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PubMed1990

Accelerated age-related decline in replicative life-span of Duchenne muscular dystrophy myoblasts: implications for cell and gene therapy.

An assessment of the replicative life-span of myoblasts is of fundamental importance in designing treatment strategies for Duchenne muscular dystrophy (DMD) based on cell or gene therapy. To ascertain myoblast life-span, or the total number of cell divisions of which a myoblast was capable, we serially passaged and counted the progeny of individual myoblasts until they senesced. We compared the life-span of myoblasts from eight DMD patients with controls: three individuals with no known neuromuscular disease, three DMD carriers, and three patients with other muscle degenerative diseases. A decline in replicative capacity was observed with increasing donor age, which was markedly accelerated for DMD relative to control myoblasts. The average myoblast from a 5-year-old control was capable of 56 doublings, or a potential yield of approximately 10(17) cells per cell. By contrast, at 2 years of age, the typical age at clinical onset, only 6% of DMD myoblasts had a life-span of 50 doublings in tissue culture, and by age 7 DMD myoblasts capable of 10 doublings were rare. Our results suggest that the myoblasts (satellite cells) of even the youngest DMD patients have undergone extensive division in an attempt to regenerate degenerating myofibers. These findings have implications for therapeutic intervention in DMD involving genetic engineering and myoblast implantation.

Age Factors↗
PubMed1984

Comparison between the growth pattern of cell cultures from normal and Duchenne dystrophy muscle.

The growth "in vitro" of muscle cells from 12 patients with Duchenne muscular dystrophy (DMD) was compared with that of muscle cells from 20 age-matched controls. In the DMD explants, the lag phase (3 days) was shorter than in controls (6 days). In dissociated cells, plating efficiency (20%) and doubling time (30 h) were identical in DMD and controls. In cultures from three DMD patients, cell clusters were occasionally observed. Myotube morphometry showed significant abnormalities in DMD cultures: the number of myotubes per field was 8.2 +/- 0.8 and 26.7 +/- 0.6 in controls, P less than 0.001; myotube length (151 +/- 20 micron) and diameter (8.2 +/- 0.9 micron) in DMD cultures were half the control values (312 +/- 46 micron and 15.6 +/- 1.2 micron, respectively, P less than 0.001). The number of nuclei per myotube in DMD was one-quarter of that in control muscle (4.0 +/- 0.2 vs 15.8 +/- 2.2, P less than 0.001). It is concluded that DMD cultures show cellular heterogeneity with the presence of fibroblasts and non-fusing myoblasts; furthermore they show delayed myoblast fusion and poor myotube differentiation.

Cell Differentiation↗
PubMed1985

Adhesive interactions between normal and dystrophic human skin fibroblasts.

The adhesive properties of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) were studied by analysing cell aggregate formation in suspensions consisting of normal and DMD fibroblasts. By the use of aggregation kinetics and fluorescent labelling, the genotypic composition of aggregates in mixed-cell suspensions could be visualised. The distribution of normal and DMD cells within these aggregates could then be compared to theoretical binomial distributions which assume no difference in the specific adhesiveness between the two genotypes. Analysis of the 3- and 4-cell aggregates which were produced by co-aggregating normal and DMD cells demonstrate that there is no qualitative (specific) difference in the adhesiveness between normal and DMD fibroblasts. However, quantitative changes in the cell-cell adhesion of DMD fibroblasts may be present, and this is supported by the relatively small proportion of intermediate size heterotypic aggregates which were formed in mixed-genotype cell suspensions. In such mixtures, fewer aggregates consisting of 5 or more cells were formed compared to fibroblast suspensions derived from pairs of normal individuals. Furthermore, cell suspensions from pairs of DMD patients produced even less greater than or equal to 5-cell aggregates than were found in the mixed-genotype experiments. These findings are considered in relation to previous reports of abnormal cell adhesiveness and other adhesion-related mechanisms in DMD cells.

