Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.
Hum Mol Genet 2006 Mar 15
PMID:An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. 1646 36

A promising approach for treating Duchenne muscular dystrophy (DMD) is by autologous cell transplantation of myogenic stem cells transduced with a therapeutic expression cassette. Development of this method has been hampered by a low frequency of cellular engraftment, the difficulty of tracing transplanted cells, the rapid loss of autologous cells carrying marker genes that are unable to halt muscle necrosis and the difficulty of stable transfer of a large dystrophin gene into myogenic stem cells. We engineered a 5.7 kb miniDys-GFP fusion gene by replacing the dystrophin C-terminal domain (DeltaCT) with an eGFP coding sequence and removing much of the dystrophin central rod domain (DeltaH2-R19). In a transgenic mdx(4Cv) mouse expressing the miniDys-GFP fusion protein under the control of a skeletal muscle-specific promoter, the green fusion protein localized on the sarcolemma, where it assembled the dystrophin-glycoprotein complex and completely prevented the development of dystrophy in transgenic mdx(4Cv) muscles. When myogenic and other stem cells from these mice were transplanted into mdx(4Cv) recipients, donor cells can be readily identified in skeletal muscle by direct green fluorescence or by using antibodies against GFP or dystrophin. In mdx(4Cv) mice reconstituted with bone marrow cells from the transgenic mice, we monitored engraftment in various muscle groups and found the number of miniDys-GFP(+) fibers increased with time. We suggest that these transgenic mdx(4Cv) mice are highly useful for developing autologous cell therapies for DMD.
Hum Mol Genet 2006 May 15
PMID:A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy. 1659 9

Duchenne muscular dystrophy and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene (Xp21). In two-thirds of DMD/BMD cases, the mutation is a large deletion of one or several exons. We have established PGD for DMD/BMD using interphase fluorescence in situ hybridization (FISH) analysis on single nuclei from blastomeres for the detection of deletions of specific exons in the dystrophin gene. We performed PGD for two carrier females; one had a deletion of exons 45-50 (DMD), and the other had a deletion of exons 45-48 (BMD). An exon 45-specific probe was used in combination with probes for the X and Y centromeres. Using this straightforward approach, we can distinguish affected and unaffected male embryos as well as carrier female and normal female embryos. Three cycles were performed for each patient, which resulted in a pregnancy and the birth of a healthy girl. To the best of our knowledge, this approach for PGD has not been previously reported. The use of interphase FISH is an attractive alternative to sexing or PCR-based mutation detection for PGD patients with known deletions of the dystrophin gene.
Mol Hum Reprod 2006 May
PMID:PGD for dystrophin gene deletions using fluorescence in situ hybridization. 1660 4

Antisense oligonucleotides are emerging as exciting therapeutic agents with the potential to overcome disease-causing mutations in the dystrophin gene. The size and complexity of the dystrophin gene allows for intervention during pre-mRNA processing, where selected exon excision can remove nonsense mutations or restore the reading frame disrupted by genomic deletions or duplications. This review summarizes some of the events leading up to forthcoming clinical trials in 2006, and speculates on some of the challenges facing targeted exon skipping as a therapy for Duchenne muscular dystrophy.
Curr Opin Mol Ther 2006 Apr
PMID:Modification of pre-mRNA processing: application to dystrophin expression. 1661 Jul 65

