Identification of point mutations in Turkish DMD/BMD families using multiplex-single stranded conformation analysis (SSCA).
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Small mutations are the cause of the disease in one third of cases of Duchenne and Becker muscular dystrophy (DMD/BMD). The identification of point mutations in the dystrophin gene is considered to be very important, because it may provide new insights into the function of dystrophin and direct information for genetic counselling. In this study, we have screened 18 deletion-prone exons (25.5% of the coding region) of the dystrophin gene by using a modified non-isotopic multiplex single-stranded conformation analysis (SSCA). Mutations responsible for the disease phenotype could be identified in five out of 56 unrelated DMD/BMD patients without detectable deletions. Two of these mutations, 980-981delCC and 719G > C, are novel mutations which have not been described previously. Four of the five mutations, including 980-981delCC detected in this study are found to be nonsense or frameshift mutations leading to the synthesis of a truncated dystrophin protein. The missense mutation, 719G > C, causing the substitution of highly conserved alanine residue at 171 with proline in the actin binding domain of the dystrophin, is associated with a BMD phenotype. This study also revealed the presence of six polymorphisms in Turkish DMD/BMD patients. (+info)
Utrophin lacks the rod domain actin binding activity of dystrophin.
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We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419-28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI = 4. 86) lacks the basic character of the repeats found in DYSR11-17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly alpha-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K(d) of 7.3 +/- 1.3 microM and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 microM. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin. (+info)
Patterns of repair of dystrophic mouse muscle: studies on isolated fibers.
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Repair of damaged skeletal muscle fibers by muscle precursor cells (MPC) is central to the regeneration that occurs after injury or disease of muscle and is vital to the success of myoblast transplantation to treat inherited myopathies. However, we lack a detailed knowledge of the mechanisms of this muscle repair. Here, we have used a novel combination of techniques to study this process, marking MPC with nuclear-localizing LacZ and tracing their contribution to regeneration of muscle fibers after grafting into preirradiated muscle of the mdx nu/nu mouse. In this model system, there is muscle degeneration, but little or no regeneration from endogenous MPC. Incorporation of donor MPC into injected muscles was analyzed by preparing single viable muscle fibers at various times after cell implantation. Fibers were either stained immediately for beta-gal, or cultured to allow their associated satellite cells to migrate from the fiber and then stained for beta-gal. Marked myonuclei were located in discrete segments of host muscle fibers and were not incorporated preferentially at the ends of the fibers. All branches on host fibers were also found to be composed of myonuclei carrying the beta-gal marker. There was no significant movement of donor myonuclei within myofibers for up to 7 weeks after MPC implantation. Although donor-derived dystrophin was usually located coincidentally with donor myonuclei, in some fibers, the dystrophin protein had spread further along the mosaic myofibers than had the myonuclei of donor origin. In addition to repairing segments of the host fiber, the implanted MPC also gave rise to satellite cells, which may contribute to future muscle repair. (+info)
Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.
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We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons. (+info)
Mutations in the dystrophin-like dys-1 gene of Caenorhabditis elegans result in reduced acetylcholinesterase activity.
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Mutations of the Caenorhabditis elegans dystrophin/utrophin-like dys-1 gene lead to hyperactivity and hypercontraction of the animals. In addition dys-1 mutants are hypersensitive to acetylcholine and acetylcholinesterase inhibitors. We investigated this phenotype further by assaying acetylcholinesterase activity. Total extracts from three different dys-1 alleles showed significantly less acetylcholinesterase-specific activity than wild-type controls. In addition, double mutants carrying a mutation in the dys-1 gene plus a mutation in either of the two major acetylcholinesterase genes (ace-1 and ace-2) display locomotor defects consistent with a strong reduction of acetylcholinesterases, whereas none of the single mutants does. Therefore, in C. elegans, disruption of the dystrophin/utrophin-like dys-1 gene affects acetylcholinesterase activity. (+info)
Hypoxia on hippocampal slices from mice deficient in dystrophin (mdx) and isoforms (mdx3cv).
