Duchenne Muscular Dystrophy Gene Product Research Articles

Duchenne muscular dystrophy gene expression is an independent prognostic marker for IDH mutant low-grade glioma

Alterations in the expression of the Duchenne muscular dystrophy (DMD) gene have been associated with the development, progression and survival outcomes of numerous cancers including tumours of the central nervous system. We undertook a detailed bioinformatic analysis of low-grade glioma (LGG) bulk RNAseq data to characterise the association between DMD expression and LGG survival outcomes. High DMD expression was significantly associated with poor survival in LGG with a difference in median overall survival between high and low DMD groups of over 7 years (P = < 0.0001). In a multivariate model, DMD expression remained significant (P = 0.02) and was an independent prognostic marker for LGG. The effect of DMD expression on overall survival was only apparent in isocitrate dehydrogenase (IDH) mutant cases where non-1p/19q co-deleted LGG patients could be further stratified into high/low DMD groups. Patients in the high DMD group had a median overall survival time almost halve that of the low DMD group. The expression of the individual DMD gene products Dp71, Dp71ab and Dp427m were also significantly associated with overall survival in LGG which have differential biological effects relevant to the pathogenesis of LGG. Differential gene expression and pathway analysis identifies dysregulated biological processes relating to ribosome biogenesis, synaptic signalling, neurodevelopment, morphogenesis and immune pathways. Genes spanning almost the entirety of chromosome 1p are upregulated in patients with high overall DMD, Dp71 and Dp427m expression which worsens survival outcomes for these patients. We confirmed dystrophin protein is variably expressed in LGG tumour tissue by immunohistochemistry and, overall, demonstrate that DMD expression has potential utility as an independent prognostic marker which can further stratify IDH mutant LGG to identify those at risk of poor survival. This knowledge may improve risk stratification and management of LGG.

Specific Dystrophins Selectively Associate with Inhibitory and Excitatory Synapses of the Mouse Cerebellum and their Loss Alters Expression of P2X7 Purinoceptors and Pro-Inflammatory Mediators

Duchenne muscular dystrophy (DMD) patients, having mutations of the DMD gene, present with a range of neuropsychiatric disorders, in addition to the quintessential muscle pathology. The neurobiological basis remains poorly understood because the contributions of different DMD gene products (dystrophins) to the different neural networks underlying such symptoms are yet to be fully characterised. While full-length dystrophin clusters in inhibitory synapses, with inhibitory neurotransmitter receptors, the precise subcellular expression of truncated DMD gene products with excitatory synapses remains unresolved. Furthermore, inflammation, involving P2X purinoceptor 7 (P2RX7) accompanies DMD muscle pathology, yet any association with brain dystrophins is yet to be established. The aim of this study was to investigate the comparative expression of different dystrophins, alongside ionotropic glutamate receptors and P2RX7s, within the cerebellar circuitry known to express different dystrophin isoforms. Immunoreactivity for truncated DMD gene products was targeted to Purkinje cell (PC) distal dendrites adjacent to, or overlapping with, signal for GluA1, GluA4, GluN2A, and GluD2 receptor subunits. P2X7R immunoreactivity was located in Bergmann glia profiles adjacent to PC-dystrophin immunoreactivity. Ablation of all DMD gene products coincided with decreased mRNA expression for Gria2, Gria3, and Grin2a and increased GluD2 immunoreactivity. Finally, dystrophin-null mice showed decreased brain mRNA expression of P2rx7 and several inflammatory mediators. The data suggest that PCs target different dystrophin isoforms to molecularly and functionally distinct populations of synapses. In contrast to muscle, dystrophinopathy in brain leads to the dampening of the local immune system.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46–55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46–55 deletion.

Dystrophin Dp71, a novel tumor suppressor?