Animals↗
PubMed1995

Abnormal calcium homeostasis in Duchenne muscular dystrophy myotubes contracting in vitro.

Resting intracellular calcium activity was recorded in three kinds of human muscle cells in culture: normal (control) and dystrophic (DMD and FSH), by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. DMD cells are characterized by a lack of dystrophin whereas FSH cells express normal dystrophin. The aim of this study was to determine whether, in dystrophin-deficient muscle cells (DMD), contraction destabilized internal calcium homeostasis. Muscle cells were cocultured with rat spinal cord explants to improve the maturation of human myotubes up to the stage where contraction appears. The resting intracellular calcium level was significantly higher in contracting DMD cells (107 +/- 8 nM; n = 44) compared to control cells (66 +/- 6 nM; n = 43) or in FSH cells (56 +/- 6 nM; n = 35). DMD myotubes cocultured in the presence of TTX which inhibited contractile activity, did not develop an increase in free cytosolic Ca2+ concentration. The amplitudes of calcium transients elicited by exposure to acetylcholine (ACh) or high K+ medium (100K) were significantly higher in contracting DMD myotubes than in control ones. The extra-responses were not observed in DMD myotubes cocultured with TTX. This study strongly suggest that: (i) contraction is a dominant factor contributing to Ca2+ abnormalities in DMD cells; and (ii) contracting dystrophin-deficient cells have defective calcium handling mechanisms during electrical events which involve sarcolemma.

Acetylcholine↗
PubMed2002

The effect of duration of macular detachment on results after the scleral buckle repair of primary, macula-off retinal detachments.

PURPOSE: To present a current series that determined the effect of duration of macular detachment (DMD) and patient age on postscleral buckle (SB) visual acuity (VA) and anatomic results. DESIGN: Retrospective, noncomparative, observational case series. PARTICIPANTS: Ninety-four consecutive patients (94 eyes) with primary, uncomplicated, macula-off retinal detachments, a preoperative VA of 20/200 or worse, and a precise history of when macular function was lost. INTERVENTION: Standard explant scleral buckle technique performed by multiple surgeons. MAIN OUTCOME MEASURES: Visual acuity, anatomic reattachment, and proliferative vitreoretinopathy. RESULTS: Visual acuity after SB of 20/40 or better was seen in 71% of eyes with a DMD of 10 days or fewer, 27% of eyes with a DMD of 11 days to 6 weeks, and 14% of eyes with a DMD of more than 6 weeks. Eyes achieved a mean VA after SB of 20/41 after a DMD of 10 days or fewer, 20/121 after a DMD of 11 days to 6 weeks, and 20/178 after a DMD of more than 6 weeks. No decrease in VA was seen within the 1- to 10-day period or the 11 days to 6 week period of DMD. Patients 60 years of age or less achieved a mean VA after SB of 20/47 compared with 20/81 for patients between 61 to 75 years of age and 20 of 96 in patients more than 76 years of age. Duration of macular detachment and patient age had no statistically significant effect on final anatomic reattachment after SB, reoperation rate, or proliferative vitreoretinopathy. CONCLUSIONS: Eyes with primary, uncomplicated, macula-off retinal detachment repaired with SB achieve excellent postoperative VA if repaired within the first 10 days of macular detachment. These results are better than the VA in eyes repaired after 11 days to 6 weeks and more than 6 weeks of macular detachment. Patients 60 years of age and younger obtained better postoperative VA than older patients. Duration of macular detachment and patient age did not significantly effect anatomic outcomes.

Adolescent↗
PubMedDate not supplied

Detection of deletions spanning the Duchenne muscular dystrophy locus using a tightly linked DNA segment.

