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     

肌营养不良蛋白在假肥大肌营养不良症肌组织中的表达研究

目的检测假肥大肌营养不良症肌组织中肌营养不良蛋白（ｄｙｓｔｒｏｐｈｉｎ）的表达。方法用针对ｄｙｓｔｒｏｐｈｉｎ棒状区第１５～１８重复区域的多克隆抗血清Ａｎｔｉ５～７，对２２例Ｄｕｃｈｅｎｎｅ型（ＤＭＤ）和４例Ｂｅｃｋｅｒ型肌营养不良症（ＢＭＤ）患者及１１例无神经肌肉疾病的急诊外伤患者（作为对照）的肌组织进行免疫组化分析。结果在对照组肌细胞中ｄｙｓｔｒｏｐｈｉｎ存在着可达检测水平的表达，并特异地定位于肌细胞膜上。１９例ＤＭＤ没有可达检测水平的ｄｙｓｔｒｏｐｈｉｎ表达，３例ＤＭＤ存在着ｄｙｓｔｒｏｐｈｉｎ表达。４例ＢＭＤ肌细胞膜上则呈现出斑片状、不连续ｄｙｓｔｒｏｐｈｉｎ弱阳性表达。结论ｄｙｓ－ｔｒｏｐｈｉｎ的缺乏是造成ＤＭＤ／ＢＭＤ表型的基本生化因素，此方法为临床上对ＤＭＤ／ＢＭＤ患者作出确诊提供了直接的特异生化测试指标。     

Developmental lag of visuospatial attention in Duchenne muscular dystrophy

Children with Duchenne muscular dystrophy (DMD) present a specific deficit of voluntary attention but to date there has been no clear characterization of their attentional skills. The present study investigated the hypothesis that DMD patients present deficits of both voluntary and automatic visuospatial attention systems and that their performance could be equivalent to that of younger healthy males. Twenty males (mean age 10 years) with diagnosis of DMD, 20 age-matched healthy males (10 years 3 months) and 20 healthy younger males (7 years 6 months) were required to perform two visuospatial attention tasks: voluntary and automatic. In the voluntary task, the performance of the DMD group was significantly worse than that of the age-matched group, and equal to that of the younger controls. In the automatic attention task also, the performance of the DMD patients was less efficient than that of the age-matched controls and equal to that of the younger children. This study supports the previous report of voluntary attention deficit in DMD and extends the evidence to include also an automatic attention system deficit. The development level of attention in DMD patients is below that expected for their age and corresponds to a delay of about three years.     

A manifesting carrier of Duchenne muscular dystrophy with severe myocardial symptoms

Summary A 42-year-old so-called manifesting carrier of Duchenne muscular dystrophy (DMD), whose first complaints were severe myocardial symptoms, is described. Immunohistochemical study using anti-dystrophin antiserum and analysis of cloned segments of X chromosome DNA were performed. Her two sons and one of her brothers appear to have had the same disease. She was admitted to hospital complaining of dyspnoea, back pain and palpitations and was first diagnosed as having myocardial infarction. However, this diagnosis was excluded. The echocardiogram showed diffuse abnormalities of myocardial function. Serum enzymes were increased. Minimal weakness and decreased deep tendon reflexes were detected in her left lower extremity. Muscle biopsy revealed a small number of necrotic fibres. Immunohistochemical study using anti-dystrophin antiserum showed a mosaic pattern of the surface membrane. Analysis of cloned segments of X chromosome DNA from the patient and her son showed the XmnI(Asp) alleles of pERT 87-15 and the TaqI alleles of pERT 87-8 in both patients.     