Dystrophins belong to a group of proteins encoding by the Duchenne muscular dystrophy ( DMD ) gene. DMD gene generates full-length dystrophin (427 kDa) and different C-terminal truncated isoforms through alternative usage of internal promoters. Short isoforms are termed in accordance with their molecular mass as Dp140, Dp116, Dp71 and Dp40. Dystrophin Dp71, is ubiquitously expresses, with the exception of skeletal muscle, and is the predominant DMD gene product in nervous system. In addition, Dp71 undergoes alternative splicing of exons 71 and/or 78 to generate different splicing variants, being Dp71f (−71/−78) and Dp71d (−71/+78) the most studied isoforms. While deficiency of dystrophin causes muscle degeneration, absence of Dp71 has been implicated in cognitive impairment and abnormal retinal physiology, two well-defined non-muscular alterations of DMD patients, reviewed in (1). As dystrophin does, Dp71 associates with sarcoglycans, dystroglycans, syntrophins and dystrobrevin, to conform the plasma membrane-associated dystrophin associated protein complex. Such protein assembly provides stability to the plasma membrane and modulates cell signaling. Interestingly, Dp71 undergoes nuclear import trough recognition of an atypical nuclear localization signal by the importin α2/β1 system. Nuclear Dp71 in turn, is involved in the maintenance of nuclear architecture, via its interaction with nuclear envelope proteins, including emerin and lamins A/C and B1 (2). Thus, it appears that Dp71 is a multifunctional protein that interacts with different partners in both the cytoplasm and nucleus, which enables it to modulate a variety of cell functions.

Altered astrocyte morphology and vascular development in dystrophin-Dp71-null mice.

Understanding retinal vascular development is crucial because many retinal vascular diseases such as diabetic retinopathy (in adults) or retinopathy of prematurity (in children) are among the leading causes of blindness. Given the localization of the protein Dp71 around the retinal vessels in adult mice and its role in maintaining retinal homeostasis, the aim of this study was to determine if Dp71 was involved in astrocyte and vascular development regulation. An experimental study in mouse retinas was conducted. Using a dual immunolabeling with antibodies to Dp71 and anti-GFAP for astrocytes on retinal sections and isolated astrocytes, it was found that Dp71 was expressed in wild-type (WT) mouse astrocytes from early developmental stages to adult stage. In Dp71-null mice, a reduction in GFAP-immunopositive astrocytes was observed as early as postnatal day 6 (P6) compared with WT mice. Using real-time PCR, it was showed that Dp71 mRNA was stable between P1 and P6, in parallel with post-natal vascular development. Regarding morphology in Dp71-null and WT mice, a significant decrease in overall astrocyte process number in Dp71-null retinas at P6 to adult age was found. Using fluorescence-conjugated isolectin Griffonia simplicifolia on whole mount retinas, subsequent delay of developing vascular network at the same age in Dp71-null mice was found. An evidence that the Dystrophin Dp71, a membrane-associated cytoskeletal protein and one of the smaller Duchenne muscular dystrophy gene products, regulates astrocyte morphology and density and is associated with subsequent normal blood vessel development was provided.

Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein

Even though the Duchenne muscular dystrophy (DMD) gene product Dystrophin Dp71d is involved in various key cellular processes through its role as a scaffold for structural and signalling proteins at the plasma membrane as well as the nuclear envelope, its subcellular trafficking is poorly understood. Here we map the nuclear import and export signals of Dp71d by truncation and point mutant analysis, showing for the first time that Dp71d shuttles between the nucleus and cytoplasm mediated by the conventional nuclear transporters, importin (IMP) α/β and the exportin CRM1. Binding was confirmed in cells using pull-downs, while in vitro binding assays showed direct, high affinity (apparent dissociation coefficient of c. 0.25nM) binding of Dp71d to IMPα/β. Interestingly, treatment of cells with the microtubule depolymerizing reagent nocodazole or the dynein inhibitor EHNA both decreased Dp71d nuclear localization, implying that Dp71d nuclear import may be facilitated by microtubules and the motor protein dynein. The role of Dp71d in the nucleus appears to relate in part to interaction with the nuclear envelope protein emerin, and maintenance of the integrity of the nuclear architecture. The clear implication is that Dp71d's previously unrecognised nuclear transport properties likely contribute to various, important physiological roles.

