Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.
Hum Mol Genet 1992 Apr
PMID:Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. 130 Nov 31

Cognitive impairment occurs in one-third of patients with Duchenne muscular dystrophy, a lethal X-linked, recessive disease caused by mutations in the dystrophin gene which is expressed in both brain and muscle, the two transcripts having alternative first exons. Previous reports have indicated that the 'brain-type' dystrophin transcript predominates in brain. Using in situ hybridisation with antisense oligonucleotides, expression of four distinct mRNAs in specific brain areas is demonstrated here; the 14 kb muscle-type and brain-type transcripts were found to coexist in cortical and hippocampal neurons and two new transcripts have been identified in dentate gyrus and cerebellar Purkinje neurons, respectively. The latter has a novel first exon which was isolated and sequenced from mouse and human, and which would encode a protein with a different amino-terminus from the known muscle- and brain-type isoforms. Mapping in human located this exon in a large intron between the muscle-type promoter and second exon of the dystrophin gene. This finding of four alternative transcripts regulated by different promoters in brain reveals a new complexity to dystrophin expression that may have important insights for mental retardation mechanisms.
Hum Mol Genet 1992 Oct
PMID:Expression of four alternative dystrophin transcripts in brain regions regulated by different promoters. 130 51

A combination of multiplex PCR with the single strand conformation polymorphism (SSCP) technique was employed to screen for point mutations in the human dystrophin gene. Co-amplification of 11 exons from genomic DNA of Duchenne and Becker muscular dystrophy (DMD/BMD) patients with no deletion or duplication was performed and the samples subjected to multiple SSCP analysis. We report the case of a nonsense mutation in a Duchenne patient identified by this approach. The mutation introduces a termination codon within exon 8 of the dystrophin gene. It is predicted to cause a very premature translational termination accounting for the severe phenotype observed. The patient inherited this mutation from his mother. In addition the analysis revealed 5 polymorphisms useful for internal control.
Hum Mol Genet 1992 Oct
PMID:Detection of a nonsense mutation in the dystrophin gene by multiple SSCP. 130 53

Genetic mapping has indicated that meiotic recombination occurs about 4 time more frequently in the dystrophin gene than expected on the basis of its length. To detect where recombinations occur within the gene, we have studied the CEPH families panel using highly polymorphic microsatellite markers located at the ends of the gene or flanking the major deletion hot spot in intron 44. We found a major hot spot of recombination between markers STR44 and STR50(1), i.e., between exons 44 and 51. Within this hot spot, a peak of recombination was located in the large intron 44. A second minor recombination prone region was found between DXS 206, (XJ, in the large intron 7) and the 5' end of the DMD gene. The distribution of the recombination events in the gene of healthy individuals was very similar to that of deletion breakpoints in DMD/BMD patients, suggesting that the two phenomenon may share a common mechanism. These results should also improve efficiency and accuracy of linkage analysis applied to carrier detection and prenatal diagnosis. In particular, if markers located at the very 3' end of the gene are not informative, the highly polymorphic ones located between exons 50 and 60 can be used instead of presently available extragenic markers, with a very low risk of diagnostic error due to recombination.
Hum Mol Genet 1992 Nov
PMID:Two hot spots of recombination in the DMD gene correlate with the deletion prone regions. 136 82

The 33 patients suffering from the Duchenne muscular dystrophy (DMD), 7 healthy donors and a DMD risk family were studied by means of polymerase multiplex chain reaction (MPCR) with 6 oligoprimer pairs for 6 different exons of dystrophin gene. The deletions varying in sizes from 1 to 6 exons were detected in 12 out of 33 DMD patients studied (36.3%). The prenatal diagnosis of DMD was carried out by chorionic villus biopsy on the 1st trimester of pregnancy. Contrary to earlier findings, in elder brother with sever DMD manifestation, no visible deletion was detected in the DNA sample from the male foetus and thus the diagnosis of DMD in foetus was rejected. The perspectives of MPCR in pre and postnatal diagnosis of DMD are discussed.
Mol Gen Mikrobiol Virusol 1991 Sep
PMID:[Analysis of deletion mutations of the dystrophin gene by the multiplex polymerase chain reaction method in the diagnosis of Duchenne muscular dystrophy]. 174 68

