Duchenne/Becker muscular dystrophy: A report on clinical,biochemical, and genetic study in Gujarat population,India

Objective:

In India, various groups have studied different regions to find out deletion pattern of dystrophin gene. We have investigated its deletion pattern among Duchenne/Becker muscular dystrophy (D/BMD) patients across Gujarat. Moreover, in this study we also correlate the same with reading frame rule. However, we too consider various clinicopathological features to establish as adjunct indices when deletion detection fails.

Materials and Methods:

In this pilot study, a total of 88 D/BMD patients consulting at our centers in Gujarat, India were included. All patients were reviewed on basis of their clinical characteristics, tested by three primer sets of 10-plex, 9-plex, and 7-plex polymerase chain reaction (PCR) for genetic analysis; whereas, biochemical indices were measured using automated biochemical analyzers.

Results:

The diagnosis of D/BMD was confirmed by multiplex-PCR (M-PCR) in D/BMD patients. A number of 65 (73.86%) out of 88 patients showed deletion in dystrophin gene. The exon 50 (58.46%) was the most frequent deletion found in our study. The mean age of onset of DMD and BMD was 4.09 ± 0.15 and 7.14 ± 0.55 years, respectively. In patients, mean creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and myoglobin levels were elevated significantly (P < 0.05) in comparison to controls. Addition to CPK, LDH and myoglobin are good adjunct when deletion detection failed. These data are further in accordance with world literature when correlated with frame rule.

Conclusion:

The analysis has been carried out for the first time for a total of 88 D/BMD patients particularly from Gujarat, India. More research is essential to elucidate specific mutation pattern in association with management and therapies of proband.     

干细胞移植治疗Duchenne型肌营养不良症的研究进展

Duchenne型肌营养不良症（duchenne muscular dystrophy，DMD）是最常见的X连锁隐性遗传性肌病，由位于Xp21的抗肌萎缩蛋白（dystrophy）基因突变引起dystrophy完全或部分缺失所致。该病主要见于男孩，发病率约3／10万活男婴。女性为致病基因携带者，所生男孩50％发病。患者一般3～5岁发病，主要表现为全身骨骼肌进行性无力、萎缩和小腿腓肠肌假性肥大。随着病情逐渐加重，12岁丧失行走能力，20岁左右因呼吸肌萎缩、无力，呼吸循环衰竭而死亡，迄今尚无有效的治疗方法。对症治疗、支持治疗、药物治疗、物理疗法和矫形治疗虽可预防及适度改善关节的畸形和挛缩，却未能有效的阻止病程的进展。对进行性肌营养不良患者而言，基因治疗和干细胞移植可望成为有效的治疗方法。近年来不少学者用骨髓或脐血干细胞移植治疗DMD模型鼠，其病理、生理、生化、抗肌萎缩蛋白表达和运动功能均有改善。     

Exon‐skipping therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the DMD gene for which no mutation‐targeted therapy has been available thus far. However, exon‐skipping mediated by antisense oligonucleotides (AOs), which are short single‐strand DNAs, has considerable potential for DMD therapy, and clinical trials in DMD patients are currently underway. This exon‐skipping therapy changes an out‐of‐frame mutation into an in‐frame mutation, aiming at conversion of a severe DMD phenotype into a mild phenotype by restoration of truncated dystrophin expression. Recently, stable and less‐toxic AOs have been developed, and their higher efficacy was confirmed in mice and dog models of DMD. In this review, we briefly summarize the genetic basis of DMD and the potential and perspectives of exon skipping as a promising therapy for this disease.     

Myoblast transplantation between monozygotic twin girl carriers of Duchenne muscular dystrophy

Monozygotic twin girls, both carriers of Duchenne muscular dystrophy, only one a severe symptomatic carrier and the other asymptomatic due to opposite lyonization, were studied. Myoblast clones were obtained from a muscle biopsy of the asymptomatic carrier. PCR amplification showed that most (94%) of these clones produced normal dystrophin mRNA. Roughly 704 million myoblasts were produced from 119 clones. These myoblasts were transplanted into the extensor carpi radialis (ECR) and in the biceps of one arm of the manifesting carrier while the other arm acted as the control. The strength of the patient was evaluated in a series of pre- and post-tests and a biopsy was obtained about 1 yr after the transplantation. The myoblast injections produced a significant force gain (12%–31%) in wrist extension but no force gain for elbow flexion. Muscle biopsies on the injected and control muscles obtained 1 yr after the injections showed only a small increase in the number of dystrophin positive fibers and the presence of numerous small type II fibers. The small beneficial effect of this transplantation cannot be attributed to immune problems, the donor and the recipient being identical twins, but may be due to a low level of spontaneous muscle regeneration.     