The Duchenne muscular dystrophy (DMD) locus has been localized to the short arm of the human X chromosome (Xp21) by detection of structural abnormalities and by genetic linkage studies. A library highly enriched for human DNA from Xp21 was constructed using DNA isolated from a male patient who had a visible deletion and three X-linked disorders (DMD, retinitis pigmentosa and chronic granulomatous disease). Seven cloned DNA probes from this library and the probe 754 (refs 5, 8) are used in the present study to screen for deletions in the DNA isolated from 57 unrelated males with DMD. Five of these DMD males are shown to exhibit deletions for one of the cloned DNA segments and at least 38 kb of surrounding DNA. In addition, two subclones from the same region detect four restriction fragment length polymorphisms which exhibit no obligate recombination with DMD in 34 meiotic events. These new DNA segments will complement the existing Xp21 probes for use in carrier detection and prenatal diagnosis of DMD. Elucidation of the end points of the five deletions will help delineate the extent of the DMD locus and ultimately lead to an understanding of the specific sequences involved in DMD.

Alleles↗
PubMedDate not supplied

Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder for which the biochemical defect is as yet unknown. Recently, two cloned segments of human X-chromosome DNA have been described which detect structural alterations within or near the genetic locus responsible for the disorder. Both of these cloned segments were described as tightly linked to the locus and were capable of detecting deletions in the DNA of boys affected with DMD. In an attempt to determine more precisely the occurrence of these deletions within a large population of DMD patients and the accuracy of one of the segments, DXS164 (pERT87), in determining the inheritance of the DMD X chromosome, the subclones 1, 8 and 15 were made available to many investigators throughout the world. Here we describe the combined results of more than 20 research laboratories with respect to the occurrence of deletions at the DXS164 locus in DNA samples isolated from patients with DMD and Becker muscular dystrophy (BMD). The results indicate that the DXS164 locus apparently recombines with DMD 5% of the time, but is probably located between independent sites of mutation which yield DMD. The breakpoints of some deletions are delineated within the DXS164 locus, and it is evident that the deletions at the DMD locus are frequent and extremely large.

Chromosome Deletion↗
PubMed1988

Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide.

Duchenne muscular dystrophy (DMD) is a debilitating X-linked muscle disease. We have used sequence information from complementary DNA clones, derived from the gene that is deleted in DMD patients, to generate an antiserum that stains the surface membrane of intact human and mouse skeletal muscle, but not that of DMD patients and mdx mice. Here we identify the protein reacting with this antiserum as a single component of relative molecular mass 210,000 (Mr = 210K) that fractionates with a low-ionic strength extract of intact human and mouse skeletal muscle. It is therefore distinct from the 400 K protein found in the heavy microsomal fraction of normal muscle and identified as a putative product of the DMD gene. We also analyse further the disease specificity of the antiserum. Positive staining is seen in normal controls, and in samples from patients with a wide range of muscular dystrophies other than DMD. Becker muscular dystrophy, which is allelically related to DMD, was the only other exception, and gave a sporadic staining pattern. The demonstration of a specific defect in the surface membrane of DMD muscle fibres substantiates the hypothesis that membrane lesions may initiate muscle degradation in DMD.

Animals↗
PubMed1992

Demonstration of cathepsins B, H and L in xenografts of normal and Duchenne-muscular-dystrophy muscles transplanted into nude mice.

The activities and contents of the lysosomal cysteine proteinases cathepsins B, H and L were examined in xenografts of biopsied muscles transplanted from age-matched normal subjects and Duchenne-muscular-dystrophy (DMD) patients into nude mice. The activity of cathepsin B increased 9-fold and that of B-plus-L increased 24-fold in the first week after transplantation in normal muscle xenografts. By the third week, the activity of cathepsin B increased a total of 20-fold and B-plus-L increased to 36-fold the original level. The activity levels of cathepsin B, B-plus-L, H and D, and acid phosphatase in normal and DMD xenografts were not significantly different when compared 2 weeks after transplantation. However, the protein content of cathepsin B in DMD muscle xenografts was more than 3-fold that of normal xenografts at 2 weeks. The profile of cathepsin H activity in normal muscle xenografts was different than those of cathepsins B and B-plus-L. In the first week, the cathepsin H diminished sharply to about one-third of the biopsied muscle level and then, by 3 weeks after transplantation, it had increased slightly to about half the original level. The amount of endogenous cysteine-proteinase inhibitor changed in parallel with the activity of cathepsins B and B-plus-L. Cathepsins B and H, but not cathepsin L, were found immunohistochemically in regenerating muscle fibres of normal and DMD xenografts 2 weeks after transplantation. Staining of cathepsin B in DMD xenografts was slightly stronger than that in normal subjects. There was no immunostaining in degenerating or necrotic muscle fibres 2 weeks after transplantation. Western-blot analysis revealed that the cathepsin B band at 29 kDa was increased in normal xenografts 2 and 3 weeks after transplantation. Also, 2 weeks after transplantation the staining intensity of this band was slightly stronger in DMD xenografts than in normal xenografts. These results suggest that cathepsin B participates in the regeneration of transplanted muscle, both normal and DMD, and in the DMD muscle fibre-wasting processes, during regeneration.