Molecular deletion patterns in Duchenne and Becker muscular dystrophy patients from KwaZulu Natal

There exists much phenotypic heterogeneity in Duchenne muscular dystrophy and its allelic variant, Becker muscular dystrophy. The molecular findings on 53 patients with Duchenne and 15 patients with Becker type muscular dystrophy in KwaZulu Natal, South Africa are reported. Multiplex PCR was performed using primers targeting 18 hot-spot exons throughout the dystrophin gene. Analysis of the multiplex PCR data revealed that 39/68 (57.0%) patients included in the study showed a deletion (33 DMD and 6 BMD patients). Twenty-five patients were Black, 4 were White and 10 were Indian. Using the Chamberlain and Beggs multiplex PCR assays, the region of the genome most frequently affected by a deletion includes exons 47-51. The distal region of the dystrophin gene was most frequently affected by the deletion in both Black and Indian patients. There were too few White patients for conclusions to be drawn concerning the most frequently affected part of the gene. Although the numbers are insufficient to determine whether ethnic differences are present, the Chamberlain and Beggs multiplex PCR assays detect deletions with the same frequency in South African DMD/BMD patients as that reported in the literature.     

Screening of gene deletions by polymerase chain reaction in Japanese patients with Duchenne muscular dystrophy

Summary Gene deletions were screened in 49 Japanese Duchenne muscular dystrophy patients from 43 families, using the polymerase chain reaction. Enzymatic amplification was carried out on six regions prone to deletion. Fifteen of 43 families (33%) had gene deletions in at least one of the six regions. This frequency was almost the same as that previously reported in Caucasians. The mid-part of the dystrophin gene was the location most frequently deleted. The frequency of deletion of the region encompassing exon 45 was higher in Japanese families (18.4%) than in Caucasians.     

Dystrophin and dystrophin-related protein in the central nervous system of normal controls and Duchenne muscular dystrophy

To clarify the localization and characterization of dystrophin and dystrophin-related protein (DRP) in the human central nervous system (CNS), we carried out immunoblotting and immunostaining studies using three region-specific anti-dystrophin and one anti-DRP antibodies. With immunostaining, punctate immuno-reactivity of dystrophin was seen along the cell bodies and dendrites of the cerebral cortical neurons and cerebellar Purkinje cells in the normal controls autopsied. By contrast, dystrophin was not detected at all in the CNS of Duchenne muscular dystrophy (DMD) patients with intellectual disturbance. Immunoreactivity of DRP was observed in the vascular walls of both normal and DMD brains, but not in the neuronal cells. Compensatory increase of DRP was not noted in DMD brains. This study suggests that in DMD the brain-type dystrophin originally present in neurons is absent and may be related to the intellectual disturbance.Supported by a grant (2-A) from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan     

自噬在Duchenne型肌营养不良中的研究

目的检测Duchenne型肌营养不良症(DMD)患者骨骼肌中LC3和p62的表达情况,分析自噬在DMD骨骼肌细胞坏死中的作用。方法收集2008年1月~2015年5月在我院就诊的病理确诊为DMD的患者(DMD组,81例),另以怀疑为肌病,但肌肉病理未见明显病变者为对照组(6例)。所有入选者均行心肌酶学、肌电图、骨骼肌活检常规组织学和酶学染色、抗dystrophin-N,-C,-R和抗dysferlin免疫组织化学染色。检测其中6例DMD患者及对照组骨骼肌中LC3和p62的表达。结果 81例DMD患者均为男性,起病年龄(4.60±2.35)岁,首发症状多以双下肢起病为主。血清肌酸激酶值的高峰出现在患者年龄的6~8岁,随着肌细胞明显坏死,肌酸激酶水平下降,但仍高于正常。在DMD患者骨骼肌中,组织病理均示典型肌营养不良改变。半定量Western blot提示DMD患者骨骼肌中LC3-II的表达降低,而p62表达显著升高。结论自噬功能障碍可能参与了DMD骨骼肌细胞坏死的病理生理过程。     

Scoliosis in Duchenne muscular dystrophy (DMD)

Scoliosis is a frequent complication in the non-ambulant patient with Duchenne muscular dystrophy (DMD). Weakness of the paraspinal muscles leads to trunk and body positional changes facilitating the development of a progressive collapsing scoliosis which inevitably interferes with comfortable sitting and may exacerbate deteriorating respiratory function. The recommended international standard of care for management of DMD includes strategies to prolong ambulation which may delay the onset of scoliosis. In the non-ambulant child there should be regular monitoring for scoliosis and, when present, surgical treatment should undertaken at an early stage. Careful multi-disciplinary pre-operative assessment and peri-operative care are essential.     