Detection of deletions within the dystrophin gene in Polish families affected with Duchenne/Becker muscular dystrophy

DNA analysis was performed in 190 cases of Duchenne and Becker muscular dystrophies (DMD/BMD), including 150 cases with DMD and 40 cases with BMD, using Southern blotting and PCR multiplex techniques with application of 25 pairs of primers. Deletions in the overall material were found in 109 cases: 81 (54%) in patients with DMD and 28 (70%) in patients with BMD. All the deletions in DMD were out of frame with the exception of two cases, whereas in BMD all the deletions but two were in frame. Junction fragments were detected in 12 cases of DMD. In five cases duplications were found: four in patients with DMD and one in a patient with BMD.     

Origins and early descriptions of "Duchenne muscular dystrophy"

One of the seminal events in the history of neurology was the identification of primary diseases of muscle and their separation from diseases in which muscle weakness was secondary to injury involving the anterior horns of the spinal cord ("progressive muscular atrophy"). Not surprisingly, one of the first groups of primary muscle diseases to be satisfactorily characterized belonged to what would today be classified as muscular dystrophies. Pride of place in this group belongs to Duchenne muscular dystrophy (DMD). DMD's primacy as the first well-characterized muscular dystrophy was due both to the fact that it is relatively common, as well as to the clinically striking feature, apparent in many cases, of apparent paradoxical enlargement of severely weakened muscles ("pseudo-hypertrophy"). This review traces the historical roots of DMD in the 19th century, from the early papers by Conte, Bell, Partridge, and Meryon through the classic monographs by Duchenne and Gowers. In addition, the first American contributions to DMD are reviewed, including those by Pepper, Hammond, and S. Weir Mitchell. Many of the original papers describing this disease are now unavailable outside of major medical libraries, and several important contributions, excepting those of Duchenne, which are recognized eponymously, are now virtually forgotten.     

序贯式干细胞移植治疗Duchenee型肌营养不良症

背景：Duchenne型肌营养不良症是一种累及肌肉系统的致死性遗传疾病,迄今尚无有效治疗方法。近年来学者们针对干细胞治疗Duchenne型肌营养不良症进行了基础研究和动物实验,在此基础上又进行了有意义的临床试验。 目的：观察Duchenne型肌营养不良症患儿接受序贯式干细胞移植治疗前后,其运动功能、肌细胞修复与再生、抗肌萎缩蛋白表达和缺失基因替代的变化,评价治疗的可行性和安全性。 方法：于2009-05应用序贯式干细胞移植治疗1例8岁男性Duchenne型肌营养不良症患儿,多重连接探针扩增方法基因分析13外显子缺失。序贯式干细胞移植,即依次行脐带间充质干细胞经静脉内移植-肌肉内移植-单倍体异基因造血干细胞移植。定期检测血清酶学变化、供者HLA植入证据、缺陷基因表达、肌细胞膜抗肌萎缩蛋白表达、运动功能改善情况。 结果与结论：序贯式干细胞移植治疗Duchenne型肌营养不良症,可使缺失基因替代,肌细胞膜dystrophin阳性表达,血清酶学显著降低,进一步提高运动功能。可阻止Duchenne型肌营养不良症患儿疾病进展,有望获得持续性改善。     

Inefficient dystrophin expression after cord blood transplantation in Duchenne muscular dystrophy

We report a boy who received two allogeneic stem cell transplantations from umbilical cord donors to treat chronic granulomatous disease (CGD). The CGD was cured after the second transplantation, but 2.5 years later he was diagnosed with Duchenne muscular dystrophy (DMD). Examinations of his DNA, muscle tissue, and myoblast cultures derived from muscle tissue were performed to determine whether any donor dystrophin was being expressed. The boy was found to have a large‐scale deletion on the X chromosome that spanned the loci for CYBB and DMD. The absence of dystrophin led to muscle histology characteristic of DMD. Analysis of myofibers demonstrated no definite donor cell engraftment. This case suggests that umbilical cord–derived hematopoietic stem cell transplantation will not be efficacious in the therapy of DMD without additional interventions that induce engraftment of donor cells in skeletal muscle. Muscle Nerve, 2010     