Dystrophin is the protein whose defect underlies Duchenne Muscular Dystrophy, DMD, a common (1:3500 male births) and fatal condition in which muscle tissue deteriorates leading to death in the second or third decade of life. Dystrophin is coded for by the largest human gene, and one of the most complex. It is translated from at least 7 distinct promoters, with the largest transcripts (which are the ones involved in DMD) containing 79 exons over >2.5 Mbp [K.F. O'Brien, L.M. Kunkel, Dystrophin and muscular dystrophy: past, present, and future, Mol. Genet. Metab. 74 (2001) 75-88, H.M. Sadoulet-Puccio, L.M. Kunkel, Dystrophin and its isoforms, Brain Pathol. 6 (1996) 25-35]. Exacerbating this complexity, it has recently been shown that dystrophin is subject to extensive alternative RNA processing, potentially producing a wide variety dystrophin variants [M. Sironi, R. Cagliani, U. Pozzoli, A. Bardoni, G.P. Comi, R. Giorda, N. Bresolin, The dystrophin gene is alternatively spliced throughout its coding sequence FEBS Lett 517 (2002) 163-166]. The structure of the dystrophin protein is highly modular, with the most common module being a motif termed the spectrin type repeat, or STR, of which there are 24. Each STR is roughly coded for by two exons, and the most common type of multiple exon-skipping events start and end at introns in the middle of STRs [R.G. Roberts, A.J. Coffey, M. Bobrow, D.R. Bentley, Exon structure of the human dystrophin gene Genomics 16 (1993) 536-538, M. Koenig, L.M. Kunkel, Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility, J. Biol. Chem. 265 (1990) 4560-4566]. This would produce fractional STR modules, however, the concept of STRs as proteins domains makes the viability of such fractional motifs questionable. However, certain of these events produce pairs of potentially complementary fractional domain that might reassemble into a hybrid STR motif. We have constructed model fragment corresponding to one such exon-skipping event, and show that the hybrid STR so produced is viable, and furthermore that some of the properties of the protein containing it differ substantially of the native, un-skipped parent.
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PMID:Hybrid spectrin type repeats produced by exon-skipping in dystrophin. 1671 78

Successful gene therapy for Duchenne muscular dystrophy (DMD) requires the restoration of dystrophin protein in skeletal muscles. To achieve this goal, appropriate regulatory elements that impart tissue-specific transgene expression need to be identified. Currently, most muscle-directed gene therapy studies utilize the muscle creatine kinase promoter. We have previously described a muscle enhancer element (mDME-1) derived from the mouse dystrophin gene that increases transcription from the mouse dystrophin muscle promoter. Here, we explore the use of this native mouse dystrophin muscle promoter/enhancer to drive expression of a human dystrophin minigene in transgenic mice. We show that the dystrophin promoter can provide tissue-specific transgene expression and that the mini-dystrophin protein is expressed at the sarcolemma of skeletal muscles from mdx mice, where it restores the dystrophin-associated glycoprotein complex. The level of transgene expression obtained is sufficient to protect mdx muscles from the morphological and physiological symptoms of muscular dystrophy, as well as from exercise-induced damage. Therefore, the dystrophin muscle promoter/enhancer sequence represents an alternative for use in gene therapy vectors for the treatment of DMD.
Mol Ther 2006 Nov
PMID:The mouse dystrophin muscle promoter/enhancer drives expression of mini-dystrophin in transgenic mdx mice and rescues the dystrophy in these mice. 1680 18

Mutations in the dystrophin gene that prevent synthesis of a functional protein lead to Duchenne muscular dystrophy (DMD), the most common serious childhood muscular dystrophy. The major isoform is produced in skeletal muscle and the size of the dystrophin gene and complexity of expression have posed great challenges to the development of a therapy for DMD. Considerable progress has been made in the areas of gene and cell replacement, yet it appears that any potential therapy for DMD is still some years away. Other approaches are being considered, and one that has generated substantial interest over the last few years is induced exon skipping. Antisense oligonucleotides have been used to block abnormal splice sites and force pre-mRNA processing back to the normal patterns. This approach is re-interpreted to address the more common dystrophin mutations, where normal splice sites are targeted to induce abnormal splicing, resulting in specific exon exclusion. Selected exon removal during processing of the dystrophin pre-mRNA can by-pass nonsense mutations or restore a disrupted reading frame arising from genomic deletions or duplications. Attributes of the dystrophin gene that have hampered gene replacement therapy may be regarded as positive features for induced exon skipping, which may be regarded as a form of by-pass surgery at the molecular level. In humans, antisense oligonucleotides have been more generally applied to down-regulate specific gene expression, for the treatment of acquired conditions such as malignancies and viral infections. From interesting in vitro experiments several years ago, the dystrophin exon-skipping field has progressed to the stage of planning for clinical trials.
Prog Mol Subcell Biol 2006
PMID:Redirecting splicing to address dystrophin mutations: molecular by-pass surgery. 1707 69