IQ, prognosis and Duchenne muscular dystrophy

The verbal scales (VS-IQ) of the IQs of 25 boys with Duchenne muscular dystrophy, the total population in Western Australia older than five yrs, were recorded. All the results of those 20 yrs of age and older lay within the normal range. The mean VS-IQ of those less than 20 yrs and less than 18 yrs is compared with the mean VS-IQ of those 20 yrs and older, and 18 yrs and older, respectively. The differences between the groups are statistically significant, and it is postulated that an active management programme has revealed a population of boys with normal intelligence who have an increased chance of prolonged survival.     

Deletion screening in patients with Duchenne muscular dystrophy

Summary DNA of 35 patients with Duchenne muscular dystrophy (DMD) from 27 unrelated families from the northern part of GDR, Czechoslovakia and Hungary were analysed by means of 9 genomic probes and cDNA probes Cf 23a and Cf 56a, which detect exons of the central part of the DMD gene. Of the unrelated DMD patients, 63% have deletions for one or more intragenic and/or cDNA probes and 33% have deletions for genomic probes, mostly for pERT 87 (15%) and P 20 (15%). 48% of the DMD patients have deletions for one or more exon regions detected by Cf 56a and Cf 23a. The deletions were mapped. The genomic probe P 20 and the distal part of the cDNA probe Cf 23a detected the same part in the centre of the DMD gene. The deletions are heterogeneous in size and extent. In patients of the same family, identical deletions were detected in the DMD gene. The detection of deletions is useful for prenatal diagnosis and carrier detection.     

Cardiac involvement in Fukuyama muscular dystrophy is less severe than in Duchenne muscular dystrophy

Background

One of the main complications in patients with muscular dystrophies is cardiac dysfunction. The literature on cardiac involvement in patients with Fukuyama congenital muscular dystrophy (FCMD) is limited.

Assessment of locomotor function in young boys with Duchenne muscular dystrophy

Thirty-three young boys (mean age 3.42 years) with Duchenne muscular dystrophy (DMD) and 21 normal control boys (mean age 3.51 years) were studied prospectively to determine whether it is possible to objectively assess locomotor function in young boys with DMD so that they can be included in treatment trials. An initial reproducibility study using a hand-held myometer demonstrated that this method was not useful. The Hammersmith Motor Ability Score demonstrated an increase in developmental abilities with age which was markedly different from normal. The locomotor quotient of the Griffiths' Scales demonstrated a deterioration of quotient scores and is a useful method of assessment that could be used in treatment trials involving young boys with DMD. Sample size planning for treatment trials is discussed.     

Challenges in Duchenne muscular dystrophy

The last seven years has witnessed an explosion in our understanding of the muscular dystrophies. In the early 1980s, prenatal diagnosis of Duchenne muscular dystrophy was developed. The cloning of the gene, in 1996, resulted in a better understanding of the disease process and led to the identification of a novel complex at the membrane. This information led to the cloning of other genes responsible for the autosomally inherited dystrophies. As we approach the millenium, the challenge is shifting to the development of therapy of these diseases. This review, in honour of Professor Alan Emery, explains how these advances have had an impact in the clinical management of pateints and the promise the progress holds for the future.     