Duchenne muscular dystrophy: an updated review of common available therapies

Background and purpose: Duchenne muscular dystrophy (DMD) is a lethal progressive pediatric muscle disorder and genetically inherited as an X-linked disease that caused by mutations in the dystrophin gene. DMD leads to progressive muscle weakness, degeneration, and wasting; finally, follows with the premature demise in affected individuals due to respiratory and/or cardiac failure typically by age of 30. For decades, scientists tried massively to find an effective therapy method, but there is no absolute cure currently for patients with DMD, nevertheless, recent advanced progressions on the treatment of DMD will be hopeful in the future. Several promising gene therapies are currently under investigation. These include gene replacement, exon skipping, suppression of stop codons. More recently, a promising gene editing tool referred to as CRISPR/Cas9 offers exciting perspectives for restoring dystrophin expression in patients with DMD. This review intents to briefly describe these methods and comment on their advances. Since DMD is a genetic disorder, it should be treated by replacing the deficient DMD copy with a functional one. However, there are different types of mutations in this gene, so such therapeutic approaches are highly mutation specific and thus are personalized. Therefore, DMD has arisen as a model of genetic disorder for understanding and overcoming of the challenges of developing personalized genetic medicines, consequently, the lessons learned from these approaches will be applicable to many other disorders.

Conclusions: This review provides an update on the recent gene therapies for DMD that aim to compensate for dystrophin deficiency and the related clinical trials.     

脐带间充质干细胞治疗Duchenne型肌营养不良3例报告

Due to their relative abundance,stable biological properties and excellent reproductive activity,umbilical cord mesenchymal stem cells have previously been utilized for the treatment of Duchenne muscular dystrophy,which is a muscular atrophy disease.Three patients who were clinically and pathologically diagnosed with Duchenne muscular dystrophy were transplanted with umbilical cord mesenchymal stem cells by intravenous infusion,in combination with multi-point intramuscular injection.They were followed up for 12 months after cell transplantation.Results showed that clinical symptoms significantly improved,daily living activity and muscle strength were enhanced,the sero-enzyme,electromyogram,and MRI scans showed improvement,and dystrophin was expressed in the muscle cell membrane.Hematoxylin-eosin staining of a muscle biopsy revealed that muscle fibers were well arranged,fibrous degeneration was alleviated,and fat infiltration was improved.These pieces of evidence suggest that umbilical cord mesenchymal stem cell transplantation can be considered as a new regimen for Duchenne muscular dystrophy.     

Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule

The severe Duchenne and milder Becker muscular dystrophy are both caused by mutations in the DMD gene. This gene codes for dystrophin, a protein important for maintaining the stability of muscle-fiber membranes. In 1988, Monaco and colleagues postulated an explanation for the phenotypic difference between Duchenne and Becker patients in the reading-frame rule: In Duchenne patients, mutations induce a shift in the reading frame leading to prematurely truncated, dysfunctional dystrophins. In Becker patients, in-frame mutations allow the synthesis of internally deleted, but largely functional dystrophins. Currently, over 4700 mutations have been reported in the Leiden DMD mutation database, of which 91% are in agreement with this rule. In this study we provide an update of the mutational variability in the DMD gene, particularly focusing on genotype-phenotype correlations and mutations that appear to be exceptions to the reading-frame rule.     

Heterogeneity of dystrophin expression in patients with Duchenne and Becker muscular dystrophy

Summary This report documents the results of an integrated biochemical and immunocytochemical investigation into the expression of dystrophin (the protein product of the Duchenne muscular dystrophy gene) in muscle biopsies from 226 patients. It is the first study in which dystrophin has been analysed on blots and on tissue sections in such a large number of patients using the same (monoclonal) antibody. The 140 patients with Xp21 muscular dystrophy who were included in this study represent a continuous spectrum of disease severity and this range was reflected in the heterogeneity of dystrophin expression which was observed with respect to abundance, size and the pattern of tissue localisation. Approximately 40% of biopsies obtained from patients diagnosed as having Duchenne muscular, dystrophy (DMD) contained isolated clearly positive fibres and a further 20% had very weak labelling on a large number of fibres. Biopsies from patients with Becker muscular dystrophy (BMD) showed labelling patterns which varied from weak labelling on the majority of fibres to clear labelling on all fibres. Typically, however, there was inter-and intra-fibre variation in labelling intensity. Approximately 85% of the 52 BMD and 54 DMD patients who had unequivocal labelling on blots demonstrated a protein of abnormal size. The remaining 15% had a protein of normal size but reduced abundance. Overall, the estimated abundance of dystrophin correlated well with clinical assessments of the disease severity expressed in patients: We conclude that dystrophin analysis is an essential and dependable technique for the differential diagnosis of patients with Xp21 muscular dystrophy.Supported by the University of Newcastle-upon-Tyne Research Committee, the Muscular Dystropy Group of Great Britain and the Medical Research Council     

