of obesity and hyperphagia and the overlap of manifestations with Prader-Willi syndrome.     

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.     

von Recklinghausen's disease and its pathogenesis

von Recklinghausen's disease was first described in 1882. Formerly, it was considered a single disease, but is now known to be two distinct disease, neurofibromatosis 1 (NF 1, peripheral form of neurofibromatosis) and neurofibromatosis 2 (NF 2, bilateral acoustic neurofibromatosis). Neurofibromatosis is inherited as an autosomal dominant with a high rate of penetrance. NF 1 gene is located in the pericentromeric region of chromosome 17. NF 2 gene is localized to chromosome 22. Clinically, there are some characteristic signs and symptoms. The typical NF 1 patient has café-au-lait spots, melanin pigmentation and palpable neurofibromas, while NF 2 has its onset with the development of tinnitus or hearing loss, due to the presence of bilateral acoustic neuroma.     

The role of p53 gene during the development of human oral malignant lesions: a comparative study of p53 gene mutation with P53 protein positive immunostaining]

We report on a patient with a deletion of 18q23. At both 2 and 4 years of age, she displayed few of the facial features or other clinical features associated with the 18q- syndrome. Fluorescent in situ hybridisation and microsatellite marker and RFLP analysis were performed to characterise the extent of the deletion, and a terminal deletion of 18q23 was confirmed. The deleted region includes the gene for myelin basic protein, suggesting that hemizygosity of this gene does not invariably lead to mental and developmental delay. The clinical presentation of this patient suggests that either she is not deleted for the genes involved in the 18q- clinical phenotype or this patient represents one end of the spectrum of the clinical variability seen with 18q terminal deletions.     

The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11

A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.     

cis-Acting sequences required for simian foamy virus type 1 vectors

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum cis-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial lacZ gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5' long terminal repeat and the start of the gag gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second cis-acting element located at the 3' end of the pol gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important cis-acting elements required for SFV-1 vector construction, and the finding of a cis-acting element in the pol gene is unique among retroviruses.     

Homologous recombination between p53 and its pseudogene in a radiation-induced mouse tumor

Genome recombination is essential for life; however, its dysfunction causes cancer. Here we report the formation of a chimera structure of the p53 gene due to homologous recombination with the p53 pseudogene in tumors produced by repeated local beta-irradiation of the backs of mice. The recombination occurred near the 5' end of exon 5. Because this tumor carried a 5-bp deletion in exon 6 of the expressed p53 allele, and the defect in p53 is reported to elevate the cellular recombination activity, this chimera formation is thought to be initiated by a radiation-induced DNA double strand break in the p53-mutated cell with enhanced recombination. The abundance of this chimera structure was estimated to be 8% of the total of tumor p53, and the functional p53 side of this chimera had no deletion in exon 6. The indication is that the recombination occurred before the loss of heterozygosity of the mutated p53 allele took place but after a few divisions of the original heterozygous p53-mutated cell toward monoclonal expansion. A novel mechanism of cancer induction is suggested.