Acid Phosphatase↗
PubMed1996

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.

Animals↗
PubMed1992

Measurement of isometric force in children with and without Duchenne's muscular dystrophy.

The purpose of this study was to examine the consistency of measurements of maximal isometric force production in children using an electronic strain gauge. A standardized protocol of force testing from the Tufts Quantitative Neuromuscular Examination was adapted to obtain isometric force measurements in seven muscle groups bilaterally. Ten children with Duchenne's muscular dystrophy (DMD) and 10 age-matched children without DMD were recruited for the study. A generalizability framework was used to examine variations in measurements attributable to group differences, test occasions within the same day, and extremity side. Intertester reliability estimates were also completed on a subsample of children with and without DMD. A three-way analysis of variance between groups, sides, and test occasions demonstrated a significant group main effect for all muscle groups. In general, significant main effects were not present for side or test occasion. Test-retest intraclass correlation coefficients (ICCs) for all muscle groups ranged from .88 to .99 for the children with DMD and from .85 to .98 for the children without DMD. Intertester ICCs were slightly lower, ranging from .74 to .97 for the children with DMD and from .71 to .98 for the children without DMD. The results indicate that the electronic strain-gauge protocol produces reproducible force measurements in seven selected muscle groups in children with and without DMD. Brussock CM, Haley SM, Munsat TL, Bernhardt DB. Measurement of isometric force in children with and without Duchenne's muscular dystrophy.

Adolescent↗
PubMed1998

Reading ability and processing in Duchenne muscular dystrophy and spinal muscular atrophy.

We analysed the reading abilities and processing of 21 children with Duchenne muscular dystrophy (DMD), 11 matched children suffering from spinal muscular atrophy (SMA) and 42 children receiving normal education. The principal result observed was that the DMD children exhibited a reading age which was significantly lower than the SMA children compared with their chronological age. These learning disabilities were not related to a deficit in non-verbal performance intelligence, but psycholinguistic evaluation showed a deficit in verbal intelligence, especially in the Similarities and Arithmetic WISC-R subtests, in phonological abilities, oral word repetition, and in digit span score. The results for the DMD children were heterogeneous, and ranged from normal to greater or lesser involvement. In an attempt to clarify the nature of this reading impairment in DMD children, the three groups (DMD, SMA, and normal control children) were tested by reading aloud a list of single words and non-words. The DMD children were significantly impaired in reading non-words, suggesting reading disability similar to dysphonetic dyslexia, the most frequent subtype of developmental dyslexia. These results are discussed in the light of psychometric data available for our DMD population and in the light of previous studies. The practical consequences of diagnosis on rehabilitation are very important. The precise description of the cognitive deficits seen in DMD is of value for future clinical and genetic studies.

Adolescent↗
PubMed1980

Decreased lysosomal dipeptidyl aminopeptidase I activity in cultured human skin fibroblasts in Duchenne's muscular dystrophy.