Gene and cell‐mediated therapies for muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder that affects 1 in 3,500 boys. Despite years of research and considerable progress in understanding the molecular mechanism of the disease and advancement of therapeutic approaches, there is no cure for DMD. The current treatment options are limited to physiotherapy and corticosteroids, and although they provide a substantial improvement in affected children, they only slow the course of the disorder. On a more optimistic note, more recent approaches either significantly alleviate or eliminate muscular dystrophy in murine and canine models of DMD and importantly, many of them are being tested in early phase human clinical trials. This review summarizes advancements that have been made in viral and nonviral gene therapy as well as stem cell therapy for DMD with a focus on the replacement and repair of the affected dystrophin gene. Muscle Nerve 47: 649–663, 2013     

Duchenne muscular dystrophy: a study of wrist and hand function

The wrist and hands of 18 Duchenne muscular dystrophy (DMD) patients were assessed for abnormalities. The subjects were divided into three groups by age. Flexion and ulnar deviation contractures of the wrist began in the youngest age group, 8-14 years. Other abnormalities, present in all age groups, included extrinsic and intrinsic digital muscle shortness, boutonniere and swan neck deformities, and hyperextension of the digital interphalangeal joints. Pain found with passive proximal interphalangeal joint flexion has not been reported previously. This study supports the importance of early assessment of the wrist and hand in DMD and suggests intervention techniques to possibly retard the deforming process.     

Ion channels in a skeletal muscle cell line from a Duchenne muscular dystrophy patient

A cell Iine (RCDMD), derived from a muscle biopsy taken from a 7-yearold patient with Duchenne muscular dystrophy (DMD), was established in vitro using conditioned media from the UCHT1 thyroid cell line as described elsewhere (Biochim Biophys Acta 1992;1134:247–255). Unlike other cell lines established by the same procedure, RCDMD cells were highly refractory to transformation and the resulting cell line grew slowly with a doubling time of approximately 72 h. Further, cells continue to grow after more than 20 doublings and 15 passages. Some of the characteristics of the cell line include lack of reaction with antidystrophin antibodies and the presence of receptors for the dihydropyridine PN200-110 (K_d) = 0.3 ± 0.05 nmol/L and B_max = 1.06 ± 0.03 pmol/mg protein and for α-bungarotoxin (K_d = 1.02 ± 0.17 nmol/L and B_max = 4.2 ± 0.37 pmol/mg protein). Patch clamped cells in the voltage clamp configuration lack ion currents when growing in complete medium with high serum, but they can be induced to differentiate by serum deprivation and addition of hormones and trace elements. After 5 days in differentiating medium, noninactivating, delayed rectifier potassium currents are seen. At day 12, A-type, inactivating potassium currents as well as transient inward currents are seen. In conditions in which sodium and potassium currents are absent, a very fast activating and fast inactivating calcium current was evident. The cell line offers the possibility of studying cellular mechanisms in the pathophysoplogy of DMD. © 1994 John Wiley & Sons, Inc.     

Myoblast implantation in Duchenne muscular dystrophy: The San Francisco study

We evaluated myoblast implantation in 10 boys with Duchenne muscular dystrophy (DMD) and absent dystrophin (age 5–10 years) who were implanted with 100 million myoblasts in the anterior tibial muscle of one leg and placebo in the other. Cyclosporine (5 mg/kg/day) was administered for 7 months. Pre- and postimplantation (after 1 and 6 months) muscle biopsies were analyzed. Force generation (tetanic tension and maximum voluntary contraction) was measured monthly in a double-blind design. There was increased force generation in both legs of all boys, probably due to cyclosporine. Using the polymerase chain reaction, evidence of myoblast survival and dystrophin mRNA expression was obtained in 3 patients after 1 month and in 1 patient after 6 months. These studies suggest a salutary effect of cyclosporine upon muscular force generation in Duchenne muscular dystrophy; however, myoblast implantation was not effective in replacing clinically significant amounts of dystrophin in DMD muscle. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 469–478, 1997.     

Molecular analysis of the duchenne muscular dystrophy gene in patients with becker muscular dystrophy presenting with dilated cardiomyopathy

Molecular analysis of the Duchenne muscular dystrophy (DMD) gene was performed on 4 unrelated patients with Becker muscular dystrophy (BMD) presenting with dilated cardiomyopathy. Two patients with a deletion involving exon 1 were quite unique in that they developed fatal myocardial involvement in their teens, despite the absence of significant muscular weakness. The deletion found in these patients comprised the 3′-end of exon 1 and the greater part of intron 1. Two other patients with a deletion of exon 47 showed progressive muscular atrophy and weakness; they were considered to be typical BMD in both clinical features and the type of gene deletion. We speculate that a deletion around exon 1 may severely damage the expression and/or the function of dystrophin selectively in cardiac muscle, but not in skeletal muscle. © 1993 John Wiley & Sons, Inc.