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Slices from control C57, mdx, and mdx3cv mice were made hypoxic until both field excitatory postsynaptic potential (fEPSP) and presynaptic afferent volley (AV) disappeared (H1). After reoxygenation and recovery of fEPSP, a second and longer hypoxic test (H2) lasted 3 minutes beyond the time required to block AV. When slices were kept in 10 mmol/L glucose, HI abolished AV 37 and 19% earlier in slices from mdr and mdx3cv mutants than in control slices (where HI = 12 +/- 4.6 minutes, mean +/- SD). During H2 or when slices were kept in 4 mmol/L glucose, AV vanished even more quickly, but the times to block did not differ significantly between slices from controls and mutants. After reoxygenation, AV fully recovered in most slices. Rates of blockade of fEPSPs were comparable in all slices, and most fEPSPs recovered fully after HI. But even in the presence of 10 mmol/L glucose, the second hypoxia suppressed fEPSPs irreversibly in some slices: 2 of 10 from control, 3 of 7 from mdx, and 1 of 6 from mdx3cv mice. Most slices in 4 mmol/L glucose showed no recovery at all: six of seven from control, three of five from mdx, and four of five from mdx3cv mice. Thus, slices from mdx mice were more susceptible than other slices to irreversible hypoxic failure when slices were kept in 10 mmol/L glucose, but they were less susceptible than other slices when kept in 4 mmol/L glucose. In conclusion, the lack of full-length dystrophin (427 kDa) predisposes to quicker loss of nerve conduction in slices from mdx and mdx3cv mutants and improved posthypoxic recovery of fEPSPs in 4 mmol/L glucose in slices from mdx but not mdx3cv mutants, perhaps because the 70-kDa and other C-terminal isoforms are still present in mdx mice. (+info)
Expression of Dp71 in Muller glial cells: a comparison with utrophin- and dystrophin-associated proteins.
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PURPOSE: The abnormal retinal electrophysiology observed in patients with Duchenne muscular dystrophy (DMD) has been attributed to an altered expression of C-terminal products of the dystrophin gene. It has been shown that Dp260 is expressed by photoreceptor cells, whereas Dp71 is present in glial cells. The present study was intended to identify all known members of the dystrophin superfamily and their associated proteins expressed in Muller glial cells (MGC). METHODS: The expression of the proteins and of their messengers was studied in MGC cultures from 2-week-old rats, by polymerase chain reaction amplification, Western blot analysis, and immunocytochemistry. An immunocytochemical localization of the proteins was also performed on enzymatically dissociated Muller cells from adult rat retinas. RESULTS: MGCs expressed a spliced isoform of Dp71 called Dp71f, as well as utrophin, beta-dystroglycan, delta and gamma-sarcoglycans, and alpha1-syntrophin. In morphologically preserved differentiated Muller cells, Dp71f was localized in clusters, utrophin was diffusely distributed in the cytoplasm, and dystrophin-associated proteins (DAPs) were membrane-bound. Most of these proteins were preferentially expressed in the vitread portion of the cells. Dp71f and utrophin expression was restricted to MGCs, whereas all DAPs were also present in other retinal cell types. CONCLUSIONS: The exclusive localization of Dp71f and utrophin in MGCs suggests that these proteins, together with DAPs, play a specific role in these cells. Further knowledge of possible interactions of these proteins within a functional complex may provide new insights into the molecular basis of the electroretinogram phenotype in DMD. (+info)
Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.
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Two young males with limb-girdle muscular dystrophy (LGMD) resulting from sarcoglycan deficiency died at 27 (patient 1) and 18 years (patient 2) of severe cardiomyopathy. Genetic analysis showed that they were compound heterozygotes for mutations in the beta sarcoglycan gene. One of these mutations, an 8 bp duplication in exon 3, was common to both patients. The second mutation in patient 2 was a 4 bp deletion at the splice donor site of intron 2, not reported previously. Patient 2 had more severe heart and skeletal muscle defects with faster deterioration; no sarcoglycans were detected in his skeletal muscle. The second mutation in patient 1, inferred because the unaffected father carries the 8 bp duplication, was not found. In patient 1, both heart and skeletal muscle were analysed and showed reduction of all sarcoglycans in both tissues and incorrect localisation of alpha and gamma sarcoglycans in heart. Therefore mutations in one sarcoglycan gene can disrupt the entire sarcoglycan complex in both skeletal and cardiac muscle. Differing expression patterns of sarcoglycan components in heart and skeletal muscle could be the result of alternatively spliced transcripts in these tissues. By sequencing an alternative transcript, highly expressed in the heart and skeletal muscle of patient 1, we found an 87 bp cryptic exon not previously reported. Although cardiomyopathy can result from mutations in alpha and gamma sarcoglycans, we show for the first time that the condition can also be caused by mutations in the beta sarcoglycan gene. This report therefore expands the phenotype of sarcoglycanopathies and suggests that cardiac function in LGMD patients with defective sarcoglycan expression should be monitored. (+info)