Detection of Duchenne muscular dystrophy carriers by dosage analysis using the DMD cDNA clone 8

Deletion screening in 11 unrelated DMD patients has been performed using DMD cDNA clones 1-8. Of these 11 patients, 6 exhibit deletions of the cDNA clone 8. The carriership of 18 female relatives from these six DMD families has been investigated by dosage analysis. It is shown that dosage analysis is an available method to determine the carrier status of the female relatives of DMD patients showing a deletion within a DMD cDNA clone.     

DNA deletions in mild and severe Becker muscular dystrophy

Summary The DNA of 33 patients diagnosed as suffering from Becker muscular dystrophy (BMD) has been probed with cloned DNA sequences from Xp21, known to reveal DNA deletions in patients suffering from the more severe Duchenne muscular dystrophy (DMD). Two BMD cases showed clear deletions. A third case gave aberrant band sizes, which further analysis showed to be caused by a small deletion. This suggests that deletions in DXS164 occur approximately as frequently in BMD as they do in DMD. Of the two cases showing large deletions, one is at the severe end of the Becker clinical spectrum, whilst the other is a classical Becker-type dystrophy. The fact that loci defined by probes commonly deleted in classical DMD patients are also deleted in BMD patients of varying severity is strong additional evidence that these disorders are allelic, and further justifies the use of probes with defined linkage relationships to DMD also being used for counselling in BMD families.     

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.     

Characterization of patients with glycerol kinase deficiency utilizing cDNA probes for the Duchenne muscular dystrophy locus

Summary Genomic DNA from five previously unreported patients with glycerol kinase deficiency (GKD), dystrophic myopathy, and adrenal insufficiency were studied with genomic probes and cDNA probes for the Duchenne muscular dystrophy (DMD) locus. These individuals, together with those reported by ourselves and others, show that patients with a contigous gene syndrome involving the DMD, GK, and adrenal hypoplasia congenita (AHC) loci have a broader distribution of microdeletion breakpoints than those observed among patients with classical DMD. This study demonstrates the use of the DMD cDNA probes to delineate the centromeric deletion breakpoints for patients with Xp21 microdeletions extending beyond the DMD locus. It also shows the practical diagnostic application of the DMD cDNA probes when the diagnosis of GKD is entertained in a patient with known DMD and only DNA is available for study.     

Gene therapy for muscle disease

The molecular mechanisms of Duchenne muscular dystrophy (DMD) have been extensively investigated since the discovery of the dystrophin gene in 1986. Nonetheless, there is currently no effective treatment for DMD. Recent reports, however, indicate that adenoassociated viral (AAV) vector-mediated transfer of a functional dystrophin cDNA into the affected muscle is a promising strategy. In addition, antisense-mediated exon skipping technology has been emerging as another promising approach to restore dystrophin expression in DMD muscle. Ongoing clinical trials show restoration of dystrophin in DMD patients without serious side effects. Here, we summarize the recent progress in gene therapy, with an emphasis on exon skipping for DMD.     

Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies

Comprehensive molecular testing for mutations in the DMD gene causing Duchenne and Becker muscular dystrophy (DMD/BMD) is challenging because of the large size of the gene and the variety of mutation types. There is an increasing demand for comprehensive DMD gene molecular testing, including deletion/duplication testing of 79 exons and direct sequencing of the 14-kb coding region from genomic DNA, to provide confirmation of clinical diagnoses in affected patients and to determine carrier risk for family members. To determine an efficient strategy to prioritize patients for comprehensive molecular testing of the DMD gene, we tested a consecutive cohort of 165 males referred over a 4-year period because of a suspicion of DMD or BMD using: (1) a new quantitative multiplex polymerase chain reaction (PCR) assay designed to detect deletions or duplications in all exons of the gene and the brain promoter and (2) direct sequencing of the coding region and intron/exon boundaries. For the patients being tested because of a suspicion of DMD, deletion/duplication testing followed by direct sequencing detected pathogenic mutations in 98% (106/108 total patients). However, of the patients tested because of a suspicion of BMD, only 60% (34/57 total patients) had causative mutations identified, all of which were deletions or duplications. Our results suggest that direct genomic sequence analysis of the DMD gene is a useful addition to deletion/duplication testing for diagnosis of DMD, but does not provide an improved sensitivity compared to deletion/duplication analysis alone for the diagnosis of BMD. In addition, due to the relatively common finding of single exon deletions and duplications (22%, 27 of 125 total patients with deletions/duplications), methods to examine all exons of the gene for deletions/duplications should be used as the initial molecular quantitative test for DMD and BMD.     