Reevaluation of Dystrophin Localization in the Mouse Retina

PURPOSE. The roles of dystrophins in retinal physiology remain elusive. The lack of proper clustering of the potassium channel Kir4.1 and of the aquaporin AQP4 was proposed to be the basis of the ERG abnormality observed in many Duchenne muscular dystrophy (DMD) patients. However, the electroretinogram of Dp71-null mice, in which this clustering is disrupted, shows only a moderate reduction of the b-wave with no change in the implicit times. Additionally, the deficit in color discrimination found in DMD patients is hard to explain through the known expression of DMD gene products. The authors thus decided to reexamine their distribution in the mouse retina. METHODS. Messenger RNA distribution was assessed by PCR coupled to laser microdissection of the outer and inner nuclear layers and by in situ hybridization for Dp427. Mouse retinas were double labeled for dystrophins versus presynaptic and postsynaptic proteins or antibodies specific for Dp427 or Dp427+Dp260. RESULTS. Messengers for Dp427, Dp260, and Dp140 were present in the inner nuclear layer. Dp427 mRNA was further detected in bipolar cells and in some amacrine cells by in situ hybridization. Comparative labeling in wild-type and mdx(5Cv) retinas (lacking Dp427) indicated a differential distribution of Dp427 and Dp260 between rod and cone terminals. CONCLUSIONS. In addition to their localization in photoreceptor terminals, Dp427, Dp260, and Dp140 are expressed in inner nuclear layer neurons, notably in bipolar cells for Dp427. Dp427 was proportionally more expressed in cone- than in rod-associated synapses compared with Dp260.

A deficit of brain dystrophin 71 impairs hypothalamic osmostat

Patients with Duchenne muscular dystrophy (DMD) and mdx mice, devoid of dystrophin proteins, show altered ionic homeostasis. To clarify dystrophin's involvement in the central control of osmotic stimuli, we investigated the effect of the disruption of Dp71, the major form of dystrophin in the brain, on the hypothalamoneurohypophysis system (HNHS) osmoregulatory response. Dp71 and Dp140 are the principal DMD gene products in the supraoptic nucleus (SON) and neurohypophysis (NH). They are present in astrocyte and pituicyte end-feet, suggesting involvement in both intrinsic osmosensitivity of the SON and vasopressin (AVP) release from the NH. In Dp71-null mice, the cellular distribution of Dp140 was modified, this protein being detected on the membrane of magnocellular soma. The plasma osmolality of Dp71-null mice was lower than that of wild-type mice under normal conditions, and this difference was maintained after salt loading, indicating a change in the set point for osmoregulation in the absence of Dp71. The increase in AVP levels detected in the SON and NH of the wild-type was not observed in Dp71-null mice following salt loading, and the increase in AVP mRNA levels in the SON was smaller in Dp71-null than in wild-type mice. This suggests that Dp71 may be involved in the functional activity of the HNHS. Its astrocyte end-feet localization emphasizes the importance of neuronal-vascular-glial interactions for the central detection of osmolality. In the SON, Dp71 may be involved in osmosensitivity and definition of the "osmostat," whereas, in the neurohypophysis, it may be involved in fine-tuning AVP release.

Molecular cloning and protein expression of Duchenne muscular dystrophy gene products in porcine retina

Due to the difference between rodent and human retinal circuitry, we characterize a new animal model of retinal perturbation in neurotransmission in Duchenne Muscular Dystrophy (DMD) patients. We investigated the expression and localization of dystrophin proteins and dystrophin associated proteins in porcine retina by reverse transcription polymerase chain reaction, Western blot analysis and immunohistochemistry. Homologues of human DMD gene products and alternative spliced isoforms of Dp71 were identified. We observed that dystrophins were expressed in the outer plexiform layer, around blood vessels and at the inner limiting membrane as previously described in human and mouse retinae. Moreover, by double immunostaining we showed that β-dystroglycan co-localizes with dystrophin in the outer plexiform layer whereas α1-syntrophin labeling differs from that for dystrophins. Using confocal laser microscopy we observed that dystrophins labeling co-localizes with pre- and post-synaptic cell markers in the outer plexiform layer. We suggest that porcine retina constitutes a good model to study the role of dystrophins in retinal neurotransmission and should be used to investigate the physiological roles of dystrophins in signal transduction.

