Cases Of Duchenne Muscular Dystrophy Research Articles

Aspects génétiques et moléculaires des dystrophinopathies

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene that encodes the cytoskeletal protein, dystrophin. Dystrophinopathies are inherited in an X-linked recessive manner. Due to the tremendous size of the gene (2.2 megabases), the DMD locus has a high spontaneous mutation rate, and one third of sporadic cases of DMD are due to a de novo mutation. There are seven tissue-specific promoters in the gene. The skeletal muscular transcript contains 79 exons and encode the full-length protein (427-kDa) located at the inner face of the sarcolemma of muscle fibers. DMD gene mutations are highly heterogeneous. Large rearrangements (deletions or duplications of one or more exons) are most frequently involved while point mutations account for 20 %-30 % of cases. A survey of current strategies of molecular diagnosis is presented here. In particular, the role of muscle biopsy (for dystrophin and RNA analyses) in the diagnosis of dystrophinopathies is discussed. In more than 90 % of cases, the clinical severity is correlated with the impact of the mutations on the reading frame and the expression of the dystrophin (absence or residual amount of mutated protein). Various mechanisms contribute to the exceptions. Besides the clinical interest for the patient, the identification of the mutation allows accurate genetic counseling in the familles, and is a necessary prerequisite for the inclusion of the patient in the genotype-based clinical trials.

Neuronal SH-SY5Y cells use the C-dystrophin promoter coupled with exon 78 skipping and display multiple patterns of alternative splicing including two intronic insertion events.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in the dystrophin gene. One-third of DMD cases are complicated by mental retardation. Here, we used reverse transcription PCR to analyze the pattern of dystrophin transcripts in neuronal SH-SY5Y cells. Among the three alternative promoters/first exons at the 5'-end, only transcripts containing the brain cortex-specific C1 exon could be amplified. The C-transcript appeared as two products: a major product of the expected size and a minor larger product that contained the cryptic exon 1a between exons C1 and 2. At the 3'-end there was complete exon 78 skipping. Together, these findings indicate that SH-SY5Y cells have neuron-specific characteristics with regard to both promoter activation and alternative splicing. We also revealed partial skipping of exons 9 and 71. Four amplified products were obtained from a fragment covering exons 36-41: a strong expected product, two weak products lacking either exon 37 or exon 38, and a second strong larger product with a 568-bp insertion between exons 40 and 41. The inserted sequence matched the 3'-end of intron 40 perfectly. We concluded that a cryptic splice site was activated in SH-SY5Y cells to create the novel, unusually large, exon 41e (751bp). In total, we identified seven alternative splicing events in neuronal SH-SY5Y cells, and calculated that 32 dystrophin transcripts could be produced. Our results may provide clues in the analysis of transcriptype-phenotype correlations as regards mental retardation in DMD.

Autonomic dysfunction: a driving force for myocardial fibrosis in young Duchenne muscular dystrophy patients?

Cardiac manifestations of Duchenne muscular dystrophy (DMD) include progressive cardiac dysfunction and an elevated resting heart rate (HR). We hypothesized this elevated HR reflects autonomic dysfunction that can be identified by heart rate variability (HRV) analyses which will be associated with myocardial fibrosis by cardiac magnetic resonance imaging (cMR). DMD patients (N=74) and controls (N=17) had time and frequency domain HRV analyses calculated via Holter monitoring. Cardiac magnetic resonance imaging was performed on DMD cases only. χ (2) test, T test, ANOVA, and logistic regression were used to perform comparisons between groups. A p value of <0.05 was used for statistical significance. DMD cases had higher resting average HR than controls (99.4±8.9, 85.4+6.2, p<0.001). Among HRV variables, decreases were seen in the following: standard deviation of R to R intervals, the percent RR intervals differing by >50ms from previous RR interval, the root-meansquare of successive differences of RR intervals, the standard deviation of the mean R to R segment (SDANN), low frequency, and high frequency domain, all p values 0.001. Maximum HR and SDANN most significantly associated with positive LGE on cMR (p=0.008, p=0.016). DMD cases on beta blocker had an average HR lower than those not on beta blocker (p=0.009), but with no difference in HRV analysis. DMD patients have reduced HRV and therefore autonomic dysfunction prior to the onset of heart failure which is associated with myocardial fibrosis.