Discovery of Metabolic Biomarkers for Duchenne Muscular Dystrophy within a Natural History Study

Serum metabolite profiling in Duchenne muscular dystrophy (DMD) may enable discovery of valuable molecular markers for disease progression and treatment response. Serum samples from 51 DMD patients from a natural history study and 22 age-matched healthy volunteers were profiled using liquid chromatography coupled to mass spectrometry (LC-MS) for discovery of novel circulating serum metabolites associated with DMD. Fourteen metabolites were found significantly altered (1% false discovery rate) in their levels between DMD patients and healthy controls while adjusting for age and study site and allowing for an interaction between disease status and age. Increased metabolites included arginine, creatine and unknown compounds at m/z of 357 and 312 while decreased metabolites included creatinine, androgen derivatives and other unknown yet to be identified compounds. Furthermore, the creatine to creatinine ratio is significantly associated with disease progression in DMD patients. This ratio sharply increased with age in DMD patients while it decreased with age in healthy controls. Overall, this study yielded promising metabolic signatures that could prove useful to monitor DMD disease progression and response to therapies in the future.     

Hypokalemia complicating Duchenne muscular dystrophy

Although patients with Duchenne muscular dystrophy (DMD) have been shown to have decreased total body potassium levels, serum potassium levels have generally been though to be within normal limits. We report two siblings with DMD noted to be hypokalemic in conjunction with a respiratory illness. Hypokalemia may have exacerbated the pre-existing pulmonary insufficiency in these patients. The literature concerning hypokalemia and DMD is reviewed, and recommendations for the closer monitoring of serum potassium levels in patients with DMD are presented.     

Comparative Genomics of X-linked Muscular Dystrophies: The Golden Retriever Model

Duchenne muscular dystrophy (DMD) is a devastating disease that dramatically decreases the lifespan and abilities of affected young people. The primary molecular cause of the disease is the absence of functional dystrophin protein, which is critical to proper muscle function. Those with DMD vary in disease presentation and dystrophin mutation; the same causal mutation may be associated with drastically different levels of disease severity. Also contributing to this variation are the influences of additional modifying genes and/or changes in functional elements governing such modifiers. This genetic heterogeneity complicates the efficacy of treatment methods and to date medical interventions are limited to treating symptoms. Animal models of DMD have been instrumental in teasing out the intricacies of DMD disease and hold great promise for advancing knowledge of its variable presentation and treatment. This review addresses the utility of comparative genomics in elucidating the complex background behind phenotypic variation in a canine model of DMD, Golden Retriever muscular dystrophy (GRMD). This knowledge can be exploited in the development of improved, more personalized treatments for DMD patients, such as therapies that can be tailor-matched to the disease course and genomic background of individual patients.     

(Na++K+)-ATPase of Duchenne muscular dystrophy erythrocyte ghosts.

Adenosine triphosphatase (ATPase) activity of erythrocyte ghosts from Duchenne muscular dystrophy (DMD) patients and carriers was stimulated by ouabain while nonmyopathic donor preparations were inhibited. Ethacrynic acid was an effective inhibitor in both DMD patients and carriers as well as in controls. However, responsiveness of the enzyme to suramin and harmaline generally differed in DMD groups from that of nonmyopathic donors. These data are consistent with the hypothesis that modified affinity of Na binding site(s) may account for some properties of the (Na⁺+K⁺)-ATPase activity of DMD erythrocytes.     

Cerebellar-Dependent Associative Learning Is Preserved in Duchenne Muscular Dystrophy: A Study Using Delay Eyeblink Conditioning

ObjectiveBesides progressive muscle weakness cognitive deficits have been reported in patients with Duchenne muscular dystrophy (DMD). Cerebellar dysfunction has been proposed to explain cognitive deficits at least in part. In animal models of DMD disturbed Purkinje cell function has been shown following loss of dystrophin. Furthermore there is increasing evidence that the lateral cerebellum contributes to cognitive processing. In the present study cerebellar-dependent delay eyeblink conditioning, a form of associative learning, was used to assess cerebellar function in DMD children.MethodsDelay eyeblink conditioning was examined in eight genetically defined male patients with DMD and in ten age-matched control subjects. Acquisition, timing and extinction of conditioned eyeblink responses (CR) were assessed during a single conditioning session.ResultsBoth groups showed a significant increase of CRs during the course of learning (block effect p < 0.001). CR acquisition was not impaired in DMD patients (mean total CR incidence 37.4 ± 17.6%) as compared to control subjects (36.2 ± 17.3%; group effect p = 0.89; group by block effect p = 0.38; ANOVA with repeated measures). In addition, CR timing and extinction was not different from controls.ConclusionsDelay eyeblink conditioning was preserved in the present DMD patients. Because eyeblink conditioning depends on the integrity of the intermediate cerebellum, this older part of the cerebellum may be relatively preserved in DMD. The present findings agree with animal model data showing that the newer, lateral cerebellum is primarily affected in DMD.     