We developed a general method of quantifying relative copy numbers of specific DNA sequences based on the theoretical accumulation of polymerase chain reaction (PCR) products when two DNA sequences are amplified together (co-amplified). Our experiments illustrate the development and theory of the technique. The precision of our estimates is demonstrated by statistical confidence intervals. Tests for effects introduced by experimental factors were performed. The precision of the technique was established by examining the relative gene dosage of the X-linked dystrophin gene in human genomic DNAs from a male, a normal female, a 47,XXX female, and a 48,XXXX cell line. The sensitivity was sufficient to distinguish three copies of the gene from four copies; equivalent to detecting loss of heterozygosity in half the cells of a tumour. Confidence intervals allowed us to reject the hypothesis that there was no difference between DNA samples. Four sample pairs would be required to demonstrate relative gene dosage ratios of 2.0 to 1.0; eight sample pairs would be required to demonstrate a relative gene dosage ratio of 1.3 to 1.0. This method should be useful in detecting gene amplification and deletion in a variety of situations.
Mol Cell Probes 1991 Aug
PMID:Precise gene dosage determination by polymerase chain reaction: theory, methodology, and statistical approach. 179 51

The protein dystrophin is absent in muscles of patients with Duchenne muscular dystrophy (DMD) as well as in mdx mice. The mdx mouse diaphragm closely resembles the human DMD phenotype and should serve as an appropriate model for future studies of dystrophin gene replacement. In this regard, recombinant adenovirus (AV) holds great promise as a vector for delivering a functional dystrophin gene to muscle. However, the use of AV is hampered by the development of an immune response against transduced cells, resulting in short-lived transgene expression as well as possible adverse effects on organ function. In the present study, sensitive reporter genes were employed to determine the efficiency and functional consequences of AV-mediated gene transfer to the diaphragm in both normal and mdx adult mice. One week after direct intramuscular injection of AV into the diaphragm, the level of transgene expression was significantly increased in mdx compared with normal diaphragms. In addition, small-caliber fibers (< 500 microns2) demonstrated preferential transduction in both groups of mice. Normal diaphragms receiving AV exhibited a substantial reduction in maximal twitch and tetanic force generation, whereas no significant effect on diaphragm contractility was found in the mdx group at 1 wk after injection. At 1 mo after AV administration, however, there was a significant decrease in force production by both normal and mdx diaphragms. Immunosuppression with cyclosporine A over 1 mo did not augment the level of transgene expression, but a beneficial effect on diaphragm force-generating capacity was observed in both groups of animals. We conclude the following: (1) short-term transduction of the diaphragm is more efficient in mdx than in normal mice; (2) AV leads to reduced force production by the diaphragm, with this effect being more pronounced in normal than in mdx in the early (but not the late) postinjection period; and (3) immunosuppressive therapy with cyclosporine has a partially protective effect on muscle function after AV administration, which is apparently unrelated to sparing of transduced fibers from elimination by the host immune system. These findings have important implications for the application of AV-mediated dystrophin gene transfer to the treatment of DMD.
Am J Respir Cell Mol Biol 1995 Nov
PMID:Efficiency and functional consequences of adenovirus-mediated in vivo gene transfer to normal and dystrophic (mdx) mouse diaphragm. 757 85

Duchenne and Becker muscular dystrophy are caused by defects in the dystrophin gene, and are candidates for treatment by gene therapy. We have shown previously that overexpression of a full-length dystrophin cDNA prevents the development of dystrophic symptoms in mdx mice. We show here that this functional correction can be achieved by expressing the full-length muscle isoform at a lower level than is present in control animals. Gene therapy for DMD may necessitate the use of truncated dystrophin mini-genes to accommodate the limited cloning capacity of current-generation viral delivery vectors. We have constructed both murine and human mini-genes deleted for exons 17-48, and have demonstrated that expression of either mini-gene can almost completely prevent the development of dystrophic symptoms in transgenic mdx mice. These results suggest that viral-mediated expression of moderate levels of a truncated dystrophin could be an effective treatment for DMD.
Hum Mol Genet 1995 Aug
PMID:Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice. 758 61

A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence.
J Mol Evol 1995 Feb
PMID:Phylogenetic relationships among transposon-like elements in human and primate DNA. 769 19

We have identified a 7.5 kb transcript from the dystrophin locus which encodes a novel 140 kDa protein (Dp140). Based on immunoblotting Dp140 consists of the distal rod domain and C-terminus of 427 kDa dystrophin and is found throughout the CNS. This protein is transcribed from an alternative promoter in the dystrophin locus upstream to exon 45. The unique 5' first exon is conserved between rat and human. The transcript has a 1 kb 5' untranslated region, and the first methionine initiation codon occurs in exon 51, predicting a protein of 140 kDa. Several studies report that Duchenne dystrophy patients with deletions in the exon 45-52 region have an increased incidence of cognitive impairment. Such deletions would affect expression of 427 kDa dystrophin and this shorter 140 kDa isoform but not the recently described small distal transcripts Dp116 or Dp71, suggesting particular importance to CNS function.
Hum Mol Genet 1995 Mar
PMID:Dp140: a novel 140 kDa CNS transcript from the dystrophin locus. 779 84


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