Duchenne/Becker muscular dystrophies (D/BMD) are X-linked recessive disorders resulting from dystrophin gene mutations. Intragenic recombination in the dystrophin gene occurs with a high frequency. Therefore, determination of the location and frequency of recombination improves D/BMD carrier detection and prenatal diagnosis in families in which the disease-causing mutation cannot be detected by most conventional methods. We describe herein a linkage analysis performed using a fast method based on capillary gel electrophoresis of fluorescent-labeled amplified alleles of 15 intragenic short tandem repeats spanning the entire dystrophin gene. On characterization of recombination events in 93 unrelated D/BMD families from southern Italy, we mapped 25 intragenic recombinations out of 273 informative meioses analyzed. The terminal regions of a gene are notoriously challenging for linkage analysis because some recombination events could be missed in case of lack of informativeness of the outermost markers. Many recombination events (10/25) identified in this study were located at the terminal regions of the dystrophin gene, and some were found by typing of several informative short tandem repeats located in these regions. Moreover, about 24% of the recombination events found in this study mapped to the 3' region of the gene, in contrast with the low frequency (4 to 15%) reported by others.
J Mol Diagn 2007 Feb
PMID:A larger spectrum of intragenic short tandem repeats improves linkage analysis and localization of intragenic recombination detection in the dystrophin gene: an analysis of 93 families from southern Italy. 1725 37

Protein-truncating mutations in the dystrophin gene lead to the most common childhood form of muscle wasting, Duchenne muscular dystrophy. Becker muscular dystrophy, a condition that typically arises from dystrophin gene lesions that do not disrupt the reading frame, clearly indicates that substantial domains of the dystrophin protein are not essential. Potential therapeutic intervention exists during pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frame-shifting mutations from the mature mRNA. Appropriately designed antisense oligonucleotides (AOs), directed at amenable splicing motifs across the dystrophin gene transcript, block exon recognition and/or spliceosome assembly so that targeted exons are removed from the mature mRNA. We describe a panel of AOs designed to induce skipping of every exon within the human dystrophin gene transcript, with the exception of the first and last exons. Every exon targeted in vitro could be removed from the dystrophin mRNA, although some exons are more efficiently excluded than others. No single motif has emerged as a universal AO annealing site for redirection of dystrophin pre-mRNA processing, although the general trend is that the most efficient compounds are directed at motifs in the first half of the target exon.
Mol Ther 2007 Jul
PMID:Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript. 1728 39

The Multiplex Ligation-dependent Probe Amplification assay (MLPA) is the method of choice for the initial mutation screen in the analysis of a large number of genes where partial or total gene deletion is part of the mutation spectrum. Although MLPA dosage probes are usually designed to bind to normal DNA sequence to identify dosage imbalance, point mutation-specific MLPA probes can also be made. Using the dystrophin gene as a model, we have designed two MLPA probe multiplexes that are specific to a number of commonly listed point mutations in the Leiden dystrophin point mutation database (http://www.dmd.nl). The point mutation probes are designed to work simultaneously with two widely used dystrophin MLPA multiplexes, allowing both full dosage analysis and partial point mutation analysis in a single test. This approach may be adapted for other syndromes with well defined common point mutations or polymorphisms.
Mol Biotechnol 2007 Feb
PMID:Simultaneous MLPA-based multiplex point mutation and deletion analysis of the dystrophin gene. 1743 79


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