Nonsense readthrough therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common form of inherited muscle disease and is characterized by progressive muscle wasting ultimately resulting in death of the patients in their twenties. DMD is characterized by a deficiency of the muscle dystrophin as a result of mutations in the dystrophin gene. Currently, no effective treatment for DMD is available. Promising molecular therapies which are mutation specific have been developed. Induction of the readthrough of nonsense mutations is expected to produce dystrophin in DMD patients with nonsense mutations, which are detected in 19% of DMD cases. Clinical effectiveness of gentamicin and PTC124 have been suggested. We have demonstrated that arbekacin-mediated readthrough can substantially ameliorate muscular dystrophy. We already have begun the clinical trial of the nonsense mutation readthrough therapy using arbekacin. We hope that this molecular therapy will contribute towards the treatment for DMD.

P.11.16 Three-dimensional gait analysis of Duchenne muscular dystrophy: A trial to evaluate the therapeutic effect of RNA/ENA chimera antisense oligonucleotide that induces dystrophin exon 45 skipping

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Antisense oligonucleotide (AO)-mediated exon skipping therapy has been proposed for the treatment of DMD, producing in-frame mRNA from the out-of-frame mRNA by inducing exon shipping. Generally, the 6-min walk test has been used for evaluating the response to newly developed therapies, whereas it is difficult to measure the fine change. In this study three-dimensional gait analysis was used for measuring the therapeutic effect of AO85, which is an 18-mer antisense RNA/2-O, 4-C ethylene-bridged nucleic acid (ENA) chimera inducing the exon 45 skipping, in DMD case. An 8-year-old DMD case with exon 44 deletion was enrolled and received a 0.5 mg/kg IV infusion of AO85, which was administered at 1-week interval for 4 weeks. In addition to the conventional tests, his kinematic and kinetic data were collected with a 10-camera MAC3D motion analysis system (Motion Analysis Corp., Santa Rosa, CA) synchronized with an embedded Kistler force plate (Kistler Instruments Corp., Amherst, NY). This system monitors the three dimensional coordinates of the markers which were placed over the anatomical landmarks. The gait parameters were extracted using nMotion musculous (nac Image Technology Inc, Tokyo, Japan). The walking distances for 6 min before and after treatment were not apparently changed (360 m and 385 m, respectively). On the other hand, three-dimensional gait analysis revealed the decreased joint torques at knee and ankle. Furthermore, the motion of his center of mass in the vertical direction during gait was reduced. These results indicate that the treatment with AO85 improved his walking motion. Three-dimensional gait analysis could find the fine change of motor function that was not able to be detected by the conventional 6-min walk test, and is the promising method to evaluate the efficacy of exon skipping therapy.

Community Structure Analysis of Gene Interaction Networks in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is an important pathology associated with the human skeletal muscle and has been studied extensively. Gene expression measurements on skeletal muscle of patients afflicted with DMD provides the opportunity to understand the underlying mechanisms that lead to the pathology. Community structure analysis is a useful computational technique for understanding and modeling genetic interaction networks. In this paper, we leverage this technique in combination with gene expression measurements from normal and DMD patient skeletal muscle tissue to study the structure of genetic interactions in the context of DMD. We define a novel framework for transforming a raw dataset of gene expression measurements into an interaction network, and subsequently apply algorithms for community structure analysis for the extraction of topological communities. The emergent communities are analyzed from a biological standpoint in terms of their constituent biological pathways, and an interpretation that draws correlations between functional and structural organization of the genetic interactions is presented. We also compare these communities and associated functions in pathology against those in normal human skeletal muscle. In particular, differential enhancements are observed in the following pathways between pathological and normal cases: Metabolic, Focal adhesion, Regulation of actin cytoskeleton and Cell adhesion, and implication of these mechanisms are supported by prior work. Furthermore, our study also includes a gene-level analysis to identify genes that are involved in the coupling between the pathways of interest. We believe that our results serve to highlight important distinguishing features in the structural/functional organization of constituent biological pathways, as it relates to normal and DMD cases, and provide the mechanistic basis for further biological investigations into specific pathways differently regulated between normal and DMD patients. These findings have the potential to serve as fertile ground for therapeutic applications involving targeted drug development for DMD.