Patterns of exon deletions in Duchenne and Becker muscular dystrophy

Summary A panel of patients with Duchenne and Becker muscular dystrophy (DMD and BMD) has been screened with the cDNA probes Cf56a and Cf23a, which detect exons in the central part of the DMD gene. One or more exons were deleted in 60% of patients. The deletions were mapped and prove to be heterogeneous in size and extent, particularly in DMD. Deletions specific to DMD and to BMD are described. Half of all BMD patients have a deletion of one particular small group of exons; smaller deletions within this same group produce the more severe DMD.     

Analysis of deletion mutations of the dystrophin gene by the multiplex polymerase chain reaction method in the diagnosis of Duchenne muscular dystrophy]

The 33 patients suffering from the Duchenne muscular dystrophy (DMD), 7 healthy donors and a DMD risk family were studied by means of polymerase multiplex chain reaction (MPCR) with 6 oligoprimer pairs for 6 different exons of dystrophin gene. The deletions varying in sizes from 1 to 6 exons were detected in 12 out of 33 DMD patients studied (36.3%). The prenatal diagnosis of DMD was carried out by chorionic villus biopsy on the 1st trimester of pregnancy. Contrary to earlier findings, in elder brother with sever DMD manifestation, no visible deletion was detected in the DNA sample from the male foetus and thus the diagnosis of DMD in foetus was rejected. The perspectives of MPCR in pre and postnatal diagnosis of DMD are discussed.     

Cardiac assessment of patients with late stage Duchenne muscular dystrophy

Background. Duchenne muscular dystrophy (DMD) patients used to die mainly from pulmonary problems. However, as advances in respiratory care increase life expectancy, mortality due to cardiomyopathy rises. Echocardiography remains the standard diagnostic modality for cardiomyopathy in DMD patients, but is hampered by scoliosis and poor echocardiographic acoustic windows in adult DMD patients. Multigated cardiac radionuclide ventriculography (MUGA) does not suffer from these limitations. N-terminal proBNP (NTproBNP) has shown to be a diagnostic factor for heart failure. We present our initial experience with plasma NT-proBNP measurement in the routine screening and diagnosis of cardiomyopathy in adult mechanically ventilated DMD patients. Methods. Retrospective study, 13 patients. Echocardiography classified left ventricular (LV) function as preserved or depressed. NT-proBNP was determined using immunoassay. LV ejection fraction (LVEF) was determined using MUGA. Results. Median (range) NT-proBNP was 73 (25 to 463) ng/l. Six patients had an NT-proBNP >125 ng/l. Seven patients showed an LVEF <45% on MUGA. DMD patients with depressed LV function (n=4) as assessed by echocardiography had significantly higher median NT-proBNP than those (n=9) with preserved LV function: 346 (266 to 463) ng/l versus 69 (25 to 257) ng/l (p=0.003). NT-proBNP significantly correlated with depressed LV function on echocardiogram and with LVEF determined by MUGA. Conclusion. Although image quality of MUGA is superior to echocardiography, the combination of echocardiography and NT-proBNP achieves similar results in the evaluation of left ventricular function and is less time consuming and burdensome for our patients. We advise to add NT-proBNP to echocardiography in the routine cardiac assessment of DMD patients. (Neth Heart J 2009;17:232–7.)     

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus.     

Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

Dystromirs as Serum Biomarkers for Monitoring the Disease Severity in Duchenne Muscular Dystrophy

Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls.We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials.Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.     

Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface

Dystrophin is the altered gene product in Duchenne muscular dystrophy (DMD). We used polyclonal antibodies against dystrophin to immunohistochemically localize the protein in human muscle. In normal individuals and in patients with myopathies other than DMD, dystrophin was localized to the sarcolemma of the fibers. The protein was absent or markedly deficient in DMD. The sarcolemmal localization of dystrophin is consistent with other evidence that there are structural and functional abnormalities of muscle surface membranes in DMD.     

Detection and identification of myoglobin in serum by immunoblotting. Effect of exercise on patients with Duchenne muscular dystrophy

The diurnal changes and the effect of pre- and postexercise on serum myoglobin (Mb) levels and creatine kinase (CK) activity were investigated and compared in 11 male patients with Duchenne muscular dystrophy (DMD) and 11 normal male controls. The Mb levels and the CK activity in patients with DMD were significantly higher (p less than 0.001) than in the controls under both resting and exercise conditions. No correlation was found between serum Mb levels and CK activity in patients with DMD. The results indicate that Mb is also a useful parameter in both the RIA and the immunoblotting technique for diagnosing muscle-fiber degeneration and screening DMD.