Muscle hypertrophy in Duchenne muscular dystrophy

Summary In order to investigate the pathological basis of muscle hypertrophy in Duchenne dystrophy, 9 biopsy specimens of the lateral gastrocnemius and 7 of the vastus lateralis were compared. All patients had calf hypertrophy and normal strength in gastrocnemius-soleus, whereas the quadriceps biopsied were all atrophied and weak. The patients' ages ranged from 4 to 11 years. The pathological and histochemical changes were assessed semi-quantitatively. Comparison of the gastrocnemius and quadriceps groups showed that the number of hypercontracted fibres, the degree of endomysial fibrosis and the degree of fat infiltration were significantly higher in the quadriceps. The fibre type differentiation was better in the gastrocnemius group. The mean fibre diameter was above normal in all gastrocnemius biopsies and showed no increase with age. In the quadriceps, fibre hypertrophy was found early in the disease but had changed into fibre atrophy in the three oldest patients. When present, fibre hypertrophy involved both fibre types. The amount of fat-fibrosis per unit area was increased in both groups, but more severely so in the quadriceps. These results indicate that there is no true muscle hypertrophy in the gastrocnemius, in which the fat-fibrosis component was increased in all patients and that the dystrophic process is more active in the quadriceps. The finding of persistent fibre hypertrophy in the gastrocnemius is discussed with respect to the postural abnormalities observed in the lower limbs in Duchenne dystrophy.     

Reliability of the Performance of Upper Limb assessment in Duchenne muscular dystrophy

The Performance of Upper Limb was specifically designed to assess upper limb function in Duchenne muscular dystrophy. The aim of this study was to assess (1) a cohort of typically developing children from the age of 3 years onwards in order to identify the age when the activities assessed in the individual items are consistently achieved, and (2) a cohort of 322 Duchenne children and young adults to establish the range of findings at different ages. We collected normative data for the scale validation on 277 typically developing subjects from 3 to 25 years old. A full score was consistently achieved by the age of 5 years. In the Duchenne cohort there was early involvement of the proximal muscles and a proximal to distal progressive involvement. The scale was capable of measuring small distal movements, related to activities of daily living, even in the oldest and weakest patients. Our data suggest that the assessment can be reliably used in both ambulant and non ambulant Duchenne patients in a multicentric setting and could therefore be considered as an outcome measure for future trials.     

Functional evaluation of Duchenne muscular dystrophy: proposal for a protocol

A protocol for the evaluation of functional activities in subjects with Duchenne muscular dystrophy (DMD) was designed. The aim of our study was to define objective clinical criteria for the evaluation both of the clinical status of the patient and of the natural history of the illness itself. A protocol with such criteria is particularly necessary when testing the efficacy of treatment. 43 still-ambulant children with DMD between the ages of 3.10 yr and 10.4 yr were examined. Of this number 19 children were evaluated every 4 months over a period of 12 months; of these 14 formed part of a randomized double blind trial with L-carnitine (1.2–1.8 g/day) versus placebo.This work has been in part supported by a grant from the Dino and Enzo Ferrari Foundation for Muscular Dystrophy Research.     

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.     

Erythrocyte (Ca2+ + Mg2+)-ATPase activity and calcium homeostasis in Duchenne muscular dystrophy

Because the erythrocyte (RBC) in Duchenne's muscular dystrophy (DMD) is thought to be a suitable experimental paradigm for the sarcolemma, the RBC membrane-bound enzyme (Ca2+ + Mg2+)-ATPase has been investigated as to its relevance to abnormalities of calcium metabolism in DMD muscle. In this study, RBC (Ca2+ + Mg2+)-ATPase activity, intracellular calcium and potassium contents and complete hemogram were examined in 10 DMD patients and 16 age-matched controls. (Ca2+ + Mg2+)-ATPase activity was found elevated in the DMD RBC, consistent with reports from previous studies, but no abnormalities in intracellular calcium, potassium or hemograms were detected. It seems that although the (Ca2+ + Mg2+)-ATPase activity is changed, it bears no relevance to calcium homeostasis in DMD RBC. It is inferred that the increase in intramuscular calcium in DMD muscle, which is also found in other neuromuscular diseases, may be a non-specific finding in the diseased muscle and part of the final common pathway leading toward cellular degeneration and death.     

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。