Proliferation and Differentiation Potential of Adipose‐Derived Stem Cells Isolated from Dystrophin and Utrophin Double Knockout mice

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive weakening of the skeletal, cardiac, and diaphragmatic muscles. Recent evidence has emerged implicating adult stem cell dysfunction in DMD histophathogenesis. Using utrophin/dystrophin double knockout (dys−/−utro−/−, dKO) mice, a DMD model that better emulates the human phenotype of the disease in mice, we determine if adipose derived stem cells (ADSCs) from dKO mice are affected in their proliferation and differentiation capacities compared to C57BL (wt) ADSCs. ADSCs were isolated from subcutaneous fat tissue of 6 week old dKO and wt mice and flow analysis confirmed positive expressions of Sca‐1, CD34, CD44 and CD29. Cell proliferation assay using a Live Cell Imaging system showed that dKO‐ADSCs were more active in proliferation than wt‐ADSCs. dKO‐ADSCs showed increased adipogenic, osteogenic, and chondrogenic potentials compared to wt‐ADSCs in vitro. Additional experiments revealed that transplanted dKO‐ADSCs displayed increased proliferation and survival and formed/accumulated more adipocytes in dKO gastrocnemius muscle (GM) compared to that in wt GM. These results suggest that dystrophic muscle milieu affects the stem cell niche, impacting stem cell function to be more active in proliferation and adipogenesis and this may partially explain ectopic fat deposition in skeletal muscle seen in advanced cases of DMD.

Evolution of Molecular Diagnosis of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the commonest of the muscular dystrophies. The DMD gene (DMD) is the biggest human gene and the most common molecular defect in the DMD gene, accounting for approximately 65 % of cases of DMD, is the deletion of one or more exons. The most basic method still in regular use involves multiplex PCR of the exons, known to be most commonly deleted. The multiplex is relatively simple. Quantitative analysis of all exons of the gene and multiplex ligation-dependent probe amplification have brought about an improvement in mutation detection rate, as they will detect all exon scale deletions as well as duplications, widely used to detect exonic and intronic mutations. As a sensitive and discriminative tool, MLPA can be used for prenatal testing. A more recent development in quantitative analysis is the use of oligonucleotide-based array comparative genomic hybridization.

Molecular characterization of an X(p21.2;q28) chromosomal inversion in a Duchenne muscular dystrophy patient with mental retardation reveals a novel long non-coding gene on Xq28

Duchenne muscular dystrophy (DMD) is the most common inherited muscular disease and is characterized by progressive muscle wasting. DMD is caused by mutations in the dystrophin gene on Xp21.2. One-third of DMD cases are complicated by mental retardation, but the pathogenesis of this is unknown. We have identified an intrachromosomal inversion, inv(X)(p21.2;q28) in a DMD patient with mental retardation. We hypothesized that a gene responsible for the mental retardation in this patient would be disrupted by the inversion. We localized the inversion break point by analysis of dystrophin complementary DNA (cDNA) and fluorescence in situ hybridization. We used 5' and 3' rapid amplification of cDNA ends to extend the known transcripts, and reverse transcription-PCR to analyze tissue-specific expression. The patient's dystrophin cDNA was separated into two fragments between exons 18 and 19. Exon 19 was dislocated to the long arm of the X-chromosome. We identified a novel 109-bp sequence transcribed upstream of exon 19, and a 576-bp sequence including a poly(A) tract transcribed downstream of exon 18. Combining the two novel sequences, we identified a novel gene, named KUCG1, which comprises three exons spanning 50 kb on Xq28. The 685-bp transcript has no open-reading frame, classifying it as a long non-coding RNA. KUCG1 mRNA was identified in brain. We cloned a novel long non-coding gene from a chromosomal break point. It was supposed that this gene may have a role in causing mental retardation in the index case.