Dystrophin Dp71 Expression Is Down-regulated during Myogenesis: ROLE OF Sp1 AND Sp3 ON THE Dp71 PROMOTER ACTIVITY

Dp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5'-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3.

Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina.

The abnormal retinal neurotransmission observed in Duchenne muscular dystrophy (DMD) patients and in some genotypes of mice lacking dystrophin has been attributed to altered expression of short products of the dystrophin gene. We have investigated the potential role of Dp71, the most abundant C-terminal dystrophin gene product, in retinal electrophysiology. Comparison of the scotopic electroretinograms (ERG) between Dp71-null mice and wild-type (wt) littermates revealed a normal ERG in Dp71-null mice with no significant changes of the b-wave amplitude and kinetics. Analysis of DMD gene products, utrophin and dystrophin-associated proteins (DAPs), showed that Dp71 and utrophin were localized around the blood vessels, in the ganglion cell layer (GCL), and the inner limiting membrane (ILM). Dp71 deficiency was accompanied by an increased level of utrophin and decreased level of beta-dystroglycan localized in the ILM, without any apparent effect on the other DAPs. Dp71 deficiency was also associated with an impaired clustering of two Müller glial cell proteins-the inwardly rectifying potassium channel Kir4.1 and the water pore aquaporin 4 (AQP4). Immunostaining of both proteins decreased around blood vessels and in the ILM of Dp71-null mice, suggesting that Dp71 plays a role in the clustering and/or stabilization of the two proteins. AQP4 and Kir4.1 may also be involved in the regulation of the ischemic process. We found that a transient ischemia resulted in a greater damage in the GCL of mice lacking Dp71 than in wt mice. This finding points at a crucial role played by Dp71 in retinal function.

Increased vulnerability to kainate-induced seizures in utrophin-knockout mice.

Utrophin, the autosomal homologue of dystrophin, the Duchenne muscular dystrophy gene product, is a cytoskeletal protein found in many tissues. In muscle fibers, the level and localization of utrophin depend on their state of differentiation and innervation. Transgenic overexpression of utrophin prevents degeneration of dystrophin-deficient muscle fibers. In brain, in addition to its enrichment in blood vessels, utrophin is associated primarily with the plasma membrane of large sensory and motor brainstem neurons, suggesting a contribution to their structural stability. Here, we examined the role of utrophin for long-term survival of dentate granule cells, which become markedly hypertrophic in a mouse model of temporal lobe epilepsy. This morphogenetic change is induced several weeks after a unilateral intrahippocampal injection of kainic acid (KA), while mice experience chronic focal seizures. Using in situ hybridization and immunohistochemistry, we show that dispersion and hypertrophy of granule cells in KA-treated wildtype mice are accompanied by a strong and long-lasting expression of utrophin in somata and proximal dendrites. Utrophin knockout mice had a normal hippocampal cytoarchitecture but were more sensitive to KA-induced excitotoxicity, as shown by increased mortality and faster progression of the lesion. At 6 weeks post-KA, the numerical density of granule cells and thickness of the granule cell layer were significantly reduced ipsilaterally in mutant mice, indicating a profound reduction in total cell number in the absence of utrophin. These findings suggest that utrophin contributes to protect CNS neurons against pathological insults, in particular, stimuli leading to massive neuronal hypertrophy.

Interleukin 6 induces overexpression of the sarcolemmal utrophin in neonatal mdx skeletal muscle.

Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by a defect in the DMD gene, which encodes the cytoskeletal protein dystrophin. Utrophin is an autosomal homolog of the DMD gene product dystrophin, and augmented expression of endogenous utrophin is expected to provide an alternative therapeutic approach to DMD. We previously reported that an immune response against a beta-galactosidase-expressing adenovirus vector, AxCALacZ, resulted in an accumulation of endogenous utrophin on the extrasynaptic sarcolemma in dystrophin-deficient mdx mice. To determine which cytokine is involved in the regulation of utrophin expression, we directly injected several cytokines separately into neonatal mdx muscles and tested whether the expression of utrophin is increased on the sarcolemma. Importantly, among the cytokines tested, solely interleukin 6 (IL-6) successfully increased expression of utrophin. Moreover, the increase in utrophin mRNA was detected in recombinant IL-6-injected mdx muscles by quantitative real-time reverse transcriptase-polymerase chain reaction. Further, IL-6 expression was elevated in AxCALacZ-infected mdx muscle at an early stage, and anti-IL-6 receptor (IL-6R) antibody treatment blocked enhanced utrophin expression in AxCALacZ-infected mdx muscle. We should point out, however, that overexpression of utrophin due to recombinant IL-6 treatment lasted only 1 week. In addition, expression of utrophin was not evident in normal C57BL/10 neonatal muscles injected with IL-6. Taken together, these results suggest that IL-6 can induce overexpression of utrophin on the extrasynaptic sarcolemma but requires preexisting factors in neonatal mdx muscle to fully regulate utrophin expression.

Gene therapy for muscular dystrophies: current status and future prospects.

Since the identification in 1987 of the gene for Duchenne muscular dystrophy (DMD), research on the molecular pathogenesis of muscular dystrophy has progressed extensively. In particular, discovery of the DMD gene product, dystrophin, led to the identification of dystrophin-associated proteins and, subsequently, the recognition of other types of muscular dystrophy caused by the defects in each of the sarcoglycan genes. On the other hand, effective therapy for DMD has not yet been established. Some of the viral vectors, such as adeno-associated virus vectors or lentiviral vector, have been proven to enable the long-term expression of the exogenous gene without overt host immune reactions. However, dystrophin cDNAs are too large (14kb) to be accommodated in these viral vectors. To solve this problem, we and other research groups succeeded in truncating full-length dystrophin cDNA to small dystrophin cDNA (4 to 5kb), the products of which protect dystrophin-deficient mdx muscle from contraction-induced membrane damage when introduced by viral vectors or as a transgene into mdx mice. The usefulness of these truncated dystrophin cDNAs should be confirmed using other animal models such as dystrophic dogs. To develop successful treatment of DMD, the authors believe that several different approaches should be used, such as cell transfer therapy, drug design to up-regulate utrophin, or a strategy to repair the mutation in vivo.

Current knowledge of dystrophin and dystrophin-associated proteins in the retina.

Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and beta-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains full-length dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). beta-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.

Expression patterns of dystrophin products, especially of apodystrophin-1/Dp71, in the neural retina of Amphibian urodele Pleurodeles waltl.

The expression patterns of the DMD (Duchenne Muscular Dystrophy) gene products, especially of Dp71 (apodystrophin-1) were investigated by immunofluorescence and immunoblotting in the retina of the Amphibian urodele Pleurodeles waltl. H-5A3 monoclonal antibody (mAb), directed against the C-terminal region of dystrophin/utrophin, and 5F3 mAb, directed against the last 31 amino acids of dystrophin and specific of Dp71, were used. Western blot analyses with H-5A3 mAb revealed distinct dystrophin-family isoforms in adult newt retinal extracts: a doublet 400-420 kDa, Dp260 isoform, a protein at about 120 kDa, and a diffuse zone at 70-80 kDa, which might correspond to Dp71. Reactivity with H-5A3 mAb appeared nearly restricted to the outer plexiform synaptic layer. On the other hand, Dp71-specific 5F3 mAb recognized trhee polypeptide bands at 70-80, 60-65 and 50-55 kDa in adult newt retina corresponding most probably to alternative spliced isoforms of Dp71. In immunohistochemistry by conventional epifluorescence microscopy, 5F3 labeling was mainly observed in the plexiform layers, the outer nuclear layer, and the photoreceptor inner segments, especially at the myoid regions. Analysis by confocal scanning laser microscopy (CSLM) revealed that 5F3 labeling was, in addition, present in the pigmented epithelium and the inner nuclear layer. Furthermore, CSLM showed that 5F3 staining at the myoids was concentrated at discrete domains underneath the plasma membrane. Our findings raised the question concerning the functional significance of Dp71 isoforms, especially at the myoid where Dp71 was detected for the first time, although it occurred here highly expressed. Putative role(s) played in this retinal compartment and other ones by Dp71 and/or other dystrophin isoforms were discussed.