Several lysosomal enzymes were assayed in cultured human skin fibroblasts from patients with Duchenne's muscular dystrophy (DMD) and age- and sex-matched control patients (N). The activity of four glycosidases, cathepsin B(1), and total autoproteolysis at pH 4.0 were unchanged between the groups, but dipeptidyl aminopeptidase I (DAP-I, or cathepsin C) in the DMD cells was found to be only 30% as active as in the control cells (P < 0.003). This difference is not the result of a redistribution or loss of enzyme during homogenization because the difference occurs in all homogenate fractions. DAP-I activity existing in N and DMD fibroblasts behaves identically with respect to activation by chloride ion, activation by the sulfhydryl reducing agent dithiothreitol, changes in hydrogen ion concentration (pH), changes in substrate concentration (i.e., apparent K(m) values), and changes in temperature (i.e., apparent activation energies). Mixtures of N and DMD cell sonicates display an additivity in DAP-I activity. These results support the conclusion that the catalytic function of the DAP-I molecule is equivalent between N and DMD fibroblasts, and that the decrease in tissue-specific DAP-I activity probably results from the fact that fewer enzyme molecules are present in the DMD cells. These results are also an indication that these nonmuscle cells are expressing some of the phenotypic aspects of the genetic defect in DMD. Cultured human skin fibroblasts may therefore be a useful cellular model in DMD research.

Cathepsins↗
PubMed1989

Complementary DNA probes for the Duchenne muscular dystrophy locus demonstrate a previously undetectable deletion in a patient with dystrophic myopathy, glycerol kinase deficiency, and congenital adrenal hypoplasia.

Genomic DNA from a patient with dystrophic myopathy, glycerol kinase deficiency, and congenital adrenal hypoplasia was investigated using cDNA probes for the Duchenne muscular dystrophy (DMD) locus. Genomic probes had not detected a deletion in this patient. Southern analysis of Hind III-digested genomic DNA from this patient identified a deletion when the three distal Hinc II DMD cDNA fragments were used as probes. The deletion began in the genomic region corresponding to the 1.05-kb Hinc II cDNA fragment and extended through the 3' end of the DMD gene. This represents a centromeric breakpoint that corresponds to a position approximately 10.2-10.6 kb from the 5' end of the 14-kb DMD cDNA. These investigations demonstrate the value of the DMD cDNA probes for improved diagnoses in patients with molecular lesions involving the DMD locus. Furthermore, this novel deletion involving the coding portion of the 3' end of the DMD gene assists in the ordering of exons in this region and will provide insight into the functional role of the carboxy terminus of the DMD gene product, dystrophin.

Adrenal Insufficiency↗
PubMed2000

Poor verbal working memory across intellectual level in boys with Duchenne dystrophy.

OBJECTIVE: To determine whether all boys with Duchenne muscular dystrophy (DMD) have a similar verbal and memory profile of skills, or whether only a subset is affected, and to determine whether the weak areas in their profile are substantially different from a control group. METHODS: Performance of patients with DMD on neuropsychological tests of verbal and memory skills was examined in two ways. Standardized test scores for 80 boys with DMD (estimated IQ range, 70 to 160) were ranked individually from worst to best, and the individual rankings were compared across the group using Friedman rank analysis. Additionally, performance of 41 boys with DMD was compared with that of their sibling control subjects of similar age and estimated IQ using multivariate analysis of variance. RESULTS: Individual cognitive profiles were significantly similar among the subjects with DMD, such that for most subjects digit span, story recall, and comprehension were the tests on which each performed most poorly. This finding remained true regardless of whether they were of high or low intellectual function. In contrast, no significant cognitive profile was found among their sibling control subjects, and when compared with their siblings, the DMD group scored significantly more poorly on digit span, comprehension, and story recall, but not on other verbal and memory measures. CONCLUSIONS: Boys with DMD have a specific cognitive profile, regardless of their general level of cognitive function. Specifically, boys with DMD performed more poorly on tests requiring attention to complex verbal information than they did on other verbal or memory measures. The possibility that the missing dystrophin brain products may contribute to selective cognitive processing is considered.