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.AIM:To study the utility of MLPA in diagnosis and carrier detection for DMD.MATERIALS AND METHODS:Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.RESULTS AND CONCLUSIONS:We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.

Exon‐skipping events in candidates for clinical trials of morpholino

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial. Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction. Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event. An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

Aminoglycoside-induced mutation suppression (stop codon readthrough) as a therapeutic strategy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal, X-linked genetic disease, affecting 1 in 3500 newborn males. It is caused by mutations in the DMD gene. Owing to the large size of the gene, the mutation rate in both germline and somatic cells is very high. Nearly 13-15% of DMD cases are caused by nonsense mutations leading to premature termination codons in the reading frame that results in truncated dystrophin protein. Currently there is no cure for DMD. The only available treatment is the use of glucocorticoids that have modest beneficial effects accompanied by significant side effects. Different therapeutic strategies have been developed ranging from gene therapy to exon skipping and nonsense mutation suppression to produce the full-length protein. These strategies have shown promise in the mdx mouse model of muscular dystrophy where they have been reported to ameliorate the dystrophic phenotype and correct the physiological defects in the membrane. Each of these molecular approaches are being investigated in clinical trials. Here we review nonsense mutation suppression by aminoglycosides as a therapeutic strategy to treat DMD with special emphasis on gentamicin-induced readthrough of disease-causing premature termination codons.

Therapeutic Potential of Proteasome Inhibition in Duchenne and Becker Muscular Dystrophies

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, alpha-sarcoglycan, and beta-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

High resolution NMR based analysis of serum lipids in Duchenne muscular dystrophy patients and its possible diagnostic significance

Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular dystrophy (DMD) (age, mean +/- SD; 8.0 +/- 3.0 years) patients and 22 healthy subjects (age, mean +/- SD; 9.0 +/- 4.0 years) were performed in the northern Indian population. The concentration of triglycerides, phospholipids, free cholesterol, cholesterol esters and total cholesterol was significantly higher in DMD patients as compared to healthy subjects. Ratio of free-cholesterol to cholesterol-esters was also significantly higher in DMD patients. Among the individual lipids, concentration of phospholipids was found to be consistently higher in DMD patients compared to healthy subjects, with a discriminatory index of 87.5%. The highest discriminatory index of 92% was found along with the ratio of PL (phospholipids) to CHOL (cholesterol), i.e. PL/CHOL. No significant quantitative difference was observed in the serum lipid constituents of positive and negative gene deletion cases of DMD. The inferences drawn from this study may provide the possibility of the diagnostic importance for DMD, especially in cases where genetic analysis fails to provide the diagnosis.

Re-examination of the Electrocardiogram in Boys With Duchenne Muscular Dystrophy and Correlation With Its Dilated Cardiomyopathy

Duchenne muscular dystrophy (DMD) results in dilated cardiomyopathy (DC). Characteristic electrocardiographic (ECG) changes include short PR interval, right ventricular hypertrophy (RVH), prolonged QTc interval, and prominent Q waves in leads I, aVL, V5, and V6 or in leads II, III, aVF, V5, and V6. We re-examined the prevalence and correlation of ECG changes with DC in DMD. Electrograms of 115 patients with DMD were evaluated. DC was defined as an echocardiographic ejection fraction<55%. PR interval and RVH were based on age-based normal values. Abnormal Q waves were >or=4 mm. Abnormal QTc interval was >or=450 ms. ST-segment depression was defined as >0.5 mm. Fisher's exact test evaluated significant differences between groups and logistic regression determined whether number of ECG changes predicted DC. Forty had DC. No significant differences existed between the number of ECG changes in DC and non-DC groups (p=0.279). Distribution of findings included short PR interval (43%), RVH (37%), prominent Q waves in leads V5 (34%) and V6 (33%), prominent Q waves in leads I, aVL, V5, and V6 (3, 1 with DC), prominent Q waves in leads II, III, aVF, V5, and V6 (9, 4 with DC), long QTc interval (0), ST depression (2, 1 with DC), and flat/biphasic ST segments (38, 15 with DC). In conclusion, ECG changes are similar in patients with DMD regardless of presence of DC. Previously reported characteristic ECG changes are seen in a minority of DMD cases. The most common findings are short PR interval and RVH. Prominent Q waves in leads II, III, aVF, V5, and V6 are more likely.

