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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

Duchenne and the less severe Becker form of muscular dystrophy (DMD,BMD) result from genetic deficiency in the level and/or activity of the protein dystrophin. The recent availability of cDNA based minigenes encoding recombinant dystrophin polypeptides has raised the possibility of somatic gene transfer as a therapeutic approach to treat dystrophin deficiency. In this respect, the mdx mouse provides a useful model of DMD exhibiting features characteristic of both the early myopathic and later fibrotic phases of the human disease. Using a mutated human cDNA, compatible in size with virus-based somatic gene transfer vectors, the pathophysiological consequences of restoring dystrophin expression have been examined in transgenic mdx mice. Transgene expression was correlated with a marked reduction of the skeletal myofibre necrosis and regeneration which is a major feature of the dystrophin-deficient phenotype in young mdx mice. The cDNA construct which is based on a very mild BMD phenotype thus encodes a highly functional dystrophin molecule whose reduced size renders it an attractive candidate for development as a therapeutic gene transfer reagent.
Hum Mol Genet 1992 Apr
PMID:Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice. 130 Nov 34

The 14kb dystrophin transcript from the Duchenne muscular dystrophy (DMD) locus, which encodes a 427kDa protein, is differentially spliced at the amino terminal end giving rise to alternative transcripts expressed in muscle and brain. Here we present evidence for a 4.8kb transcript from the DMD locus which is ubiquitously expressed but is particularly abundant in Schwannoma cells where dystrophin could not be detected. The hybridisation of Western blots with dystrophin antibodies also identifies a protein of approximately 80kDa of variable abundance in different human and mdx tissues. Immunocytochemistry studies confirm the expression of this protein in nerve cells, a tissue in which full length dystrophin is not detected. Sequencing of the 5' end of a clone isolated from a rat Schwannoma cDNA library, shows that the 4.8kb transcript shares exons with the carboxy terminal end of dystrophin but the 5' untranslated region is not contained within the dystrophin transcript. We propose that the 4.8kb gene product be called apodystrophin-1 as its expression is distinct from the dystrophin 14kb mRNA but it is transcribed from the same locus.
Hum Mol Genet 1992 May
PMID:Characterization of a 4.8kb transcript from the Duchenne muscular dystrophy locus expressed in Schwannoma cells. 130 Nov 45

The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions. We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD. A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands. Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes. Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb. In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion. Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb. This mapping provides the basis for the identification of the AHC and GK genes.
Hum Mol Genet 1992 Nov
PMID:A YAC contig in Xp21 containing the adrenal hypoplasia congenita and glycerol kinase deficiency genes. 130 Nov 66

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

Multiplex polymerase chain reaction was carried out with the material from 68 patients suffering from Duchenne muscular dystrophy in Moscow and Leningrad clinics. Six pairs of oligoprimers were used. Deletions were detected in the material from 22 patients. A new type of deletion was found. Data on deletion frequencies and spectrum were compared with the results published by other authors.
Mol Gen Mikrobiol Virusol
PMID:[Analysis of deletions in the dystrophin gene by the multiplex amplification method in patients suffering from Duchenne's muscular dystrophy]. 145 81

Two prenatal diagnoses were carried out by the technique of intragenic polymorphous marker detecting heterozygosity in pregnant women in the families with cases of Duchenne muscular dystrophy. In both cases the DNA fragment from pERT87-15 region was amplified. This fragment includes a polymorphous site in BamHI region of recognition. DNA analyses of the families members have been made and the genetical risk has been calculated by the Bayes method. The prognoses for both fetuses are good.
Mol Gen Mikrobiol Virusol 1991 Feb
PMID:[Prenatal DNA-diagnosis of Duchenne muscular dystrophy]. 167 55

Both normal and pathological transcripts of tissue-specific genes may be detected by polymerase chain reaction (PCR) amplification in tissues not normally considered to express the gene product. The exploitation of constitutive basal mRNA levels ("ectopic" transcription) would be a major boon to diagnostic medicine since it promises both to simplify the analysis of complex genes and to avoid the requirement for an expressing tissue that is sometimes obtainable only by biopsy. We have demonstrated the feasibility of this novel strategy by characterizing a mutation in the X-chromosomal Duchenne (or Becker) muscular dystrophy (DMD/BMD) gene encoding dystrophin. The massive size of this gene has in the past often hindered carrier detection due to the high frequency of recombination and the high proportion of new mutations. In this study a deletion was identified in both a BMD patient and a heterozygous carrier using only a minimal volume of peripheral blood. Following specifically primed reverse transcription of lymphocyte RNA, the relevant region of the pathological cDNA was PCR-amplified. Sequence analysis indicated an in-frame deletion of exons 45 to 47.
Mol Biol Med 1990 Dec
PMID:Characterization of pathological dystrophin transcripts from the lymphocytes of a muscular dystrophy carrier. 170 53


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