Electron microscopic observations of triple immunogold labelling for dystrophin, beta-dystroglycan and adhalin in human skeletal myofibers.

Dystrophin is the Duchenne muscular dystrophy gene product and is a membrane cytoskeletal protein present in the network of the plasma membrane undercoat. Adhalin (50 kDa dystrophin-associated glycoprotein) and beta-dystroglycan (43 kDa dystrophin-associated glycoprotein) are the transmembrane components of the normal muscle plasma membrane, and beta-dystroglycan has been demonstrated to bind dystrophin at the inside surface of normal muscle plasma membrane. This investigation was undertaken to test whether the epitopes of dystrophin, beta-dystroglycan and adhalin are closely associated with each other by using triple immunogold labelling electron microscopy on normal human skeletal myofibers. Although closely associated signals of triplet immunogold particles were observed, there were less numerous than expected. However, closely associated signals of two epitopes of dystrophin and beta-dystroglycan, dystrophin and adhalin, or adhalin and beta-dystroglycan were frequently observed. These ultrastructural findings are consistent with biochemical evidence implying that dystrophin, beta-dystroglycan and adhalin are closely associated with each other at the normal muscle plasma membrane.

Reduced levels of dystrophin associated proteins in the brains of mice deficient for Dp71.

Duchenne muscular dystrophy (DMD) is a progressive degenerative lethal muscle disease. A significant proportion of DMD affected children suffer also from mental retardation. The rod shaped protein, dystrophin, which is absent from or defective in the muscle of DMD patients, binds to a number of membrane associated proteins (known collectively as dystrophin associated proteins [DAPs]). The levels of DAPs is greatly reduced in the muscle of DMD patients and mdx mice, which lack dystrophin. In addition to dystrophin isoforms, the DMD gene codes also for several smaller proteins. One of the small proteins, Dp71, is expressed in most or all non-muscle tissues and is the major DMD gene product in the brain. The function of the small DMD gene products is unknown. Here we show that mutant mice which do not express the smaller non-muscle products of the DMD gene have a reduced level of DAPs in their brain. This suggests that Dp71 is important for the formation and/or stabilization of a DAPs complex in brain.

Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells — Possible application for prenatal diagnosis

Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells — Possible application for prenatal diagnosis

Characterization of the Ocular Phenotype of Duchenne and Becker Muscular Dystrophy

Dystrophin, the Duchenne muscular dystrophy gene product, has been localized to the outer plexiform layer of normal human retina. The purpose of this study is to define completely the ocular phenotype associated with mutations at Xp21, the Duchenne muscular dystrophy gene locus. Twenty-one patients with a diagnosis of Duchenne muscular dystrophy and five patients with Becker muscular dystrophy had ophthalmologic examinations, including electroretinograms (ERGs). Electroretinogram results were correlated with respect to patient DNA analysis. Twenty-three (88%) patients had reduced scotopic b-wave amplitudes to bright-white flash stimulus, including nine with negative-shaped ERGs. Rod-isolated responses were reduced or not recordable above noise in 14 (67%) patients. Most isolated cone responses (92%) were normal. Flicker amplitudes were reduced in seven patients. Two of these patients with proximal (5' end) deletions had normal scotopic b-waves to dim blue and bright-white flash stimulus. Patients with deletions toward the middle of the gene had greater reductions in their scotopic b-wave amplitudes than patients with deletions located toward the 5' end. Most patients had normal color vision, extraocular muscle function, and Snellen visual acuity. Increased macular pigmentation was seen in 16 patients with Duchenne muscular dystrophy. Most patients with Duchenne or Becker muscular dystrophy have evidence of abnormal scotopic ERGs. Patients with deletions in the central region of the gene had the most severe ERG changes. This study supports previous suggestions that dystrophin may play a role in retinal neurotransmission. The presence of increased macular pigmentation and normal photopic ERGs distinguishes patients with Duchenne muscular dystrophy mutations from other X-linked retinal disorders with negative-shaped ERGs.