G.P.12.05 Gastrostomy tube feeding in patients with severe Duchenne muscular dystrophy

To evaluate effects, risks and the indication of percutaneous endoscopic gastrostomy (PEG) for severe Duchenne muscular dystrophy (DMD), we reviewed clinical records of 21 patients with advanced DMD with gastrostomy in National Hospital Organization Nishitaga Hospital, Sendai, Japan. Mean age and mean body weight of the patients at the time of procedure were 29.3 years old and 26.2kg, respectively. All patients were advanced and severe cases of DMD who had feeding problems such as swallowing difficulties, prolonged meal time, and weight loss.

Corticosteroid treatment retards development of ventricular dysfunction in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized by a predictable decline in cardiac function with age that contributes to early death. Although corticosteroids are a clinically effective pharmacologic therapy for skeletal muscle function, there is limited published work documenting the impact on cardiac function. The primary objective of this work is to determine benefit from steroid treatment on the development of ventricular dysfunction in DMD. We performed a historical cohort study of DMD cases undergoing serial cardiac evaluations from 1998–2006. In addition to the history of steroid use, basic medical characteristics and serial echocardiographic measures were obtained for each identified case meeting inclusion criteria. Data from initial (7.5 ± 0.8 years) and follow-up (12 ± 0.7 years) evaluation was collected from untreated ( n = 23) and steroid treated ( n = 14) DMD cases. Kaplan–Meier freedom from ventricular dysfunction was 93% for steroid treated cases versus 53% for untreated cases at 1500 days. Treatment with steroids was protective against ventricular dysfunction (Hazard ratio 0.16 95% CI 0.04, 0.70). We demonstrate here that steroid treatment, begun prior to ventricular dysfunction retards the anticipated development of ventricular dysfunction.

Studies Point Way to New Therapeutic Prospects for Muscular Dystrophy

AL T H O U G H 20 Y E A R S H A V E elapsed since scientists identified the protein encoded by the defective gene that causes Duchenne muscular dystrophy (DMD), treatment options to help slow the disease’s progressive muscle degeneration have remained limited. In recent years, however, therapeutic prospects have become brighter. Scientists focusing on the disease have developed some promising potential treatments, some of which are in clinical trials. In addition, a few researchers from outside the field have made some fortuitous discoveries about muscle maintenance and regeneration that are providing insights that may one day lead to new treatments. DMD is the most prevalent type of muscular dystrophy, affecting about 1 of every 3500 boys, according to the National Human Genome Research Institute. The accepted treatment is prednisone, but the adverse effects of the drug, such as weight gain, behavioral changes, bone fractures, and cataracts, almost outweigh its benefits for the boys and young men with the disease, explained Sharon Hesterlee, PhD, vice president of translational research at the Muscular Dystrophy Association. (Girls are rarely affected but may carry the mutated gene responsible for DMD, which is on the X chromosome.) “Steroids keep the boys walking longer,” Hesterlee said. “They still get weaker, but the drug slows the progression of the disease.” Scientists are looking for an intervention that halts disease progression or even reverses it, she said. Approaches targeting the disease’s genetic underpinnings are currently being explored. Patients with DMD do not produce sufficient amounts of dystrophin, a protein that is essential for proper muscle maintenance and regeneration. The gene that instructs the cells to produce dystrophin is a very long molecule that is particularly vulnerable to alterations. About 10% to 15% of DMD cases involve a nonsense mutation that prematurely stops translation of the dystrophin gene. But a team of scientists from the University of Pennsylvania School of Medicine in Philadelphia, the University of Massachusetts Medical School in Worcester, and PTC Therapeutics, Inc, in South Plainfield, NJ, have developed a drug that allows the ribosome to read through these genetic errors and produce dystrophin, yet still obey normal signals to stop translation. Recent tests of the drug, PTC124, in mice with a DMD-like disorder have shown it can lead to the production of enough dystrophin to restore muscle function (Welch EM et al. Nature. 2007;447[7140]:87-91). H. Lee Sweeney, PhD, professor and chair of the department of physiology at the University of Pennsylvania School of Medicine, explained that the advantage of PTC124 over stem cell–based therapies or other gene therapy approaches is that it is a small molecule that could easily be delivered to muscles throughout the body. Additionally, there is a large margin between the dose of the drug necessary to restore muscle function and the amount that would be toxic, so delivering a dose that is both safe and effective should be feasible. Previously, the researchers had considered the therapeutic potential of gentamicin, which has a similar effect but is much more toxic.