Adolescent↗
PubMed1985

Duchenne muscular dystrophy: studies of cell motility in vitro.

Duchenne muscular dystrophy (DMD) is a severe degenerative disorder of skeletal muscle. It has been suggested that an abnormality of the plasma membrane may be responsible for the pathogenesis of DMD, and a number of cell surface changes have been described in DMD muscle fibres and other cell types. Alterations in cell-to-cell and cell-to-substratum adhesiveness have been reported for DMD cells and we have determined whether these alterations in cell adhesiveness affect migration of cells from DMD muscle explants. DMD cells move more rapidly and spend less time at rest than do normal or DMD carrier cells, although the differences were statistically significant only for the latter cells. An inverse relationship between cell speed and contact with surrounding cells was not observed. All cells tended to persist in their direction of movement, and there were no differences between the types of cells studied. Our results support the view that there may be a cell surface defect in DMD.

Cell Adhesion↗
PubMed2005

Swiss physicians' practices of long-term mechanical ventilatory support of patients with Duchenne Muscular Dystrophy.

INTRODUCTION: Previous studies have shown that long-term ventilation (LTV) is still not routinely offered to or discussed with patients with Duchenne Muscular Dystrophy (DMD). This is in contrast to the increasing evidence that these interventions can successfully improve quality of life and prolong survival of affected individuals. AIM: The aim of this survey was to describe the clinical practice and the attitudes of Swiss physicians in the respiratory follow up of DMD individuals and to understand better the current provision of respiratory services in Switzerland. Another aim was to explore the contentment of the patients about the provision of information on LTV. METHODS: Postal questionnaires requesting information on the current practice of respiratory care were sent to physicians, including pulmonologists and neurologists known or suspected to be involved in the care of children and adults with DMD. 26 of 30 (87%) physicians returned the questionnaire. A second questionnaire was sent to 90 DMD patients of whom 43 (48%) returned the questionnaire. RESULTS: 88% of the physicians indicated to discuss the option of LTV with their patients and families, but 50% of the surveyed physicians start such discussions only after the patient has already developed respiratory failure. Regular pulmonary function assessments are performed by the majority physicians in DMD patients, but only 50% of the physicians perform regular sleep studies to detect sleep disordered breathing. CONCLUSION: The majority of Swiss physicians involved in the care of DMD patients disclose information on LTV to DMD patients, but commonly late in the disease course after respiratory problems have developed. The current routine of respiratory follow-up of DMD patients seems insufficient, at least with respect to early detection of nocturnal hypoventilation.

Adolescent↗
PubMedDate not supplied

Prenatal diagnosis of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is one of the most common X-linked genetic disorders seen in children. Mutations in the DMD gene coding for the protein dystrophin causes the severe muscle-wasting disorder leading to death in the second decade of life. In the absence of a cure, prenatal diagnosis (PND) appears to be the best approach to reduce the burden of this disease on the individual family and ultimately on society. There are few published reports worldwide on PND and very few from the developing countries. We report our experience with PND for families with DMD using multiplex polymerase chain reaction (PCR) and microsatellite polymorphic marker analysis. METHODS: From August 1997 to October 1999, PND was offered on request to 23 families with one or two boys affected with DMD. A total of 26 foetuses were screened for DMD. Initially the deletions in the DMD gene in the affected child were identified by multiplex PCR screening for 23 exons in 6 sets. In patients where deletions were not identified, microsatellite repeat analysis was carried out to follow the inheritance of the mutant allele. DNA was extracted from chorionic villus samples obtained by chorionic villus biopsy performed at 10-15 weeks of gestation in 17 families, and at 16-20 weeks in 6 families. RESULTS: Deletions were identified in 20 affected boys. In 2 families, microsatellite repeat analysis was done to identify the mutant allele. Of the 26 foetuses, 5 were found to be affected with DMD and the parents opted for termination of pregnancies. CONCLUSIONS: Multiplex PCR technology and microsatellite repeat analysis can be used effectively for PND of DMD.

Female↗