Two novel missense mutations in the myostatin gene identified in Japanese patients with Duchenne muscular dystrophy

BackgroundMyostatin is a negative regulator of skeletal muscle growth. Truncating mutations in the myostatin gene have been reported to result in gross muscle hypertrophy. Duchenne muscular dystrophy (DMD), the most common lethal muscle wasting disease, is a result of an absence of muscle dystrophin. Although this disorder causes a rather uniform pattern of muscle wasting, afflicted patients display phenotypic variability. We hypothesized that genetic variation in myostatin is a modifier of the DMD phenotype.MethodsWe analyzed 102 Japanese DMD patients for mutations in the myostatin gene.ResultsTwo polymorphisms that are commonly observed in Western countries, p.55A>T and p.153K>R, were not observed in these Japanese patients. An uncommon polymorphism of p.164E>K was uncovered in four cases; each patient was found to be heterozygous for this polymorphism, which had the highest frequency of the polymorphism observed in the Japanese patients. Remarkably, two patients were found to be heterozygous for one of two novel missense mutations (p.95D>H and p.156L>I). One DMD patient carrying a novel missense mutation of p.95D>H was not phenotypically different from the non-carriers. The other DMD patient was found to carry both a novel mutation (p.156L>I) and a known polymorphism (p.164E>K) in one allele, although his phenotype was not significantly modified. Any nucleotide change creating a target site for micro RNAs was not disclosed in the 3' untranslated region.ConclusionOur results indicate that heterozygous missense mutations including two novel mutations did not produce an apparent increase in muscle strength in Japanese DMD cases, even in a patient carrying two missense mutations.

Duchenne and Becker muscular dystrophy: a molecular and immunohistochemical approach

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene. We studied 106 patients with a diagnosis of probable DMD/BMD by analyzing 20 exons of the dystrophin gene in their blood and, in some of the cases, by immunohistochemical assays for dystrophin in muscle biopsies. In 71.7% of the patients, deletions were found in at least one of the exons; 68% of these deletions were in the hot-spot 3' region. Deletions were found in 81.5% of the DMD cases and in all the BMD cases. The cases without deletions, which included the only woman in the study with DMD, had dystrophin deficiency. The symptomatic female carriers had no deletions but had abnormal dystrophin distribution in the sarcolemma (discontinuous immunostains). The following diagnoses were made for the remaining cases without deletions with the aid of a muscle biopsy: spinal muscular atrophy, congenital myopathy; sarcoglycan deficiency and unclassified limb-girdle muscular dystrophy. Dystrophin analysis by immunohistochemistry continues to be the most specific method for diagnosis of DMD/BMD and should be used when no exon deletions are found in the dystrophin gene in the blood.